WEBVTT 00:00:00.000 --> 00:00:03.310 00:00:03.310 --> 00:00:05.120 - Hello, everyone. 00:00:05.120 --> 00:00:10.730 Thank you for joining us in this unprecedented time. 00:00:10.730 --> 00:00:15.380 We started this JoVE series of protocols 00:00:15.380 --> 00:00:22.040 at about the same time we began updating the WHO laboratory 00:00:22.040 --> 00:00:24.740 techniques volumes. 00:00:24.740 --> 00:00:27.530 And our rationale at that time was ideally, 00:00:27.530 --> 00:00:32.479 in the future that all of, or at least 00:00:32.479 --> 00:00:38.870 most of or many of the protocols in the WHO laboratory 00:00:38.870 --> 00:00:43.700 techniques book would eventually become visual experiments 00:00:43.700 --> 00:00:50.310 to assist with not only reading, but actual operational details. 00:00:50.310 --> 00:00:54.500 So our objectives today during this pandemic 00:00:54.500 --> 00:00:59.930 are to try and provide a glimpse into some of those for you, 00:00:59.930 --> 00:01:03.710 one, because of the limitations in travel, 00:01:03.710 --> 00:01:08.660 and the decrease in the opportunities 00:01:08.660 --> 00:01:12.830 for actual wet laboratories. 00:01:12.830 --> 00:01:18.170 Our second objective is for each of our presenters 00:01:18.170 --> 00:01:21.620 to update any of the techniques that they 00:01:21.620 --> 00:01:27.020 have presented in the JoVE format special rabies series. 00:01:27.020 --> 00:01:30.890 And thirdly, to place in perspective 00:01:30.890 --> 00:01:35.240 where their particular presentation fits in 00:01:35.240 --> 00:01:40.880 regards to rabies prevention, control, research, et cetera, 00:01:40.880 --> 00:01:46.940 as we recognize that we're already well into, in 2021, 00:01:46.940 --> 00:01:52.010 the ZBT, the Zero by 30, the elimination of human rabies 00:01:52.010 --> 00:01:55.910 caused by dogs and how these protocols may 00:01:55.910 --> 00:01:58.970 assist in that endeavor. 00:01:58.970 --> 00:02:03.170 But not only in regards to canine rabies, which 00:02:03.170 --> 00:02:05.610 is our major burden throughout the world, 00:02:05.610 --> 00:02:08.509 particularly in Asia and Africa, but we 00:02:08.509 --> 00:02:14.170 recognize that also threatens the progress in the Americas, 00:02:14.170 --> 00:02:16.450 particularly in Latin America, which 00:02:16.450 --> 00:02:20.680 serves as one of the greatest regional demonstrations 00:02:20.680 --> 00:02:24.310 that canine rabies can be eliminated. 00:02:24.310 --> 00:02:28.600 Obviously, that has been impacted by the COVID pandemic 00:02:28.600 --> 00:02:31.840 because surveillance is down. 00:02:31.840 --> 00:02:37.600 As surveillance is down, we know that the evaluation of programs 00:02:37.600 --> 00:02:38.830 falter. 00:02:38.830 --> 00:02:44.260 But not only surveillance, but also sometimes vaccination 00:02:44.260 --> 00:02:47.110 programs are also on hold. 00:02:47.110 --> 00:02:51.280 So that when we consider the impacts 00:02:51.280 --> 00:02:54.880 of the pandemic upon both surveillance as well as dog 00:02:54.880 --> 00:03:01.100 vaccination, cases are going to rise, as we see from the news, 00:03:01.100 --> 00:03:05.380 for example, in Arequipa, Peru, and in other parts 00:03:05.380 --> 00:03:07.540 of Latin America. 00:03:07.540 --> 00:03:11.560 And in those parts of the world that have already well achieved 00:03:11.560 --> 00:03:13.840 the elimination of canine rabies, 00:03:13.840 --> 00:03:16.840 we've had the epidemiological luxury 00:03:16.840 --> 00:03:21.040 of wildlife rabies discovery. 00:03:21.040 --> 00:03:23.440 And some of the opportunities that we'll 00:03:23.440 --> 00:03:25.420 have to talk about today are directly 00:03:25.420 --> 00:03:28.540 focused upon enhanced surveillance, which 00:03:28.540 --> 00:03:30.910 could be applied to canine rabies, 00:03:30.910 --> 00:03:35.980 but are of obvious importance to finding, detecting, preventing, 00:03:35.980 --> 00:03:38.770 and controlling wildlife rabies. 00:03:38.770 --> 00:03:41.890 And not the least of which are generated 00:03:41.890 --> 00:03:46.390 by bats, which are some of the global reservoirs 00:03:46.390 --> 00:03:48.660 for lyssaviruses. 00:03:48.660 --> 00:03:53.610 So I'm calling in from the Atlanta area. 00:03:53.610 --> 00:03:58.110 I wanted to say a happy equinox, whether you're 00:03:58.110 --> 00:04:00.760 in the north or south temperate zones. 00:04:00.760 --> 00:04:04.170 We'll have each of our presenters 00:04:04.170 --> 00:04:08.430 speak for about 10 minutes, introducing their topic, 00:04:08.430 --> 00:04:11.280 talking about any of the salient details, 00:04:11.280 --> 00:04:14.490 giving any operational updates. 00:04:14.490 --> 00:04:17.100 If you have questions, we have a couple 00:04:17.100 --> 00:04:20.760 of ways to resolve those. 00:04:20.760 --> 00:04:22.440 One, by raising your hand. 00:04:22.440 --> 00:04:24.810 You can type into the text box. 00:04:24.810 --> 00:04:28.140 And the presenter has the option of either answering 00:04:28.140 --> 00:04:31.320 the question if convenient when it comes up, 00:04:31.320 --> 00:04:34.000 or at the end of their session. 00:04:34.000 --> 00:04:37.860 We will have a short break at about halfway 00:04:37.860 --> 00:04:40.570 through the program. 00:04:40.570 --> 00:04:45.030 After that, we'll resume and questions either directly 00:04:45.030 --> 00:04:47.490 during or after each presentation, 00:04:47.490 --> 00:04:49.980 or if we have time, if our presenters, all 00:04:49.980 --> 00:04:52.410 being good professionals, stay on time, 00:04:52.410 --> 00:04:56.190 at the end for any wrap-up discussions, any questions that 00:04:56.190 --> 00:04:59.550 didn't get answered, or any questions 00:04:59.550 --> 00:05:03.810 the participants may have of their colleagues. 00:05:03.810 --> 00:05:05.250 We're happy to go ahead and start 00:05:05.250 --> 00:05:11.320 with our first presentation from Dr. Wei-Cheng Hsu, coming in 00:05:11.320 --> 00:05:14.440 from the Animal Health Research Institute, 00:05:14.440 --> 00:05:19.150 the Epidemiology Research Department in Taipei, 00:05:19.150 --> 00:05:23.230 and will be sharing with us issues of great importance, 00:05:23.230 --> 00:05:27.010 particularly in the Asian area, for the discovery 00:05:27.010 --> 00:05:30.850 of lyssaviruses associated with bats. 00:05:30.850 --> 00:05:31.608 Dr. Hsu. 00:05:31.608 --> 00:05:38.940 00:05:38.940 --> 00:05:40.240 You have the floor. 00:05:40.240 --> 00:05:43.270 I'm going to mute myself. 00:05:43.270 --> 00:05:44.635 And we don't hear-- 00:05:44.635 --> 00:05:47.335 [AUDIO OUT] 00:05:47.335 --> 00:05:59.570 00:05:59.570 --> 00:06:02.660 Perhaps try unmuting your microphone. 00:06:02.660 --> 00:06:03.920 Hello. 00:06:03.920 --> 00:06:05.830 Hello. 00:06:05.830 --> 00:06:07.233 Hi, that's-- Hi. 00:06:07.233 --> 00:06:07.900 We can hear you. 00:06:07.900 --> 00:06:08.690 Thank you. 00:06:08.690 --> 00:06:09.770 OK, thank you. 00:06:09.770 --> 00:06:10.270 Hello. 00:06:10.270 --> 00:06:12.460 I am Wei-Cheng Hsu from Taiwan. 00:06:12.460 --> 00:06:15.850 My talk is about standard operating procedure 00:06:15.850 --> 00:06:20.290 for lyssavirus surveillance of the bat population in Taiwan. 00:06:20.290 --> 00:06:30.680 00:06:30.680 --> 00:06:34.070 And this is my outline. 00:06:34.070 --> 00:06:36.620 And the introduction. 00:06:36.620 --> 00:06:39.860 Lyssaviruses do not take pathogens. 00:06:39.860 --> 00:06:44.660 And now we know we have in [INAUDIBLE] 00:06:44.660 --> 00:06:48.770 there are 16 species of lyssavirus 00:06:48.770 --> 00:06:56.550 and 3 more new lyssaviruses have been found recently. 00:06:56.550 --> 00:07:04.620 And this is the map of lyssavirus distribution. 00:07:04.620 --> 00:07:08.190 You can see the gray color. 00:07:08.190 --> 00:07:12.460 The gray color represent there are no surveillance data. 00:07:12.460 --> 00:07:16.220 00:07:16.220 --> 00:07:25.190 And in Taiwan, we have 35 species of bats. 00:07:25.190 --> 00:07:31.625 And bat diversity is very high in Taiwan. 00:07:31.625 --> 00:07:35.110 00:07:35.110 --> 00:07:43.960 And the SOP of lyssavirus is [INAUDIBLE] in Taiwan, 00:07:43.960 --> 00:07:47.680 including first our target animal, 00:07:47.680 --> 00:07:53.890 our dead bats, or bats dying of weakness, 00:07:53.890 --> 00:07:58.090 and this case will be submitted to our laboratory, 00:07:58.090 --> 00:08:03.580 and we will perform the necropsy and the sample collection, 00:08:03.580 --> 00:08:09.680 and we will do an FAT and RT-PCR at the same time, 00:08:09.680 --> 00:08:19.250 and we will run the Fujirebio and the Millipore 5100. 00:08:19.250 --> 00:08:22.010 These two conjugates. 00:08:22.010 --> 00:08:26.525 In RT-PCR, we will run two set of primers. 00:08:26.525 --> 00:08:29.105 00:08:29.105 --> 00:08:35.720 If either FAT or RT-PCR show the positive, we follow up, 00:08:35.720 --> 00:08:41.770 we will do a virus isolation, and phylogenetic analysis. 00:08:41.770 --> 00:08:47.140 In any fresh specimen, we will do 00:08:47.140 --> 00:08:49.480 a histopathology examination. 00:08:49.480 --> 00:08:52.450 00:08:52.450 --> 00:08:56.080 In Taiwan we are very lucky, because in Taiwan we 00:08:56.080 --> 00:09:00.640 have 22 animal disease control center, 00:09:00.640 --> 00:09:08.770 and we also have four bird conservation NGO, 00:09:08.770 --> 00:09:11.140 non-government organizations. 00:09:11.140 --> 00:09:22.370 So 22 ADCC and four NGO will send some bat carcass to us. 00:09:22.370 --> 00:09:30.120 And about the result, we start our lyssavirus surveillance 00:09:30.120 --> 00:09:36.300 in 2008, and in the first year we 00:09:36.300 --> 00:09:40.620 capture the health bat and euthanasia, 00:09:40.620 --> 00:09:45.030 and all case is negative. 00:09:45.030 --> 00:09:48.960 And after the first year, we just 00:09:48.960 --> 00:09:53.550 consulted with Dr. [INAUDIBLE],, and he gave us 00:09:53.550 --> 00:09:56.190 a very good suggestion. 00:09:56.190 --> 00:10:01.620 He think we need to start to do these kind of surveillance, 00:10:01.620 --> 00:10:02.550 so we change. 00:10:02.550 --> 00:10:08.490 We just collect dead bat. 00:10:08.490 --> 00:10:14.820 So we stop to do the live bat surveillance. 00:10:14.820 --> 00:10:21.690 In 2008 to 2015, we do the lyssavirus surveillance only 00:10:21.690 --> 00:10:26.600 by FAT. 00:10:26.600 --> 00:10:33.110 And after eight years of surveillance, 00:10:33.110 --> 00:10:41.420 we change our SOP. 00:10:41.420 --> 00:10:48.410 We do the FAT and the RT-PCR at the same time. 00:10:48.410 --> 00:10:51.800 And after we change our SOP, we start 00:10:51.800 --> 00:10:56.170 to find new novel lyssaviruses. 00:10:56.170 --> 00:11:00.230 00:11:00.230 --> 00:11:04.820 There are final novel lyssaviruses. 00:11:04.820 --> 00:11:08.570 Belongs to two species of bat in Taiwan. 00:11:08.570 --> 00:11:17.352 During 2016 to 2020, the positive rate 00:11:17.352 --> 00:11:28.190 of lyssavirus of the Japanese Pipistrelle is about 1%, 00:11:28.190 --> 00:11:32.540 and the positive rate of lyssaviruses 00:11:32.540 --> 00:11:36.950 in Mountain Noctule is about 100%, 00:11:36.950 --> 00:11:43.430 because this species we only got one sample, 00:11:43.430 --> 00:11:46.920 and this sample is positive. 00:11:46.920 --> 00:11:52.790 And you can see a blue color character and the red color 00:11:52.790 --> 00:11:54.260 character. 00:11:54.260 --> 00:11:58.970 The blue means the Japanese Pipistrelle. 00:11:58.970 --> 00:12:01.340 You can see four cases. 00:12:01.340 --> 00:12:06.740 And in all case, the location is different. 00:12:06.740 --> 00:12:13.990 And we also find one positive case of Mountain Noctule, 00:12:13.990 --> 00:12:19.045 and it's in a red color. 00:12:19.045 --> 00:12:23.830 00:12:23.830 --> 00:12:32.200 And the phylogenetic analysis show, in Japanese Pipistrelle, 00:12:32.200 --> 00:12:36.290 we just named it. 00:12:36.290 --> 00:12:44.370 It's a novel-- it's a new lyssavirus, 00:12:44.370 --> 00:12:50.440 and we named it as Taiwan bat lyssavirus type I. 00:12:50.440 --> 00:12:59.182 In the Mountain Noctule, after a phylogenetic analysis, 00:12:59.182 --> 00:13:03.120 it also new lyssaviruses. 00:13:03.120 --> 00:13:07.160 We named it Taiwan bat lyssavirus type II, 00:13:07.160 --> 00:13:09.460 and this is unpublished data. 00:13:09.460 --> 00:13:16.120 00:13:16.120 --> 00:13:20.480 You can see the genetic tree. 00:13:20.480 --> 00:13:26.000 Taiwan bat lyssavirus type I and type II are clustered together, 00:13:26.000 --> 00:13:28.880 and both belong to the [INAUDIBLE] group 1. 00:13:28.880 --> 00:13:32.160 00:13:32.160 --> 00:13:37.140 And as I mentioned before, we do RT-PCR, 00:13:37.140 --> 00:13:41.010 and the type I and type II Taiwan bat lyssavirus 00:13:41.010 --> 00:13:43.440 can work well with two primer. 00:13:43.440 --> 00:13:46.620 And this is our primer sets. 00:13:46.620 --> 00:13:52.890 But we found something stranger in Taiwan bat lyssavirus type 00:13:52.890 --> 00:14:03.425 I. Because if we do [INAUDIBLE] and if we scan 00:14:03.425 --> 00:14:05.360 we saw Fujirebio conjugate. 00:14:05.360 --> 00:14:07.890 00:14:07.890 --> 00:14:16.790 We got a negative result. But if we do the Millipore conjugate, 00:14:16.790 --> 00:14:21.170 we can find a positive signal. 00:14:21.170 --> 00:14:25.790 But type II bat lyssavirus, both conjugate works well. 00:14:25.790 --> 00:14:32.787 00:14:32.787 --> 00:14:39.570 In virus isolation, we found if we use the mouse neuroblastoma 00:14:39.570 --> 00:14:44.340 cell, MNA cell, it's almost impossible 00:14:44.340 --> 00:14:48.930 to isolate the Taiwan bat lyssavirus type II. 00:14:48.930 --> 00:14:55.170 So in Taiwan lyssavirus type II, we do a mouse inoculation. 00:14:55.170 --> 00:15:03.660 We [INAUDIBLE] three times, and after the mouse inoculation, 00:15:03.660 --> 00:15:08.220 we use the BHK cell to do the virus isolation. 00:15:08.220 --> 00:15:10.350 It still [INAUDIBLE] three times. 00:15:10.350 --> 00:15:12.930 00:15:12.930 --> 00:15:16.200 So after three times in the mouse and after three times 00:15:16.200 --> 00:15:22.920 the BHK cell, we can reach 100% infectivity. 00:15:22.920 --> 00:15:27.660 So it is very difficult to isolate the Taiwan bat 00:15:27.660 --> 00:15:31.450 lyssavirus type II. 00:15:31.450 --> 00:15:32.960 So, discussion. 00:15:32.960 --> 00:15:39.010 00:15:39.010 --> 00:15:46.960 So during our 10 more years of surveillance, 00:15:46.960 --> 00:15:52.300 we found these SOP can work well in Taiwan. 00:15:52.300 --> 00:15:58.585 We use this SOP, we found five positive lyssavirus cases. 00:15:58.585 --> 00:16:01.870 00:16:01.870 --> 00:16:06.868 We [INAUDIBLE] would find every lyssavirus 00:16:06.868 --> 00:16:10.060 has different characters. 00:16:10.060 --> 00:16:15.220 And a variation of lyssavirus antigen in a sample, 00:16:15.220 --> 00:16:18.760 recommend first two different conjugates 00:16:18.760 --> 00:16:21.430 should be used in FAT. 00:16:21.430 --> 00:16:27.190 Second, more than one primer set in the RT-PCR should be used. 00:16:27.190 --> 00:16:35.850 Third, the more detecting tools, the less false negativity. 00:16:35.850 --> 00:16:42.390 Nonetheless, cost performance ratio should be considered. 00:16:42.390 --> 00:16:45.030 00:16:45.030 --> 00:16:48.810 And how to find your own new lyssaviruses? 00:16:48.810 --> 00:16:54.050 00:16:54.050 --> 00:17:00.800 In my personal point of view, I would say be patient. 00:17:00.800 --> 00:17:06.680 Because, in our experience, we found our first lyssavirus 00:17:06.680 --> 00:17:10.084 case, we spent more than eight years. 00:17:10.084 --> 00:17:12.589 00:17:12.589 --> 00:17:15.950 Second, do the right things. 00:17:15.950 --> 00:17:19.160 We only collect dead or dying bats, 00:17:19.160 --> 00:17:22.579 and with cooperation with the animal disease control 00:17:22.579 --> 00:17:27.810 center and the NGO of bat conservation at the same time, 00:17:27.810 --> 00:17:31.760 so we can expand our surveillance area 00:17:31.760 --> 00:17:38.170 and we employment both FAT and RT-PCR. 00:17:38.170 --> 00:17:43.010 And we routinely do virus isolation. 00:17:43.010 --> 00:17:47.550 00:17:47.550 --> 00:17:49.830 Finally, the most important. 00:17:49.830 --> 00:17:53.280 you need to have a good luck. 00:17:53.280 --> 00:17:55.060 So that's my presentation. 00:17:55.060 --> 00:17:57.665 Thank you. 00:17:57.665 --> 00:18:01.000 - Thank you very much, Dr. Hsu. 00:18:01.000 --> 00:18:07.910 And we do have a question. 00:18:07.910 --> 00:18:12.590 One question is, could it be that, 00:18:12.590 --> 00:18:15.650 because you're an Island, that you 00:18:15.650 --> 00:18:19.340 have more apparent diversity of lyssaviruses 00:18:19.340 --> 00:18:22.380 compared to other locations? 00:18:22.380 --> 00:18:26.840 And they wonder why, for example, in the US 00:18:26.840 --> 00:18:31.480 there is such a low diversity, which I would disagree with, 00:18:31.480 --> 00:18:32.480 but they wanted to know. 00:18:32.480 --> 00:18:34.647 Dr. Hsu, do you have any thoughts on their question? 00:18:34.647 --> 00:18:37.562 00:18:37.562 --> 00:18:38.950 - Yes. 00:18:38.950 --> 00:18:43.730 I think it's a very good question, 00:18:43.730 --> 00:18:46.450 but I don't have the answer. 00:18:46.450 --> 00:18:50.950 And in Taiwan, from 2016 to 2020, 00:18:50.950 --> 00:18:57.880 almost every year we test about 100 cases, 00:18:57.880 --> 00:19:04.640 and the positive rate is just very close to 1% every year. 00:19:04.640 --> 00:19:06.730 So it's very strange. 00:19:06.730 --> 00:19:12.010 Very strange, a very high positive rate, but I 00:19:12.010 --> 00:19:12.880 don't know why. 00:19:12.880 --> 00:19:13.770 Thank you. 00:19:13.770 --> 00:19:16.750 - And do you have any human exposures 00:19:16.750 --> 00:19:19.561 to these lyssaviruses? 00:19:19.561 --> 00:19:25.180 - We do have some bat bite cases. 00:19:25.180 --> 00:19:32.680 The bat comes into the house of somebody, and bite the people, 00:19:32.680 --> 00:19:34.090 bite human. 00:19:34.090 --> 00:19:43.090 And until now, human contact cases 00:19:43.090 --> 00:19:47.350 will go to do the [INAUDIBLE],, and we will diagnose 00:19:47.350 --> 00:19:52.100 this bat in less than 24 hours. 00:19:52.100 --> 00:19:55.440 So until now we don't have human cases. 00:19:55.440 --> 00:19:57.100 - Excellent. 00:19:57.100 --> 00:20:00.470 Thank you very much for that presentation, 00:20:00.470 --> 00:20:04.720 and I hope that you won't have any human cases. 00:20:04.720 --> 00:20:06.760 You can stop sharing your screen, 00:20:06.760 --> 00:20:09.950 and we'll have our next presenter. 00:20:09.950 --> 00:20:12.110 Thank you. 00:20:12.110 --> 00:20:15.790 Our next presenter will be Dr. Ye Feng. 00:20:15.790 --> 00:20:19.090 Dr. Feng is an associate professor and epidemiologist 00:20:19.090 --> 00:20:21.970 at the National Reference Laboratory 00:20:21.970 --> 00:20:25.900 for Animal Rabies [INAUDIBLE]. 00:20:25.900 --> 00:20:27.940 Research interests focus on rabies 00:20:27.940 --> 00:20:32.530 epidemiology, diagnostic surveillance, and also 00:20:32.530 --> 00:20:35.980 responsible for training of individuals 00:20:35.980 --> 00:20:39.280 in China and other countries. 00:20:39.280 --> 00:20:41.500 Dr. Feng will be talking with us today 00:20:41.500 --> 00:20:44.980 about the evaluation of the universal nested reverse 00:20:44.980 --> 00:20:49.030 transcription polymerase chain reaction of the detection 00:20:49.030 --> 00:20:50.440 of lyssaviruses. 00:20:50.440 --> 00:20:51.990 Dr. Feng? 00:20:51.990 --> 00:20:53.370 - Hello, chairs. 00:20:53.370 --> 00:20:59.130 Thank you, and I'm afraid I can't [INAUDIBLE] 00:20:59.130 --> 00:21:06.178 because someone has flagged their [INAUDIBLE].. 00:21:06.178 --> 00:21:08.666 - Can you share your screen? 00:21:08.666 --> 00:21:12.920 - I'm afraid someone is still share-- 00:21:12.920 --> 00:21:14.460 - Ah. 00:21:14.460 --> 00:21:19.665 Dr. Hsu, have you stopped sharing your screen? 00:21:19.665 --> 00:21:30.860 00:21:30.860 --> 00:21:33.290 Dr. Hsu, can you hear me? 00:21:33.290 --> 00:21:36.230 Have you stopped sharing your screen? 00:21:36.230 --> 00:21:37.346 - OK. 00:21:37.346 --> 00:21:39.110 - Ah, now we'll try. 00:21:39.110 --> 00:21:45.410 00:21:45.410 --> 00:21:46.580 Thank you, Dr. Feng. 00:21:46.580 --> 00:21:47.590 The floor is yours. 00:21:47.590 --> 00:21:50.966 00:21:50.966 --> 00:21:56.340 - I'm Ye Feng, work site OIE reference laboratory in China. 00:21:56.340 --> 00:22:00.210 It's my pleasure to be given the opportunity to join you here 00:22:00.210 --> 00:22:02.220 for the [INAUDIBLE]. 00:22:02.220 --> 00:22:04.260 The purpose of my presentation is 00:22:04.260 --> 00:22:08.970 to give you a shot into real [INAUDIBLE] universal RT-nested 00:22:08.970 --> 00:22:14.040 PCR for the detection of lyssavirus. 00:22:14.040 --> 00:22:18.720 To detect rabies virus other member species opportunist 00:22:18.720 --> 00:22:23.400 lyssavirus, we have developed a [INAUDIBLE] lyssavirus nested 00:22:23.400 --> 00:22:27.000 RT-PCR in 2007. 00:22:27.000 --> 00:22:30.690 The principle of this method is the reverse transcription 00:22:30.690 --> 00:22:36.963 of [INAUDIBLE] RNA into to CPA by using only [INAUDIBLE].. 00:22:36.963 --> 00:22:39.670 [INAUDIBLE] 00:22:39.670 --> 00:22:42.880 The CDA [INAUDIBLE] two rounds of PCR. 00:22:42.880 --> 00:22:45.790 The two rounds of PCR significantly 00:22:45.790 --> 00:22:49.840 increased the sensitivity of PCR. 00:22:49.840 --> 00:22:52.750 The CDA undergoes goes the first round PCR 00:22:52.750 --> 00:22:59.740 without primers to amplify on 940 [INAUDIBLE] BP fragment. 00:22:59.740 --> 00:23:03.820 Then the second round PCR used a first round PCR product 00:23:03.820 --> 00:23:10.420 as a template to amplify a 371 BP fragment [INAUDIBLE] 00:23:10.420 --> 00:23:12.250 the primers. 00:23:12.250 --> 00:23:15.460 The primers of the first round and second round PCR 00:23:15.460 --> 00:23:17.890 was designed based on the [INAUDIBLE] region 00:23:17.890 --> 00:23:22.450 of the [? engine ?] of seven major lyssavirus species, 00:23:22.450 --> 00:23:27.880 because only seven lyssavirus species was found in 2007. 00:23:27.880 --> 00:23:33.010 This method was patented in 2009. 00:23:33.010 --> 00:23:35.635 The lyssavirus species was [INAUDIBLE] increase 00:23:35.635 --> 00:23:37.585 in recent-- 00:23:37.585 --> 00:23:44.520 [AUDIO OUT] --lyssavirus species was approved by the ICTV. 00:23:44.520 --> 00:23:47.680 00:23:47.680 --> 00:23:51.490 In order to detect the sensitivity and the specificity 00:23:51.490 --> 00:24:00.880 of these 18 lyssavirus species, the other 18 [INAUDIBLE] 00:24:00.880 --> 00:24:06.090 for [INAUDIBLE] of each lyssavirus for PCR. 00:24:06.090 --> 00:24:11.816 Result are listed RT-PCR to detect an 18 lyssavirus 00:24:11.816 --> 00:24:14.860 [INAUDIBLE] on the left. 00:24:14.860 --> 00:24:19.165 We can see all 18 lyssavirus putting the same two 00:24:19.165 --> 00:24:25.790 [INAUDIBLE],, indicating that the nested RT-PCR detect all 18 00:24:25.790 --> 00:24:27.280 lyssavirus. 00:24:27.280 --> 00:24:31.030 16 of classmates [INAUDIBLE] application 00:24:31.030 --> 00:24:33.220 in two rounds of PCR. 00:24:33.220 --> 00:24:39.520 It is also [INAUDIBLE] virus in [INAUDIBLE] was amplified, 00:24:39.520 --> 00:24:41.950 unlike in the second round PCR. 00:24:41.950 --> 00:24:44.800 And [? coronavirus ?] [INAUDIBLE] 00:24:44.800 --> 00:24:48.250 was applied only in the first round PCR. 00:24:48.250 --> 00:24:50.650 The sensitivity of the measure will 00:24:50.650 --> 00:24:52.670 show in the table on the right. 00:24:52.670 --> 00:24:56.110 The sensitivity was varied out in the detection 00:24:56.110 --> 00:25:09.390 of [INAUDIBLE] values ranging from 2.24 to 22,400 [INAUDIBLE] 00:25:09.390 --> 00:25:15.060 per [INAUDIBLE],, and as shown in the table on the right. 00:25:15.060 --> 00:25:21.030 To [INAUDIBLE] the [INAUDIBLE] the cause of this discrepancy, 00:25:21.030 --> 00:25:25.230 I think with cooperation of all 18 lyssavirus 00:25:25.230 --> 00:25:28.590 with full primer regions who contact. 00:25:28.590 --> 00:25:32.220 With the result showing that [INAUDIBLE] and nucleotide 00:25:32.220 --> 00:25:35.370 T are autoprimers. 00:25:35.370 --> 00:25:40.350 And it to die in the first round PCR. 00:25:40.350 --> 00:25:47.610 And the second nucleotide T at three [INAUDIBLE] in the primer 00:25:47.610 --> 00:25:52.680 and 371F in the second round PCR was not 00:25:52.680 --> 00:25:58.830 identical to the responding position of the Aravan virus 00:25:58.830 --> 00:26:01.350 and Ikoma virus. 00:26:01.350 --> 00:26:06.450 They have tried to change the [INAUDIBLE] of the primer to T 00:26:06.450 --> 00:26:12.330 of [INAUDIBLE] and change the G of the primer to A-- 00:26:12.330 --> 00:26:15.330 to C, sorry, of the coronavirus. 00:26:15.330 --> 00:26:19.710 Both virus was in the PCR. 00:26:19.710 --> 00:26:23.850 This result shows that the critical roles 00:26:23.850 --> 00:26:28.020 of the three [INAUDIBLE] and nucleotides 00:26:28.020 --> 00:26:29.505 of the primary [INAUDIBLE] success 00:26:29.505 --> 00:26:33.510 following application of the [INAUDIBLE] region. 00:26:33.510 --> 00:26:35.340 The result was unsure. 00:26:35.340 --> 00:26:38.730 It means this difficulty can be attributed 00:26:38.730 --> 00:26:42.240 to the dismatch between the primers and templates 00:26:42.240 --> 00:26:46.810 due to the [INAUDIBLE] sequence that was attained. 00:26:46.810 --> 00:26:51.220 To test the validation of the RT nested PCR, 00:26:51.220 --> 00:26:59.320 we use 9,624 branches of eight domestic animal species 00:26:59.320 --> 00:27:02.950 in 10 years of clinical, [INAUDIBLE] technologies 00:27:02.950 --> 00:27:04.870 and surveillance in China. 00:27:04.870 --> 00:27:07.320 But the result shows that RT nested 00:27:07.320 --> 00:27:13.220 PCR has 100 sensitivity and over 90 [INAUDIBLE] 00:27:13.220 --> 00:27:18.340 in corporation with the direct fluorescent antibody test. 00:27:18.340 --> 00:27:22.960 300 decayed practical samples will test the positive, 00:27:22.960 --> 00:27:30.340 but RT-PCR was up by nested PCR [INAUDIBLE] at FAT. 00:27:30.340 --> 00:27:33.550 This result indicated that the nested RTP [INAUDIBLE] 00:27:33.550 --> 00:27:36.880 is most of the two, that FAT in detection 00:27:36.880 --> 00:27:39.870 of [INAUDIBLE] samples. 00:27:39.870 --> 00:27:42.420 00:27:42.420 --> 00:27:44.730 In addition, 10 other [INAUDIBLE] 00:27:44.730 --> 00:27:47.340 laboratory was invited to [INAUDIBLE] 00:27:47.340 --> 00:27:51.800 the test to further write [INAUDIBLE] the nested RT-PCR. 00:27:51.800 --> 00:27:56.190 All 10 laboratories are [INAUDIBLE] nested RT-PCR 00:27:56.190 --> 00:28:03.210 with 100% in constant with FAT with no false positive or false 00:28:03.210 --> 00:28:07.650 negative, indicating that this nested RT-PCR has 00:28:07.650 --> 00:28:11.540 the high price of [INAUDIBLE]. 00:28:11.540 --> 00:28:14.960 Our nested RT-PCR has been approved 00:28:14.960 --> 00:28:18.860 as the national standards of previous technologies in China 00:28:18.860 --> 00:28:20.780 in 2018. 00:28:20.780 --> 00:28:24.170 This method was published in the Journal of [INAUDIBLE] 00:28:24.170 --> 00:28:29.220 Experiment in 2018. 00:28:29.220 --> 00:28:32.470 Finally, I would like to end my presentation with each 00:28:32.470 --> 00:28:34.500 produce how my laboratory. 00:28:34.500 --> 00:28:38.100 My director is Professor of [INAUDIBLE],, 00:28:38.100 --> 00:28:42.300 [INAUDIBLE] Laboratory, rabies classical [INAUDIBLE],, 00:28:42.300 --> 00:28:44.490 and [INAUDIBLE] this group. 00:28:44.490 --> 00:28:47.880 My laboratory would like to cooperate with all of you 00:28:47.880 --> 00:28:49.650 to make rabies history. 00:28:49.650 --> 00:28:50.250 Thank you. 00:28:50.250 --> 00:28:52.860 00:28:52.860 --> 00:28:57.920 - Thank you for that very nice presentation, Doctor Feng. 00:28:57.920 --> 00:29:03.110 And I thought I saw one question. 00:29:03.110 --> 00:29:08.540 The person wants to how easy is it 00:29:08.540 --> 00:29:12.170 to adjust your primers when looking 00:29:12.170 --> 00:29:14.720 for different lyssaviruses? 00:29:14.720 --> 00:29:19.910 For example, do you think you ever might have a case where 00:29:19.910 --> 00:29:25.950 you think there might be a lyssavirus but cannot find it? 00:29:25.950 --> 00:29:28.140 So I think they're speaking, for example, 00:29:28.140 --> 00:29:34.020 of having some disparate ones as we go beyond filo group three 00:29:34.020 --> 00:29:37.230 on our clock. 00:29:37.230 --> 00:29:40.890 How easy is it, do you think, to find new lyssaviruses 00:29:40.890 --> 00:29:45.280 based upon your primer design? 00:29:45.280 --> 00:29:46.790 - Thank you. 00:29:46.790 --> 00:29:49.810 00:29:49.810 --> 00:29:58.030 My thought was our primer was designed based on the seven 00:29:58.030 --> 00:29:59.950 lyssavirus species. 00:29:59.950 --> 00:30:07.630 And after that, we [INAUDIBLE] how 18 lyssavirus species now, 00:30:07.630 --> 00:30:16.060 so we just test a classmate to test whether our primer works 00:30:16.060 --> 00:30:19.490 now or not. 00:30:19.490 --> 00:30:21.580 - Thank you. 00:30:21.580 --> 00:30:24.100 - OK, thank you. 00:30:24.100 --> 00:30:30.250 - And do you think there's a greater diversity 00:30:30.250 --> 00:30:36.490 of bat lyssaviruses in Asia given the results that you've 00:30:36.490 --> 00:30:38.710 found so far That. 00:30:38.710 --> 00:30:42.320 Haven't been discovered yet? 00:30:42.320 --> 00:30:48.260 - Maybe because they [INAUDIBLE] enough surveillance in China 00:30:48.260 --> 00:30:51.210 and other Asian countries. 00:30:51.210 --> 00:30:57.110 And if we operate more rabies technologies, 00:30:57.110 --> 00:31:01.460 maybe we can find more lyssavirus maybe, I think. 00:31:01.460 --> 00:31:02.510 - Yes. 00:31:02.510 --> 00:31:09.660 And do people who are exposed to bats in China, 00:31:09.660 --> 00:31:12.630 do they submit the bats for diagnosis 00:31:12.630 --> 00:31:15.000 or receive prophylaxis? 00:31:15.000 --> 00:31:20.010 Or do you think because our major burden is with dogs 00:31:20.010 --> 00:31:25.290 that the education is focused more upon rabies 00:31:25.290 --> 00:31:28.500 in dogs as it should be? 00:31:28.500 --> 00:31:31.020 Do people get exposed to bats in China 00:31:31.020 --> 00:31:34.810 and receive prophylaxis after a bite? 00:31:34.810 --> 00:31:36.310 - Yes, of course. 00:31:36.310 --> 00:31:40.570 In China, our work is [INAUDIBLE] dog rabies 00:31:40.570 --> 00:31:49.900 because dog [INAUDIBLE] is a major transfer source in China. 00:31:49.900 --> 00:31:56.050 It's over 99 human cases was caused by dogs, 00:31:56.050 --> 00:32:05.090 so we have lots of dog rabies. 00:32:05.090 --> 00:32:10.600 And I don't think they have enough money and enough people 00:32:10.600 --> 00:32:14.050 to operate [? with ?] this rabies research. 00:32:14.050 --> 00:32:20.240 Maybe in the future we can operate more research on that. 00:32:20.240 --> 00:32:21.140 - Yes. 00:32:21.140 --> 00:32:27.650 And you probably saw some recent papers from our colleagues 00:32:27.650 --> 00:32:34.850 in Europe talking about the diversity of lyssaviruses 00:32:34.850 --> 00:32:38.730 in Eurasia as well as in Africa. 00:32:38.730 --> 00:32:42.680 For example, the finding of a west Caucasian bat 00:32:42.680 --> 00:32:46.980 virus in a cat in Italy. 00:32:46.980 --> 00:32:51.410 And I'm wondering, considering beyond [INAUDIBLE] 00:32:51.410 --> 00:32:55.550 group one, such as the African and some of the Eurasian 00:32:55.550 --> 00:32:57.590 lyssaviruses, do you believe we need 00:32:57.590 --> 00:33:01.640 to have new vaccines for the future to cover the broader 00:33:01.640 --> 00:33:04.100 spectrum of these lyssaviruses? 00:33:04.100 --> 00:33:08.260 00:33:08.260 --> 00:33:15.550 - I think they have three groups lyssavirus species. 00:33:15.550 --> 00:33:21.415 And our vaccine can cover group one and group two. 00:33:21.415 --> 00:33:24.070 00:33:24.070 --> 00:33:28.450 Maybe in the future we can operate-- 00:33:28.450 --> 00:33:31.820 vaccine can cover all the lyssavirus. 00:33:31.820 --> 00:33:39.580 But in China, and especially in other countries in Asia, 00:33:39.580 --> 00:33:46.270 and we just how rabies virus and work with lyssavirus 00:33:46.270 --> 00:33:49.660 and Thailand bat lyssavirus, I think. 00:33:49.660 --> 00:34:00.310 So the vaccine ratio is enough for rabies control 00:34:00.310 --> 00:34:01.950 and [INAUDIBLE]. 00:34:01.950 --> 00:34:03.750 - Yes. 00:34:03.750 --> 00:34:05.670 And I don't see any other questions, 00:34:05.670 --> 00:34:10.300 so I thank you for your time and your presentation. 00:34:10.300 --> 00:34:14.250 And if you'd be so kind to stop screen sharing, 00:34:14.250 --> 00:34:17.409 we'll go on with our next participant. 00:34:17.409 --> 00:34:19.570 Thank you very much. 00:34:19.570 --> 00:34:23.760 We're going to stay on the theme about lyssaviruses 00:34:23.760 --> 00:34:26.610 and their detection, and in particular, 00:34:26.610 --> 00:34:29.190 in regards to molecular detection, 00:34:29.190 --> 00:34:33.630 our next participant will be Megan Golding speaking to us 00:34:33.630 --> 00:34:38.159 about pan- lyssavirus, a real-time PCR for rabies 00:34:38.159 --> 00:34:42.480 diagnosis, recognizing that rabies is caused by lyssavirus 00:34:42.480 --> 00:34:44.670 or lyssaviruses cause rabies. 00:34:44.670 --> 00:34:48.900 And Megan is coming to us from the Animal and Plant Health 00:34:48.900 --> 00:34:52.590 Inspection Agency Rabies and Viral Zoonoses 00:34:52.590 --> 00:34:55.290 Group from the UK. 00:34:55.290 --> 00:34:58.412 Megan, you have the floor. 00:34:58.412 --> 00:34:59.360 - Thank you. 00:34:59.360 --> 00:35:01.410 Hi, so I'm Megan. 00:35:01.410 --> 00:35:03.870 I work for the Rabies and Viral Zoonoses Group 00:35:03.870 --> 00:35:05.520 at the Animal Plant Health Agency 00:35:05.520 --> 00:35:08.400 and I'm going to be talking about the real-time PCR 00:35:08.400 --> 00:35:10.860 that we use primarily in rabies diagnosis 00:35:10.860 --> 00:35:13.470 and comparing it to the other [INAUDIBLE] that we also use. 00:35:13.470 --> 00:35:19.150 00:35:19.150 --> 00:35:20.553 There we go. 00:35:20.553 --> 00:35:21.970 Just a little bit about our group. 00:35:21.970 --> 00:35:24.670 First of all, we're a UK and international reference 00:35:24.670 --> 00:35:26.830 laboratory for rabies and we carry out 00:35:26.830 --> 00:35:29.440 a range of functions and research and diagnostics 00:35:29.440 --> 00:35:32.020 by serology and molecular and today I'll 00:35:32.020 --> 00:35:34.990 be focusing on our molecular and comparing the assays 00:35:34.990 --> 00:35:38.500 we use in lyssavirus diagnostics. 00:35:38.500 --> 00:35:41.770 There are a range of ways that we use PCR and our diagnostics 00:35:41.770 --> 00:35:43.480 and it's become a really valuable tool 00:35:43.480 --> 00:35:46.720 in our infectious disease. 00:35:46.720 --> 00:35:48.640 We use it in suspect cases to confirm 00:35:48.640 --> 00:35:50.980 disease in an animal or human exhibiting symptoms 00:35:50.980 --> 00:35:52.750 of that disease. 00:35:52.750 --> 00:35:55.730 We use it primarily in our passive surveillance scheme. 00:35:55.730 --> 00:35:57.370 So we have a passive bat surveillance 00:35:57.370 --> 00:36:00.820 scheme where we test at bat submitted to us by the public. 00:36:00.820 --> 00:36:04.000 This allows us to possibly monitor current lyssaviruses 00:36:04.000 --> 00:36:06.040 in the UK, like European bat lyssavirus 00:36:06.040 --> 00:36:08.680 too, as well as monitor when you want to merge, 00:36:08.680 --> 00:36:10.630 like European bat lyssavirus one, 00:36:10.630 --> 00:36:15.100 which we detected for the first time in 2018. 00:36:15.100 --> 00:36:18.040 And we also use it in targeted surveillance. 00:36:18.040 --> 00:36:19.690 We haven't done this in a long time, 00:36:19.690 --> 00:36:23.020 but we can do swabs of bats and test those 00:36:23.020 --> 00:36:25.780 by PCR of targeted species or in areas where we've 00:36:25.780 --> 00:36:28.370 had a lot of positive cases. 00:36:28.370 --> 00:36:30.730 And we also then send our positive PCR products 00:36:30.730 --> 00:36:33.760 for sequencing to confirm the species 00:36:33.760 --> 00:36:36.940 and to investigate phylogeny. 00:36:36.940 --> 00:36:40.630 So the widely accepted PCR as a primary diagnostic tool 00:36:40.630 --> 00:36:43.450 for the first time in 2018, recognizing 00:36:43.450 --> 00:36:45.890 its advantages in that there's no requirement 00:36:45.890 --> 00:36:46.780 for a live virus. 00:36:46.780 --> 00:36:48.730 And it's particularly useful in cases 00:36:48.730 --> 00:36:50.890 where the sample is sub-optimal. 00:36:50.890 --> 00:36:53.320 Those are the three assays that we use listed there 00:36:53.320 --> 00:36:56.500 and the one highlighted in green is our primary go-to assay 00:36:56.500 --> 00:36:57.880 for initial screening. 00:36:57.880 --> 00:37:01.180 And the other two are mainly used as confirmatory assays. 00:37:01.180 --> 00:37:03.250 All of our assets are based on the N gene 00:37:03.250 --> 00:37:05.290 region of the lyssavirus genome because it 00:37:05.290 --> 00:37:06.850 is the most conserved. 00:37:06.850 --> 00:37:10.360 And also we do tend to use the molecular assays in combination 00:37:10.360 --> 00:37:11.515 with serology, like F80. 00:37:11.515 --> 00:37:14.100 00:37:14.100 --> 00:37:16.770 So just to break down the assay that we use initially. 00:37:16.770 --> 00:37:19.290 First of all, of our assays have an RT step 00:37:19.290 --> 00:37:21.600 because the lyssavirus genome is RNA 00:37:21.600 --> 00:37:24.120 and the [? san ?] material PCR cDNA. 00:37:24.120 --> 00:37:26.100 They're also all one step because this 00:37:26.100 --> 00:37:28.860 makes them simpler and more convenient to carry out. 00:37:28.860 --> 00:37:30.930 It also reduces how much manipulation 00:37:30.930 --> 00:37:34.660 is involved to reduce the chance of contamination. 00:37:34.660 --> 00:37:35.910 It is a real time assay. 00:37:35.910 --> 00:37:39.330 So the assay works by a intercalataing dye called SYBR 00:37:39.330 --> 00:37:42.550 green that binds to double stranded DNA. 00:37:42.550 --> 00:37:45.240 And then once found, it emits fluorescence, 00:37:45.240 --> 00:37:47.830 which is measured by the real time machine at each cycle. 00:37:47.830 --> 00:37:51.450 So it detects and quantifies target DNA or RNA in real time. 00:37:51.450 --> 00:37:53.400 And also gives us a CT value, which 00:37:53.400 --> 00:37:55.950 is the number which the PCR product crosses 00:37:55.950 --> 00:37:57.390 the threshold of detection. 00:37:57.390 --> 00:38:00.390 A lower CT would mean that you have a viable nucleic acid 00:38:00.390 --> 00:38:02.460 load. 00:38:02.460 --> 00:38:04.242 The assay is also pan-lyssavirus, 00:38:04.242 --> 00:38:05.700 so we've developed primers that are 00:38:05.700 --> 00:38:08.490 successful at detecting all known lyssaviruses, 00:38:08.490 --> 00:38:10.680 making this assay great for general lyssavirus 00:38:10.680 --> 00:38:15.130 surveillance monitoring current and emerging disease. 00:38:15.130 --> 00:38:17.850 However, it isn't capable of differentiating 00:38:17.850 --> 00:38:20.490 between the species it detects, so you would need to carry out 00:38:20.490 --> 00:38:22.740 further analysis to do that. 00:38:22.740 --> 00:38:24.390 This is the thermal profile we use. 00:38:24.390 --> 00:38:26.490 The key bit to know is that the assay is rapid. 00:38:26.490 --> 00:38:30.760 And [INAUDIBLE] the reverse transcription step is step one. 00:38:30.760 --> 00:38:33.990 To make it a one step assay, we amplify for 40 cycles 00:38:33.990 --> 00:38:37.200 and we carry out with dissociation curve analysis. 00:38:37.200 --> 00:38:39.300 This is the kind of output that we would get. 00:38:39.300 --> 00:38:41.370 So on the left, you've got the amplification plot 00:38:41.370 --> 00:38:43.800 where you can see which of your samples is amplified 00:38:43.800 --> 00:38:45.030 and what their CT is. 00:38:45.030 --> 00:38:47.460 So just for example, the blue curve 00:38:47.460 --> 00:38:52.770 would have the lower CT value, approximately 20. 00:38:52.770 --> 00:38:55.373 But that would also mean it has the highest viral load. 00:38:55.373 --> 00:38:57.540 And on the right, you've got the dissociation curve. 00:38:57.540 --> 00:38:59.550 Because the dye bind is non-specific, 00:38:59.550 --> 00:39:02.010 we analyze this curve to check the melting temperature 00:39:02.010 --> 00:39:04.710 is specific to lyssavirus, which we know to be 00:39:04.710 --> 00:39:07.440 78 to 79.5 degrees Celsius. 00:39:07.440 --> 00:39:09.300 And amplification isn't due to non 00:39:09.300 --> 00:39:13.920 specific binding like the formation of primer timers. 00:39:13.920 --> 00:39:17.050 So just to summarize, it is a pan-lyssavirus assay, 00:39:17.050 --> 00:39:18.870 so it can detect all lyssavirus species 00:39:18.870 --> 00:39:19.980 but can't differentiate. 00:39:19.980 --> 00:39:22.210 You'd need to carry out further analysis. 00:39:22.210 --> 00:39:24.030 The project length is a bit short, 00:39:24.030 --> 00:39:27.210 120 base pairs, so it's not ideal for sanger sequencing. 00:39:27.210 --> 00:39:30.120 So you'd likely need to carry on another PCR after this. 00:39:30.120 --> 00:39:32.520 It comes in real time, so you get CT values. 00:39:32.520 --> 00:39:34.370 And it's within a closed tube system, 00:39:34.370 --> 00:39:36.730 so there's a minimal risk of contamination. 00:39:36.730 --> 00:39:37.410 It's rapid. 00:39:37.410 --> 00:39:41.010 And you can use the dissociation curve to confirm specificity. 00:39:41.010 --> 00:39:47.130 So I'll just compare that to a conventional hemi-nested PCR. 00:39:47.130 --> 00:39:48.990 Because it's a conventional PCR, you 00:39:48.990 --> 00:39:51.480 need to carry out post PCR analysis, like gel 00:39:51.480 --> 00:39:53.460 electrophoresis, in order to determine 00:39:53.460 --> 00:39:55.200 what it is you detected. 00:39:55.200 --> 00:39:57.030 It also makes it an open tube system, 00:39:57.030 --> 00:40:00.240 so it's much more vulnerable to contamination than our SYBR. 00:40:00.240 --> 00:40:03.480 Like our SYBR, it's pan-lyssavirus, 00:40:03.480 --> 00:40:05.730 but you still can't differentiate between species. 00:40:05.730 --> 00:40:08.490 But the product is much larger at 600 base pairs, which 00:40:08.490 --> 00:40:10.450 is ideal for sanger sequencing. 00:40:10.450 --> 00:40:13.140 So this assay is one we would typically carry out 00:40:13.140 --> 00:40:15.150 if we've had a positive SYBR result 00:40:15.150 --> 00:40:18.240 and we want to send the product for sequencing. 00:40:18.240 --> 00:40:20.610 It's also a nested PCR, so we carry out 00:40:20.610 --> 00:40:24.173 two rounds and with the same forward primer 00:40:24.173 --> 00:40:26.340 in the second round but a slightly different reverse 00:40:26.340 --> 00:40:29.880 primer to amplify the round one product to confirm specificity 00:40:29.880 --> 00:40:32.760 of the first round and improve sensitivity. 00:40:32.760 --> 00:40:34.560 It's also much slower than a SYBR 00:40:34.560 --> 00:40:36.330 and it takes over five hours to run both 00:40:36.330 --> 00:40:39.360 of the rounds and the analysis. 00:40:39.360 --> 00:40:41.610 And then to compare that to our other real time 00:40:41.610 --> 00:40:43.980 assay, which is our TaqMan. 00:40:43.980 --> 00:40:47.190 However, instead of dye, it uses highly specific probes 00:40:47.190 --> 00:40:49.080 to detect target sequences. 00:40:49.080 --> 00:40:51.060 So we use three probes. 00:40:51.060 --> 00:40:54.570 These are EBLV-1 and EBLV-2 because these 00:40:54.570 --> 00:40:57.750 are the two lyssavirus species in the UK currently. 00:40:57.750 --> 00:41:00.270 And they only concern in certain bat species, 00:41:00.270 --> 00:41:01.830 so this assay is great if we have 00:41:01.830 --> 00:41:05.250 a preliminary positive result in one of our bat species 00:41:05.250 --> 00:41:09.290 and we want to quickly confirm it's the expected lyssavirus. 00:41:09.290 --> 00:41:12.410 We also use a RABV probe mainly as an indication 00:41:12.410 --> 00:41:15.800 of contamination, because it's extremely unlikely to get 00:41:15.800 --> 00:41:18.290 RABV in our bats in the UK. 00:41:18.290 --> 00:41:21.800 So it's a differential PCR so you can see what 00:41:21.800 --> 00:41:23.890 lyssavirus you have amplified. 00:41:23.890 --> 00:41:25.670 You also still get CT values. 00:41:25.670 --> 00:41:27.480 And it occurs within a closed system, 00:41:27.480 --> 00:41:30.230 so there's a minimized risk of contamination. 00:41:30.230 --> 00:41:32.832 It uses the same primers as our SYBR PCR, 00:41:32.832 --> 00:41:35.040 so it's the same product length, too small for sanger 00:41:35.040 --> 00:41:37.010 sequencing, but this is less needed 00:41:37.010 --> 00:41:40.550 than it is with SYBR because you have the probes. 00:41:40.550 --> 00:41:43.880 And it's not as fast as SYBR, but still a lot faster 00:41:43.880 --> 00:41:46.270 than a hemi-nested. 00:41:46.270 --> 00:41:48.430 Controls are a critical part of any PCR, 00:41:48.430 --> 00:41:50.020 particularly in diagnostics. 00:41:50.020 --> 00:41:52.510 We typically use three in our assays. 00:41:52.510 --> 00:41:56.320 Firstly, we have the positive PCR control, which is CVS. 00:41:56.320 --> 00:41:57.970 Prepared from infected mouse brain. 00:41:57.970 --> 00:42:01.690 Validate and calibrate it to be within a predetermined range. 00:42:01.690 --> 00:42:04.660 We track the CT of this control across all of our PCRs 00:42:04.660 --> 00:42:07.150 to show good inter-comparability and provide 00:42:07.150 --> 00:42:10.480 assurance of the robustness and reproducibility of the assay. 00:42:10.480 --> 00:42:13.240 When the control CT deviates from what we would expect, 00:42:13.240 --> 00:42:16.670 this would be investigated in the PCR and repeated if needed. 00:42:16.670 --> 00:42:19.210 We also have a PCR negative control, like water, 00:42:19.210 --> 00:42:22.930 to confirm negative samples and show there's no contamination. 00:42:22.930 --> 00:42:25.530 We also use beta-actin, which is a housekeeping gene, 00:42:25.530 --> 00:42:27.880 as an internal control system in our samples to show 00:42:27.880 --> 00:42:30.160 that extraction was successful. 00:42:30.160 --> 00:42:32.350 Any samples with a negative beta-actin 00:42:32.350 --> 00:42:34.000 would be investigated and repeated, 00:42:34.000 --> 00:42:36.530 or re-extracted as needed. 00:42:36.530 --> 00:42:38.560 The other key thing to consider is cost. 00:42:38.560 --> 00:42:40.690 Real-time machines-- real-time assays, 00:42:40.690 --> 00:42:42.790 sorry, require optic consumables so the machine 00:42:42.790 --> 00:42:44.428 can measure through the plastic, which 00:42:44.428 --> 00:42:45.970 can be more expensive than what would 00:42:45.970 --> 00:42:47.535 be fine with the conventional. 00:42:47.535 --> 00:42:49.660 The real-time machines are also much more expensive 00:42:49.660 --> 00:42:51.490 than their conventional counterparts. 00:42:51.490 --> 00:42:53.740 And just a general consideration of reagents, primers, 00:42:53.740 --> 00:42:54.580 and probes. 00:42:54.580 --> 00:42:56.800 Especially probes, if you want to design a lot 00:42:56.800 --> 00:42:59.140 and you're carrying out low throughput PCR, 00:42:59.140 --> 00:43:01.690 the costs can begin to add up. 00:43:01.690 --> 00:43:04.520 And also just consider the cost of resources like staff time. 00:43:04.520 --> 00:43:06.850 While real-time machines are more expensive, 00:43:06.850 --> 00:43:08.380 the assay is much faster. 00:43:08.380 --> 00:43:11.950 And nowadays, you can also remote access software 00:43:11.950 --> 00:43:13.570 so you could report results from home. 00:43:13.570 --> 00:43:17.245 Whereas with conventional, you're a lot more restricted. 00:43:17.245 --> 00:43:19.120 So my take home point would be that if you're 00:43:19.120 --> 00:43:20.828 needing to choose an assay, make sure you 00:43:20.828 --> 00:43:23.590 choose one is best according to your needs, what 00:43:23.590 --> 00:43:24.490 you're using it for. 00:43:24.490 --> 00:43:26.500 Is it general surveillance or targeted? 00:43:26.500 --> 00:43:29.110 Is it high or low throughput? 00:43:29.110 --> 00:43:31.090 All of these have implications on cost. 00:43:31.090 --> 00:43:32.890 And always carry out local validation 00:43:32.890 --> 00:43:35.920 of your essay to make sure it's doing what you need it to do. 00:43:35.920 --> 00:43:37.710 Be aware of the impact of false results. 00:43:37.710 --> 00:43:39.910 Rabies is obviously incredibly fatal disease, 00:43:39.910 --> 00:43:41.740 so getting a wrong diagnostic result 00:43:41.740 --> 00:43:43.420 can have serious implications. 00:43:43.420 --> 00:43:46.720 Carry follow up investigations of inconclusive or positive 00:43:46.720 --> 00:43:48.370 results to confirm them. 00:43:48.370 --> 00:43:50.950 And while the real-time PCR does have a reduced 00:43:50.950 --> 00:43:53.350 risk of contamination compared to the conventional, 00:43:53.350 --> 00:43:55.780 it can still happen. 00:43:55.780 --> 00:43:58.720 And always, quality is essential to being 00:43:58.720 --> 00:44:01.240 able to rely on your assay and the results produced. 00:44:01.240 --> 00:44:02.860 Monitoring [INAUDIBLE] controls, watch 00:44:02.860 --> 00:44:05.723 for trends and signs of degradation or contamination. 00:44:05.723 --> 00:44:07.390 Always follow good [INAUDIBLE] practice, 00:44:07.390 --> 00:44:11.890 like ISO17025 is the international standard for test 00:44:11.890 --> 00:44:14.110 accreditation and one that YE requires 00:44:14.110 --> 00:44:15.830 assets that are accredited to. 00:44:15.830 --> 00:44:17.650 Also continuously validate your assays 00:44:17.650 --> 00:44:20.102 to show that it's the one that's most appropriate for you. 00:44:20.102 --> 00:44:22.060 All this works towards minimizing contamination 00:44:22.060 --> 00:44:24.310 and getting the right result. 00:44:24.310 --> 00:44:25.310 Thank you for listening. 00:44:25.310 --> 00:44:27.872 You can check out our blog release on World Rabies 00:44:27.872 --> 00:44:29.080 Day every year for more info. 00:44:29.080 --> 00:44:33.260 00:44:33.260 --> 00:44:34.640 - Great. 00:44:34.640 --> 00:44:43.710 Thank you, Megan, for that very concise, and yet engrossing, 00:44:43.710 --> 00:44:45.280 presentation. 00:44:45.280 --> 00:44:50.360 And we have two questions so far. 00:44:50.360 --> 00:44:56.430 One, obviously you can apply your SOP 00:44:56.430 --> 00:44:58.560 to post-mortem samples. 00:44:58.560 --> 00:45:04.890 Suppose you had a suspect human suspected of rabies in the UK. 00:45:04.890 --> 00:45:07.800 Would you apply this particular protocol 00:45:07.800 --> 00:45:09.930 for antimortem diagnostics? 00:45:09.930 --> 00:45:12.850 00:45:12.850 --> 00:45:15.040 - We can. 00:45:15.040 --> 00:45:19.030 I don't-- just trying to think. 00:45:19.030 --> 00:45:19.810 We haven't. 00:45:19.810 --> 00:45:23.680 Our suspects usually-- it's post-mortem usually. 00:45:23.680 --> 00:45:29.348 But I think if you have tissue that we can extract, yeah, 00:45:29.348 --> 00:45:29.890 I don't know. 00:45:29.890 --> 00:45:31.060 Because it's normally brain tissue 00:45:31.060 --> 00:45:32.102 that it would be done on. 00:45:32.102 --> 00:45:35.750 So I don't know if it would be the most appropriate one. 00:45:35.750 --> 00:45:39.430 - So suppose you did have a suspect human case 00:45:39.430 --> 00:45:44.020 and they were submitting saliva to your laboratory. 00:45:44.020 --> 00:45:46.780 Would you be using this or a different protocol 00:45:46.780 --> 00:45:47.363 for diagnoses? 00:45:47.363 --> 00:45:47.863 - Yeah. 00:45:47.863 --> 00:45:48.490 Sorry, yeah. 00:45:48.490 --> 00:45:50.525 Yeah, we could extract the saliva 00:45:50.525 --> 00:45:51.900 and analyze that with this assay. 00:45:51.900 --> 00:45:52.750 Yeah. 00:45:52.750 --> 00:45:53.950 - Very good. 00:45:53.950 --> 00:45:58.210 We have a secondary follow up question as well. 00:45:58.210 --> 00:46:02.350 Do you ever come across submitted specimens 00:46:02.350 --> 00:46:08.030 that might have more than one lyssavirus in the sample? 00:46:08.030 --> 00:46:08.810 - So we haven't. 00:46:08.810 --> 00:46:11.990 So we only have to lyssaviruses in UK and the 00:46:11.990 --> 00:46:15.080 also only occur in two bat groups. 00:46:15.080 --> 00:46:16.740 And they're very specific. 00:46:16.740 --> 00:46:20.750 So EBLV-2 two we've only ever detected in [INAUDIBLE] bats. 00:46:20.750 --> 00:46:24.290 And we've only ever detected EBLV-1 in our [INAUDIBLE] bats. 00:46:24.290 --> 00:46:27.440 And I think hypothetically it's definitely possible, 00:46:27.440 --> 00:46:28.910 so we always test the both. 00:46:28.910 --> 00:46:31.130 But no, we haven't yet 00:46:31.130 --> 00:46:31.770 - Thank you. 00:46:31.770 --> 00:46:35.570 And simpler question, which I think you 00:46:35.570 --> 00:46:37.040 may have already answered. 00:46:37.040 --> 00:46:40.370 Do you think a person could be infected with more than one 00:46:40.370 --> 00:46:43.210 lyssavirus at the same time? 00:46:43.210 --> 00:46:43.710 - Yeah. 00:46:43.710 --> 00:46:44.210 Yeah. 00:46:44.210 --> 00:46:46.000 Again, I think definitely possible 00:46:46.000 --> 00:46:47.690 and we would always test all of them. 00:46:47.690 --> 00:46:50.480 But nothing we've encountered. 00:46:50.480 --> 00:46:50.980 - Yes. 00:46:50.980 --> 00:46:54.650 I don't think in any of our collective experiences 00:46:54.650 --> 00:46:58.130 on this call from the panelists and their colleagues, 00:46:58.130 --> 00:46:59.810 I don't think we've ever detected 00:46:59.810 --> 00:47:03.320 more than a single variant in a person 00:47:03.320 --> 00:47:08.150 at the same time, which is going to be rare unto itself. 00:47:08.150 --> 00:47:10.040 A related question. 00:47:10.040 --> 00:47:14.450 What's-- do you know what the sensitivity of your test is 00:47:14.450 --> 00:47:19.550 in regards to antemortem samples such as saliva? 00:47:19.550 --> 00:47:24.230 - Unfortunately, off the top of my head, I don't. 00:47:24.230 --> 00:47:26.490 But it is a really sensitive assay. 00:47:26.490 --> 00:47:29.730 So I think it is good, because we have tested saliva before. 00:47:29.730 --> 00:47:33.380 So it's definitely still a good assay to choose in most cases. 00:47:33.380 --> 00:47:34.100 - Sure. 00:47:34.100 --> 00:47:38.270 And do you have any experience with degraded samples, 00:47:38.270 --> 00:47:42.120 for example, from animals that may have been dead for a while? 00:47:42.120 --> 00:47:44.000 - Oh yeah, definitely. 00:47:44.000 --> 00:47:45.750 The bats that we get in are often 00:47:45.750 --> 00:47:49.850 ones that have been left to the elements for weeks. 00:47:49.850 --> 00:47:52.190 Yet we've had some really- ones in really bad shape. 00:47:52.190 --> 00:47:55.402 There's no brain left, in which case serology isn't possible. 00:47:55.402 --> 00:47:57.110 Again, it's is still a really good assay. 00:47:57.110 --> 00:48:00.770 And we still pick up [INAUDIBLE] in these samples. 00:48:00.770 --> 00:48:03.200 It's still the strong sensitive assay. 00:48:03.200 --> 00:48:05.960 - In which element would they have been exposed to? 00:48:05.960 --> 00:48:07.588 Hydrogen or helium? 00:48:07.588 --> 00:48:10.130 - Oh sorry, I mean just they've been left in the environment. 00:48:10.130 --> 00:48:13.550 So just left in the rain and the wind for weeks on end. 00:48:13.550 --> 00:48:16.400 - Sorry, that was just a subtle attempt at humor 00:48:16.400 --> 00:48:17.805 from this side of the-- 00:48:17.805 --> 00:48:18.580 - Oh, sorry. 00:48:18.580 --> 00:48:19.080 [LAUGH] 00:48:19.080 --> 00:48:22.280 - --pond in regards to Americana. 00:48:22.280 --> 00:48:26.540 Do any of our panelists have any additional questions? 00:48:26.540 --> 00:48:30.830 00:48:30.830 --> 00:48:32.970 Otherwise, we thank you very much. 00:48:32.970 --> 00:48:36.640 And if you'd be so kind to stop your screen sharing 00:48:36.640 --> 00:48:39.430 and stick around, if you would be so kind, so 00:48:39.430 --> 00:48:43.170 that we could have a more engaging discussion at the end. 00:48:43.170 --> 00:48:44.920 Otherwise, thank you very much Megan. 00:48:44.920 --> 00:48:47.330 - Thank you. 00:48:47.330 --> 00:48:53.540 - Our next presentation will be by Dr. Luka Zaeck coming 00:48:53.540 --> 00:48:57.380 from us to us from Europe from the Institute of Molecular 00:48:57.380 --> 00:49:02.510 Virology and Cell Biology from our teutonic colleagues 00:49:02.510 --> 00:49:07.370 at the Frederick Loeffler Institute in the Isle of Rehm's 00:49:07.370 --> 00:49:09.290 in Germany. 00:49:09.290 --> 00:49:14.030 And Dr. Luka will be speaking with us today 00:49:14.030 --> 00:49:20.690 in regards to a high resolution 3D imaging of rabies virus 00:49:20.690 --> 00:49:25.030 infection insolvent cleared brain. 00:49:25.030 --> 00:49:26.490 Dr. Zaeck, you have the floor. 00:49:26.490 --> 00:49:36.500 00:49:36.500 --> 00:49:38.270 - OK now, can you hear me? 00:49:38.270 --> 00:49:41.312 Thank you very much for the introduction. 00:49:41.312 --> 00:49:42.770 As mentioned, my name is Luka Zaeck 00:49:42.770 --> 00:49:45.890 and I am coming from a slightly different angle 00:49:45.890 --> 00:49:48.350 in the previous talks because I'm coming more 00:49:48.350 --> 00:49:50.395 from the basic research angle on this matter 00:49:50.395 --> 00:49:52.520 because I want to talk to you about high resolution 00:49:52.520 --> 00:49:54.650 3D imaging of rabies virus infection 00:49:54.650 --> 00:49:57.110 in solvent-cleared tissue. 00:49:57.110 --> 00:50:05.770 So the question there often is when 00:50:05.770 --> 00:50:07.960 trying to understand rabies pathogenesis 00:50:07.960 --> 00:50:10.690 is that we have different models. 00:50:10.690 --> 00:50:12.220 For example, with in vitro models 00:50:12.220 --> 00:50:15.370 using, for example, ex vivo cultures, primary neurons, 00:50:15.370 --> 00:50:17.980 primary astrocytes, or looking at material 00:50:17.980 --> 00:50:21.010 from infected animals, we have various caveats 00:50:21.010 --> 00:50:22.390 that we have to keep in mind when 00:50:22.390 --> 00:50:23.680 looking at primary cultures. 00:50:23.680 --> 00:50:26.200 We are leaving out other populations 00:50:26.200 --> 00:50:29.170 of the central nervous system cell populations. 00:50:29.170 --> 00:50:31.660 And we look at conventional immunohistochemistry 00:50:31.660 --> 00:50:33.760 and 2-D IHC. 00:50:33.760 --> 00:50:36.010 So in animal samples, while we can perfectly 00:50:36.010 --> 00:50:37.660 see whether or not there is infection, 00:50:37.660 --> 00:50:39.952 we were having trouble asserting this kind of infection 00:50:39.952 --> 00:50:43.210 to particular cell types because of limited multicolor 00:50:43.210 --> 00:50:45.820 or multiplexing capabilities, as well 00:50:45.820 --> 00:50:48.430 as we aren't really able to say anything about the larger 00:50:48.430 --> 00:50:51.190 morphology of cells because IHC, convention HC, 00:50:51.190 --> 00:50:55.760 deals with small slices of about five to 10 micrometers. 00:50:55.760 --> 00:50:58.340 So how do we get a broader context of infection? 00:50:58.340 --> 00:51:01.360 How are we able to look at more of the infection 00:51:01.360 --> 00:51:04.120 than just a small layer? 00:51:04.120 --> 00:51:05.860 We can do that by taking bigger samples. 00:51:05.860 --> 00:51:07.943 Which comes with the problem that a bigger sample, 00:51:07.943 --> 00:51:10.180 as you can see here in the image and the photo, 00:51:10.180 --> 00:51:14.000 that the light from microscopy cannot go into it anymore 00:51:14.000 --> 00:51:15.770 and it doesn't come out anymore. 00:51:15.770 --> 00:51:17.390 In order to alleviate this problem, 00:51:17.390 --> 00:51:20.050 we can use a protocol called tissue optical clearing, which 00:51:20.050 --> 00:51:22.240 in the end, makes our tissue transparent, 00:51:22.240 --> 00:51:24.430 or see through, as I hope you can appreciate 00:51:24.430 --> 00:51:26.470 because you can now actually read the word 00:51:26.470 --> 00:51:28.425 rabies below our sample. 00:51:28.425 --> 00:51:29.800 That works in the basic principle 00:51:29.800 --> 00:51:32.410 that the organ, or tissues, generally, 00:51:32.410 --> 00:51:34.480 are made up of several biomolecules. 00:51:34.480 --> 00:51:36.490 There's water, there's fat, there's proteins. 00:51:36.490 --> 00:51:39.490 They all refract light differently 00:51:39.490 --> 00:51:42.040 and this different reflection and refraction of the light 00:51:42.040 --> 00:51:45.580 is causing tissue opacity, so we can't image deeply 00:51:45.580 --> 00:51:46.690 into the sample. 00:51:46.690 --> 00:51:48.470 By using this specialized protocol, 00:51:48.470 --> 00:51:50.680 we can draw those factors out of the sample. 00:51:50.680 --> 00:51:53.880 For example, we can draw water and lipids 00:51:53.880 --> 00:51:56.900 so that we are left with a primarily proteinacious sample, 00:51:56.900 --> 00:51:58.900 which has a high refractive index. 00:51:58.900 --> 00:52:01.930 If we immerse that sample in an organic solvent that 00:52:01.930 --> 00:52:03.460 also has a higher refractive index, 00:52:03.460 --> 00:52:06.160 we are left with a so-called see-through, or transparent, 00:52:06.160 --> 00:52:09.580 organ, which is what you just saw in the picture before. 00:52:09.580 --> 00:52:12.280 And our workflow basically consists out of that. 00:52:12.280 --> 00:52:14.680 We take organs, for example, from experimentally infected 00:52:14.680 --> 00:52:15.180 animals. 00:52:15.180 --> 00:52:17.150 But equally feasible, as for example, 00:52:17.150 --> 00:52:19.300 human clinical samples, which are fixed. 00:52:19.300 --> 00:52:22.870 We dissect them to a millimeter thickness, which 00:52:22.870 --> 00:52:25.660 is about 50 to 100-fold thicker than this conventional 00:52:25.660 --> 00:52:27.370 immunohistochemistry that I mentioned, 00:52:27.370 --> 00:52:31.330 we apply our protocol, which consists of several steps, 00:52:31.330 --> 00:52:34.150 of treatment steps, of the immunostaining and clearing. 00:52:34.150 --> 00:52:36.280 And then finally, we can do confocal laser scanning 00:52:36.280 --> 00:52:37.070 microscopy. 00:52:37.070 --> 00:52:39.880 So high resolution images. 00:52:39.880 --> 00:52:44.860 This allows us to actually get deep informations 00:52:44.860 --> 00:52:47.590 on the infection context because we are looking 00:52:47.590 --> 00:52:49.210 at a three dimensional volume. 00:52:49.210 --> 00:52:52.090 We can assess relations between cell populations. 00:52:52.090 --> 00:52:56.350 We can go do multicolor assays to actually pinpoint 00:52:56.350 --> 00:52:59.650 the infection of cells to specific cell types. 00:52:59.650 --> 00:53:01.930 Because we're at a confocal level here, 00:53:01.930 --> 00:53:04.420 we can actually go at a sub-cellular resolution 00:53:04.420 --> 00:53:08.540 where we can look at the cells at this mentioned sub-celluar 00:53:08.540 --> 00:53:09.170 resolution. 00:53:09.170 --> 00:53:10.900 So we can go from a broad overview 00:53:10.900 --> 00:53:15.220 to an in-depth analysis on a single cell level. 00:53:15.220 --> 00:53:17.130 And of course, the protocol the images 00:53:17.130 --> 00:53:18.880 I showed you before were in mouse samples, 00:53:18.880 --> 00:53:19.755 experimental animals. 00:53:19.755 --> 00:53:23.560 It's equally applicable to basically any kind of tissue. 00:53:23.560 --> 00:53:24.460 Here as an example. 00:53:24.460 --> 00:53:27.130 There is a RABV infected ferret. 00:53:27.130 --> 00:53:29.590 And as you can see, if we take our samples 00:53:29.590 --> 00:53:31.540 from different sides of the brain, 00:53:31.540 --> 00:53:34.780 we find a different abundance of rabies virus infection 00:53:34.780 --> 00:53:36.160 in different areas of the brain. 00:53:36.160 --> 00:53:39.550 This is not a real surprise yet. 00:53:39.550 --> 00:53:43.180 The strong suit of this way, for example, is also true. 00:53:43.180 --> 00:53:45.160 If we look at sub figure d here, we 00:53:45.160 --> 00:53:47.695 can find rare and very localized events 00:53:47.695 --> 00:53:49.570 because we were looking at such a high volume 00:53:49.570 --> 00:53:52.360 that we can miss it by only looking at the small micrometer 00:53:52.360 --> 00:53:54.580 thick section and conventional IHC 00:53:54.580 --> 00:53:57.520 so that we are able to find those two single infected cells 00:53:57.520 --> 00:53:59.380 in this large volume here. 00:53:59.380 --> 00:54:02.977 Again much more visual for us to see 00:54:02.977 --> 00:54:05.560 if we look at three dimensional reconstructions and renderings 00:54:05.560 --> 00:54:09.130 of those volumes because it is allowing us to actually grasp 00:54:09.130 --> 00:54:11.680 the spatial morphological distribution, and also just 00:54:11.680 --> 00:54:15.790 the neuromorphology of those infected cells. 00:54:15.790 --> 00:54:19.210 And as mentioned, the protocol is fully multiplexable 00:54:19.210 --> 00:54:22.480 so we can use multiple standings. 00:54:22.480 --> 00:54:25.000 Apart from the rabies virus antigens of course, 00:54:25.000 --> 00:54:27.400 we can stain for marker proteins like GFAP, 00:54:27.400 --> 00:54:29.350 which is a marker for [INAUDIBLE] 00:54:29.350 --> 00:54:31.930 in the central nervous system, in this case, astrocytes. 00:54:31.930 --> 00:54:34.005 Or we can look at neurons by staining 00:54:34.005 --> 00:54:36.580 for neuronal markers like MAP2 and then pinpoint 00:54:36.580 --> 00:54:40.450 our location, our infection, to specific cellular 00:54:40.450 --> 00:54:42.070 subpopulations. 00:54:42.070 --> 00:54:45.760 So to sum this experimental approach up 00:54:45.760 --> 00:54:48.490 that we have explained in this protocol that 00:54:48.490 --> 00:54:52.450 was used this special issue, it works on the basic principle 00:54:52.450 --> 00:54:54.340 that we use refractive index matching 00:54:54.340 --> 00:54:58.480 to yield optically clear tissue, which is allowing us to use it 00:54:58.480 --> 00:55:00.720 for high volume microscopy. 00:55:00.720 --> 00:55:02.603 This is very important because when 00:55:02.603 --> 00:55:04.020 we're looking at rabies virus that 00:55:04.020 --> 00:55:06.540 is affecting neuronal tissues, we 00:55:06.540 --> 00:55:09.890 need to understand the distribution in it's 00:55:09.890 --> 00:55:13.520 complex morphology of the neuron with projecting axons 00:55:13.520 --> 00:55:15.440 in all directions, not only to be 00:55:15.440 --> 00:55:17.510 able to actually understand pathogenesis 00:55:17.510 --> 00:55:20.150 at a level of basic research. 00:55:20.150 --> 00:55:22.460 This gives us this large spectrum of logical context 00:55:22.460 --> 00:55:24.980 so we can actually understand how the infection is 00:55:24.980 --> 00:55:26.640 distributed in space. 00:55:26.640 --> 00:55:28.370 And it's also an unbiased approach 00:55:28.370 --> 00:55:30.890 to detect rare and localized events because we're not 00:55:30.890 --> 00:55:32.730 restricting ourselves to several layers 00:55:32.730 --> 00:55:35.630 but we're looking at an entire volume. 00:55:35.630 --> 00:55:38.460 The question then arises is OK, this is a basic protocol. 00:55:38.460 --> 00:55:41.210 What can we actually do with this in a real world scenario? 00:55:41.210 --> 00:55:44.660 And I would like to show you two applications of this protocol 00:55:44.660 --> 00:55:46.790 in a real world basic research scenario 00:55:46.790 --> 00:55:49.685 as well as how we have advanced and further optimized 00:55:49.685 --> 00:55:51.165 these kind of protocols. 00:55:51.165 --> 00:55:52.790 So first I would like to talk about how 00:55:52.790 --> 00:55:56.150 we can use this to in-depth quantify different cells. 00:55:56.150 --> 00:55:59.810 00:55:59.810 --> 00:56:02.360 It comes as no surprise that rabies virus is 00:56:02.360 --> 00:56:04.250 infecting neurons in the brain. 00:56:04.250 --> 00:56:07.530 But what about other cell types in the central nervous system? 00:56:07.530 --> 00:56:09.770 When we look at pathogenic viruses 00:56:09.770 --> 00:56:11.690 with this lab adapted viruses, so basically 00:56:11.690 --> 00:56:13.610 strain-dependent differences, we can actually 00:56:13.610 --> 00:56:16.880 see that here on the right hand side, all of the viruses 00:56:16.880 --> 00:56:19.850 are more or less equally able to infect neurons 00:56:19.850 --> 00:56:21.770 while only the pathogenic [INAUDIBLE] 00:56:21.770 --> 00:56:23.870 viruses, the wild viruses so to speak, 00:56:23.870 --> 00:56:27.140 are able to cause a non-abortive infection of astrocytes. 00:56:27.140 --> 00:56:29.840 While lab-adapter strains, or vaccine strains like ERA, 00:56:29.840 --> 00:56:33.050 are not able to infect astrocytes 00:56:33.050 --> 00:56:38.480 in the brains of IM-inoculated mice non-abortively. 00:56:38.480 --> 00:56:40.850 This the same picture for intracranial inoculation. 00:56:40.850 --> 00:56:44.870 I'm not showing this year for time reasons. 00:56:44.870 --> 00:56:47.030 But by using those large volumes, 00:56:47.030 --> 00:56:49.970 we are able to quantify the full volume, giving us 00:56:49.970 --> 00:56:53.210 a high and robust quantification to really say 00:56:53.210 --> 00:56:56.990 that there is no non-abortive infection within lab adapted 00:56:56.990 --> 00:56:59.705 virus restraints in IM-infected mice. 00:56:59.705 --> 00:57:03.330 00:57:03.330 --> 00:57:07.020 The other application is that besides being 00:57:07.020 --> 00:57:09.960 able to use the high volume for qualification purposes, 00:57:09.960 --> 00:57:12.930 that we use this high volume for visualization purposes. 00:57:12.930 --> 00:57:16.510 And here we optimize our protocols in several ways. 00:57:16.510 --> 00:57:18.540 First of all, we take larger tissue samples. 00:57:18.540 --> 00:57:20.070 But second of all, we implemented 00:57:20.070 --> 00:57:22.020 an additional technical platform that 00:57:22.020 --> 00:57:24.420 allows us to purchase this lychee fluorescence 00:57:24.420 --> 00:57:27.600 microscope, which in comparison to the confocal that 00:57:27.600 --> 00:57:29.760 has a high resolution capacity, this 00:57:29.760 --> 00:57:33.870 is a high volumetric capacity allowing us to acquire not just 00:57:33.870 --> 00:57:36.420 a region of interest for our high volume sample 00:57:36.420 --> 00:57:40.230 but the full high volume sample as in total. 00:57:40.230 --> 00:57:42.360 And here we can see what this looks 00:57:42.360 --> 00:57:44.820 like for central nervous tissues for the brain. 00:57:44.820 --> 00:57:46.680 Again, no surprise. 00:57:46.680 --> 00:57:50.530 The brain is positively full of rabies virus infection 00:57:50.530 --> 00:57:53.620 as you can see here in the red color. 00:57:53.620 --> 00:57:56.790 This is equally true for other CNS parts like the spinal cord. 00:57:56.790 --> 00:57:59.760 Again you can here see the spinal cord 00:57:59.760 --> 00:58:01.980 inside the spinal column still, which 00:58:01.980 --> 00:58:04.530 is positively full of rabies virus antigen, 00:58:04.530 --> 00:58:07.623 also visible in detail shots. 00:58:07.623 --> 00:58:09.540 But the interesting thing that we can see here 00:58:09.540 --> 00:58:12.030 is that in an intramuscularly-inoculated 00:58:12.030 --> 00:58:14.310 animal, we can also visualize the nerve strands 00:58:14.310 --> 00:58:17.220 from the periphery that allow the virus to actually 00:58:17.220 --> 00:58:19.150 infiltrate the central nervous system. 00:58:19.150 --> 00:58:23.160 And this arises the question, or bears the question, 00:58:23.160 --> 00:58:25.710 what is the [INAUDIBLE] for rabies virus in the periphery? 00:58:25.710 --> 00:58:27.250 Besides peripheral nerves of course, 00:58:27.250 --> 00:58:30.067 because it has to somehow get to the central nervous system. 00:58:30.067 --> 00:58:32.400 But are there other cell types in the periphery that are 00:58:32.400 --> 00:58:34.240 being affected by the virus? 00:58:34.240 --> 00:58:37.140 So we looked at the inoculation site but looked at hind legs. 00:58:37.140 --> 00:58:40.710 This is actually a slice, or a section of a hind leg. 00:58:40.710 --> 00:58:44.220 The circular center here is the bone. 00:58:44.220 --> 00:58:46.200 And again, we can see the peripheral nerves 00:58:46.200 --> 00:58:48.240 that the rabies virus uses to infiltrate 00:58:48.240 --> 00:58:49.780 the central nervous system. 00:58:49.780 --> 00:58:51.960 And if we go into closer detail here, 00:58:51.960 --> 00:58:55.110 we can actually see that in green, you can see neurons. 00:58:55.110 --> 00:58:58.630 In Anorectic and rabies virus, we can see infected neurons. 00:58:58.630 --> 00:59:00.990 But we can also see that the virus is often 00:59:00.990 --> 00:59:03.510 associated with the RABV antigens often associated 00:59:03.510 --> 00:59:05.790 around neurons. 00:59:05.790 --> 00:59:09.040 In a tomography, again shown here, for example, 00:59:09.040 --> 00:59:12.120 that the antigen surrounded around the neurons, 00:59:12.120 --> 00:59:14.550 showing us that it doesn't seem to be affecting neurons 00:59:14.550 --> 00:59:16.170 exclusively in the periphery. 00:59:16.170 --> 00:59:20.340 If we stained for an additional marker, MBP, 00:59:20.340 --> 00:59:23.400 myelin basic protein, we can see that it does coalocalize 00:59:23.400 --> 00:59:26.940 with this marker, showing us that the virus seems 00:59:26.940 --> 00:59:29.910 to be infecting cells in the periphery surrounding neurons, 00:59:29.910 --> 00:59:32.130 expressing myelin sheaths. 00:59:32.130 --> 00:59:33.930 What kind of cells do that? 00:59:33.930 --> 00:59:37.680 The insulators of the periphery, so-called Schwann cells. 00:59:37.680 --> 00:59:40.500 So using this high volumetric approach because we 00:59:40.500 --> 00:59:42.300 aren't restricted to small volumes , 00:59:42.300 --> 00:59:45.750 we're able to screen a high volume of tissue, 00:59:45.750 --> 00:59:48.540 allowing the identification of a new target subpopulation 00:59:48.540 --> 00:59:50.580 of virus in peripheral tissues. 00:59:50.580 --> 00:59:53.680 In this case, Schwann cells. 00:59:53.680 --> 00:59:57.930 So as a global summary of what this basic research-- 00:59:57.930 --> 01:00:00.000 of course it's a basic research method-- 01:00:00.000 --> 01:00:02.370 but of course, this three dimensional tissue imaging 01:00:02.370 --> 01:00:06.030 can yield is that it can help in elucidating global virus 01:00:06.030 --> 01:00:09.510 tropism by, for example, allowing us to gain insights 01:00:09.510 --> 01:00:11.460 into strain-dependent differences 01:00:11.460 --> 01:00:13.080 in the central nervous system. 01:00:13.080 --> 01:00:16.890 By identifying new target cell populations like Schwann 01:00:16.890 --> 01:00:20.100 cells in the periphery with pathogenic [INAUDIBLE] rabies 01:00:20.100 --> 01:00:20.880 viruses. 01:00:20.880 --> 01:00:23.190 Or also by giving additional evidence 01:00:23.190 --> 01:00:25.170 for anterograde transport of rabies virus, 01:00:25.170 --> 01:00:26.490 which I haven't shown here. 01:00:26.490 --> 01:00:28.470 But just to quickly give an insight, 01:00:28.470 --> 01:00:30.900 we can also see those infected Schwann cells 01:00:30.900 --> 01:00:34.080 after intracranial-inoculated animals giving us 01:00:34.080 --> 01:00:35.520 this supporting evidence. 01:00:35.520 --> 01:00:37.650 Not just for the exclusive retrograde, 01:00:37.650 --> 01:00:40.450 but also for anterograde transport. 01:00:40.450 --> 01:00:42.780 This I would like to close my talk. 01:00:42.780 --> 01:00:44.500 I am part of the [INAUDIBLE] Group, 01:00:44.500 --> 01:00:46.530 [INAUDIBLE] Group at the [INAUDIBLE] 01:00:46.530 --> 01:00:48.120 Institute in Germany. 01:00:48.120 --> 01:00:50.370 And I would like to thank all of the people 01:00:50.370 --> 01:00:52.110 in our rabies group that have been involved with this, 01:00:52.110 --> 01:00:53.340 particularly would like to highlight 01:00:53.340 --> 01:00:55.470 Madlin Portratz, who's done a lot of the imaging work. 01:00:55.470 --> 01:00:57.428 And I'd love to answer any questions there are. 01:00:57.428 --> 01:01:00.400 Thank you very much. 01:01:00.400 --> 01:01:01.360 - Thank you, Luka. 01:01:01.360 --> 01:01:05.980 And I'm still trying to get over my amazement 01:01:05.980 --> 01:01:09.700 from your kaleidoscopic investigations, which 01:01:09.700 --> 01:01:12.610 have really helped to revolutionize 01:01:12.610 --> 01:01:14.920 our understanding of pathobiology 01:01:14.920 --> 01:01:17.080 from these techniques. 01:01:17.080 --> 01:01:17.830 Very nice job. 01:01:17.830 --> 01:01:19.150 Thank you. 01:01:19.150 --> 01:01:24.160 We do have some questions, one from our colleague Dr. Tudo who 01:01:24.160 --> 01:01:26.440 wanted to know that given that there are 01:01:26.440 --> 01:01:28.360 previous articles in the literature 01:01:28.360 --> 01:01:31.960 that not only street rabies viruses 01:01:31.960 --> 01:01:35.200 but other lyssaviruses may have the capability 01:01:35.200 --> 01:01:38.230 to infect astrocytes. 01:01:38.230 --> 01:01:42.400 Were you able to compare different species 01:01:42.400 --> 01:01:47.390 of lyssaviruses besides rabies virus? 01:01:47.390 --> 01:01:49.470 - We haven't done that yet. 01:01:49.470 --> 01:01:51.240 So the studies that I've shown you 01:01:51.240 --> 01:01:53.575 are exclusively done on RTBV. 01:01:53.575 --> 01:01:54.950 But of course, different strains, 01:01:54.950 --> 01:01:56.990 pathogenic versus non-pathogenic or field work. 01:01:56.990 --> 01:01:59.420 [INAUDIBLE] lab adapted or vaccine viruses. 01:01:59.420 --> 01:02:01.070 We're actually working on looking 01:02:01.070 --> 01:02:05.370 at astrocyte tropism in the lyssavirus spectrum as a whole. 01:02:05.370 --> 01:02:08.990 So we are hopefully able to give an insight 01:02:08.990 --> 01:02:11.200 on that in the future. 01:02:11.200 --> 01:02:13.842 But concerning astrocyte tropism that has been shown for lab 01:02:13.842 --> 01:02:15.550 adapted viruses, that has actually mostly 01:02:15.550 --> 01:02:20.270 been done either in vitro cultures 01:02:20.270 --> 01:02:24.460 so that we do not have this additional layer 01:02:24.460 --> 01:02:26.590 of the entire organism. 01:02:26.590 --> 01:02:29.890 We can also see with another nice publication 01:02:29.890 --> 01:02:33.820 that those lab adapted viruses seem 01:02:33.820 --> 01:02:35.880 to infect astrocytes, but abortively, 01:02:35.880 --> 01:02:37.255 because the infection is cleared. 01:02:37.255 --> 01:02:40.200 01:02:40.200 --> 01:02:43.110 - And do you have any opportunities to look 01:02:43.110 --> 01:02:45.105 at species beyond rodents? 01:02:45.105 --> 01:02:50.110 01:02:50.110 --> 01:02:53.260 - As I showed of course, it is possible to apply the protocol 01:02:53.260 --> 01:02:55.210 the variety of tissues. 01:02:55.210 --> 01:02:57.650 We are an animal health facility. 01:02:57.650 --> 01:03:01.480 Of course, we are mostly working with animal tissue, 01:03:01.480 --> 01:03:03.560 not likely human clinical, samples of course. 01:03:03.560 --> 01:03:05.800 That would be an extremely interesting endeavor 01:03:05.800 --> 01:03:08.260 to also look at those particular questions 01:03:08.260 --> 01:03:11.392 in human clinical samples but also with naturally 01:03:11.392 --> 01:03:12.850 infected animals, like for example, 01:03:12.850 --> 01:03:16.420 bats would be a very interesting endeavor to do. 01:03:16.420 --> 01:03:16.960 - Yes. 01:03:16.960 --> 01:03:22.990 And one of our participants asks beyond neural cells, 01:03:22.990 --> 01:03:25.030 astrocytes, et cetera, what about muscle 01:03:25.030 --> 01:03:26.260 cells in the periphery? 01:03:26.260 --> 01:03:29.320 Aren't they infected also? 01:03:29.320 --> 01:03:30.730 - Literature tells us yes. 01:03:30.730 --> 01:03:33.790 We haven't particularly put our focus to that yet. 01:03:33.790 --> 01:03:37.540 Also, very interesting question to look at this amplification 01:03:37.540 --> 01:03:40.420 stage of what has always been described as an amplification 01:03:40.420 --> 01:03:42.133 stage in muscle cells. 01:03:42.133 --> 01:03:44.050 But we haven't specifically looked at that yet 01:03:44.050 --> 01:03:45.970 and first focused on whether we can 01:03:45.970 --> 01:03:49.660 find additional cells of the peripheral nervous system that 01:03:49.660 --> 01:03:51.840 are infected by the virus. 01:03:51.840 --> 01:03:52.340 - Very good. 01:03:52.340 --> 01:03:56.690 And we know that one of our late German colleagues Dr. Leuchter 01:03:56.690 --> 01:04:00.440 Schneider did some very nice comparative pathobiological 01:04:00.440 --> 01:04:01.340 studies. 01:04:01.340 --> 01:04:07.070 And we have one participant ask how based on your techniques 01:04:07.070 --> 01:04:11.030 is rabies virus transferred from the periphery 01:04:11.030 --> 01:04:12.560 to the central nervous system? 01:04:12.560 --> 01:04:15.470 01:04:15.470 --> 01:04:19.840 - I don't know if it's possible to ask for clarification here. 01:04:19.840 --> 01:04:22.030 What does the person mean by how? 01:04:22.030 --> 01:04:25.030 So what we can see from our images 01:04:25.030 --> 01:04:29.340 is that we infect the mice at peripheral sites. 01:04:29.340 --> 01:04:31.720 So for example, the hind leg muscles, in all case 01:04:31.720 --> 01:04:34.180 those experimental mice. 01:04:34.180 --> 01:04:40.030 And we see the nerves from the infection site being infected 01:04:40.030 --> 01:04:41.920 up until the spinal cord where it then 01:04:41.920 --> 01:04:44.860 enters into the central nervous system. 01:04:44.860 --> 01:04:45.430 - Excellent. 01:04:45.430 --> 01:04:47.050 I hope that answers the question. 01:04:47.050 --> 01:04:47.650 - Yes. 01:04:47.650 --> 01:04:50.890 And our colleague Dr. Thomas Muller 01:04:50.890 --> 01:04:53.140 mentions that recent studies show 01:04:53.140 --> 01:04:57.280 that bat-associated lyssaviruses are also 01:04:57.280 --> 01:05:02.320 able to infect astrocytes. 01:05:02.320 --> 01:05:05.330 We do have a question from one of our colleagues 01:05:05.330 --> 01:05:08.200 that I believe this is from Dr. Satoshi Inoway, 01:05:08.200 --> 01:05:09.910 perhaps from Japan. 01:05:09.910 --> 01:05:14.700 What about the salivary glands? 01:05:14.700 --> 01:05:19.770 - Also very interesting to look at salivary glands in terms 01:05:19.770 --> 01:05:21.750 of transmission of the virus. 01:05:21.750 --> 01:05:25.290 I mean we have to, of course, focus here, or stage here, 01:05:25.290 --> 01:05:26.760 that we are dealing with mice. 01:05:26.760 --> 01:05:31.920 And I personally am not so sure on the role of mice 01:05:31.920 --> 01:05:35.130 with the upkeep of rabies virus transmission cycles. 01:05:35.130 --> 01:05:39.900 I would assume they have a rather minor role. 01:05:39.900 --> 01:05:45.585 Well we have found some rabies [INAUDIBLE] antigen 01:05:45.585 --> 01:05:47.140 in salivary glands. 01:05:47.140 --> 01:05:48.650 We are still working on this. 01:05:48.650 --> 01:05:52.830 It didn't seem as much as we expected to be there. 01:05:52.830 --> 01:05:53.730 - Yes. 01:05:53.730 --> 01:05:55.770 And I think you hit that right on the head 01:05:55.770 --> 01:05:59.142 that this has been one of our pet peeves for many of us 01:05:59.142 --> 01:06:00.600 that no, you're absolutely correct. 01:06:00.600 --> 01:06:03.090 Rodents are not a reservoir. 01:06:03.090 --> 01:06:06.810 However, whenever we have larger bodied rodents 01:06:06.810 --> 01:06:12.150 in contact with, for example, carnivores, in North America, 01:06:12.150 --> 01:06:17.220 we have spillover infection from carnivore rabies virus 01:06:17.220 --> 01:06:20.670 to large body rodents like beavers, 01:06:20.670 --> 01:06:22.590 or groundhogs, woodchucks. 01:06:22.590 --> 01:06:26.310 So perhaps some of our erstwhile investigators in the field 01:06:26.310 --> 01:06:29.100 might be able to send some of these naturally 01:06:29.100 --> 01:06:31.990 infected animals, preserved appropriately, to you 01:06:31.990 --> 01:06:34.800 and we might be able to get some answers to questions 01:06:34.800 --> 01:06:39.450 if they're differential impacts of these viruses and carnivores 01:06:39.450 --> 01:06:42.120 or non-reservoir species. 01:06:42.120 --> 01:06:45.340 - Thank you for your very excellent presentation. 01:06:45.340 --> 01:06:50.490 I see that we are now in time for a very short break. 01:06:50.490 --> 01:06:52.980 For some of us who have been hydrating because we've 01:06:52.980 --> 01:06:55.500 been glued to our screens, we don't 01:06:55.500 --> 01:07:00.540 want to take any time away from listening to these 01:07:00.540 --> 01:07:03.480 so I know I'm going to take a quick bathroom break, 01:07:03.480 --> 01:07:08.310 and we will be back in 3 minutes. 01:07:08.310 --> 01:07:10.480 Please stay with us until that time. 01:07:10.480 --> 01:07:12.296 Be back shortly. 01:07:12.296 --> 01:07:16.240 [MUSIC - TALKING HEADS, "ONCE IN A LIFETIME"] 01:07:16.240 --> 01:07:21.170 01:07:21.170 --> 01:07:25.930 You may find yourself living in a shotgun shack. 01:07:25.930 --> 01:07:29.940 You may find yourself in another part of the world. 01:07:29.940 --> 01:07:32.830 And you may find yourself behind the wheel 01:07:32.830 --> 01:07:35.015 of a large automobile. 01:07:35.015 --> 01:07:39.150 And you may find yourself in a beautiful house 01:07:39.150 --> 01:07:41.460 with a beautiful wife. 01:07:41.460 --> 01:07:46.025 And you may ask yourself, well, how did I get here? 01:07:46.025 --> 01:07:50.140 Letting the days go by, let the water hold me down. 01:07:50.140 --> 01:07:54.390 Letting the days go by, water flowing underground. 01:07:54.390 --> 01:07:58.074 Into the blue again after the money's gone. 01:07:58.074 --> 01:08:02.900 Once in a lifetime, water flowing underground. 01:08:02.900 --> 01:08:06.544 You may ask yourself, how do I work this? 01:08:06.544 --> 01:08:11.200 And you may ask yourself, where is that large automobile? 01:08:11.200 --> 01:08:15.373 And you may tell yourself, this is not my beautiful house. 01:08:15.373 --> 01:08:18.930 And you may tell yourself, this is not my beautiful wife. 01:08:18.930 --> 01:08:23.177 Letting the days go by, let the water hold me down. 01:08:23.177 --> 01:08:27.520 Letting the days go by, water flowing underground. 01:08:27.520 --> 01:08:31.520 Into the blue again after the money's gone. 01:08:31.520 --> 01:08:36.246 Once in a lifetime, water flowing underground. 01:08:36.246 --> 01:08:40.405 Same as is it ever was, same as it ever was, same 01:08:40.405 --> 01:08:46.890 as it ever was, same as it ever was, same as it ever was, same 01:08:46.890 --> 01:08:52.070 as it ever was, same as it ever was, same as it ever was. 01:08:52.070 --> 01:08:56.503 Letting the days go by, let the water hold me down. 01:08:56.503 --> 01:09:00.904 Letting the days go by, water flowing underground. 01:09:00.904 --> 01:09:04.327 Into the blue again after the moneys gone. 01:09:04.327 --> 01:09:08.776 Once in a lifetime, water flowing underground. 01:09:08.776 --> 01:09:13.332 You may ask yourself, what is that beautiful house? 01:09:13.332 --> 01:09:16.870 You may ask yourself, where does that highway go to? 01:09:16.870 --> 01:09:20.890 And you may ask yourself, am I right, am I wrong? 01:09:20.890 --> 01:09:25.463 And you may say to yourself, my God, what have I done? 01:09:25.463 --> 01:09:29.927 Letting the days go by, let the water hold me down. 01:09:29.927 --> 01:09:33.895 Letting the days go by, water flowing underground. 01:09:33.895 --> 01:09:38.136 Into the blue again, into the silent water. 01:09:38.136 --> 01:09:42.040 Under the rocks and stone, there is water underground. 01:09:42.040 --> 01:09:46.432 Letting the days go by, let the water hold me down. 01:09:46.432 --> 01:09:50.830 Letting the days go by, water flowing underground. 01:09:50.830 --> 01:09:54.680 Into the blue again after the money's gone. 01:09:54.680 --> 01:10:00.180 Once in a lifetime, water flowing under-- 01:10:00.180 --> 01:10:04.260 - Hello, everyone, and welcome back from our short break. 01:10:04.260 --> 01:10:07.875 You are at the JoVE rabies webinar. 01:10:07.875 --> 01:10:10.740 01:10:10.740 --> 01:10:18.430 We're going to be rejoining with our next presenter, Dr. Laurent 01:10:18.430 --> 01:10:22.680 Dacheux, who is one of the deputy directors 01:10:22.680 --> 01:10:27.210 at the National Reference Center for Rabies at the Institut 01:10:27.210 --> 01:10:28.530 Pasteur. 01:10:28.530 --> 01:10:31.710 Some of the things he does is to supervise the activities 01:10:31.710 --> 01:10:35.520 of diagnosis in humans and other animals 01:10:35.520 --> 01:10:40.560 in a very high quality assurance environment. 01:10:40.560 --> 01:10:45.900 And he's very much invested in the development and validation 01:10:45.900 --> 01:10:49.770 of diagnostic techniques related to rabies, particularly 01:10:49.770 --> 01:10:52.320 in regards to their WHO collaborating 01:10:52.320 --> 01:10:54.150 center activities. 01:10:54.150 --> 01:11:00.150 He's a very active researcher, collaborator, and publisher. 01:11:00.150 --> 01:11:05.190 And he's going to be sharing with us today 01:11:05.190 --> 01:11:07.170 some of his experience in the field 01:11:07.170 --> 01:11:11.730 post-mortem rabies rapid immunochromatographic 01:11:11.730 --> 01:11:15.690 diagnostic test for resource-limited settings 01:11:15.690 --> 01:11:19.590 with further molecular applications. 01:11:19.590 --> 01:11:21.510 Dr. Dacheux, the floor is yours. 01:11:21.510 --> 01:11:33.950 01:11:33.950 --> 01:11:35.000 - Sorry. 01:11:35.000 --> 01:11:35.830 Excuse me. 01:11:35.830 --> 01:11:37.640 My microphone went off. 01:11:37.640 --> 01:11:38.730 So it's OK now? 01:11:38.730 --> 01:11:40.250 Yeah, OK. 01:11:40.250 --> 01:11:44.310 So thank you, Dr. Rupprecht, For this nice introduction. 01:11:44.310 --> 01:11:45.740 So as you indicated, I would like 01:11:45.740 --> 01:11:51.140 to share with you some results and experience about the field 01:11:51.140 --> 01:11:53.900 post-mortem diagnosis with a RIDT or Rapid 01:11:53.900 --> 01:11:56.220 Immunochromatographic Diagnosis Test, 01:11:56.220 --> 01:12:00.140 which was published in the special edition of JoVE. 01:12:00.140 --> 01:12:03.690 So the context. 01:12:03.690 --> 01:12:06.100 We know that, unfortunately, rabies, 01:12:06.100 --> 01:12:08.940 and especially canine rabies underreporting, especially 01:12:08.940 --> 01:12:12.840 in enzootic countries and low- and middle-income countries. 01:12:12.840 --> 01:12:16.055 And this fact is that the this impact 01:12:16.055 --> 01:12:18.660 impairs disease surveillance and control 01:12:18.660 --> 01:12:22.320 due to the lack of reliable data. 01:12:22.320 --> 01:12:24.720 We know also that to have some reliable data, 01:12:24.720 --> 01:12:28.710 we need to confirm rabies cases, especially animal cases, 01:12:28.710 --> 01:12:31.446 by biological diagnosis tests which are done 01:12:31.446 --> 01:12:33.750 on post-mortem brain tissue. 01:12:33.750 --> 01:12:38.100 And the references methods are provided by WHO and OIE-- 01:12:38.100 --> 01:12:40.830 of course, the classical one with a direct fluorescence 01:12:40.830 --> 01:12:43.920 antibody test, but also with a Direct Rapid 01:12:43.920 --> 01:12:48.420 Immunohistochemistry Test, DRIT, and also 01:12:48.420 --> 01:12:51.450 the molecular technique such as the one 01:12:51.450 --> 01:12:55.930 you will see in this webinar, like qPCR and RT-qPCR. 01:12:55.930 --> 01:12:58.740 However, these techniques, these reference techniques, 01:12:58.740 --> 01:13:00.690 have some limitations. 01:13:00.690 --> 01:13:02.660 Because there is a fact that many-- 01:13:02.660 --> 01:13:04.680 [CLEARS THROAT] excuse me-- they are mainly 01:13:04.680 --> 01:13:06.780 restricted to central veterinary laboratories. 01:13:06.780 --> 01:13:10.800 Indeed, they have some different technical and material 01:13:10.800 --> 01:13:14.760 constraints, like it should be done in equipped laboratories 01:13:14.760 --> 01:13:19.680 with a trained staff with power suppliers 01:13:19.680 --> 01:13:26.020 or with the [INAUDIBLE] maintenance, and so on. 01:13:26.020 --> 01:13:27.600 So what are the needs? 01:13:27.600 --> 01:13:30.690 What we are looking in, for example, in Africa and Asia, 01:13:30.690 --> 01:13:34.830 is to have a rapid and reliable diagnostic tests and protocols 01:13:34.830 --> 01:13:38.010 also [INAUDIBLE] low technical expertise. 01:13:38.010 --> 01:13:40.710 And these diagnosis methods should be accessible 01:13:40.710 --> 01:13:44.310 to decentralized laboratories, especially 01:13:44.310 --> 01:13:48.720 in the rural area for example, so even in the field, 01:13:48.720 --> 01:13:51.480 like a Point-Of-Care Test or POCT. 01:13:51.480 --> 01:13:53.790 And we know that several diagnosis protocols were 01:13:53.790 --> 01:13:55.410 recently developed and tested. 01:13:55.410 --> 01:13:57.450 And the one I would like to focus on 01:13:57.450 --> 01:14:01.540 is Rapid Immunochromatographic Diagnosis Test, or RIDT, 01:14:01.540 --> 01:14:04.520 which also is referred as Lateral Flow Device, LFD, 01:14:04.520 --> 01:14:08.310 or LFA, Lateral Flow Immunoassay. 01:14:08.310 --> 01:14:10.620 So this is the aim of this short presentation, 01:14:10.620 --> 01:14:15.340 is to present you an application of RIDT in an African context, 01:14:15.340 --> 01:14:17.610 for example, from the brain sample 01:14:17.610 --> 01:14:20.220 collection to the analysis of the results, 01:14:20.220 --> 01:14:26.820 and also further, to the medical diagnosis based on this RIDT. 01:14:26.820 --> 01:14:30.780 Different rapid immunochromatographic tests 01:14:30.780 --> 01:14:34.230 are available on the market for the detection of rabies. 01:14:34.230 --> 01:14:36.870 But my purpose of my talk will be 01:14:36.870 --> 01:14:38.550 focused on only one which has been 01:14:38.550 --> 01:14:43.080 tested in this purpose, the rapid rabies antigen from 01:14:43.080 --> 01:14:44.080 [INAUDIBLE]. 01:14:44.080 --> 01:14:46.080 And you can see here the content of the kit, 01:14:46.080 --> 01:14:49.650 where have all inside which can be used on the file. 01:14:49.650 --> 01:14:53.250 What you need to have access to have a tube 01:14:53.250 --> 01:14:55.710 to preserve brain samples. 01:14:55.710 --> 01:14:58.080 So rapidly, what is the principle of the lateral flow 01:14:58.080 --> 01:14:58.740 device? 01:14:58.740 --> 01:15:01.290 So it's based on the immunochromatographic 01:15:01.290 --> 01:15:04.980 techniques using gold-conjugated detector antibodies. 01:15:04.980 --> 01:15:08.070 So you have here the device with a sample pad, 01:15:08.070 --> 01:15:10.470 the conjugate pad, the nitrocellulose membrane, 01:15:10.470 --> 01:15:14.694 the test line of T-line, the control line, C-line, 01:15:14.694 --> 01:15:15.930 and the absorbent pad. 01:15:15.930 --> 01:15:18.810 And what are very important here are the antibodies. 01:15:18.810 --> 01:15:21.480 We have here the gold-labeled IgG 01:15:21.480 --> 01:15:24.900 to detect the antibodies which are called detector antibodies. 01:15:24.900 --> 01:15:27.810 Here on the test line you have the sensor antibodies, 01:15:27.810 --> 01:15:29.820 IgG sensor antibodies. 01:15:29.820 --> 01:15:32.400 And the control line, you have the antibodies, 01:15:32.400 --> 01:15:34.530 again the IgG antibodies. 01:15:34.530 --> 01:15:37.080 And so the principle is quite-- they are very simple, 01:15:37.080 --> 01:15:38.533 like a pregnancy test. 01:15:38.533 --> 01:15:40.700 So you have the brain samples, crushed brain sample, 01:15:40.700 --> 01:15:42.090 as I specified before. 01:15:42.090 --> 01:15:44.610 You put it on the pad, and then you 01:15:44.610 --> 01:15:48.810 have a mixture between the antigens in red here 01:15:48.810 --> 01:15:51.870 and the detector antibodies. 01:15:51.870 --> 01:15:55.230 If you have antigens, you have an immune complex 01:15:55.230 --> 01:15:56.340 which are formed. 01:15:56.340 --> 01:15:58.500 And then we [INAUDIBLE] in addition 01:15:58.500 --> 01:16:01.860 with a free IgG detector. 01:16:01.860 --> 01:16:04.560 And then if you have the immune complex, 01:16:04.560 --> 01:16:08.950 they will be fixed at the T-line by the sensor antibody. 01:16:08.950 --> 01:16:13.170 And the three IgG detector antibodies [INAUDIBLE] 01:16:13.170 --> 01:16:16.440 and will be kept fixed under control line 01:16:16.440 --> 01:16:19.560 by the anti-IgG antibodies. 01:16:19.560 --> 01:16:23.190 For this test, one key point is the collection 01:16:23.190 --> 01:16:25.080 of the brain samples. 01:16:25.080 --> 01:16:28.080 In the lab, you can make an autopsy of the total brain 01:16:28.080 --> 01:16:29.670 and the head of the animals. 01:16:29.670 --> 01:16:31.600 But in the field, it's not very practical. 01:16:31.600 --> 01:16:34.760 So what way [INAUDIBLE] need to have access 01:16:34.760 --> 01:16:36.870 directly to the brain stem. 01:16:36.870 --> 01:16:39.630 You can see here, we have the occipital lobe. 01:16:39.630 --> 01:16:41.400 And you will see a presentation of that, 01:16:41.400 --> 01:16:43.620 I think, later on in the webinar. 01:16:43.620 --> 01:16:46.170 It is compatible with the field application. 01:16:46.170 --> 01:16:50.920 And these are to be done quite freshly on fresh samples. 01:16:50.920 --> 01:16:54.630 And you can see here the foramen magnum and the access 01:16:54.630 --> 01:16:57.950 to the brain stem. 01:16:57.950 --> 01:16:58.860 Here we can see that. 01:16:58.860 --> 01:17:00.860 And what is very important here, as a key point, 01:17:00.860 --> 01:17:02.480 is to have the good sample, because we 01:17:02.480 --> 01:17:06.530 know that brain stem reference samples for the diagnosis, 01:17:06.530 --> 01:17:08.930 which is quite rich in terms presence of antigens 01:17:08.930 --> 01:17:12.375 and viruses, that the teams or the staff 01:17:12.375 --> 01:17:14.000 have to be trained for this step, which 01:17:14.000 --> 01:17:16.010 is highly recommended. 01:17:16.010 --> 01:17:18.440 And after all, here you can see that on the field 01:17:18.440 --> 01:17:22.520 we can collect the brain stem with different disposal things, 01:17:22.520 --> 01:17:25.800 like a plastic pipette, a drinking straw 01:17:25.800 --> 01:17:28.250 or plastic drinking straw, clamps, 01:17:28.250 --> 01:17:32.750 even with a disposable droppers, provided in the kits. 01:17:32.750 --> 01:17:34.520 And for the protocol of the [INAUDIBLE],, 01:17:34.520 --> 01:17:37.900 the initial protocol provided by the [INAUDIBLE] 01:17:37.900 --> 01:17:41.020 for the manufacturer, you have the brain samples. 01:17:41.020 --> 01:17:43.670 You put that in-- 01:17:43.670 --> 01:17:47.480 you put that in the PBS, with a dilution of 10%. 01:17:47.480 --> 01:17:51.725 You mix it with a swab provided in the kit. 01:17:51.725 --> 01:17:53.900 PBS are not provided in the kit, so you 01:17:53.900 --> 01:17:56.630 have to carry it with you, as well as a tube. 01:17:56.630 --> 01:17:59.600 And then after, you put this dilution 01:17:59.600 --> 01:18:02.120 on the buffer contained in the kit. 01:18:02.120 --> 01:18:06.410 You mix with a swab provided in the kit, 01:18:06.410 --> 01:18:09.500 take some drops here, and put on the pad 01:18:09.500 --> 01:18:10.930 and wait for the [INAUDIBLE]. 01:18:10.930 --> 01:18:13.680 [INAUDIBLE] That's quite simple. 01:18:13.680 --> 01:18:15.800 But the problem is that you have to put some PBS, 01:18:15.800 --> 01:18:18.600 and that also you have to [? resolve ?] a clear PBS 01:18:18.600 --> 01:18:19.100 and tube. 01:18:19.100 --> 01:18:21.810 So in our protocol, it was very simple, 01:18:21.810 --> 01:18:24.860 we just skipped these dilution steps to have 01:18:24.860 --> 01:18:29.215 a more rapid diagnosis method. 01:18:29.215 --> 01:18:30.590 And in fact, we also demonstrated 01:18:30.590 --> 01:18:34.190 that this technique was able to increase the level 01:18:34.190 --> 01:18:36.380 of sensitivity and specificity. 01:18:36.380 --> 01:18:38.390 And here are just the [INAUDIBLE] results. 01:18:38.390 --> 01:18:40.190 I mean, here we have a positive result 01:18:40.190 --> 01:18:42.500 with a tester line and a control line. 01:18:42.500 --> 01:18:44.120 So two [INAUDIBLE],, this is positive. 01:18:44.120 --> 01:18:46.610 One [INAUDIBLE] is the control line, it's negative. 01:18:46.610 --> 01:18:49.380 And here if you have no [INAUDIBLE],, 01:18:49.380 --> 01:18:51.560 no migrations, for only the test, 01:18:51.560 --> 01:18:53.750 like no control [INAUDIBLE],, that 01:18:53.750 --> 01:18:56.120 means that you have invalid results, 01:18:56.120 --> 01:18:58.490 and the test has to be repeated. 01:18:58.490 --> 01:19:01.460 And here is an example of a representative result 01:19:01.460 --> 01:19:04.350 we had from our experiments. 01:19:04.350 --> 01:19:07.070 We performed that in five laboratories. 01:19:07.070 --> 01:19:08.750 And what was interesting, it [INAUDIBLE] 01:19:08.750 --> 01:19:13.640 being done in three laboratories in the field in Africa. 01:19:13.640 --> 01:19:18.900 And here we performed that in only more than 250 samples. 01:19:18.900 --> 01:19:21.470 And you can see, the results-- 01:19:21.470 --> 01:19:25.100 01:19:25.100 --> 01:19:27.980 their sensitivity and specificity are not so bad. 01:19:27.980 --> 01:19:30.620 For the sensitivity we have nearly 98%, 01:19:30.620 --> 01:19:35.940 and for the specificity 95%. 01:19:35.940 --> 01:19:37.770 Another interesting point of these kits 01:19:37.770 --> 01:19:39.460 is that if you have the device here, 01:19:39.460 --> 01:19:42.330 so you already use it for the detection of antigens, 01:19:42.330 --> 01:19:44.310 you can also use it for the detection 01:19:44.310 --> 01:19:47.520 of RNA, viral RNA, which are fixed 01:19:47.520 --> 01:19:50.110 in storage under the membrane. 01:19:50.110 --> 01:19:56.010 So you have to open, when it is finished, the device carefully. 01:19:56.010 --> 01:19:59.100 You take the membrane, just get [INAUDIBLE] 01:19:59.100 --> 01:20:03.210 the part where you put the samples, 01:20:03.210 --> 01:20:06.660 and perform to an extraction with different protocol. 01:20:06.660 --> 01:20:10.170 Here we use a protocol based on TRIzol extraction. 01:20:10.170 --> 01:20:13.900 And you can use whatever techniques, validity technique, 01:20:13.900 --> 01:20:15.810 for the detection of viral RNA-- 01:20:15.810 --> 01:20:19.680 RT-qPCR or [INAUDIBLE] PCR. 01:20:19.680 --> 01:20:22.980 And this could be also done for the Sanger sequencing 01:20:22.980 --> 01:20:24.240 by genotyping. 01:20:24.240 --> 01:20:26.105 And here we have some examples which 01:20:26.105 --> 01:20:31.920 were obtained in a study conducted in Chad in 2016. 01:20:31.920 --> 01:20:35.040 And you see here, we have the samples 01:20:35.040 --> 01:20:38.123 which have been tested directly in the lab or samples which 01:20:38.123 --> 01:20:39.540 have been collected from the field 01:20:39.540 --> 01:20:42.060 and sent to the lab at room temperature. 01:20:42.060 --> 01:20:44.220 And here are the combined results. 01:20:44.220 --> 01:20:48.510 And we were able to have a positive detection by PCR 01:20:48.510 --> 01:20:51.690 when combining of nearly 96%. 01:20:51.690 --> 01:20:55.050 Of course it was lower for the samples from the field 01:20:55.050 --> 01:20:56.370 than in the lab. 01:20:56.370 --> 01:20:59.730 And from these positive samples, which are in total 18, 01:20:59.730 --> 01:21:05.150 we are able to make the sequencing and typing for 14. 01:21:05.150 --> 01:21:09.200 These results have-- interesting results have been confirmed, 01:21:09.200 --> 01:21:13.160 or at least observed, in several other studies. 01:21:13.160 --> 01:21:15.410 Here I just put some example, which is 01:21:15.410 --> 01:21:18.560 the first one here published. 01:21:18.560 --> 01:21:21.260 This one is interesting, because it demonstrated also 01:21:21.260 --> 01:21:23.740 that it's working for other lyssaviruses 01:21:23.740 --> 01:21:25.040 and rabies viruses. 01:21:25.040 --> 01:21:28.910 This is the one from 2016-- 01:21:28.910 --> 01:21:30.500 sorry, it's a mistake-- 01:21:30.500 --> 01:21:31.760 conducted in Chad. 01:21:31.760 --> 01:21:36.350 This one was one conducted on interlaboratory trial 01:21:36.350 --> 01:21:37.070 with RIDT. 01:21:37.070 --> 01:21:41.498 And this one is the last one published by Kimitsuki in 2020. 01:21:41.498 --> 01:21:43.040 And they're interesting, because it's 01:21:43.040 --> 01:21:48.176 comparing other kits like this one from ADTEC, Bionote, 01:21:48.176 --> 01:21:50.310 and even Elabscience. 01:21:50.310 --> 01:21:53.494 They demonstrated a good sensitivity and specificity-- 01:21:53.494 --> 01:21:56.790 a specificity [INAUDIBLE] and specificity of 88%, 01:21:56.790 --> 01:22:00.770 for example, for ADTEC and 96% for Bionote. 01:22:00.770 --> 01:22:02.510 And what is interesting is that they also 01:22:02.510 --> 01:22:04.940 use this skipping the dilution step 01:22:04.940 --> 01:22:07.030 and increase that sensitivity. 01:22:07.030 --> 01:22:09.030 There were some other studies, two studies, 01:22:09.030 --> 01:22:12.230 that demonstrated also not good results. 01:22:12.230 --> 01:22:14.770 For example, for the two studies here, that 01:22:14.770 --> 01:22:16.520 evaluated some different commercial tests 01:22:16.520 --> 01:22:19.730 and demonstrated that they have very great variation 01:22:19.730 --> 01:22:21.950 between the kits, the manufacturers, 01:22:21.950 --> 01:22:24.890 and it was [INAUDIBLE] in 2020. 01:22:24.890 --> 01:22:28.760 So we've got just some key messages here for this test. 01:22:28.760 --> 01:22:31.460 It's quite simple, rapid, and quite low-cost, 01:22:31.460 --> 01:22:33.530 even though it depends how you manage to drive 01:22:33.530 --> 01:22:35.435 the price of this test. 01:22:35.435 --> 01:22:37.230 It's simple to perform and interpret. 01:22:37.230 --> 01:22:40.880 And as you can see here, so it's suitable for field surveillance 01:22:40.880 --> 01:22:42.410 applications. 01:22:42.410 --> 01:22:45.210 You can use it in different various areas, 01:22:45.210 --> 01:22:48.050 especially in Africa, for example, or even in Asia, 01:22:48.050 --> 01:22:50.990 where we need careful validation of these methods, 01:22:50.990 --> 01:22:52.897 because as [INAUDIBLE] indicated, 01:22:52.897 --> 01:22:55.230 that we can add some potential batch-to-batch variation. 01:22:55.230 --> 01:22:57.470 Also, depending on the manufacturers, 01:22:57.470 --> 01:23:00.440 you can [INAUDIBLE] low quality for the test. 01:23:00.440 --> 01:23:03.152 Today, it's not recommended by WHO and OIE 01:23:03.152 --> 01:23:05.360 for working diagnosis, and this is very [INAUDIBLE],, 01:23:05.360 --> 01:23:07.640 but from our experience, it's very interesting, 01:23:07.640 --> 01:23:11.720 because it improves the motivation of people working 01:23:11.720 --> 01:23:20.350 and who are [INAUDIBLE] viral area to be involved 01:23:20.350 --> 01:23:21.740 in the fight of rabies. 01:23:21.740 --> 01:23:25.490 And also, you can have some information from the field 01:23:25.490 --> 01:23:26.360 to the laboratory. 01:23:26.360 --> 01:23:29.420 And it could be done, also, to complete e reference 01:23:29.420 --> 01:23:30.800 steps which have been-- 01:23:30.800 --> 01:23:34.470 which are done in a centralized laboratory, for example. 01:23:34.470 --> 01:23:36.380 Thank you for your attention. 01:23:36.380 --> 01:23:38.340 I will open for questions. 01:23:38.340 --> 01:23:41.870 - Thank you, Laurent, for a very clear presentation. 01:23:41.870 --> 01:23:46.400 I had a quick question in regards to your figure. 01:23:46.400 --> 01:23:48.260 You're not suggesting that they should 01:23:48.260 --> 01:23:50.660 be collecting the samples without gloves 01:23:50.660 --> 01:23:55.320 on that hand in terms of biosafety? 01:23:55.320 --> 01:23:57.270 When we go ahead and look at the diagram-- 01:23:57.270 --> 01:23:59.272 there, the diagram-- yes. 01:23:59.272 --> 01:23:59.910 - Oh, yeah. 01:23:59.910 --> 01:24:01.440 [LAUGHTER] 01:24:01.440 --> 01:24:01.940 - Sorry. 01:24:01.940 --> 01:24:02.687 - You're right. 01:24:02.687 --> 01:24:03.610 - I couldn't resist. 01:24:03.610 --> 01:24:04.110 - Yeah. 01:24:04.110 --> 01:24:05.070 Yeah, sure, sure, sure. 01:24:05.070 --> 01:24:05.770 Of course. 01:24:05.770 --> 01:24:07.770 Even if you're vaccinated, sure. 01:24:07.770 --> 01:24:10.500 - And we do have another question. 01:24:10.500 --> 01:24:15.540 One of the participants asks how to avoid contamination 01:24:15.540 --> 01:24:17.010 when working with PBS. 01:24:17.010 --> 01:24:20.040 01:24:20.040 --> 01:24:22.520 - In fact, we just keep-- we don't use PBS. 01:24:22.520 --> 01:24:28.490 That's why in this protocol, we skip this step. 01:24:28.490 --> 01:24:30.530 So we take directly, the sample directly, 01:24:30.530 --> 01:24:35.012 in the tube here of the buffer. 01:24:35.012 --> 01:24:36.470 And after, when it is finished, you 01:24:36.470 --> 01:24:37.740 can get a closer [INAUDIBLE]. 01:24:37.740 --> 01:24:42.320 So from here, we don't have any contamination. 01:24:42.320 --> 01:24:44.700 - Excellent. 01:24:44.700 --> 01:24:48.720 Another quick question just came in. 01:24:48.720 --> 01:24:51.960 One-- this is from Andre [INAUDIBLE],, 01:24:51.960 --> 01:24:58.330 asking, seeing such a large variation in the diagnostic, 01:24:58.330 --> 01:25:01.480 let's say, results between different brands 01:25:01.480 --> 01:25:05.320 or manufacturers, is it feasible to ever 01:25:05.320 --> 01:25:09.010 have this test approved by the OIE, 01:25:09.010 --> 01:25:11.380 or would a specific brand have to be 01:25:11.380 --> 01:25:14.500 approved if all the manufacturers are not 01:25:14.500 --> 01:25:16.818 up to standard? 01:25:16.818 --> 01:25:17.860 - That's a good question. 01:25:17.860 --> 01:25:19.266 That's a good point. 01:25:19.266 --> 01:25:25.870 Currently we are making a study of that in partners 01:25:25.870 --> 01:25:28.540 in Asia and Africa to investigate that. 01:25:28.540 --> 01:25:30.580 We are focusing on this Bionote kit. 01:25:30.580 --> 01:25:35.560 But maybe other people have demonstrated that [INAUDIBLE] 01:25:35.560 --> 01:25:36.550 it would be good. 01:25:36.550 --> 01:25:39.800 01:25:39.800 --> 01:25:43.385 And colleagues from FLI demonstrated 01:25:43.385 --> 01:25:44.940 that some are not so good. 01:25:44.940 --> 01:25:47.660 But I think some are not so bad. 01:25:47.660 --> 01:25:49.310 For Bionote in our hands-- 01:25:49.310 --> 01:25:50.990 [INAUDIBLE] you were there-- 01:25:50.990 --> 01:25:55.700 if we confirm that this manufacturer or the quality 01:25:55.700 --> 01:25:58.466 of this device are not so bad if you [INAUDIBLE] 01:25:58.466 --> 01:26:01.880 some control for each batch, for example. 01:26:01.880 --> 01:26:07.380 I don't know why they cannot be used or validated. 01:26:07.380 --> 01:26:10.160 It's the same for, for example, for conjugated, 01:26:10.160 --> 01:26:12.930 when you have a antibody against rabies, 01:26:12.930 --> 01:26:14.680 you have to look at the [INAUDIBLE] check. 01:26:14.680 --> 01:26:16.305 And you have to [INAUDIBLE] some batch. 01:26:16.305 --> 01:26:19.560 But yeah, I think it's like a classical diagnosis method. 01:26:19.560 --> 01:26:22.250 And if you make the same things, it's the same control. 01:26:22.250 --> 01:26:25.122 So yeah, for sure, you have to validate them. 01:26:25.122 --> 01:26:29.660 If you validated them before, and you are checking the time, 01:26:29.660 --> 01:26:33.410 like a coefficiency [INAUDIBLE] and a comparison with FAT. 01:26:33.410 --> 01:26:34.760 - Exactly. 01:26:34.760 --> 01:26:39.530 And I think this is the whole issue with both OIE and WHO, 01:26:39.530 --> 01:26:44.330 that it has to do with the science behind the technique as 01:26:44.330 --> 01:26:47.960 opposed to a particular producer of-- 01:26:47.960 --> 01:26:51.650 a manufacturer of a conjugate, a kit, et cetera. 01:26:51.650 --> 01:26:52.880 And it's just as you said. 01:26:52.880 --> 01:26:57.920 It really comes down to quality control, and licensure issues, 01:26:57.920 --> 01:27:02.060 and the regulatory authorities, which, unfortunately, are often 01:27:02.060 --> 01:27:03.470 lacking. 01:27:03.470 --> 01:27:06.200 It seems that this has really started 01:27:06.200 --> 01:27:09.570 a whole suite of questions. 01:27:09.570 --> 01:27:12.860 What's your recommendations for saliva as opposed to brain? 01:27:12.860 --> 01:27:15.620 - Oh, saliva. 01:27:15.620 --> 01:27:18.360 For the moment, some manufacturers 01:27:18.360 --> 01:27:19.850 said it's good for saliva. 01:27:19.850 --> 01:27:24.920 We test just-- you can see in the paper, other [INAUDIBLE].. 01:27:24.920 --> 01:27:29.060 So it's very, very highly concentrated and very low 01:27:29.060 --> 01:27:31.660 in quality, because you have very few antigens. 01:27:31.660 --> 01:27:36.050 So we at all don't recommend for saliva. 01:27:36.050 --> 01:27:37.850 - And how do you recommend storing 01:27:37.850 --> 01:27:40.760 the kits with the samples for sending them 01:27:40.760 --> 01:27:43.490 to a lab for later testing? 01:27:43.490 --> 01:27:47.110 Room temperature, refrigeration? 01:27:47.110 --> 01:27:48.720 - We test that [INAUDIBLE]. 01:27:48.720 --> 01:27:51.790 And it was taking a lot of time to come. 01:27:51.790 --> 01:27:52.660 And we are still OK. 01:27:52.660 --> 01:27:56.435 But for sure, like RNA, the more time you spend at room 01:27:56.435 --> 01:27:58.810 temperature-- especially in Africa where it's quite hot-- 01:27:58.810 --> 01:28:01.990 you can degrade your RNA. 01:28:01.990 --> 01:28:06.520 If you can keep that in a freezer not a [INAUDIBLE].. 01:28:06.520 --> 01:28:08.080 One key point, also-- 01:28:08.080 --> 01:28:12.800 I think it's the same thing on the FDA paper, you know, 01:28:12.800 --> 01:28:14.290 [INAUDIBLE]. 01:28:14.290 --> 01:28:16.150 The problem is that you can have some RNA. 01:28:16.150 --> 01:28:19.810 If you store them at room temperature for a long time, 01:28:19.810 --> 01:28:21.730 probably the RNA will degrade, so you will 01:28:21.730 --> 01:28:23.290 have a little piece of RNA. 01:28:23.290 --> 01:28:25.223 So it's good for qPCR, for example. 01:28:25.223 --> 01:28:26.890 But if you want to make some genotyping, 01:28:26.890 --> 01:28:30.430 this could be more difficult. This is what we experienced. 01:28:30.430 --> 01:28:32.440 - And given the findings that you've 01:28:32.440 --> 01:28:36.730 had about the elimination of the dilution step, 01:28:36.730 --> 01:28:40.360 do you anticipate that the Bionote manufacturers will 01:28:40.360 --> 01:28:44.410 change their recommendations in the directions for the kit? 01:28:44.410 --> 01:28:47.060 01:28:47.060 --> 01:28:49.310 - I don't know. 01:28:49.310 --> 01:28:53.200 - I think that's probably the best answer. 01:28:53.200 --> 01:28:53.700 - Yeah. 01:28:53.700 --> 01:28:55.540 so maybe-- that depends on the data. 01:28:55.540 --> 01:29:00.250 Maybe if we have some robust and reliable data on that, maybe. 01:29:00.250 --> 01:29:02.310 But probably, they will not change 01:29:02.310 --> 01:29:05.180 [INAUDIBLE] their quality control and test again. 01:29:05.180 --> 01:29:07.480 Probably they will wait for other people to do that. 01:29:07.480 --> 01:29:08.320 No. 01:29:08.320 --> 01:29:09.410 - Very good. 01:29:09.410 --> 01:29:12.170 Thank you very much for your presentation. 01:29:12.170 --> 01:29:14.920 If you'd be so kind to stop screen sharing, 01:29:14.920 --> 01:29:19.210 we'll move on to our next presenter who's 01:29:19.210 --> 01:29:23.740 a relative novitiate in the lyssavirus group-- no, 01:29:23.740 --> 01:29:25.240 I'm just kidding. 01:29:25.240 --> 01:29:29.800 This is another colleague from Institute Pasteur 01:29:29.800 --> 01:29:31.030 who'll be joining us. 01:29:31.030 --> 01:29:37.900 It's Dr. Noel Tordo from the antiviral unit. 01:29:37.900 --> 01:29:44.020 And also, maybe joining us today from the Pasteur Institute 01:29:44.020 --> 01:29:46.530 in Guinea in Conakry. 01:29:46.530 --> 01:29:49.460 Dr. Tordo, you have the floor. 01:29:49.460 --> 01:29:51.070 - Thank you very much, Chuck. 01:29:51.070 --> 01:29:53.680 And hello to everybody there. 01:29:53.680 --> 01:29:54.420 Can you hear me? 01:29:54.420 --> 01:29:57.110 01:29:57.110 --> 01:30:00.830 - Yes, I think you can do your screen share, too, 01:30:00.830 --> 01:30:03.260 to make the slides bigger. 01:30:03.260 --> 01:30:06.170 01:30:06.170 --> 01:30:09.190 So when you go to Presentation mode at the bottom right-hand 01:30:09.190 --> 01:30:10.490 screen-- 01:30:10.490 --> 01:30:11.565 - I did. 01:30:11.565 --> 01:30:12.560 - Ah, good. 01:30:12.560 --> 01:30:13.280 - That's good. 01:30:13.280 --> 01:30:14.310 That's good, right? 01:30:14.310 --> 01:30:15.640 - That's good, thanks. 01:30:15.640 --> 01:30:16.300 - OK. 01:30:16.300 --> 01:30:19.960 So I will talk about an in-vitro ELISA test 01:30:19.960 --> 01:30:21.760 to evaluate rabies vaccine potency. 01:30:21.760 --> 01:30:23.770 This job has been done essentially 01:30:23.770 --> 01:30:27.740 by Corinne Jallet, a technician in my lab in Paris, 01:30:27.740 --> 01:30:30.110 which Antiviral Strategy Unit. 01:30:30.110 --> 01:30:32.600 And as you said, Chuck, I'm now in Guinea, 01:30:32.600 --> 01:30:37.000 in a real rabies country, leading the Pasteur Institute 01:30:37.000 --> 01:30:39.190 that I'm building there. 01:30:39.190 --> 01:30:45.100 So the purpose of the test is that animal welfare globally 01:30:45.100 --> 01:30:48.370 is encouraging manufacturers and national control 01:30:48.370 --> 01:30:53.530 laboratories to follow the 3Rs strategy for replacement, 01:30:53.530 --> 01:30:58.300 reduction, and refinement of the laboratory animal testing. 01:30:58.300 --> 01:31:02.080 And this consists to try to avoid using animals 01:31:02.080 --> 01:31:05.800 and replace them by in-vitro, and WHO 01:31:05.800 --> 01:31:09.010 and the regulatory authorities require the development 01:31:09.010 --> 01:31:13.600 of these in-vitro approaches as alternatives to the NIH 01:31:13.600 --> 01:31:17.680 for validating rabies vaccine potency. 01:31:17.680 --> 01:31:21.080 So as you know, the lyssavirus glycoprotein, 01:31:21.080 --> 01:31:23.350 so the rabies glycoprotein, is a trimer. 01:31:23.350 --> 01:31:25.390 And you can see [INAUDIBLE] here, 01:31:25.390 --> 01:31:29.290 the trimer form of the glycoprotein which 01:31:29.290 --> 01:31:31.600 is on the surface of the virus. 01:31:31.600 --> 01:31:34.900 And indeed, this is the more immunogenic form 01:31:34.900 --> 01:31:37.870 to which more of the antibodies are going. 01:31:37.870 --> 01:31:41.955 And G protein is the main viral antigen. Triangles of G 01:31:41.955 --> 01:31:44.320 are the most immunogenic form. 01:31:44.320 --> 01:31:47.500 And G induces neutralizing antibodies, 01:31:47.500 --> 01:31:51.200 and G carries most of the antigenic sides. 01:31:51.200 --> 01:31:54.890 This is a monomer of this trimer that you can see here. 01:31:54.890 --> 01:31:59.875 And you can see in brown, then site II is located here, and-- 01:31:59.875 --> 01:32:03.190 site III, sorry, and in pink site II. 01:32:03.190 --> 01:32:08.150 I will talk about these two sites later. 01:32:08.150 --> 01:32:13.600 So we are talking about one antibody, which name is D1-25. 01:32:13.600 --> 01:32:16.460 I will say D1 in the following lines. 01:32:16.460 --> 01:32:19.790 And D1 has been obtained by Balb-C mice immunized 01:32:19.790 --> 01:32:23.580 with inactivated rabies vaccine PV strain. 01:32:23.580 --> 01:32:28.330 [INAUDIBLE] IgG 1 isotype, which is recognizing the site III. 01:32:28.330 --> 01:32:30.670 And this antibody is neutralizing. 01:32:30.670 --> 01:32:32.230 As you can see in this picture, when 01:32:32.230 --> 01:32:34.390 you have a trimer of glycoprotein, 01:32:34.390 --> 01:32:37.750 you need to have the transmembrane and the central-- 01:32:37.750 --> 01:32:41.390 and the [? superplasmid ?] part of the protein to maintain it. 01:32:41.390 --> 01:32:44.560 If you cut it, and you have what we name the G ecto, 01:32:44.560 --> 01:32:46.090 they are only monomers. 01:32:46.090 --> 01:32:48.400 And you can see that D1, which is presented 01:32:48.400 --> 01:32:52.150 in the red [INAUDIBLE],, is recognizing 01:32:52.150 --> 01:32:55.150 the total G, so the triangles of glycoprotein, 01:32:55.150 --> 01:33:00.990 so the more immunogenic form, and not the monomers. 01:33:00.990 --> 01:33:06.800 So this ELISA test with a D1 antibody 01:33:06.800 --> 01:33:10.790 has been tested since a long time versus NIH. 01:33:10.790 --> 01:33:14.450 More than 1,000 vaccine lots have been tested. 01:33:14.450 --> 01:33:16.490 There is a good concordance between the two 01:33:16.490 --> 01:33:17.900 tests for vaccine potency. 01:33:17.900 --> 01:33:20.510 You can see here in blue you have 01:33:20.510 --> 01:33:22.580 the results of the ELISA test. 01:33:22.580 --> 01:33:25.230 In red you have the NIH test. 01:33:25.230 --> 01:33:27.620 And you can see that basically, from one 01:33:27.620 --> 01:33:29.600 to the other of the vaccines, we have 01:33:29.600 --> 01:33:32.180 approximately the same fluctuation 01:33:32.180 --> 01:33:34.010 in the quality of the test. 01:33:34.010 --> 01:33:37.280 And the ELISA test with the D1 antibody 01:33:37.280 --> 01:33:39.300 is more sensitive, more rapid-- 01:33:39.300 --> 01:33:41.900 it's only one day-- and less fluctuant. 01:33:41.900 --> 01:33:45.000 You can see that in this second part of the test, 01:33:45.000 --> 01:33:47.900 the NIH test was done only once. 01:33:47.900 --> 01:33:50.570 And you can see that we have a large fluctuation 01:33:50.570 --> 01:33:55.565 for each batch, while the blue line is staying more 01:33:55.565 --> 01:33:58.310 into the variation. 01:33:58.310 --> 01:34:02.480 So rapidly, the different steps of the test 01:34:02.480 --> 01:34:05.720 with the monoclonal antibody D1-25. 01:34:05.720 --> 01:34:11.700 So the sensitization, passivation, ELISA test assay. 01:34:11.700 --> 01:34:14.570 And I will talk about results and analysis of the results 01:34:14.570 --> 01:34:17.360 after talking about security precaution, and conclusion 01:34:17.360 --> 01:34:19.800 and perspectives first. 01:34:19.800 --> 01:34:21.470 The sensitization. 01:34:21.470 --> 01:34:24.830 So you can see that we take, of course, a plate, and to put 01:34:24.830 --> 01:34:28.820 on the plate the antibody [INAUDIBLE] to fix 01:34:28.820 --> 01:34:30.810 on the plate these antibodies. 01:34:30.810 --> 01:34:33.590 And this is done by just putting-- 01:34:33.590 --> 01:34:36.100 diluting the antibody in carbonate buffer 01:34:36.100 --> 01:34:40.100 at a concentration of about 1 microgram per mL. 01:34:40.100 --> 01:34:43.220 3 hours' incubation in humidity, you 01:34:43.220 --> 01:34:47.060 remove the supernatant, and dry with absorbent 01:34:47.060 --> 01:34:48.980 paper for 5 minutes. 01:34:48.980 --> 01:34:52.640 Then is starting the passivation. 01:34:52.640 --> 01:34:55.160 So for the passivation, it consists 01:34:55.160 --> 01:34:58.790 to avoid that any vaccine after will 01:34:58.790 --> 01:35:02.030 have the nonspecific binding with the plate. 01:35:02.030 --> 01:35:06.920 And so you fill up the plate with a passivating proteins, 01:35:06.920 --> 01:35:10.730 like BSA for example, that are occupying nonspecifically 01:35:10.730 --> 01:35:11.820 the plate. 01:35:11.820 --> 01:35:14.480 And this is, again, the same type of experiment, 01:35:14.480 --> 01:35:18.830 very simple, 30 minutes at 37 degrees in humidity. 01:35:18.830 --> 01:35:21.320 Remove the supernatant, dry. 01:35:21.320 --> 01:35:26.090 And this plate can be kept for approximately 3 months 01:35:26.090 --> 01:35:31.640 at minus 20 degrees before being used for ELISA. 01:35:31.640 --> 01:35:35.060 So the ELISA is done by two different steps. 01:35:35.060 --> 01:35:39.560 First, the capture-- so after washing extensively the plate, 01:35:39.560 --> 01:35:43.070 we are preparing serial two-fold dilution of the reference 01:35:43.070 --> 01:35:47.300 vaccine and two-fold dilution of the tested vaccine. 01:35:47.300 --> 01:35:50.210 And then we distribute 200 microliters 01:35:50.210 --> 01:35:53.810 in duplicates of this dilution, of the reference 01:35:53.810 --> 01:35:57.050 and the tested vaccine, plus, of course, the blank, which 01:35:57.050 --> 01:35:58.730 is a diluent buffer. 01:35:58.730 --> 01:36:03.380 Incubation for 1 hour at 37 degrees, discard supernatant, 01:36:03.380 --> 01:36:04.400 wash. 01:36:04.400 --> 01:36:07.980 And then the revelation. 01:36:07.980 --> 01:36:10.940 The revelation is done within our hands. 01:36:10.940 --> 01:36:13.640 We can change-- I will talk about that later on-- 01:36:13.640 --> 01:36:16.220 with the same antibody, D1, which 01:36:16.220 --> 01:36:19.170 is conjugated with peroxydase. 01:36:19.170 --> 01:36:22.040 So we grow this antibody, we incubate, we wash. 01:36:22.040 --> 01:36:26.000 And then you put the substrate-chromogen solution, 01:36:26.000 --> 01:36:30.680 which after incubation for 30 minutes at room temperature, 01:36:30.680 --> 01:36:34.010 allows to go to the stop solution. 01:36:34.010 --> 01:36:36.560 And you can read the optical density 01:36:36.560 --> 01:36:40.940 at 492 nanometers with a spectrophotometer. 01:36:40.940 --> 01:36:46.280 So this is a typical result. So you calculate the mean-- 01:36:46.280 --> 01:36:50.360 the optical density of each duplicate for reference vaccine 01:36:50.360 --> 01:36:52.880 by subtracting the blank, of course. 01:36:52.880 --> 01:36:57.740 And you plot the OD value on the vertical axis, 01:36:57.740 --> 01:37:00.110 and the G protein trimer concentration 01:37:00.110 --> 01:37:01.910 on the horizontal axis. 01:37:01.910 --> 01:37:04.230 And you draw the reference curve. 01:37:04.230 --> 01:37:08.030 And once you have that, you can estimate the tested vaccine 01:37:08.030 --> 01:37:10.520 concentration, essentially trying 01:37:10.520 --> 01:37:14.670 to look at the linear places here with your tested vaccine. 01:37:14.670 --> 01:37:18.515 And you can estimate it in terms of glycoprotein, trimeric 01:37:18.515 --> 01:37:21.210 glycoprotein concentration. 01:37:21.210 --> 01:37:25.650 So we are providing a reference vaccine in the test, 01:37:25.650 --> 01:37:29.150 which is a purified inactivated rabies virus vaccine, 01:37:29.150 --> 01:37:31.124 the lot 09. 01:37:31.124 --> 01:37:35.100 Its G protein content is well known. 01:37:35.100 --> 01:37:37.310 It has been measured by different tests, BCA 01:37:37.310 --> 01:37:39.830 and also by estimation on SDS. 01:37:39.830 --> 01:37:43.430 And the result that we got are in the concentration. 01:37:43.430 --> 01:37:47.000 And they can be transformed in equivalent international units 01:37:47.000 --> 01:37:50.540 by comparison to the WHO International Standard 01:37:50.540 --> 01:37:54.440 that we can test in parallel. 01:37:54.440 --> 01:37:57.470 So just to say that we have to highlight 01:37:57.470 --> 01:38:00.020 some security and precautions. 01:38:00.020 --> 01:38:04.550 You have to wear adequate personal protective equipment-- 01:38:04.550 --> 01:38:06.890 coats, gloves, mask, glasses. 01:38:06.890 --> 01:38:08.930 I'm sorry, she's not Corinne. 01:38:08.930 --> 01:38:12.050 I didn't-- I wasn't able to capture the picture that we 01:38:12.050 --> 01:38:14.770 have on the job article. 01:38:14.770 --> 01:38:18.080 It's someone else, but it is basically what we can do. 01:38:18.080 --> 01:38:20.360 When this is live virus that you are titrating, 01:38:20.360 --> 01:38:23.480 you work on the biosafety cabinet. 01:38:23.480 --> 01:38:25.940 And what I said since the beginning is 01:38:25.940 --> 01:38:28.660 that we are removing many times supernatant. 01:38:28.660 --> 01:38:31.160 And this supernatant, if they are contaminated, 01:38:31.160 --> 01:38:35.795 should be put in a bleach solution, 2.5 sodium 01:38:35.795 --> 01:38:38.870 hydrochloride, in order to avoid any contamination. 01:38:38.870 --> 01:38:44.870 And of course, you do that by using good laboratory practice. 01:38:44.870 --> 01:38:47.530 So some conclusions. 01:38:47.530 --> 01:38:52.690 This ELISA test is recognizing the trimeric immunogenic form 01:38:52.690 --> 01:38:54.250 of the glycoprotein. 01:38:54.250 --> 01:38:57.460 And it is a suitable alternative to the NIH test. 01:38:57.460 --> 01:39:01.720 And this is the first paper in which we showed that in 2014. 01:39:01.720 --> 01:39:05.290 It is also able to discriminate sub-potent vaccine lots. 01:39:05.290 --> 01:39:09.290 In this paper, it is shown that you can heat up, for example, 01:39:09.290 --> 01:39:13.200 a vaccine for degradation and the level of the response 01:39:13.200 --> 01:39:15.350 is going down. 01:39:15.350 --> 01:39:19.180 It is used, indeed, by human and veterinary vaccine producers 01:39:19.180 --> 01:39:23.530 since more than 20 years, despite the fact that it is not 01:39:23.530 --> 01:39:26.620 recognized officially, because they are using it to follow 01:39:26.620 --> 01:39:30.430 production before submitting their batch release 01:39:30.430 --> 01:39:34.310 to their national authorities. 01:39:34.310 --> 01:39:37.670 And this antibody can also be used 01:39:37.670 --> 01:39:40.390 in competition for titration of anti-rabies antibodies 01:39:40.390 --> 01:39:44.410 with the anti-rabies antibodies in equine and human sera, 01:39:44.410 --> 01:39:46.940 for example, if you want to test the quality 01:39:46.940 --> 01:39:51.440 of your anti-rabies sera. 01:39:51.440 --> 01:39:53.730 Perspectives. 01:39:53.730 --> 01:39:57.960 Working with monoclonal antibodies poses all the time 01:39:57.960 --> 01:40:01.130 the risk that your vaccine strain 01:40:01.130 --> 01:40:05.600 is disappearing because it has some mutated epitopes. 01:40:05.600 --> 01:40:08.570 And I would like to come back on the first presentation 01:40:08.570 --> 01:40:11.630 of this webinar, where, if I remember correctly, 01:40:11.630 --> 01:40:20.600 one of the [? TW ?] BLB1 was not recognized by one of the-- 01:40:20.600 --> 01:40:22.468 I don't remember what is the conjugate. 01:40:22.468 --> 01:40:24.010 And so this is the risk that you have 01:40:24.010 --> 01:40:26.300 when you're working with monoclonal antibodies. 01:40:26.300 --> 01:40:29.300 So in our case, we are using the same monoclonal antibodies, 01:40:29.300 --> 01:40:34.225 the D1, recognizing the site III for both capture 01:40:34.225 --> 01:40:34.850 and revelation. 01:40:34.850 --> 01:40:36.620 You have seen that. 01:40:36.620 --> 01:40:39.350 This method is applicable, of course, with other antibodies. 01:40:39.350 --> 01:40:42.710 And this antibody is an antibody of the [? Wistar. ?] 01:40:42.710 --> 01:40:46.040 And currently, an ELISA combining 01:40:46.040 --> 01:40:52.640 D1 site III for capture and this [? Wistar ?] antibody 01:40:52.640 --> 01:40:55.880 for site II for revelation is currently 01:40:55.880 --> 01:41:00.170 tested by vaccine companies from many countries 01:41:00.170 --> 01:41:03.470 in the world and the National Control Laboratories 01:41:03.470 --> 01:41:05.780 under the umbrella of the European Directorate 01:41:05.780 --> 01:41:08.390 for Quality and Medicine and Healthcare, 01:41:08.390 --> 01:41:11.450 and the Biological Standardization Program. 01:41:11.450 --> 01:41:13.610 And of course, the goal of this is 01:41:13.610 --> 01:41:17.600 to end up with an ELISA which could be recognized 01:41:17.600 --> 01:41:22.760 by the different authorities, national and international 01:41:22.760 --> 01:41:25.550 authorities, and to have a sort of ELISA test 01:41:25.550 --> 01:41:30.770 that will be proposed in the end to replace the NIH test. 01:41:30.770 --> 01:41:36.230 So I have to thank, apart from Corinne Jallet Sylvie 01:41:36.230 --> 01:41:37.360 Morgeaux at ANSM-- 01:41:37.360 --> 01:41:40.490 this is a French authority in Lyon; 01:41:40.490 --> 01:41:43.460 Jean-Michel Chapsal from Sanofi Pasteur in Lyon; 01:41:43.460 --> 01:41:46.220 Sabrina Kali from my lab in Paris [INAUDIBLE];; 01:41:46.220 --> 01:41:48.410 and Pierre Perrin was indeed the guy 01:41:48.410 --> 01:41:52.280 who created this 20 years ago, and he's now retired. 01:41:52.280 --> 01:41:53.870 And thank you, Pierre. 01:41:53.870 --> 01:41:56.250 And thank you for your attention. 01:41:56.250 --> 01:41:59.840 - Thank you, Dr. Tordo, for a very clear presentation. 01:41:59.840 --> 01:42:04.010 And obviously, these monoclonals in this particular recipe 01:42:04.010 --> 01:42:08.000 has great utility for determining 01:42:08.000 --> 01:42:10.850 the potency of biologics in the future 01:42:10.850 --> 01:42:15.090 and hopefully putting to rest, finally, this NIH test. 01:42:15.090 --> 01:42:16.530 I have one question. 01:42:16.530 --> 01:42:20.450 What about application towards veterinary vaccines 01:42:20.450 --> 01:42:23.390 that might be adjuvanted? 01:42:23.390 --> 01:42:26.690 - So we are currently testing that. 01:42:26.690 --> 01:42:29.060 One of the difficulties is that when 01:42:29.060 --> 01:42:32.510 you are talking about human vaccine, as you know, 01:42:32.510 --> 01:42:34.730 there is only a lot of vaccine strains 01:42:34.730 --> 01:42:36.410 that are used up to now. 01:42:36.410 --> 01:42:37.830 This could increase. 01:42:37.830 --> 01:42:39.800 And so most of them are strains that 01:42:39.800 --> 01:42:45.500 are recognized by D1 antibody or your antibody, Chuck. 01:42:45.500 --> 01:42:49.190 By the way, it is possible that some vaccine strain, even 01:42:49.190 --> 01:42:52.700 the human part, could be not recognized by one 01:42:52.700 --> 01:42:54.740 or the other of the antibodies. 01:42:54.740 --> 01:42:57.650 If we are talking about the veterinary world, 01:42:57.650 --> 01:42:58.445 it's more complex. 01:42:58.445 --> 01:43:00.410 We know that we have more diversity. 01:43:00.410 --> 01:43:03.680 So not only the adjuvant, but also the diversity. 01:43:03.680 --> 01:43:08.960 So we are making some tests now with some veterinary companies 01:43:08.960 --> 01:43:12.870 in order to enlarge that to the veterinary one. 01:43:12.870 --> 01:43:14.840 But the problem that I can see is 01:43:14.840 --> 01:43:17.150 that we have to be certain that this 01:43:17.150 --> 01:43:24.890 is covering exactly the strain that is of interest, of course. 01:43:24.890 --> 01:43:25.670 - Very good. 01:43:25.670 --> 01:43:28.700 And do you see any utility to the use 01:43:28.700 --> 01:43:34.550 of one versus the two monoclonal antibodies used in the test? 01:43:34.550 --> 01:43:39.950 01:43:39.950 --> 01:43:42.320 Is there an advantage to just use D1 01:43:42.320 --> 01:43:46.120 for both capture and detection? 01:43:46.120 --> 01:43:49.590 - The kit that we are-- because we are selling this kit already 01:43:49.590 --> 01:43:51.356 on MTA-- 01:43:51.356 --> 01:43:52.650 - Ah, so you're selling. 01:43:52.650 --> 01:43:55.020 It so there's a potential conflict. 01:43:55.020 --> 01:43:55.900 [LAUGHS] 01:43:55.900 --> 01:43:57.210 - No, no, no. 01:43:57.210 --> 01:43:59.280 We are selling since 20 years. 01:43:59.280 --> 01:44:02.340 So it's not really a problem, because we are participating 01:44:02.340 --> 01:44:07.740 also to this trial of having a test which will combine, 01:44:07.740 --> 01:44:13.490 indeed, the two antibodies, the D1 and the [INAUDIBLE] 01:44:13.490 --> 01:44:14.670 antibody. 01:44:14.670 --> 01:44:18.180 The interest that I saw at the beginning in that one 01:44:18.180 --> 01:44:21.420 is that it is recognizing this trimeric form. 01:44:21.420 --> 01:44:24.340 And it is clear that this is a form which is 01:44:24.340 --> 01:44:26.760 a more, let's say, immunogenic. 01:44:26.760 --> 01:44:30.360 So if you propose a vaccine along the line, 01:44:30.360 --> 01:44:33.270 you would like to have a vaccine that 01:44:33.270 --> 01:44:36.480 is inactivated vaccine, vaccine which is still keeping 01:44:36.480 --> 01:44:38.580 the native format, if you want. 01:44:38.580 --> 01:44:41.350 And in this way, D1 is interesting. 01:44:41.350 --> 01:44:45.930 But maybe-- I don't know if it has ever been looked at. 01:44:45.930 --> 01:44:50.400 Maybe also, the other antibodies can specifically 01:44:50.400 --> 01:44:54.250 fix on the trimeric form. 01:44:54.250 --> 01:44:58.890 So no, I have no problem to me with another antibody. 01:44:58.890 --> 01:45:02.070 - Thank you for your very diplomatic answer. 01:45:02.070 --> 01:45:03.780 And it's very good to see you again. 01:45:03.780 --> 01:45:04.942 Glad you're in good health. 01:45:04.942 --> 01:45:06.150 Thanks for your presentation. 01:45:06.150 --> 01:45:07.480 - Thank you very much. 01:45:07.480 --> 01:45:08.700 - Thank you, sir. 01:45:08.700 --> 01:45:12.540 We're going to move to our next presentation now. 01:45:12.540 --> 01:45:16.440 We'll be with Jodie Jarvis. 01:45:16.440 --> 01:45:19.740 Jodie received her degree in wildlife biology 01:45:19.740 --> 01:45:22.230 from the University of Wisconsin. 01:45:22.230 --> 01:45:26.430 She's been a member of the New York State Rabies Laboratory 01:45:26.430 --> 01:45:33.690 since 2001 and the deputy director since 2019. 01:45:33.690 --> 01:45:37.890 The New York state Rabies Lab is one of our premiere ones 01:45:37.890 --> 01:45:39.870 here, now that we've gone over the pond 01:45:39.870 --> 01:45:46.080 from Asia, from Europe, and now into North America. 01:45:46.080 --> 01:45:48.000 New York, Texas, and California are 01:45:48.000 --> 01:45:52.290 three of our major laboratories here in the United States 01:45:52.290 --> 01:45:54.270 detecting-- 01:45:54.270 --> 01:45:57.870 having submissions throughout the country of sometimes 01:45:57.870 --> 01:46:01.440 in excess of 100,000 samples-- 01:46:01.440 --> 01:46:03.600 surveillance, surveillance, surveillance. 01:46:03.600 --> 01:46:06.060 And Jodie will give us some insights, 01:46:06.060 --> 01:46:10.950 as we've talked about, that these tests are marvelous, 01:46:10.950 --> 01:46:14.160 as long as we have the proper sample. 01:46:14.160 --> 01:46:19.710 And so will be sharing with us some rabies necropsy techniques 01:46:19.710 --> 01:46:24.630 in both small as well as mega or large animals, 01:46:24.630 --> 01:46:28.440 to ensure that we actually have the proper sample 01:46:28.440 --> 01:46:31.240 from the CNS for our tests. 01:46:31.240 --> 01:46:32.640 Jodie, the floor is yours. 01:46:32.640 --> 01:46:37.430 01:46:37.430 --> 01:46:40.040 - Sorry, I had to unmute myself. 01:46:40.040 --> 01:46:41.675 Do I have it on the correct screen? 01:46:41.675 --> 01:46:48.320 01:46:48.320 --> 01:46:48.820 Sorry. 01:46:48.820 --> 01:46:51.530 Is that-- am I doing the correct screen? 01:46:51.530 --> 01:46:53.330 - Looks good from here. 01:46:53.330 --> 01:46:55.520 If you want to move it to Presentation mode 01:46:55.520 --> 01:46:57.500 in the bottom right hand, you may. 01:46:57.500 --> 01:47:07.380 01:47:07.380 --> 01:47:08.970 - Is it on presentation mode? 01:47:08.970 --> 01:47:09.810 I'm sorry. 01:47:09.810 --> 01:47:12.160 This is my first time doing a Zoom. 01:47:12.160 --> 01:47:15.936 01:47:15.936 --> 01:47:19.240 Is the [INAUDIBLE] right one? 01:47:19.240 --> 01:47:20.200 OK. 01:47:20.200 --> 01:47:22.390 There we go. 01:47:22.390 --> 01:47:22.890 OK. 01:47:22.890 --> 01:47:25.780 Sorry about that. 01:47:25.780 --> 01:47:28.510 So I'm Jodie Jarvis. 01:47:28.510 --> 01:47:31.660 And as Dr. Rupprecht has said, I've 01:47:31.660 --> 01:47:36.070 been working at the Rabies Lab for about 18 years now. 01:47:36.070 --> 01:47:40.780 And all of the other authors on this paper 01:47:40.780 --> 01:47:42.520 have been here for quite a while, too. 01:47:42.520 --> 01:47:46.397 I'm actually one of the newer people. 01:47:46.397 --> 01:47:48.730 And I wanted to give you just a little bit of background 01:47:48.730 --> 01:47:49.990 on the Wadsworth Lab. 01:47:49.990 --> 01:47:53.120 01:47:53.120 --> 01:47:55.560 The Wadsworth Center is the main rabies laboratory 01:47:55.560 --> 01:47:57.220 for New York State. 01:47:57.220 --> 01:47:59.230 New York City has its own laboratory 01:47:59.230 --> 01:48:02.020 that tests just human exposure cases within the city. 01:48:02.020 --> 01:48:03.700 But we do all of the other testing 01:48:03.700 --> 01:48:05.380 for the rest of the state. 01:48:05.380 --> 01:48:09.070 We receive over 6,000 specimens per year. 01:48:09.070 --> 01:48:12.730 In 2019, the year that this article is published, 01:48:12.730 --> 01:48:17.380 the Wadsworth Rabies Lab tested 7,225 samples. 01:48:17.380 --> 01:48:19.480 And during our busiest days, we can receive 01:48:19.480 --> 01:48:22.330 between 1 and 200 samples. 01:48:22.330 --> 01:48:26.620 And anything arriving before 2:00 PM gets same-day results. 01:48:26.620 --> 01:48:29.680 So our testing methods follow the guidance 01:48:29.680 --> 01:48:33.070 given by the protocol for post-mortem diagnosis of rabies 01:48:33.070 --> 01:48:35.920 in animals by direct fluorescence antibody 01:48:35.920 --> 01:48:39.310 testing, the minimum standard for rabies diagnosis 01:48:39.310 --> 01:48:41.200 in the United States. 01:48:41.200 --> 01:48:42.880 Basically, the takeaway from this 01:48:42.880 --> 01:48:45.970 is that our laboratory has a lot of experience in necropsy 01:48:45.970 --> 01:48:48.360 and doing it in a safe and time-efficient manner. 01:48:48.360 --> 01:48:51.800 01:48:51.800 --> 01:48:54.410 Every sample package we open is like a surprise. 01:48:54.410 --> 01:48:56.630 We receive submissions from veterinarians 01:48:56.630 --> 01:48:59.720 to private citizens, all with different capabilities 01:48:59.720 --> 01:49:03.350 and resources available to them to perform a decapitation. 01:49:03.350 --> 01:49:06.590 And that's why we have several different methods 01:49:06.590 --> 01:49:09.440 dependent on the decapitation And just to note, 01:49:09.440 --> 01:49:13.310 we do not ask private citizens to perform their decapitations, 01:49:13.310 --> 01:49:17.660 but we can't control what they do on their own. 01:49:17.660 --> 01:49:19.460 Most of our samples are delivered to us 01:49:19.460 --> 01:49:22.580 by mail, UPS, or FedEx, in shipping boxes supplied 01:49:22.580 --> 01:49:24.340 by Wadsworth Center. 01:49:24.340 --> 01:49:27.380 Our large box is suitable for a large dog head. 01:49:27.380 --> 01:49:30.140 But if larger, like most livestock, other methods 01:49:30.140 --> 01:49:32.120 are needed to get the submission to the lab. 01:49:32.120 --> 01:49:35.240 01:49:35.240 --> 01:49:38.070 And that was the initial reason for doing this video-- 01:49:38.070 --> 01:49:42.140 a source for us to recommend to large animal veterinarian that 01:49:42.140 --> 01:49:44.450 need to collect samples in the field. 01:49:44.450 --> 01:49:46.220 We added the small animal portion 01:49:46.220 --> 01:49:49.130 to show a safer alternative for tissue collection that doesn't 01:49:49.130 --> 01:49:51.096 create as much aerosol as saws. 01:49:51.096 --> 01:49:55.560 01:49:55.560 --> 01:49:57.690 This picture shows the only type of tools 01:49:57.690 --> 01:49:59.520 that we use for necropsy-- 01:49:59.520 --> 01:50:02.730 no saws or power tools of any kind. 01:50:02.730 --> 01:50:06.840 From left to right, we have short blunt scissors; 01:50:06.840 --> 01:50:11.160 forceps for manipulating smaller items without direct contact; 01:50:11.160 --> 01:50:15.480 hammer and chisel for going through bone; 01:50:15.480 --> 01:50:19.020 tenaculars for moving the head into position and holding 01:50:19.020 --> 01:50:21.710 muscle when cutting with the knife-- 01:50:21.710 --> 01:50:24.200 and at no time do we directly touch the head 01:50:24.200 --> 01:50:27.920 with our gloved hand; a carving knife 01:50:27.920 --> 01:50:32.210 to cut through skin and muscle to get to the skull. 01:50:32.210 --> 01:50:35.480 These last three items are not added unless we're 01:50:35.480 --> 01:50:37.700 using the large animal method. 01:50:37.700 --> 01:50:41.580 And they are a long knife ground down to a thinner blade; 01:50:41.580 --> 01:50:44.940 a chemistry spoon; and a long-handled tablespoon 01:50:44.940 --> 01:50:47.820 with the spoon part ground down to be just a little bit wider 01:50:47.820 --> 01:50:49.980 than the handle. 01:50:49.980 --> 01:50:51.990 In addition to well-maintained tools, 01:50:51.990 --> 01:50:54.810 the technician has safety protocols to follow. 01:50:54.810 --> 01:50:57.840 A minimum of eye protection, a mask, and gloves 01:50:57.840 --> 01:51:00.720 are worn, as well as being rabies vaccinated. 01:51:00.720 --> 01:51:04.560 In the lab, we actually wear scrubs and surgical gowns 01:51:04.560 --> 01:51:09.528 as well, but in the field that might not be available. 01:51:09.528 --> 01:51:12.298 01:51:12.298 --> 01:51:14.340 So I know there are other websites available that 01:51:14.340 --> 01:51:16.890 show how to do the brain collection through the foramen, 01:51:16.890 --> 01:51:21.030 particularly for transmissible spongiform encephalopathy. 01:51:21.030 --> 01:51:24.270 However, when asked by large animal veterinarians for advice 01:51:24.270 --> 01:51:26.310 on how to perform it, we preferred 01:51:26.310 --> 01:51:28.470 to have this video to [INAUDIBLE].. 01:51:28.470 --> 01:51:30.240 Eliminates any confusion if there's 01:51:30.240 --> 01:51:34.980 any differences in tissue collected or safety protocols. 01:51:34.980 --> 01:51:37.220 The large animal method is best for livestock 01:51:37.220 --> 01:51:39.530 with cervical vertebrae removed. 01:51:39.530 --> 01:51:41.900 The requirement is that the foramen is large enough 01:51:41.900 --> 01:51:44.580 for the tools to pass through. 01:51:44.580 --> 01:51:48.140 Using the thin bladed knife, circle 01:51:48.140 --> 01:51:51.000 along between the meninges and the brain stem. 01:51:51.000 --> 01:51:54.240 When it feels loose, hold onto the brain stem with forceps 01:51:54.240 --> 01:51:58.510 and insert the thin long-handled spoon 01:51:58.510 --> 01:52:01.385 or the chemistry spoon between the meninges and the brain 01:52:01.385 --> 01:52:02.320 stem. 01:52:02.320 --> 01:52:05.050 You can then use the spoon to disconnect the brain stem 01:52:05.050 --> 01:52:08.740 and cerebellum at the back end of the cylinder shape created 01:52:08.740 --> 01:52:09.760 by the knife. 01:52:09.760 --> 01:52:13.120 You can then use the forceps and spoon simultaneously 01:52:13.120 --> 01:52:16.030 to remove the brain stem in one piece. 01:52:16.030 --> 01:52:18.590 As our lab requires pieces of all three 01:52:18.590 --> 01:52:20.690 lobes of the cerebellum for testing, 01:52:20.690 --> 01:52:23.180 it usually requires going back in with the spoon 01:52:23.180 --> 01:52:24.530 to scoop out the cerebellum. 01:52:24.530 --> 01:52:28.090 01:52:28.090 --> 01:52:30.100 The ventral method is an easy method 01:52:30.100 --> 01:52:33.430 when the foramen is too small to do the large animal method, 01:52:33.430 --> 01:52:35.830 and there are no vertebrae attached. 01:52:35.830 --> 01:52:39.280 With this method, the ventral side of the head is facing up. 01:52:39.280 --> 01:52:41.830 With the hammer and chisel, make two cuts, 01:52:41.830 --> 01:52:45.760 forming an upside down V around the foramen. 01:52:45.760 --> 01:52:47.860 You will need to bend that V down. 01:52:47.860 --> 01:52:50.800 I personally use my chisel under the tip of the V 01:52:50.800 --> 01:52:53.380 and tap a few times with the hammer. 01:52:53.380 --> 01:52:56.620 But it can also be done with tenaculars or forceps. 01:52:56.620 --> 01:52:59.090 It mostly depends on the thickness of the skull, 01:52:59.090 --> 01:53:02.350 how much force you will need-- 01:53:02.350 --> 01:53:06.390 or how much force you will need. 01:53:06.390 --> 01:53:09.750 With the curved scissors, with the blades closed, 01:53:09.750 --> 01:53:14.280 that can be used as a scoop for scooping out 01:53:14.280 --> 01:53:16.530 the cerebellum and brain stem. 01:53:16.530 --> 01:53:20.560 It's sometimes left with the foramen, 01:53:20.560 --> 01:53:22.660 and you can use forceps to pull that out. 01:53:22.660 --> 01:53:27.240 01:53:27.240 --> 01:53:30.150 The dorsal method is the most labor intensive, 01:53:30.150 --> 01:53:33.390 but is most useful if tissues other than brain stem 01:53:33.390 --> 01:53:35.220 and cerebellum is needed. 01:53:35.220 --> 01:53:37.170 It is also used if the vertebrae are still 01:53:37.170 --> 01:53:41.210 attached to the skull, hindering the other methods. 01:53:41.210 --> 01:53:44.350 In this method, the dorsal part of the head is facing up. 01:53:44.350 --> 01:53:48.370 The muscle needs to be cut away to expose the skull. 01:53:48.370 --> 01:53:51.100 That is done with a carving knife using tenaculars 01:53:51.100 --> 01:53:52.780 to hold the head in place. 01:53:52.780 --> 01:53:54.820 Do not use your hands. 01:53:54.820 --> 01:54:00.160 Once the skull is exposed, cut two horizontal cuts 01:54:00.160 --> 01:54:03.280 on either side of the skull from the midline down 01:54:03.280 --> 01:54:05.360 towards your workspace. 01:54:05.360 --> 01:54:08.060 From that line, you want to make cuts 01:54:08.060 --> 01:54:12.370 along the sides of the skull towards the back of the skull. 01:54:12.370 --> 01:54:14.800 It will look like an upside down U. 01:54:14.800 --> 01:54:19.300 Gripping the face of the U with the teeth of one of the-- 01:54:19.300 --> 01:54:21.160 with the teeth of the tenaculars, 01:54:21.160 --> 01:54:25.150 you can use that to pry open the flap of the skull. 01:54:25.150 --> 01:54:28.120 And once opened, scissors can be used to scoop out 01:54:28.120 --> 01:54:29.710 the cerebellum and brain stem. 01:54:29.710 --> 01:54:34.440 01:54:34.440 --> 01:54:37.110 Of course, the specimens from the video and article 01:54:37.110 --> 01:54:38.982 are examples of good submissions. 01:54:38.982 --> 01:54:40.440 But this is the real world, and not 01:54:40.440 --> 01:54:42.440 everything comes in perfect shape. 01:54:42.440 --> 01:54:45.570 The animal may have full damage due to trauma, gunshot, 01:54:45.570 --> 01:54:47.780 or poor decapitation. 01:54:47.780 --> 01:54:49.680 The tissue may not scoop up nicely 01:54:49.680 --> 01:54:51.870 because of decomposition. 01:54:51.870 --> 01:54:54.480 Foreign objects like quills or packing materials 01:54:54.480 --> 01:54:57.060 may cause obstacles. 01:54:57.060 --> 01:54:59.640 Sometimes, it's just due to the species of animal-- 01:54:59.640 --> 01:55:01.470 goats and deer with their horns and antlers 01:55:01.470 --> 01:55:04.260 in the way to lay the head on the workspace, 01:55:04.260 --> 01:55:07.110 or some breeds of dogs or cats with more rounded skulls that 01:55:07.110 --> 01:55:10.470 won't stay in a position, or full-body animals that 01:55:10.470 --> 01:55:11.950 require more manipulation. 01:55:11.950 --> 01:55:15.760 01:55:15.760 --> 01:55:17.680 Those are the times when modifications need 01:55:17.680 --> 01:55:21.670 to be made to those methods. 01:55:21.670 --> 01:55:25.120 Regardless of which method or what modifications are made, 01:55:25.120 --> 01:55:27.530 safety is still top priority. 01:55:27.530 --> 01:55:30.760 With the methods demonstrated in this video, 01:55:30.760 --> 01:55:33.700 safety is worked into the procedure. 01:55:33.700 --> 01:55:35.860 By eliminating the use of saws, the amount 01:55:35.860 --> 01:55:38.840 of potentially dangerous aerosols are reduced. 01:55:38.840 --> 01:55:41.240 Using the ventral or large animal method 01:55:41.240 --> 01:55:43.820 eliminates the need for a knife, reducing the risk 01:55:43.820 --> 01:55:46.010 for a cut or puncture wound. 01:55:46.010 --> 01:55:49.400 If dorsal method is used, make sure your knife is sharp. 01:55:49.400 --> 01:55:52.310 Don't handle the head directly with your gloved hand. 01:55:52.310 --> 01:55:54.860 Tenaculars will help to avoid cuts and punctures 01:55:54.860 --> 01:55:58.160 from quills bone shards, broken blades embedded 01:55:58.160 --> 01:56:01.580 from the decapitation, or other foreign objects. 01:56:01.580 --> 01:56:05.780 And never hold the head steady with your hands when cutting. 01:56:05.780 --> 01:56:10.310 Proper PPE, and if available the use of a fume hood or BSC 01:56:10.310 --> 01:56:13.190 can help protect the technician from splashes. 01:56:13.190 --> 01:56:17.980 Proper PPE is also essential for safety from aerosol and shards. 01:56:17.980 --> 01:56:20.090 Remember that the safety protocols are not 01:56:20.090 --> 01:56:21.140 just for rabies. 01:56:21.140 --> 01:56:23.120 These animals could have a different illness, 01:56:23.120 --> 01:56:25.250 and it's best to stay protected. 01:56:25.250 --> 01:56:26.290 Thank you very much. 01:56:26.290 --> 01:56:29.980 01:56:29.980 --> 01:56:30.480 - Great. 01:56:30.480 --> 01:56:31.800 Thank you, Jodie. 01:56:31.800 --> 01:56:33.450 Quick question. 01:56:33.450 --> 01:56:39.720 Do you recall, have you ever had a case in which the brain 01:56:39.720 --> 01:56:44.860 stem was negative but the forebrain or the hippocampus 01:56:44.860 --> 01:56:45.760 was positive? 01:56:45.760 --> 01:56:50.740 01:56:50.740 --> 01:56:53.180 - With the brain stem negative, you asked? 01:56:53.180 --> 01:56:53.680 - Yes. 01:56:53.680 --> 01:56:54.180 - No. 01:56:54.180 --> 01:56:55.270 I think we've seen-- 01:56:55.270 --> 01:56:55.770 - Good. 01:56:55.770 --> 01:56:59.710 - --where the brain stem is positive and not very much 01:56:59.710 --> 01:57:01.210 in the cerebellum. 01:57:01.210 --> 01:57:02.470 - Excellent. 01:57:02.470 --> 01:57:05.390 That's the ideal answer, thank you. 01:57:05.390 --> 01:57:11.080 And a similar question-- have you had situations where-- 01:57:11.080 --> 01:57:13.120 and oftentimes this is complicated 01:57:13.120 --> 01:57:18.160 because of TSEs, mad cow, et cetera, chronic wasting-- 01:57:18.160 --> 01:57:21.970 where if you don't have a full thickness 01:57:21.970 --> 01:57:28.270 cross-section of the brain stem, that a simple may be negative. 01:57:28.270 --> 01:57:34.600 - We have seen in a lateral rabies virus in the stem. 01:57:34.600 --> 01:57:38.555 They're very strict on having a full cross-section of brain 01:57:38.555 --> 01:57:39.430 stem for that reason. 01:57:39.430 --> 01:57:42.040 01:57:42.040 --> 01:57:43.200 We've seen it in a cow. 01:57:43.200 --> 01:57:46.050 And I know there is a picture of it in the protocol 01:57:46.050 --> 01:57:47.040 that we followed. 01:57:47.040 --> 01:57:48.930 I believe it's from a donkey. 01:57:48.930 --> 01:57:49.500 - Yes. 01:57:49.500 --> 01:57:53.250 And if we think of the transmission from peripheries 01:57:53.250 --> 01:57:57.450 to the CNS, we can think of how that may be possible. 01:57:57.450 --> 01:58:01.500 It's not very common, but still it has occurred. 01:58:01.500 --> 01:58:05.340 And I also saw that we had a comment from our colleagues 01:58:05.340 --> 01:58:10.050 at in Texas that the same as with your experience, 01:58:10.050 --> 01:58:10.920 and between-- 01:58:10.920 --> 01:58:12.870 and which California was on as well-- 01:58:12.870 --> 01:58:16.410 between New York and Texas, the denominators 01:58:16.410 --> 01:58:19.500 are overwhelming in the sense that, yes, 01:58:19.500 --> 01:58:25.410 you can have other areas of the brain negative 01:58:25.410 --> 01:58:28.560 but the brain stem positive, demonstrating 01:58:28.560 --> 01:58:32.400 the anatomic pathologic criticality of having 01:58:32.400 --> 01:58:35.490 the brain stem for diagnosis. 01:58:35.490 --> 01:58:39.640 So I certainly thank you for pointing that out. 01:58:39.640 --> 01:58:41.550 And a very excellent presentation. 01:58:41.550 --> 01:58:44.880 I'm looking quickly to see in the Q&A. 01:58:44.880 --> 01:58:51.130 I don't see any open questions there or in the chat. 01:58:51.130 --> 01:58:52.220 No. 01:58:52.220 --> 01:58:57.440 What are your criteria for rejecting a specimen 01:58:57.440 --> 01:59:02.330 as not sufficient for doing, for example, DFA-- 01:59:02.330 --> 01:59:04.490 Direct Fluorescent Antibody testing? 01:59:04.490 --> 01:59:07.550 - If we don't have a full cross-section of stem, 01:59:07.550 --> 01:59:10.550 we'll still test it but we can't call it negative. 01:59:10.550 --> 01:59:15.290 We'll call it [? unsat. ?] Same with if we don't have samples 01:59:15.290 --> 01:59:19.100 from all three lobes of the cerebellum. 01:59:19.100 --> 01:59:25.520 Decomposition is a tricky one. 01:59:25.520 --> 01:59:28.730 - If you have samples that are, say, less than ideal-- 01:59:28.730 --> 01:59:32.480 if the material looks like ice cream 01:59:32.480 --> 01:59:38.580 and it's obviously denatured, decaying, has a bad odor, 01:59:38.580 --> 01:59:42.260 will you ever resort to molecular techniques 01:59:42.260 --> 01:59:43.340 for diagnostics? 01:59:43.340 --> 01:59:48.500 Or if it's not suitable for DFA, then you don't report results? 01:59:48.500 --> 01:59:50.740 - So we'll run DFA. 01:59:50.740 --> 01:59:52.840 If it's positive, we can still call it that. 01:59:52.840 --> 01:59:57.400 If it's negative, we do have it-- 01:59:57.400 --> 01:59:59.530 do molecular testing on it. 01:59:59.530 --> 02:00:03.850 Depending on the history will depend if we 02:00:03.850 --> 02:00:09.060 can release the PCR results. 02:00:09.060 --> 02:00:11.470 If it's more surveillance we'll do it. 02:00:11.470 --> 02:00:16.660 If there's actual treatment or anything involved with a human, 02:00:16.660 --> 02:00:19.420 then we prefer to steer on the side of caution, 02:00:19.420 --> 02:00:24.350 have them be treated for an [INAUDIBLE].. 02:00:24.350 --> 02:00:27.590 - Thank you, Jodie, for your very excellent presentation. 02:00:27.590 --> 02:00:29.540 Demonstrating the comparative anatomy 02:00:29.540 --> 02:00:33.275 is not only important but critical to rabies diagnostics. 02:00:33.275 --> 02:00:35.540 If you don't have the right specimen, 02:00:35.540 --> 02:00:40.440 you can't expect to get the correct true result. I'll ask, 02:00:40.440 --> 02:00:44.480 if you'd be so kind, to stop screen sharing. 02:00:44.480 --> 02:00:49.670 And we'll move on to our next panelist. 02:00:49.670 --> 02:00:54.480 Erin Patrick is the Tennessee, Kentucky rabies biologist-- 02:00:54.480 --> 02:00:55.460 [CHEERING] 02:00:55.460 --> 02:00:57.656 - Did I talk at warp speed? 02:00:57.656 --> 02:00:58.628 - No, you did a good-- 02:00:58.628 --> 02:01:00.980 - Oh, I think you're need-- 02:01:00.980 --> 02:01:03.560 I think, Jodie, I think you need to mute your microphone. 02:01:03.560 --> 02:01:05.330 [LAUGHTER] 02:01:05.330 --> 02:01:08.360 Erin is the biologist for the United States 02:01:08.360 --> 02:01:12.230 Department of Agriculture APHIS Wildlife Services. 02:01:12.230 --> 02:01:15.750 She worked for Wildlife Services for 16 years, 02:01:15.750 --> 02:01:20.930 including experience in Florida and in Kentucky 02:01:20.930 --> 02:01:24.500 as a rabies biologist before moving to Tennessee. 02:01:24.500 --> 02:01:26.840 Her current responsibilities include 02:01:26.840 --> 02:01:29.420 implementation of the National Rabies Management 02:01:29.420 --> 02:01:32.000 Plan in Tennessee and Kentucky to help 02:01:32.000 --> 02:01:36.740 stop the westward spread of raccoon rabies virus variant. 02:01:36.740 --> 02:01:40.040 She received her degree from the University of Tennessee 02:01:40.040 --> 02:01:42.510 and the University of Arkansas. 02:01:42.510 --> 02:01:45.350 She lives in Knoxville, where she's 02:01:45.350 --> 02:01:48.140 going to be sharing some of her experience 02:01:48.140 --> 02:01:50.330 on the direct rapid immunohistochemistry 02:01:50.330 --> 02:01:53.820 test, which again demonstrates that you-- 02:01:53.820 --> 02:01:56.150 it's great to be a trained laboratorian 02:01:56.150 --> 02:01:59.660 and with 10,000 hours of laboratory experience, 02:01:59.660 --> 02:02:02.450 but the utility of some of these techniques such as we 02:02:02.450 --> 02:02:06.530 heard for the lateral flow assay or now for the DRIT, 02:02:06.530 --> 02:02:09.360 that even a wildlife biologist can do it. 02:02:09.360 --> 02:02:11.240 Erin, you have the floor. 02:02:11.240 --> 02:02:11.990 - OK. 02:02:11.990 --> 02:02:14.508 Can you hear me OK? 02:02:14.508 --> 02:02:15.050 - [INAUDIBLE] 02:02:15.050 --> 02:02:15.980 - OK, great. 02:02:15.980 --> 02:02:17.510 All right, good. 02:02:17.510 --> 02:02:18.290 OK, yes. 02:02:18.290 --> 02:02:22.070 So definitely, coming from a little bit 02:02:22.070 --> 02:02:25.170 of a different background, not a lab background. 02:02:25.170 --> 02:02:30.290 But I think what we do is obviously very important. 02:02:30.290 --> 02:02:33.500 And it plays an important role with rabies management 02:02:33.500 --> 02:02:34.470 worldwide. 02:02:34.470 --> 02:02:38.630 And so it is very important for people without lab backgrounds 02:02:38.630 --> 02:02:41.780 to have the ability to kind of help diagnose 02:02:41.780 --> 02:02:45.680 and then make plans for rabies management. 02:02:45.680 --> 02:02:48.890 And that's really what this presentation is about. 02:02:48.890 --> 02:02:54.260 So I want to recognize my co-authors, who-- 02:02:54.260 --> 02:02:56.270 obviously, most of those, they work 02:02:56.270 --> 02:02:58.460 for the National Rabies Management Program 02:02:58.460 --> 02:03:02.000 and USDA Wildlife Services. 02:03:02.000 --> 02:03:05.180 But-- I'm going to make sure-- 02:03:05.180 --> 02:03:06.260 OK. 02:03:06.260 --> 02:03:09.330 So of course, I'm talking about the DRIT test, 02:03:09.330 --> 02:03:13.190 which is the Direct Rabbit Immunohistochemical Test. 02:03:13.190 --> 02:03:15.470 And I will refer to it as DRIT for the rest 02:03:15.470 --> 02:03:19.250 of this presentation, because it's a lot easier to say. 02:03:19.250 --> 02:03:23.420 And obviously, it is an OIE and WHO recognized alternative 02:03:23.420 --> 02:03:29.210 to DFA, is becoming a very important component 02:03:29.210 --> 02:03:33.380 of our national rabies management program. 02:03:33.380 --> 02:03:36.440 If you have not had a chance to watch our video 02:03:36.440 --> 02:03:40.100 that we did for JoVE, which kind of takes you 02:03:40.100 --> 02:03:43.280 through the DRIT test step by step, 02:03:43.280 --> 02:03:46.760 or if you've not had a chance to use it, 02:03:46.760 --> 02:03:54.020 just really quickly, it is a relatively straightforward test 02:03:54.020 --> 02:03:56.820 that takes just a couple hours from, 02:03:56.820 --> 02:04:00.360 you can go from getting the sample in the field-- 02:04:00.360 --> 02:04:02.810 whether that's picking up a roadkill 02:04:02.810 --> 02:04:05.270 or having a suspect animal-- 02:04:05.270 --> 02:04:06.980 going from the sample in the field 02:04:06.980 --> 02:04:09.930 to removing the brain stem-- 02:04:09.930 --> 02:04:14.060 it's just like she just explained from the New York 02:04:14.060 --> 02:04:15.260 Lab-- 02:04:15.260 --> 02:04:16.190 taking brain stem. 02:04:16.190 --> 02:04:18.320 And then we make touch impressions. 02:04:18.320 --> 02:04:20.600 Then the touch impressions are put 02:04:20.600 --> 02:04:22.790 through a series of slide staining, 02:04:22.790 --> 02:04:25.580 and the slide staining is only about an hour. 02:04:25.580 --> 02:04:29.780 So start to finish, you can do the entire thing in just 02:04:29.780 --> 02:04:34.400 a couple hours and have a result. 02:04:34.400 --> 02:04:37.370 So the significance of DRIT, it's 02:04:37.370 --> 02:04:41.660 been incredibly significant to us in the field. 02:04:41.660 --> 02:04:45.690 But it's got obviously a lot of other significance. 02:04:45.690 --> 02:04:48.260 And first of all, the accuracy is 02:04:48.260 --> 02:04:51.260 comparable to DFA, which obviously is very important. 02:04:51.260 --> 02:04:53.690 The sensitivity and specificity has 02:04:53.690 --> 02:04:58.931 proven to be comparable in multiple studies. 02:04:58.931 --> 02:05:04.820 One of the big positive things is that it is-- 02:05:04.820 --> 02:05:09.410 it can be conducted at ambient temperatures, and on benchtop-- 02:05:09.410 --> 02:05:12.950 or, as we like to say in the field of wildlife biology, 02:05:12.950 --> 02:05:14.780 on a tailgate. 02:05:14.780 --> 02:05:17.540 We don't necessarily use tailgates that often, 02:05:17.540 --> 02:05:22.550 but meaning that we don't need to be at a centralized lab 02:05:22.550 --> 02:05:25.580 to be able to conduct this test. 02:05:25.580 --> 02:05:27.800 It uses basic laboratory techniques 02:05:27.800 --> 02:05:36.590 that can be attainable and easy for people to learn. 02:05:36.590 --> 02:05:39.440 We do not need specialized equipment 02:05:39.440 --> 02:05:42.170 We can run this test, of course, with just 02:05:42.170 --> 02:05:48.770 light microscopy, which is obviously a huge benefit. 02:05:48.770 --> 02:05:52.310 And it requires training, yes, but a short time 02:05:52.310 --> 02:05:53.790 to gain proficiency. 02:05:53.790 --> 02:05:57.560 So we can take people who have a background, maybe, 02:05:57.560 --> 02:05:59.990 in biology or natural resources-- of course, 02:05:59.990 --> 02:06:01.370 wildlife biology-- 02:06:01.370 --> 02:06:05.930 and take them through the training in a couple of days, 02:06:05.930 --> 02:06:11.720 and then they can go back into their field settings 02:06:11.720 --> 02:06:16.050 and quickly have proficiency with this test. 02:06:16.050 --> 02:06:18.140 So that's a huge benefit. 02:06:18.140 --> 02:06:22.040 And overall, it's inexpensive, which 02:06:22.040 --> 02:06:27.050 is because of the simplicity of it, I guess. 02:06:27.050 --> 02:06:28.700 The DRIT test does have the ability 02:06:28.700 --> 02:06:31.370 to recognize all variants of rabies. 02:06:31.370 --> 02:06:35.030 And I do want to point out that we and this is USDA Wildlife 02:06:35.030 --> 02:06:37.880 Services, we use this as a complement 02:06:37.880 --> 02:06:39.390 to public health surveillance. 02:06:39.390 --> 02:06:40.670 This is not for-- 02:06:40.670 --> 02:06:44.090 we don't use this to, of course, diagnose a rabies 02:06:44.090 --> 02:06:47.750 in human health and safety cases or cases 02:06:47.750 --> 02:06:53.120 where an animal has exposed a human or domestic animal. 02:06:53.120 --> 02:06:56.240 We use this for enhanced rabies surveillance 02:06:56.240 --> 02:07:00.030 to get a better, bigger picture of what's going on. 02:07:00.030 --> 02:07:04.010 And over the years, the DRIT has really 02:07:04.010 --> 02:07:07.610 became a highly critical tool for us 02:07:07.610 --> 02:07:10.730 to consult to enhance rabies surveillance. 02:07:10.730 --> 02:07:14.960 So it allows us to conduct this enhanced rabies surveillance 02:07:14.960 --> 02:07:18.320 to kind of piggyback off the public health surveillance, 02:07:18.320 --> 02:07:23.750 and it allows us to have real time over the management 02:07:23.750 --> 02:07:26.300 decisions that can be made from this test. 02:07:26.300 --> 02:07:29.980 02:07:29.980 --> 02:07:32.670 So why do we want to do that? 02:07:32.670 --> 02:07:37.620 If you, excuse me, if you're familiar with our work then 02:07:37.620 --> 02:07:39.660 you probably understand, but if you're not, 02:07:39.660 --> 02:07:45.630 our agency works to prevent the spread of terrestrial rabies 02:07:45.630 --> 02:07:48.870 in the United States and to eliminate rabies variants 02:07:48.870 --> 02:07:51.140 at local, regional, and national level. 02:07:51.140 --> 02:07:54.390 So that's one of our big goals with the National Rabies 02:07:54.390 --> 02:07:56.730 Management Program. 02:07:56.730 --> 02:08:00.840 To do this, we will distribute oral rabies vaccines 02:08:00.840 --> 02:08:03.240 across a very large area. 02:08:03.240 --> 02:08:08.340 17 states where last year had oral rabies vaccines 02:08:08.340 --> 02:08:12.450 distributed, and over time we've distributed over 9 and 1/2 02:08:12.450 --> 02:08:18.000 million rabies vaccines to work on this goal of preventing 02:08:18.000 --> 02:08:20.700 and eliminating rabies variants. 02:08:20.700 --> 02:08:24.540 So obviously, there's a lot of time, effort, personnel, 02:08:24.540 --> 02:08:27.900 and money going into getting these vaccines out 02:08:27.900 --> 02:08:29.170 on the landscape. 02:08:29.170 --> 02:08:33.570 So it's very important that we get these vaccines out 02:08:33.570 --> 02:08:35.170 in the right places. 02:08:35.170 --> 02:08:40.170 So the test has allowed us to gain confidence and make 02:08:40.170 --> 02:08:47.400 management decisions with confidence over the years. 02:08:47.400 --> 02:08:48.730 So DRIT by the numbers. 02:08:48.730 --> 02:08:50.340 So how much are we talking about Here? 02:08:50.340 --> 02:08:52.870 02:08:52.870 --> 02:08:56.760 First of all in 2020, USDA Wildlife Services conducted 02:08:56.760 --> 02:09:02.910 over 7,000 tests on samples and about 2.3% of those 02:09:02.910 --> 02:09:06.030 were positive. 02:09:06.030 --> 02:09:08.850 Since the beginning of when we started doing DRIT, 02:09:08.850 --> 02:09:11.940 we have tested over 110,000 samples 02:09:11.940 --> 02:09:16.860 of using DRIT and about 2.1% have been positive. 02:09:16.860 --> 02:09:20.400 So we've been able to get a lot of data point 02:09:20.400 --> 02:09:25.560 over the years that has allowed us to make our management 02:09:25.560 --> 02:09:28.650 decisions with confidence. 02:09:28.650 --> 02:09:31.957 You know, a lot of times I get questions from people saying, 02:09:31.957 --> 02:09:33.540 well that's a lot of negatives, that's 02:09:33.540 --> 02:09:35.280 a lot of time and effort, you guys 02:09:35.280 --> 02:09:36.540 are spending on the negatives. 02:09:36.540 --> 02:09:39.030 And I always tell people that the negatives when you're 02:09:39.030 --> 02:09:41.700 doing, of course, surveillance, are just 02:09:41.700 --> 02:09:45.390 as important as the positives, so those negatives have allowed 02:09:45.390 --> 02:09:48.120 us to help make our management decisions just as 02:09:48.120 --> 02:09:50.970 much as the positives have. 02:09:50.970 --> 02:09:54.300 Today, we've had 87 Wildlife Services employees that 02:09:54.300 --> 02:09:55.890 have been trained to do DRIT. 02:09:55.890 --> 02:09:59.310 And again, most all of those are field biologists or field 02:09:59.310 --> 02:10:02.580 technicians, and they've been very successful at it. 02:10:02.580 --> 02:10:07.545 We do confirm our positives and of course, number 02:10:07.545 --> 02:10:09.700 of negatives with the confirmation lab, 02:10:09.700 --> 02:10:12.330 and we also do proficiency testing. 02:10:12.330 --> 02:10:18.840 And so, it's been a very helpful test to learn, 02:10:18.840 --> 02:10:22.320 and it's been one that has been totally doable by the field 02:10:22.320 --> 02:10:24.540 biologists including myself. 02:10:24.540 --> 02:10:27.180 We have 24 field biologists that are currently 02:10:27.180 --> 02:10:28.290 performing the test. 02:10:28.290 --> 02:10:33.040 02:10:33.040 --> 02:10:38.590 And the other big thing that has been kind of a big deal to us 02:10:38.590 --> 02:10:40.480 to the field is that we don't have 02:10:40.480 --> 02:10:43.270 to have the centralized testing locations in order 02:10:43.270 --> 02:10:45.880 to do the test. 02:10:45.880 --> 02:10:51.160 We conduct these tests in 15 different facilities currently. 02:10:51.160 --> 02:10:53.150 The majority of those are in office spaces. 02:10:53.150 --> 02:10:58.210 So in office spaces that are USDA Wildlife Services offices, 02:10:58.210 --> 02:11:02.320 and we're able to basically turn a room of that office 02:11:02.320 --> 02:11:05.680 into a DRIT testing location. 02:11:05.680 --> 02:11:07.210 Those were great. 02:11:07.210 --> 02:11:10.690 We also work with cooperating agencies 02:11:10.690 --> 02:11:13.360 at times, where we will work maybe 02:11:13.360 --> 02:11:17.140 with a University or a public health lab or something, 02:11:17.140 --> 02:11:20.000 and we will do our there. 02:11:20.000 --> 02:11:21.790 And we also have a couple of mobile labs. 02:11:21.790 --> 02:11:26.110 And those mobile labs kind of take that you 02:11:26.110 --> 02:11:29.360 can do this to the field. 02:11:29.360 --> 02:11:31.870 The second picture from the top is a picture of one 02:11:31.870 --> 02:11:35.890 of our mobile labs, and we have this dropdown table 02:11:35.890 --> 02:11:43.923 and you can do the entire test there without any power. 02:11:43.923 --> 02:11:45.340 The only time you would need power 02:11:45.340 --> 02:11:47.380 is at the end, when you are reading on a flood. 02:11:47.380 --> 02:11:51.460 So we have the ability to take either of these mobile labs 02:11:51.460 --> 02:11:54.040 into a situation or an area that we 02:11:54.040 --> 02:11:58.060 need maybe a lot of testing with a quick turnaround 02:11:58.060 --> 02:12:00.670 or just testing in general with a quick turnaround. 02:12:00.670 --> 02:12:06.010 And so that has been a pretty big deal and helpful part 02:12:06.010 --> 02:12:08.270 of that we do to test. 02:12:08.270 --> 02:12:11.410 So I want to provide just an example 02:12:11.410 --> 02:12:15.160 of how we are using today. 02:12:15.160 --> 02:12:19.780 Literally, today to make these management decisions. 02:12:19.780 --> 02:12:24.610 We've recently had a couple of positive cases of rabies 02:12:24.610 --> 02:12:29.350 that were detected through our enhanced rabies surveillance 02:12:29.350 --> 02:12:30.130 test. 02:12:30.130 --> 02:12:34.210 And they were areas that we hadn't had rabies 02:12:34.210 --> 02:12:35.920 quite in those areas before. 02:12:35.920 --> 02:12:38.360 So we're able to-- 02:12:38.360 --> 02:12:40.930 first of all, we pull in the tests 02:12:40.930 --> 02:12:42.880 that we've done in the past couple of years 02:12:42.880 --> 02:12:45.640 to kind of get an idea of what the surveillance is like 02:12:45.640 --> 02:12:49.150 and where we need to kind of look. 02:12:49.150 --> 02:12:54.490 And so, as a field biologist, that's helpful, first of all, 02:12:54.490 --> 02:12:55.760 to pull in all the negatives. 02:12:55.760 --> 02:12:57.760 Again, those negatives are really important when 02:12:57.760 --> 02:13:01.720 you're looking at rabies and potentially a different area. 02:13:01.720 --> 02:13:04.000 But pulling in all of those samples 02:13:04.000 --> 02:13:06.460 that we had done over the last year or two, 02:13:06.460 --> 02:13:11.110 and then making a plan of where we need to kind of focus 02:13:11.110 --> 02:13:15.610 and where we need to try to get some additional samples. 02:13:15.610 --> 02:13:18.610 And in this situation, we can potentially 02:13:18.610 --> 02:13:20.530 pull one of our mobile labs over, 02:13:20.530 --> 02:13:23.530 and we could have people out in the field, which are out 02:13:23.530 --> 02:13:27.040 in the field today, looking for sick animals, 02:13:27.040 --> 02:13:29.500 trying to get some surveillance numbers up. 02:13:29.500 --> 02:13:32.170 And I mean, we could literally have an animal 02:13:32.170 --> 02:13:36.100 and have a result within two hours. 02:13:36.100 --> 02:13:38.560 So that's an example of how we are 02:13:38.560 --> 02:13:42.190 using DRIT to make management decisions, 02:13:42.190 --> 02:13:44.740 because this will could potentially 02:13:44.740 --> 02:13:49.280 change how we end up putting the vaccine out and on the ground. 02:13:49.280 --> 02:13:54.490 So it's definitely a test that we are utilizing every day 02:13:54.490 --> 02:13:56.350 both in the field, and then taking 02:13:56.350 --> 02:14:00.340 that data back to our planners and the people that 02:14:00.340 --> 02:14:04.780 do the modeling, and so we're able to work together 02:14:04.780 --> 02:14:09.590 to work towards our goal of elimination. 02:14:09.590 --> 02:14:12.250 So I will summarize, there's really 02:14:12.250 --> 02:14:14.650 no changes to the standard operating procedure 02:14:14.650 --> 02:14:18.310 if you have seen the video or read our document. 02:14:18.310 --> 02:14:20.110 Number of changes. 02:14:20.110 --> 02:14:23.620 We're still doing things the way that we have been doing them 02:14:23.620 --> 02:14:26.480 and they work well, so we're thankful for that, 02:14:26.480 --> 02:14:28.900 and we'll hopefully continue that. 02:14:28.900 --> 02:14:32.680 The ongoing challenge has been availability and reliability 02:14:32.680 --> 02:14:35.860 of the antibodies to the mouse. 02:14:35.860 --> 02:14:39.670 And overall, the drug test has been 02:14:39.670 --> 02:14:42.730 and is a safe, efficient, effective, and affordable way 02:14:42.730 --> 02:14:46.480 to provide rabies testing. 02:14:46.480 --> 02:14:49.790 It's allowing field biologists to make science based, 02:14:49.790 --> 02:14:55.870 data driven management, which is obviously we're 02:14:55.870 --> 02:14:58.390 using this every day, and it's really 02:14:58.390 --> 02:15:01.390 became a game changer for effective Wildlife rabies 02:15:01.390 --> 02:15:04.550 management. 02:15:04.550 --> 02:15:07.360 So with that, we definitely want to thank 02:15:07.360 --> 02:15:11.110 we work closely with a lot of state programs 02:15:11.110 --> 02:15:14.530 within Wildlife Services for people that are conducting 02:15:14.530 --> 02:15:17.758 the DRIT, so we want to obviously acknowledge 02:15:17.758 --> 02:15:19.300 them and their hard work that they're 02:15:19.300 --> 02:15:21.730 doing not only in the lab but out in the field 02:15:21.730 --> 02:15:23.920 to collect the sample. 02:15:23.920 --> 02:15:27.730 And then CDC and the Wadsworth, and wistar. 02:15:27.730 --> 02:15:30.820 And then we have so many cooperators that are helping us 02:15:30.820 --> 02:15:34.320 with samples and have been for quite a while now, 02:15:34.320 --> 02:15:37.940 so we always want to acknowledge those people as well. 02:15:37.940 --> 02:15:40.382 So with that, I will take any questions. 02:15:40.382 --> 02:15:41.920 - Super thank you, Erin. 02:15:41.920 --> 02:15:47.680 And we do want to mention that one 02:15:47.680 --> 02:15:51.580 participates in proficiency testing for DRIT the same as 02:15:51.580 --> 02:15:52.870 for DFA. 02:15:52.870 --> 02:15:57.268 And so you participate in national DRIT proficiency 02:15:57.268 --> 02:15:58.060 testing, don't you? 02:15:58.060 --> 02:15:59.530 - Yes, we do. 02:15:59.530 --> 02:16:02.500 We do proficiency testing just like you said, like would be-- 02:16:02.500 --> 02:16:06.580 I think, twice a year now, we do not get the blood samples 02:16:06.580 --> 02:16:08.360 and do that. 02:16:08.360 --> 02:16:13.620 And overall, we've been extremely successful with that. 02:16:13.620 --> 02:16:14.120 Yeah. 02:16:14.120 --> 02:16:14.455 So yeah. 02:16:14.455 --> 02:16:14.955 - Great. 02:16:14.955 --> 02:16:16.540 - It's been a very successful thing. 02:16:16.540 --> 02:16:20.800 - And how do you considering their reportable nature, 02:16:20.800 --> 02:16:22.930 how do you share your results from 02:16:22.930 --> 02:16:26.650 enhanced surveilling with local or state public health 02:16:26.650 --> 02:16:27.860 officials? 02:16:27.860 --> 02:16:28.360 - Yeah. 02:16:28.360 --> 02:16:29.318 That's a good question. 02:16:29.318 --> 02:16:34.020 So when we have positive, a couple of different things. 02:16:34.020 --> 02:16:36.879 One is we get them confirmed by a confirmation lab 02:16:36.879 --> 02:16:40.013 either Wadsworth or the CDC. 02:16:40.013 --> 02:16:41.680 So they're going there for confirmation. 02:16:41.680 --> 02:16:45.129 And once we get confirmation from their, 02:16:45.129 --> 02:16:48.040 each state works independently, I 02:16:48.040 --> 02:16:52.690 guess, with their state health lab in Tennessee, Kentucky. 02:16:52.690 --> 02:16:57.969 When I get positive, I send them right on to the state lab. 02:16:57.969 --> 02:17:00.790 So we do work in conjunction with our state 02:17:00.790 --> 02:17:04.850 public health lab and public health agencies, 02:17:04.850 --> 02:17:07.930 so to make sure that everything is matching up. 02:17:07.930 --> 02:17:11.680 And so we're definitely in communication with them 02:17:11.680 --> 02:17:13.250 and that goes both ways. 02:17:13.250 --> 02:17:15.879 So they're communicating with us and us with them. 02:17:15.879 --> 02:17:18.820 - And I noticed that these DRIT results are now 02:17:18.820 --> 02:17:21.580 officially recognized and appear in 02:17:21.580 --> 02:17:26.000 our annual national surveillance report that CDC puts out. 02:17:26.000 --> 02:17:26.764 So it-- 02:17:26.764 --> 02:17:27.264 - Yeah. 02:17:27.264 --> 02:17:30.910 - --demonstrates the utility that Wildlife Services 02:17:30.910 --> 02:17:34.150 biologists and others have been doing. 02:17:34.150 --> 02:17:37.389 And I'm happy to point out that although-- 02:17:37.389 --> 02:17:39.879 obviously, who wouldn't rather use a lab 02:17:39.879 --> 02:17:41.799 when you don't have to use the field, 02:17:41.799 --> 02:17:46.090 but the DRIT actually started in parts of Africa, 02:17:46.090 --> 02:17:49.570 not only for dogs but a variety of wildlife. 02:17:49.570 --> 02:17:52.570 DRIT's been done all over the world, 02:17:52.570 --> 02:17:56.830 and similarly it's not so much a kit, 02:17:56.830 --> 02:18:00.219 because as you've demonstrated in your protocols 02:18:00.219 --> 02:18:05.200 and from your video, that the variety of chemicals, 02:18:05.200 --> 02:18:10.660 fixatives like Formalin and washing with Tween PBSC. 02:18:10.660 --> 02:18:14.410 Most of the parts of DRIT scalpels for collecting 02:18:14.410 --> 02:18:19.930 the correct brain stem sample, biosafety protocols, 02:18:19.930 --> 02:18:22.190 most of these things are readily available. 02:18:22.190 --> 02:18:26.680 So it's not a kit per se as you would think formerly, 02:18:26.680 --> 02:18:28.670 because one of the questions was, well, 02:18:28.670 --> 02:18:32.889 how can we get a DRIT kit to X. Or one has to do 02:18:32.889 --> 02:18:35.139 is go ahead and read the protocols. 02:18:35.139 --> 02:18:37.840 There's a variety of protocols out there. 02:18:37.840 --> 02:18:41.620 DRIT has been recognized as a companion 02:18:41.620 --> 02:18:44.620 to DFA for antigen testing. 02:18:44.620 --> 02:18:47.680 And really, I would say, the most critical part 02:18:47.680 --> 02:18:51.370 of the DRIT or the antibodies, and those antibodies 02:18:51.370 --> 02:18:52.900 can either be monoclonal. 02:18:52.900 --> 02:18:56.230 And most of the WHO collaborating centers, 02:18:56.230 --> 02:18:58.510 some of whom are on this call-- 02:18:58.510 --> 02:19:02.530 CDC, CFIA, our colleagues in Europe. 02:19:02.530 --> 02:19:04.809 There are monoclonal's that are available that 02:19:04.809 --> 02:19:07.780 have been able to demonstrate their utility in the DRIT, 02:19:07.780 --> 02:19:10.420 and our colleagues in Southern Africa 02:19:10.420 --> 02:19:14.379 have demonstrated the utility of polyclonal antibodies 02:19:14.379 --> 02:19:16.240 and performing the DRIT. 02:19:16.240 --> 02:19:18.790 Once you've demonstrated sensitivity and specificity 02:19:18.790 --> 02:19:22.629 of your biologic, all the other parts, the slides, 02:19:22.629 --> 02:19:26.530 the fixatives, the washes, the immunohistochemical stains, 02:19:26.530 --> 02:19:29.209 the microscopes, all of those are commercially available. 02:19:29.209 --> 02:19:33.490 So you don't need a kit, you just need the ability to read, 02:19:33.490 --> 02:19:35.889 the ability to learn, the ability to watch, 02:19:35.889 --> 02:19:38.650 the ability to train, and the ability to do. 02:19:38.650 --> 02:19:42.010 So I thank you very much for pointing out your experience 02:19:42.010 --> 02:19:47.920 with us today, and I hope you'll stay on for the roundtable 02:19:47.920 --> 02:19:50.860 and would you be so kind to stop your screen sharing, 02:19:50.860 --> 02:19:54.760 so we'll go on to our next panelist. 02:19:54.760 --> 02:20:00.400 Our next panelists will be closing up the presentations 02:20:00.400 --> 02:20:05.800 before our discussions, and it demonstrates 02:20:05.800 --> 02:20:08.110 that we've come full circle from talking 02:20:08.110 --> 02:20:12.190 about the utility of laboratory testing, 02:20:12.190 --> 02:20:15.880 to protocols and techniques, for diagnostics 02:20:15.880 --> 02:20:19.600 in and out of the lab, in the utility of oral vaccination, 02:20:19.600 --> 02:20:23.260 not only for Wildlife, but obviously, for the future. 02:20:23.260 --> 02:20:27.100 In real time, there have been some very nice recent papers 02:20:27.100 --> 02:20:31.280 about oral vaccinations applied to dogs, for free ranging dogs, 02:20:31.280 --> 02:20:35.720 for the utility in the ZBT, to 0 by 30 elimination 02:20:35.720 --> 02:20:38.750 of human rabies caused by dog rabies. 02:20:38.750 --> 02:20:41.780 And so our next panelist, Harry Bernstein, 02:20:41.780 --> 02:20:44.330 a biologist, also with the United States Department 02:20:44.330 --> 02:20:47.690 of Agriculture, a National Wildlife Research Center, 02:20:47.690 --> 02:20:50.870 working on the rabies project as a Wildlife biologist, 02:20:50.870 --> 02:20:54.860 had a lot of time and experience in Puerto Rico, working 02:20:54.860 --> 02:21:00.290 on another rabies reservoir, so beyond dogs, raccoons, skunks, 02:21:00.290 --> 02:21:02.270 foxes, bats. 02:21:02.270 --> 02:21:06.650 We have a non-indigenous imported reservoir vector 02:21:06.650 --> 02:21:11.090 in the Caribbean into Puerto Rico, the mongoose. 02:21:11.090 --> 02:21:14.210 And Harry will be talking about some of his experience 02:21:14.210 --> 02:21:16.100 and thinking about biomarkers that 02:21:16.100 --> 02:21:21.080 can be incorporated in debates for oral vaccination 02:21:21.080 --> 02:21:24.950 to gain some insight as to the utility of such programs 02:21:24.950 --> 02:21:28.930 in reaching those free ranging animals in the field. 02:21:28.930 --> 02:21:29.430 Harry? 02:21:29.430 --> 02:21:32.230 02:21:32.230 --> 02:21:35.280 [NON-ENGLISH SPEECH] 02:21:35.280 --> 02:21:36.450 - Thank you very much. 02:21:36.450 --> 02:21:42.680 02:21:42.680 --> 02:21:45.120 There we go. 02:21:45.120 --> 02:21:51.240 See here-- can everybody-- pardon me. 02:21:51.240 --> 02:21:53.250 Can everybody see the slide without my notes? 02:21:53.250 --> 02:21:56.150 02:21:56.150 --> 02:21:57.910 - I can. 02:21:57.910 --> 02:21:59.390 - Fantastic. 02:21:59.390 --> 02:22:01.980 Good morning, good afternoon, and good evening, 02:22:01.980 --> 02:22:03.680 depending on your time zone. . 02:22:03.680 --> 02:22:05.180 Thank you very much for including me 02:22:05.180 --> 02:22:06.800 in this talk today. 02:22:06.800 --> 02:22:10.100 I'd like to start off by acknowledging my co-authors 02:22:10.100 --> 02:22:14.410 here from the Hawaii field station, as well as the rabies 02:22:14.410 --> 02:22:16.910 project leader of the National Wildlife Research Center, Dr. 02:22:16.910 --> 02:22:19.670 M. Gilbert, oour collaborators from Germany, 02:22:19.670 --> 02:22:23.810 and in particular, Mr Stephen Volker, who is the chemist, who 02:22:23.810 --> 02:22:27.350 refined this method that I'm going to be describing today 02:22:27.350 --> 02:22:30.410 on the use of Iophenoxic acid, which I'll refer to 02:22:30.410 --> 02:22:34.850 as IPA, not to be confused with the beverage in mongoose serum 02:22:34.850 --> 02:22:40.230 using liquid chromatography and mass spectrometer techniques. 02:22:40.230 --> 02:22:42.480 For those unfamiliar, Iophenoxic IPA 02:22:42.480 --> 02:22:45.840 is a white crystalline powder, and there are multiple-- 02:22:45.840 --> 02:22:48.540 pardon me, derivatives that are typically 02:22:48.540 --> 02:22:50.000 identified by the branch chain. 02:22:50.000 --> 02:22:55.320 So there's the methyl, ethyl, propyl, butyl, isobutyl, 02:22:55.320 --> 02:23:00.150 and pentyl IPA that are available. 02:23:00.150 --> 02:23:05.250 Historically, the use of IPA as a biomarker was relied 02:23:05.250 --> 02:23:06.880 on measuring an increase in-- 02:23:06.880 --> 02:23:07.380 - Excuse me. 02:23:07.380 --> 02:23:09.180 - --an idea-- yes. 02:23:09.180 --> 02:23:11.940 - I see one of the charts, it said, are you 02:23:11.940 --> 02:23:13.860 on PowerPoint mode. 02:23:13.860 --> 02:23:17.190 So please put slides on PowerPoint mode 02:23:17.190 --> 02:23:19.170 for presentation. 02:23:19.170 --> 02:23:20.190 Yeah, there you go. 02:23:20.190 --> 02:23:21.000 That's great. 02:23:21.000 --> 02:23:21.500 Thank you. 02:23:21.500 --> 02:23:22.042 - Is that it? 02:23:22.042 --> 02:23:24.480 02:23:24.480 --> 02:23:30.740 - Good display settings again, in the PowerPoint, 02:23:30.740 --> 02:23:32.480 at the top middle. 02:23:32.480 --> 02:23:35.396 - At your top middle, Harry. 02:23:35.396 --> 02:23:36.740 - I had this earlier. 02:23:36.740 --> 02:23:39.540 02:23:39.540 --> 02:23:41.290 - If you press From Beginning-- 02:23:41.290 --> 02:23:46.470 02:23:46.470 --> 02:23:50.060 and then, here, Display Settings. 02:23:50.060 --> 02:23:52.010 At the top middle. 02:23:52.010 --> 02:23:52.580 - 1 over. 02:23:52.580 --> 02:23:56.540 02:23:56.540 --> 02:23:59.240 Display Settings. 02:23:59.240 --> 02:24:02.370 - I'm unfortunately not seeing that on my screen. 02:24:02.370 --> 02:24:02.870 - Oh. 02:24:02.870 --> 02:24:10.027 02:24:10.027 --> 02:24:11.860 - Why don't I stop sharing and try it again, 02:24:11.860 --> 02:24:12.670 is that all right? 02:24:12.670 --> 02:24:13.170 - Sure. 02:24:13.170 --> 02:24:17.640 02:24:17.640 --> 02:24:20.070 - Should be able to go to share screen. 02:24:20.070 --> 02:24:24.260 02:24:24.260 --> 02:24:24.760 - OK. 02:24:24.760 --> 02:24:27.555 Let see here. 02:24:27.555 --> 02:24:30.450 Can share screen. 02:24:30.450 --> 02:24:31.230 When we go here-- 02:24:31.230 --> 02:24:36.450 02:24:36.450 --> 02:24:39.570 - And then bottom. 02:24:39.570 --> 02:24:40.380 Oops. 02:24:40.380 --> 02:24:42.960 You've just jumped over again. 02:24:42.960 --> 02:24:46.410 Do you see at the top, it says, Show Taskbar Display Settings 02:24:46.410 --> 02:24:48.171 or End. 02:24:48.171 --> 02:24:50.730 - The taskbar, I have that. 02:24:50.730 --> 02:24:54.270 - Do you have Display Settings next to it? 02:24:54.270 --> 02:24:55.620 - No, that's what's so odd. 02:24:55.620 --> 02:24:58.910 02:24:58.910 --> 02:24:59.570 We did earlier. 02:24:59.570 --> 02:25:02.220 02:25:02.220 --> 02:25:04.290 - Yeah, I don't have control of it. 02:25:04.290 --> 02:25:09.280 Central, come in, do you have control of his screen? 02:25:09.280 --> 02:25:09.940 - No. 02:25:09.940 --> 02:25:12.490 That would be within his computer 02:25:12.490 --> 02:25:14.770 that that needs to be changed. 02:25:14.770 --> 02:25:19.060 - Yeah, but I don't think he can see that. 02:25:19.060 --> 02:25:23.220 So how do we go from this to just presentation mode resume? 02:25:23.220 --> 02:25:31.800 02:25:31.800 --> 02:25:33.060 - Maybe-- oh, there we go. 02:25:33.060 --> 02:25:34.240 - Oh, there you go. 02:25:34.240 --> 02:25:36.480 - How's that? 02:25:36.480 --> 02:25:36.985 OK. 02:25:36.985 --> 02:25:37.560 My note-- 02:25:37.560 --> 02:25:37.740 [INTERPOSING VOICES] 02:25:37.740 --> 02:25:38.370 - --OK. 02:25:38.370 --> 02:25:41.130 So just to backtrack a moment, I can only 02:25:41.130 --> 02:25:44.160 suggest that IPA, there are multiple derivatives, 02:25:44.160 --> 02:25:48.960 and they can be previously, they were used as a biomarker, 02:25:48.960 --> 02:25:53.010 and that was dependent on evaluating blood iodine 02:25:53.010 --> 02:25:55.170 levels, which required a pre and post 02:25:55.170 --> 02:25:58.800 baiting sampling to look for that elevated iodine levels. 02:25:58.800 --> 02:26:03.190 In recent years, there have been chromatography 02:26:03.190 --> 02:26:05.140 and mass spectrometer methods developed, 02:26:05.140 --> 02:26:07.930 where we can actually detect these IPA 02:26:07.930 --> 02:26:11.130 derivatives independently in serum, 02:26:11.130 --> 02:26:13.420 individually unquantified. 02:26:13.420 --> 02:26:16.180 Ethyl IPA is, as you can see on the right side, 02:26:16.180 --> 02:26:20.110 it's the most common form and derivative of IPA 02:26:20.110 --> 02:26:21.640 that's out there right now. 02:26:21.640 --> 02:26:25.780 And in our case, we used ethyl as well as methyl. 02:26:25.780 --> 02:26:28.210 And we refined to little the process, 02:26:28.210 --> 02:26:30.700 the liquid chromatography mass spectrometer method 02:26:30.700 --> 02:26:35.080 for isolating and quantifying the concentration of IPA 02:26:35.080 --> 02:26:38.560 derivatives in moongoose serum. 02:26:38.560 --> 02:26:40.960 And then we took this into the laboratory in the field 02:26:40.960 --> 02:26:43.600 as well, and used it as a biological marker 02:26:43.600 --> 02:26:49.670 for oral rabies vaccination using placebo baits. 02:26:49.670 --> 02:26:51.320 So again, thanks to Stephen Volker 02:26:51.320 --> 02:26:55.880 for providing a layman's version of this extraction procedure, 02:26:55.880 --> 02:26:58.190 since I'm not a chemist, provided in language 02:26:58.190 --> 02:27:00.650 that I can understand, and hopefully 02:27:00.650 --> 02:27:03.200 share with you clearly. 02:27:03.200 --> 02:27:05.200 So this is a very selective and highly sensitive 02:27:05.200 --> 02:27:09.280 method to quantify multiple Iophenoxic acid 02:27:09.280 --> 02:27:11.090 derivatives in moongoose serum. 02:27:11.090 --> 02:27:12.970 So an overview of the extraction procedure 02:27:12.970 --> 02:27:16.030 was the serum was fortified with an internal standard. 02:27:16.030 --> 02:27:17.975 Now in our case, we used propyl-IPA 02:27:17.975 --> 02:27:20.600 because it was not something we were going to use in the field. 02:27:20.600 --> 02:27:21.940 If you're using an internal standard, 02:27:21.940 --> 02:27:23.315 you want to use something that is 02:27:23.315 --> 02:27:27.030 separate from what's going to be used in your actual baits. 02:27:27.030 --> 02:27:28.830 The serum was fortified and extracted 02:27:28.830 --> 02:27:31.110 with an acidifide acetonitrile. 02:27:31.110 --> 02:27:33.180 Following that, excess sodium chloride 02:27:33.180 --> 02:27:36.540 was added to the sample in order to phase-separate acetonitrile 02:27:36.540 --> 02:27:38.570 in the water. 02:27:38.570 --> 02:27:41.090 Then a portion of that acetonitrile phase 02:27:41.090 --> 02:27:44.660 was removed from the sample, and essentially dried 02:27:44.660 --> 02:27:47.960 using a flow of natural gas and clean water bath. 02:27:47.960 --> 02:27:52.400 This dry extract was then reconstituted with a 75% to 25% 02:27:52.400 --> 02:27:56.000 ratio of water and acetonitrile, and transferred 02:27:56.000 --> 02:27:58.820 to the high precision liquid chromatography 02:27:58.820 --> 02:28:03.021 vial for analysis using liquid chromatography and mass 02:28:03.021 --> 02:28:05.650 spectrometry. 02:28:05.650 --> 02:28:08.700 - So to look at the workflow of this procedure-- 02:28:08.700 --> 02:28:10.860 and this is the final procedure that we ended up 02:28:10.860 --> 02:28:15.180 using after several methods to try to refine everything. 02:28:15.180 --> 02:28:17.310 We used 50 microliters of mongoose serum. 02:28:17.310 --> 02:28:19.860 And then we applied 50 microliters 02:28:19.860 --> 02:28:23.685 of our internal standard-- in this case, it was propyl-IPA. 02:28:23.685 --> 02:28:26.610 We added 950 microliters of acetonitrile 02:28:26.610 --> 02:28:30.030 with 0.5% [? trichloroacetic ?] acid. 02:28:30.030 --> 02:28:32.760 The sample was then vortex mixed, and then 02:28:32.760 --> 02:28:34.320 that excess of sodium chloride-- 02:28:34.320 --> 02:28:37.500 150 milligrams-- was added to that sample. 02:28:37.500 --> 02:28:41.910 It was mixed again, centrifuged, and then add supernatant 02:28:41.910 --> 02:28:44.700 800 microliters of supernatant was transferred again 02:28:44.700 --> 02:28:46.440 to another vial. 02:28:46.440 --> 02:28:48.060 Following that, the solvents were 02:28:48.060 --> 02:28:50.730 removed through that drying process 02:28:50.730 --> 02:28:53.310 that I described with the liquid nitrogen in a heated water 02:28:53.310 --> 02:28:54.150 bath. 02:28:54.150 --> 02:28:56.520 And the sample was reconstituted. 02:28:56.520 --> 02:28:58.140 After this entire process, it was 02:28:58.140 --> 02:29:02.490 transferred to a vial for the chromatography and mass control 02:29:02.490 --> 02:29:04.140 analysis. 02:29:04.140 --> 02:29:05.970 So basically, the entire process is 02:29:05.970 --> 02:29:07.440 reduced to a tiny vial, which gets 02:29:07.440 --> 02:29:11.257 inserted into our quote, unquote, black box machine. 02:29:11.257 --> 02:29:13.590 And unfortunately, I don't have my notes in front of me. 02:29:13.590 --> 02:29:16.830 I can't tell you the exact model of this. 02:29:16.830 --> 02:29:20.190 And it was basically set to run overnight. 02:29:20.190 --> 02:29:23.010 We had multiple samples to run, and it was set 02:29:23.010 --> 02:29:24.390 to run for a 24-hour period. 02:29:24.390 --> 02:29:27.360 02:29:27.360 --> 02:29:32.310 This provides a nice example of the chromatogram that 02:29:32.310 --> 02:29:34.440 resulted from the analysis. 02:29:34.440 --> 02:29:37.770 And the analysis of individual samples 02:29:37.770 --> 02:29:40.680 actually took less than 2 and 1/2 minutes to perform. 02:29:40.680 --> 02:29:44.790 And on the y-axis, we can see the actual molecule count 02:29:44.790 --> 02:29:48.430 at each point in time for each derivative. 02:29:48.430 --> 02:29:51.660 So we have our methyl-IPA, and then our ethyl-IPA, 02:29:51.660 --> 02:29:53.580 and that our standard or reference standard, 02:29:53.580 --> 02:29:58.463 the propyl-IPA at approximately 1 and 1/2 minutes in. 02:29:58.463 --> 02:30:00.630 So this provides some really interesting information 02:30:00.630 --> 02:30:01.560 from a single sample. 02:30:01.560 --> 02:30:05.370 We can differentiate different derivatives 02:30:05.370 --> 02:30:08.310 of IPA, which became very useful, which I'll 02:30:08.310 --> 02:30:10.245 be describing in a few slides. 02:30:10.245 --> 02:30:13.380 02:30:13.380 --> 02:30:17.640 The method validation for this particular extraction 02:30:17.640 --> 02:30:19.740 and quantification is we have a detection 02:30:19.740 --> 02:30:22.080 limit for methyl and ethyl-IPA. 02:30:22.080 --> 02:30:25.710 For methyl, it was 0.01 micrograms per milliliter, 02:30:25.710 --> 02:30:29.730 and for ethyl, it was approximately 0.14 micrograms 02:30:29.730 --> 02:30:30.390 per milliliter. 02:30:30.390 --> 02:30:32.070 And that is just the detection limit. 02:30:32.070 --> 02:30:34.080 That is a presence or absence. 02:30:34.080 --> 02:30:36.570 Following that, we also had a quantitative limit 02:30:36.570 --> 02:30:39.090 where we could actually quantify the concentration 02:30:39.090 --> 02:30:42.540 of these derivatives in that serum sample. 02:30:42.540 --> 02:30:46.080 Quantitation limit for methyl being approximately micrograms 02:30:46.080 --> 02:30:46.980 per milliliter. 02:30:46.980 --> 02:30:51.410 Ethyl IPA was higher at almost 0.5. 02:30:51.410 --> 02:30:54.920 So validating this method, we took clean mongoose serum 02:30:54.920 --> 02:30:59.870 that had no known marker in it, and fortified them 02:30:59.870 --> 02:31:03.500 with three concentrations or three levels of methyl-IPA 02:31:03.500 --> 02:31:06.800 and ethyl-IPA here to run the analysis. 02:31:06.800 --> 02:31:11.300 And the accuracy range for this ranged anywhere from 95% up 02:31:11.300 --> 02:31:12.710 to 113%. 02:31:12.710 --> 02:31:16.310 And that's very common in this type of analysis 02:31:16.310 --> 02:31:20.390 for that accuracy range to be below and above 100% 02:31:20.390 --> 02:31:23.210 with the mean being ideally around 100%. 02:31:23.210 --> 02:31:24.920 And the coefficient of variance, which 02:31:24.920 --> 02:31:28.550 is simply the standard deviation above mean was less than 5%. 02:31:28.550 --> 02:31:32.570 And that provides and the standardization of variance 02:31:32.570 --> 02:31:34.310 regardless of the accuracy. 02:31:34.310 --> 02:31:38.360 So it's suffice to say that this process was extremely 02:31:38.360 --> 02:31:43.280 accurate in quantifying the concentration of IPA residues 02:31:43.280 --> 02:31:47.930 in mongoose serum 02:31:47.930 --> 02:31:49.850 We conducted a series of laboratory trials 02:31:49.850 --> 02:31:54.110 where we offered captive mongooses, 02:31:54.110 --> 02:31:56.900 various concentrations of both ethyl and methyl 02:31:56.900 --> 02:32:01.560 iophenoxic acid in a placebo or rabies vaccine bait. 02:32:01.560 --> 02:32:03.870 A bait is approximately 5 centimeters long, 02:32:03.870 --> 02:32:05.150 weighs about 2 to 3 grams. 02:32:05.150 --> 02:32:09.230 It Consists of a foil blister pack, 02:32:09.230 --> 02:32:11.660 in which there is a liquid, which in the field 02:32:11.660 --> 02:32:13.160 would consist of the vaccine. 02:32:13.160 --> 02:32:14.660 And an external bait matrix. 02:32:14.660 --> 02:32:18.270 In this case, it's an egg flavored external bait matrix. 02:32:18.270 --> 02:32:20.510 So in the laboratory we offered these baits 02:32:20.510 --> 02:32:23.720 to captive mongooses and then collected a serum sample 02:32:23.720 --> 02:32:27.350 prior to bait offering, as well as on day one following bait 02:32:27.350 --> 02:32:28.160 offering. 02:32:28.160 --> 02:32:31.587 And then weekly for between four and eight weeks. 02:32:31.587 --> 02:32:33.170 And you can see more details about how 02:32:33.170 --> 02:32:36.470 these concentrations that are presented in the paper. 02:32:36.470 --> 02:32:42.300 You can see how they were explained and calculated. 02:32:42.300 --> 02:32:44.310 Looking at the ethyl IPA concentrations 02:32:44.310 --> 02:32:46.590 over approximately an eight week period, 02:32:46.590 --> 02:32:50.560 as you can see from day 0 there was no concentration, 02:32:50.560 --> 02:32:52.600 which is not unusual. 02:32:52.600 --> 02:32:54.270 That's what we expected to see. 02:32:54.270 --> 02:32:56.520 But what was really unexpected is 02:32:56.520 --> 02:33:01.860 looking at the longevity of this marker in serum samples. 02:33:01.860 --> 02:33:03.840 Now, this is highly variable across species. 02:33:03.840 --> 02:33:07.260 In some species it's metabolized within a few days 02:33:07.260 --> 02:33:09.540 and I believe in the dog it can be up to a year. 02:33:09.540 --> 02:33:12.330 And we were very surprised to see how long this persisted, 02:33:12.330 --> 02:33:15.743 this ethyl IPA marker persisted in mongoose samples. 02:33:15.743 --> 02:33:17.160 And look at the trend, there's not 02:33:17.160 --> 02:33:19.680 much of a decrease over an eight week period 02:33:19.680 --> 02:33:22.830 regardless of the concentration and the method 02:33:22.830 --> 02:33:24.960 by which that product was delivered. 02:33:24.960 --> 02:33:27.840 Either it was in-cooperated into the external bait matrix 02:33:27.840 --> 02:33:30.330 or within the internal blister pattern. 02:33:30.330 --> 02:33:32.970 02:33:32.970 --> 02:33:34.800 So seeing that longevity, we also 02:33:34.800 --> 02:33:37.590 evaluated methyl IPA to see if it was consistent. 02:33:37.590 --> 02:33:41.580 And we sampled for four weeks for the methyl IPA. 02:33:41.580 --> 02:33:44.670 And you can see from the figure, that there was also very little 02:33:44.670 --> 02:33:46.050 degradation over time. 02:33:46.050 --> 02:33:48.940 And the curves are pretty flat going across 02:33:48.940 --> 02:33:50.820 for that four-week period. 02:33:50.820 --> 02:33:53.430 This had a kind of unintended benefits 02:33:53.430 --> 02:33:56.730 when we conducted our field operations. 02:33:56.730 --> 02:33:59.040 Typically, what we do when we conduct 02:33:59.040 --> 02:34:00.990 more rabies vaccination in the field 02:34:00.990 --> 02:34:03.840 is collect a blood sample, a serum 02:34:03.840 --> 02:34:06.960 sample approximately five to six weeks following vaccination 02:34:06.960 --> 02:34:09.760 to look for rabies virus neutralizing antibodies, 02:34:09.760 --> 02:34:12.560 as well as a biomarker. 02:34:12.560 --> 02:34:15.630 And in this case, we were hoping to use iophenoxic acid 02:34:15.630 --> 02:34:18.600 as that biomarker. 02:34:18.600 --> 02:34:21.120 We conducted a field application, a field trial 02:34:21.120 --> 02:34:22.025 with a placebo bait. 02:34:22.025 --> 02:34:23.400 There was no vaccine in this bait 02:34:23.400 --> 02:34:29.940 but there was 2.8 milligrams of ethyl IPA in the fall of 2016. 02:34:29.940 --> 02:34:32.760 And then we used methyl IPA for a follow up trial 02:34:32.760 --> 02:34:35.400 in the spring of 2017. 02:34:35.400 --> 02:34:37.320 Baits were distributed in South West Puerto 02:34:37.320 --> 02:34:40.950 Rico in a subtropical dry forest habitat at an application 02:34:40.950 --> 02:34:44.530 rate of 200 baits per kilometer square. 02:34:44.530 --> 02:34:46.780 We gave them mongooses about five days following 02:34:46.780 --> 02:34:49.000 bating to find the baits and consume 02:34:49.000 --> 02:34:50.980 them, hopefully, consume them. 02:34:50.980 --> 02:34:53.110 And that we conducted trapping daily 02:34:53.110 --> 02:34:55.270 for 10 consecutive days after that, 02:34:55.270 --> 02:34:57.220 taking a blood sample from each animal 02:34:57.220 --> 02:34:58.560 that we captured every animal. 02:34:58.560 --> 02:35:02.040 02:35:02.040 --> 02:35:05.700 Looking at these field results from the fall of 2016, 02:35:05.700 --> 02:35:10.110 we sampled 87 mongooses across all our study sites. 02:35:10.110 --> 02:35:14.580 55 of those or 63% of those animals that we sampled, 02:35:14.580 --> 02:35:17.700 were positive for ethyl iophenoxic acid, 02:35:17.700 --> 02:35:21.360 suggesting that they had in fact consumed baits. 02:35:21.360 --> 02:35:26.790 In the spring, we captured 123 animals and sampled them. 02:35:26.790 --> 02:35:30.180 84 of those were positive for methyl IPA. 02:35:30.180 --> 02:35:32.590 It's about 68%. 02:35:32.590 --> 02:35:36.180 Now, if we pull these samples and we pull back and look at it 02:35:36.180 --> 02:35:40.710 from a larger viewpoint and account for recaptures 02:35:40.710 --> 02:35:43.710 between seasons, we sampled a total 02:35:43.710 --> 02:35:46.530 of 180 individual animals, unique animals. 02:35:46.530 --> 02:35:49.680 And 134 of those across those two seasons 02:35:49.680 --> 02:35:52.560 were positive for either one or both 02:35:52.560 --> 02:35:55.170 of those iophenoxic acid revenues. 02:35:55.170 --> 02:35:58.080 And that's a portion of approximately 74% 02:35:58.080 --> 02:35:59.893 bait consumption. 02:35:59.893 --> 02:36:01.560 And this is where it's very interesting. 02:36:01.560 --> 02:36:05.070 The longevity of that ethyl IPA worked to our advantage. 02:36:05.070 --> 02:36:09.610 Because we were able to in the spring of 2017, 02:36:09.610 --> 02:36:12.190 capture animals and find them positive 02:36:12.190 --> 02:36:15.190 for ethyl iophenoxic acid that we had not 02:36:15.190 --> 02:36:18.190 captured in the fall of 2016. 02:36:18.190 --> 02:36:22.030 So that helped bolster our sample size for that season. 02:36:22.030 --> 02:36:24.280 It also allowed us to determine that there 02:36:24.280 --> 02:36:26.170 was a certain proportion of animals 02:36:26.170 --> 02:36:28.330 that we captured in the spring that 02:36:28.330 --> 02:36:29.830 were positive for both biomarkers, 02:36:29.830 --> 02:36:34.190 suggesting they had consumed baits during both time periods. 02:36:34.190 --> 02:36:38.440 Looking at the proportion of bait consumption from the study 02:36:38.440 --> 02:36:40.930 is very encouraging when we consider 02:36:40.930 --> 02:36:42.820 that some of the literature in some species 02:36:42.820 --> 02:36:48.250 suggest that a vaccination rate of 60% to 80% to 90% 02:36:48.250 --> 02:36:51.110 annually may be required in order to interrupt 02:36:51.110 --> 02:36:56.030 virus transmission in certain species. 02:36:56.030 --> 02:36:59.200 So this is a new technique for the species, certainly not 02:36:59.200 --> 02:37:01.430 a new technique. iophenoxic acid, as I mentioned, 02:37:01.430 --> 02:37:05.110 has been used as a biomarker for a variety of reasons 02:37:05.110 --> 02:37:09.020 in the literature. 02:37:09.020 --> 02:37:12.870 Just to wrap up and to conclude that this study, 02:37:12.870 --> 02:37:15.530 we were able to refine our method 02:37:15.530 --> 02:37:19.040 to evaluate the IPA concentration 02:37:19.040 --> 02:37:22.370 and be able to quantify it in mongoose serum. 02:37:22.370 --> 02:37:26.030 And this is also important for use as a biological marker 02:37:26.030 --> 02:37:30.650 to estimate bait consumption in particular in areas where there 02:37:30.650 --> 02:37:35.570 is a natural rabies virus exposure background not 02:37:35.570 --> 02:37:38.570 from rabies virus exposure, where you have naturally 02:37:38.570 --> 02:37:42.860 occurring rabies virus neutralizing antibodies making 02:37:42.860 --> 02:37:45.360 the evaluation of these antibodies post vaccination 02:37:45.360 --> 02:37:47.960 very difficult because it's very difficult to determine 02:37:47.960 --> 02:37:51.200 whether those antibodies came from the natural exposure 02:37:51.200 --> 02:37:53.750 or from vaccination. 02:37:53.750 --> 02:37:56.750 So IPA has proved to be a useful biomarker 02:37:56.750 --> 02:38:01.040 to estimate this consumption in free ranging mongooses 02:38:01.040 --> 02:38:02.830 in our trials in Puerto Rico. 02:38:02.830 --> 02:38:05.952 02:38:05.952 --> 02:38:09.570 And with that, I'll open it up to any questions. 02:38:09.570 --> 02:38:11.390 I apologize for the technical difficulties 02:38:11.390 --> 02:38:13.110 here at the beginning. 02:38:13.110 --> 02:38:14.810 - Thank you Are. 02:38:14.810 --> 02:38:20.360 One quick question, how easy or difficult 02:38:20.360 --> 02:38:23.840 is it to perform this analysis, I'm 02:38:23.840 --> 02:38:27.320 thinking in particular for some of our colleagues 02:38:27.320 --> 02:38:33.650 around the globe as to cost, experience, technicality, et 02:38:33.650 --> 02:38:35.600 cetera? 02:38:35.600 --> 02:38:40.800 - The extraction and analysis is fairly straightforward. 02:38:40.800 --> 02:38:46.220 The work flow is a fairly standard protein extraction 02:38:46.220 --> 02:38:49.100 analysis that's been slightly modified using the salt 02:38:49.100 --> 02:38:53.180 and acid additions in order to be able to concentrate 02:38:53.180 --> 02:38:54.290 the iophenoxic acid. 02:38:54.290 --> 02:38:57.140 02:38:57.140 --> 02:39:01.790 It would be costly to farm it out to an external laboratory 02:39:01.790 --> 02:39:05.930 but it can be done in-house with a little bit of training 02:39:05.930 --> 02:39:07.460 and with the refined technique. 02:39:07.460 --> 02:39:10.950 And we've done the iterative process to refine it. 02:39:10.950 --> 02:39:12.660 I think it's fairly straightforward. 02:39:12.660 --> 02:39:14.840 It's just a little bit time-consuming 02:39:14.840 --> 02:39:17.670 to get the samples prepared. 02:39:17.670 --> 02:39:21.900 - And based upon the use of biomarkers, 02:39:21.900 --> 02:39:25.440 either iophenoxic acid or others together 02:39:25.440 --> 02:39:31.050 with say, enhanced surveillance, how would one 02:39:31.050 --> 02:39:36.830 go about determining how much vaccine would be needed 02:39:36.830 --> 02:39:40.370 per area based upon these insights, 02:39:40.370 --> 02:39:45.815 whether it's a mongoose, a fox, a raccoon, or a dog? 02:39:45.815 --> 02:39:49.870 02:39:49.870 --> 02:39:52.410 - So how much-- 02:39:52.410 --> 02:39:54.070 if you could rephrase the question to-- 02:39:54.070 --> 02:39:54.570 - Sure. 02:39:54.570 --> 02:40:01.710 So in other words, you find out that 65% of your mongoose 02:40:01.710 --> 02:40:05.520 have evidence of consuming a bait based on iophenoxic acid. 02:40:05.520 --> 02:40:09.750 Or based upon tetracycline, that we find out 02:40:09.750 --> 02:40:13.920 that 70% of our foxes are eating a bait. 02:40:13.920 --> 02:40:16.440 Or based upon enhanced surveillance, 02:40:16.440 --> 02:40:22.230 we find out that X proportion of raccoons, or skunks, or foxes, 02:40:22.230 --> 02:40:25.110 or dogs, are positive. 02:40:25.110 --> 02:40:29.280 How would you combine these laboratory techniques 02:40:29.280 --> 02:40:33.930 based on biomarkers, serology, enhanced surveillance. 02:40:33.930 --> 02:40:36.480 And I'm opening it up to all the participants 02:40:36.480 --> 02:40:37.710 based on the question. 02:40:37.710 --> 02:40:40.800 How would you use these laboratory techniques 02:40:40.800 --> 02:40:45.420 to help determine what your bait density might be for a given 02:40:45.420 --> 02:40:48.500 species in a given area? 02:40:48.500 --> 02:40:49.000 - Right. 02:40:49.000 --> 02:40:52.500 So one of the ways we determined the actual application rate 02:40:52.500 --> 02:40:55.000 was to look at the population density of the target species. 02:40:55.000 --> 02:40:58.240 And that helps drive as well as our RV 02:40:58.240 --> 02:41:02.590 in raccoon, that helps drive the application rate looking 02:41:02.590 --> 02:41:06.640 at that initial ecological information. 02:41:06.640 --> 02:41:09.090 This is particularly useful, iophenoxic acid 02:41:09.090 --> 02:41:12.760 is particularly useful in areas, somewhere like Puerto Rico 02:41:12.760 --> 02:41:15.880 where there is a naive population. 02:41:15.880 --> 02:41:17.980 But there is a level of background exposure, 02:41:17.980 --> 02:41:23.150 so you would have to conduct a free vaccination 02:41:23.150 --> 02:41:26.990 survey to look at what that background is just like you 02:41:26.990 --> 02:41:31.940 would do in ORV application in the lower 48 states 02:41:31.940 --> 02:41:37.280 as well as a follow up sampling to look at the serum conversion 02:41:37.280 --> 02:41:38.940 rate. 02:41:38.940 --> 02:41:42.510 But you also would look for that biomarker 02:41:42.510 --> 02:41:47.670 to help distinguish animals that may have been previously 02:41:47.670 --> 02:41:51.420 exposed, either through previous vaccination in the lower 02:41:51.420 --> 02:41:57.210 48 or through natural virus exposure in naive places 02:41:57.210 --> 02:42:00.740 like Puerto Rico where there is a background level of serology, 02:42:00.740 --> 02:42:01.710 is that help? 02:42:01.710 --> 02:42:02.210 - Sure. 02:42:02.210 --> 02:42:06.240 So based on your experience to date, what do you think 02:42:06.240 --> 02:42:11.100 might be a reasonable density of baits for distribution 02:42:11.100 --> 02:42:12.900 in the prevention and control of rabies 02:42:12.900 --> 02:42:18.180 and mongoose in Puerto Rico or elsewhere in the Caribbean? 02:42:18.180 --> 02:42:21.750 - Right now we're starting with 200 baits per square kilometer. 02:42:21.750 --> 02:42:25.920 That proved to be at this particular study site, a very 02:42:25.920 --> 02:42:27.780 good application rate. 02:42:27.780 --> 02:42:32.520 We do have plans to conduct a follow up trials 02:42:32.520 --> 02:42:34.530 in slightly different habitats. 02:42:34.530 --> 02:42:38.970 And that, excuse me, that bait application rate 02:42:38.970 --> 02:42:42.870 may have to vary depending on the habitat type, which 02:42:42.870 --> 02:42:46.480 can drive mongoose population densities. 02:42:46.480 --> 02:42:48.720 So in areas where you have very high densities 02:42:48.720 --> 02:42:52.120 you may have to increase that application 02:42:52.120 --> 02:42:55.840 rate because the non-target species that may consume them. 02:42:55.840 --> 02:42:59.050 In Puerto Rico those tend to be predominantly 02:42:59.050 --> 02:43:01.960 nocturnal rodents i.e. rats and/or mice 02:43:01.960 --> 02:43:03.430 that may consume those bats. 02:43:03.430 --> 02:43:05.300 That has to be taken into consideration. 02:43:05.300 --> 02:43:06.700 So it may vary by habitat. 02:43:06.700 --> 02:43:08.740 But at least to start with our research 02:43:08.740 --> 02:43:10.600 suggests so far that 200 baits per square 02:43:10.600 --> 02:43:13.420 kilometer maybe an adequate application 02:43:13.420 --> 02:43:15.370 rate for the species. 02:43:15.370 --> 02:43:18.470 - Any guesstimates on when oral vaccination 02:43:18.470 --> 02:43:24.700 studies, trials with vaccine may actually begin in Puerto Rico? 02:43:24.700 --> 02:43:29.380 - We are planning on conducting a pilot trial 02:43:29.380 --> 02:43:34.370 in a limited area in North Central Puerto Rico most likely 02:43:34.370 --> 02:43:36.920 in the spring of 2022. 02:43:36.920 --> 02:43:39.980 We had hoped that we might be able to pursue it this fall, 02:43:39.980 --> 02:43:44.040 but given the COVID pandemic and a lot of other circumstances, 02:43:44.040 --> 02:43:44.790 it may be delayed. 02:43:44.790 --> 02:43:50.510 But we do have plans to conduct that trial in a limited area 02:43:50.510 --> 02:43:55.910 in next spring using iophenoxic acid as a biomarker 02:43:55.910 --> 02:43:58.760 to again, distinguish between that natural exposure 02:43:58.760 --> 02:44:00.630 and vaccine uptake. 02:44:00.630 --> 02:44:02.300 So we're very excited about it. 02:44:02.300 --> 02:44:04.760 It's something that hasn't yet been done 02:44:04.760 --> 02:44:09.170 and I'm very proud to have been part of this team conducting 02:44:09.170 --> 02:44:10.520 this research. 02:44:10.520 --> 02:44:13.730 - Great, and I'll broadly open this up 02:44:13.730 --> 02:44:20.050 to all of our participants beyond free ranging 02:44:20.050 --> 02:44:26.180 carnivores from dogs, foxes, skunks, raccoons, raccoon dogs, 02:44:26.180 --> 02:44:31.070 ferret badgers, et cetera. 02:44:31.070 --> 02:44:34.070 And you probably have seen the literature, her discussions, 02:44:34.070 --> 02:44:35.960 what do you think about the concept 02:44:35.960 --> 02:44:39.170 of oral vaccination of bats? 02:44:39.170 --> 02:44:45.510 02:44:45.510 --> 02:44:49.770 - And Are or Erin or Noel or anybody 02:44:49.770 --> 02:44:51.718 is free to take that up. 02:44:51.718 --> 02:44:53.260 You look like you have some thoughts, 02:44:53.260 --> 02:44:55.218 but you don't want to say them. 02:44:55.218 --> 02:44:56.146 - I do. 02:44:56.146 --> 02:44:58.980 Do you remember-- 02:44:58.980 --> 02:45:00.105 - Go ahead, Noel. 02:45:00.105 --> 02:45:05.010 - Do you remember that we published a paper on that let's 02:45:05.010 --> 02:45:07.140 say 20 years ago. 02:45:07.140 --> 02:45:12.610 It was with vampire bats and together with Alvaro in Mexico. 02:45:12.610 --> 02:45:13.770 - Yes. 02:45:13.770 --> 02:45:15.270 - Yeah it was-- 02:45:15.270 --> 02:45:18.870 it was done in the lab and we captured bats. 02:45:18.870 --> 02:45:23.790 We gave them, so I think it was the Merial vaccine 02:45:23.790 --> 02:45:25.510 if I remember correctly. 02:45:25.510 --> 02:45:26.010 And-- 02:45:26.010 --> 02:45:26.593 - I think it-- 02:45:26.593 --> 02:45:28.890 yes, it was vaccinia recombinant, yes. 02:45:28.890 --> 02:45:30.630 - Yeah, it was the vaccinia recombinant. 02:45:30.630 --> 02:45:37.190 And they got some positive antibodies and that has 02:45:37.190 --> 02:45:40.220 been published and that's it. 02:45:40.220 --> 02:45:42.470 I remember to have given a talk on this 02:45:42.470 --> 02:45:46.460 just for joking, a talk of this in class, America. 02:45:46.460 --> 02:45:49.010 And in front of me was a specialist 02:45:49.010 --> 02:45:53.000 of a bat rabies from Argentina. 02:45:53.000 --> 02:45:56.780 And he was looking at me saying that you are totally crazy. 02:45:56.780 --> 02:46:01.490 We know to do with a bat rabies or vampire 02:46:01.490 --> 02:46:04.310 rabid in South America. 02:46:04.310 --> 02:46:07.730 We are just vaccinating cows around 02:46:07.730 --> 02:46:11.520 and we are just staying the same like that. 02:46:11.520 --> 02:46:14.600 And after a while there is a certain proportion 02:46:14.600 --> 02:46:18.800 of the cave, which is dying. 02:46:18.800 --> 02:46:21.950 And the rest is no more rabid for 10 years. 02:46:21.950 --> 02:46:25.580 This is what he replied like a sort 02:46:25.580 --> 02:46:31.040 of self vaccination of bats. 02:46:31.040 --> 02:46:32.990 So this is where we stopped indeed. 02:46:32.990 --> 02:46:34.790 We didn't go ahead. 02:46:34.790 --> 02:46:39.060 But it was money given by the European community. 02:46:39.060 --> 02:46:39.560 - Yes. 02:46:39.560 --> 02:46:42.470 02:46:42.470 --> 02:46:45.470 It seems how what's old is new. 02:46:45.470 --> 02:46:49.790 And if any of our colleagues from Europe or Latin America 02:46:49.790 --> 02:46:54.560 are on, particularly one of our colleagues Daniel Straker, 02:46:54.560 --> 02:46:58.730 this has now become novel again to try to think 02:46:58.730 --> 02:47:01.260 about vaccines for vampires. 02:47:01.260 --> 02:47:06.800 But not only as we did, meaning the passive transferable, 02:47:06.800 --> 02:47:11.030 but now they're thinking about transmissible vaccines. 02:47:11.030 --> 02:47:17.270 And so trying to find unique viruses only hopefully, 02:47:17.270 --> 02:47:20.810 secluded in vampires and putting in your genes 02:47:20.810 --> 02:47:24.740 and infecting the vampires and having infectious clones 02:47:24.740 --> 02:47:27.650 go from vampire to vampire to vampire. 02:47:27.650 --> 02:47:30.390 What do we think about transmissible vaccines 02:47:30.390 --> 02:47:30.890 for bats? 02:47:30.890 --> 02:47:34.928 02:47:34.928 --> 02:47:36.412 - Who is thinking? 02:47:36.412 --> 02:47:39.130 [LAUGHTER] 02:47:39.130 --> 02:47:41.070 - Erin, Are what do you think? 02:47:41.070 --> 02:47:43.730 02:47:43.730 --> 02:47:45.830 Any of the other panelists? 02:47:45.830 --> 02:47:48.380 I mean, if we're going to think about transmissible vaccines 02:47:48.380 --> 02:47:51.800 for vampires or other bats for rabies, 02:47:51.800 --> 02:47:54.500 lyssas, other diseases, why aren't we 02:47:54.500 --> 02:47:59.550 thinking about transmissible vaccines, viruses 02:47:59.550 --> 02:48:01.530 for carnivores? 02:48:01.530 --> 02:48:03.210 It would certainly make it easier 02:48:03.210 --> 02:48:09.252 than the 200 baits per square kilometer for a mongoose? 02:48:09.252 --> 02:48:10.960 - One thing that's interesting about this 02:48:10.960 --> 02:48:13.390 is, and I'm not a bat guy, so I'm going 02:48:13.390 --> 02:48:14.600 to have to punt on that one. 02:48:14.600 --> 02:48:18.558 I'm very, very new to anything bat related. 02:48:18.558 --> 02:48:20.350 One thing that is interesting that we found 02:48:20.350 --> 02:48:25.300 is with this background exposure in mongooses 02:48:25.300 --> 02:48:28.790 there is a certain level of having 02:48:28.790 --> 02:48:32.060 positive animals in some areas and that can also help 02:48:32.060 --> 02:48:34.550 guide that vaccination process. 02:48:34.550 --> 02:48:37.580 Do you decide that areas that have a high background serology 02:48:37.580 --> 02:48:40.970 are a low priority for vaccination 02:48:40.970 --> 02:48:44.420 because there may be some natural immunity, 02:48:44.420 --> 02:48:48.230 or is it a higher priority to kind of bump up 02:48:48.230 --> 02:48:52.010 what's already there in order to cross 02:48:52.010 --> 02:48:58.452 that threshold required to interrupt a virus transmission? 02:48:58.452 --> 02:48:59.910 And those are some of the questions 02:48:59.910 --> 02:49:02.340 that we're still working with answering. 02:49:02.340 --> 02:49:05.410 02:49:05.410 --> 02:49:05.950 - Yes. 02:49:05.950 --> 02:49:11.510 And I think this issue of transmissibles 02:49:11.510 --> 02:49:17.730 is worth days, if not weeks of more discussion. 02:49:17.730 --> 02:49:22.890 And I am wondering what either any of our other panel members 02:49:22.890 --> 02:49:27.720 may think about that topic or do they have any questions for any 02:49:27.720 --> 02:49:31.600 of the presenters today. 02:49:31.600 --> 02:49:35.920 It's open to our colleagues from Asia. 02:49:35.920 --> 02:49:38.680 I know it's probably getting kind of late for you. 02:49:38.680 --> 02:49:43.810 If you have any questions or comments or New York, 02:49:43.810 --> 02:49:46.980 or Luka, or-- 02:49:46.980 --> 02:49:48.570 yes, Noel. 02:49:48.570 --> 02:49:51.930 - If I remember correctly, behind this story 02:49:51.930 --> 02:49:55.950 of vampire bats that we tried to vaccinate orally, 02:49:55.950 --> 02:50:00.690 the idea behind was to sort of take 02:50:00.690 --> 02:50:04.320 a certain number of those bats and to put them 02:50:04.320 --> 02:50:08.640 on the body, the vaccine, I mean the recombinant vaccine 02:50:08.640 --> 02:50:13.920 in order to send them back in the cave for grooming 02:50:13.920 --> 02:50:15.130 each other. 02:50:15.130 --> 02:50:18.210 And so in the sense of what you were mentioning before, 02:50:18.210 --> 02:50:21.820 allowing them to inter vaccinate, let's say, 02:50:21.820 --> 02:50:23.890 to transmit the vaccination. 02:50:23.890 --> 02:50:28.860 So it was the basis of the idea at the time also. 02:50:28.860 --> 02:50:30.720 Maybe we have to rethink. 02:50:30.720 --> 02:50:37.440 If I can remember the things you knew two years ago when 02:50:37.440 --> 02:50:40.875 we were using a vaccine for humans, 02:50:40.875 --> 02:50:42.930 a recombinant vaccine the same way 02:50:42.930 --> 02:50:46.540 that we are using now in COVID didn't exist. 02:50:46.540 --> 02:50:51.000 Some cases were existing in vaccinating 02:50:51.000 --> 02:50:54.510 for example for rabies, dogs with DNA vaccine, 02:50:54.510 --> 02:50:58.130 but apart from that this new technology didn't exist. 02:50:58.130 --> 02:51:01.440 And we were staying at the inactivated or attenuated 02:51:01.440 --> 02:51:02.400 vaccine. 02:51:02.400 --> 02:51:08.400 And you have seen how a pandemic and before a pandemic, how 02:51:08.400 --> 02:51:12.120 Ebola for example as the proper use of VSV 02:51:12.120 --> 02:51:15.730 for human use or other new vaccine for human use. 02:51:15.730 --> 02:51:19.980 So maybe as you said, yes, it was old things. 02:51:19.980 --> 02:51:24.720 But we have to see old things with new eyes that sometimes 02:51:24.720 --> 02:51:28.240 help us to go ahead more rapidly. 02:51:28.240 --> 02:51:33.180 - So given that point and given how incredibly rapidly 02:51:33.180 --> 02:51:35.850 the world responded to the current pandemic 02:51:35.850 --> 02:51:40.440 with new vaccine applications, although some of us 02:51:40.440 --> 02:51:42.540 are not crazy about having to store them 02:51:42.540 --> 02:51:47.220 at minus 20 degrees, which of the techniques, technologies, 02:51:47.220 --> 02:51:52.020 both from the New vaccines as well as the ones to come, 02:51:52.020 --> 02:51:55.620 do you have any particular excitement as to how they 02:51:55.620 --> 02:51:56.955 might be applied for rabies? 02:51:56.955 --> 02:52:01.640 02:52:01.640 --> 02:52:02.765 - Move along the audience. 02:52:02.765 --> 02:52:05.570 02:52:05.570 --> 02:52:06.530 - Go ahead. 02:52:06.530 --> 02:52:09.590 You have the com, what do you think? 02:52:09.590 --> 02:52:11.510 Or do we really need them? 02:52:11.510 --> 02:52:14.390 I mean, given what we've already been able to accomplish. 02:52:14.390 --> 02:52:17.210 All developed countries have eliminated 02:52:17.210 --> 02:52:20.300 dog rabies, developing countries, if we look 02:52:20.300 --> 02:52:24.440 at the Americas look at the case of Mexico, have eliminated 02:52:24.440 --> 02:52:28.280 not only human rabies transmitted by dogs but dog 02:52:28.280 --> 02:52:29.540 to dog transmission. 02:52:29.540 --> 02:52:37.320 So it is obviously attainable in not just the ultra wealthy 02:52:37.320 --> 02:52:38.070 countries. 02:52:38.070 --> 02:52:40.860 And I'm saying this for any of our participants 02:52:40.860 --> 02:52:41.640 who are still on. 02:52:41.640 --> 02:52:44.820 I know we had quite a few from the Indian subcontinent, 02:52:44.820 --> 02:52:48.820 from Pakistan, from Africa, et cetera. 02:52:48.820 --> 02:52:52.000 Do we need new vaccines for rabies 02:52:52.000 --> 02:52:54.850 given what we've accomplished so far in either humans 02:52:54.850 --> 02:52:55.990 or domestic animals? 02:52:55.990 --> 02:52:58.580 Look at the new WHO recommendations. 02:52:58.580 --> 02:53:04.210 We don't need to have daily inoculations intracutaneously 02:53:04.210 --> 02:53:05.980 with nerve tissue based vaccines. 02:53:05.980 --> 02:53:09.700 We can use modern cell cultured dose bearing 02:53:09.700 --> 02:53:12.160 and do post exposure in a week. 02:53:12.160 --> 02:53:16.120 Do we really need new vaccine-- or is this just an excuse? 02:53:16.120 --> 02:53:20.110 Is this an excuse for us to drag our feet or can 02:53:20.110 --> 02:53:24.510 we accomplish the tools with what we have now? 02:53:24.510 --> 02:53:26.890 - In my opinion there are two things. 02:53:26.890 --> 02:53:31.230 Never stop thinking, dreaming, proposing new things. 02:53:31.230 --> 02:53:33.630 So this is one point and we are scientists 02:53:33.630 --> 02:53:35.380 and we should do that. 02:53:35.380 --> 02:53:38.730 The second is that, I'm in Africa now, I'm in Guinea. 02:53:38.730 --> 02:53:42.090 In Guinea we did the sort of overview of what 02:53:42.090 --> 02:53:43.890 was available, not available. 02:53:43.890 --> 02:53:46.170 The vaccine of course, it was it was 02:53:46.170 --> 02:53:49.290 a limited amount of vaccine, we are trying to improve that. 02:53:49.290 --> 02:53:52.810 I'm talking only about human vaccine for the moment, 02:53:52.810 --> 02:53:55.320 I'm not talking about dog vaccine. 02:53:55.320 --> 02:53:58.780 If we are looking to the WHO recommendation, 02:53:58.780 --> 02:54:02.860 WHO recommendation is 95% of the case. 02:54:02.860 --> 02:54:05.160 This is a bleeding bite, so you have 02:54:05.160 --> 02:54:09.630 to make immunoglobulin and a vaccine. 02:54:09.630 --> 02:54:14.580 There is not a single drop or immunoglobulin in Guinea. 02:54:14.580 --> 02:54:17.100 When someone from the embassy recently was bitten, 02:54:17.100 --> 02:54:20.340 I was forced to go to Morocco to find some 02:54:20.340 --> 02:54:22.330 in order to give them. 02:54:22.330 --> 02:54:25.080 So we are two sets, two ways. 02:54:25.080 --> 02:54:29.910 One the scientists are dreaming new, this is our job. 02:54:29.910 --> 02:54:34.050 And after there is a recommendation, and sometimes 02:54:34.050 --> 02:54:36.900 we have the impression that the recommendation are followed, 02:54:36.900 --> 02:54:38.830 while in many countries. 02:54:38.830 --> 02:54:44.220 They are not followed simply because the I mean 02:54:44.220 --> 02:54:46.170 monoclonal antibodies you remember, 02:54:46.170 --> 02:54:49.200 we were saying that 10 years of looking at monoclonal antibody 02:54:49.200 --> 02:54:53.640 to replace RIG or eRIG. 02:54:53.640 --> 02:54:59.045 There are still waiting on let's say developed country. 02:54:59.045 --> 02:55:00.420 But a lot of developing countries 02:55:00.420 --> 02:55:02.380 have not reached this level. 02:55:02.380 --> 02:55:05.040 So we have both to complete what is already done 02:55:05.040 --> 02:55:10.030 and what is recommended and to dream for future. 02:55:10.030 --> 02:55:10.830 - Yes. 02:55:10.830 --> 02:55:13.860 And on that topic of human and the dilemma 02:55:13.860 --> 02:55:17.370 of post exposure in RIGs or monoclonals, 02:55:17.370 --> 02:55:22.360 we have that disparity between a small fraction of the world 02:55:22.360 --> 02:55:28.330 uses RIG, the majority of the world uses vaccine only. 02:55:28.330 --> 02:55:33.940 And so we've either been incredibly fortunate and lucky 02:55:33.940 --> 02:55:36.460 or incredibly blurred in our surveillance. 02:55:36.460 --> 02:55:39.010 Because we can't explain. 02:55:39.010 --> 02:55:42.430 We don't have massive post exposure failures 02:55:42.430 --> 02:55:45.820 even though the majority of our patients 02:55:45.820 --> 02:55:48.760 out there are only receiving vaccines. 02:55:48.760 --> 02:55:52.510 And although I highly agree with you never stop dreaming, 02:55:52.510 --> 02:55:56.470 look where we've gone from since past Doris time. 02:55:56.470 --> 02:56:01.330 I also wonder from this new wave of both diagnostics 02:56:01.330 --> 02:56:04.450 and vaccines because of COVID, will we 02:56:04.450 --> 02:56:07.390 become even more neglected and not 02:56:07.390 --> 02:56:10.960 have the ability of even use the proven tools that we 02:56:10.960 --> 02:56:15.550 have because of supply chain interruptions. 02:56:15.550 --> 02:56:18.580 Because of issues of stay at home. 02:56:18.580 --> 02:56:22.840 Because of problems of not having any money 02:56:22.840 --> 02:56:29.080 left over for everyday things once this particular pandemic 02:56:29.080 --> 02:56:30.100 is curtailed. 02:56:30.100 --> 02:56:33.690 02:56:33.690 --> 02:56:34.990 This is one of my concerns. 02:56:34.990 --> 02:56:36.270 Go ahead, please. 02:56:36.270 --> 02:56:41.680 - Sorry speaking to the do we have the tools available, 02:56:41.680 --> 02:56:43.270 I think, yes. 02:56:43.270 --> 02:56:47.230 But I also think that continuing to find more efficient tools-- 02:56:47.230 --> 02:56:50.350 and since the mongoose has been the primary area of my work 02:56:50.350 --> 02:56:53.170 over the last several years, previous attempts 02:56:53.170 --> 02:56:56.770 at vaccinating mongoose using the currently available 02:56:56.770 --> 02:56:59.343 and licensed vaccines at the time, at least of the doses 02:56:59.343 --> 02:57:00.760 in which they were delivered, were 02:57:00.760 --> 02:57:03.700 found not to be immunogenic in that species. 02:57:03.700 --> 02:57:07.240 And with the advent of these newer vaccines, 02:57:07.240 --> 02:57:09.910 we have found them to be immunogenic 02:57:09.910 --> 02:57:11.690 at least in laboratory trials. 02:57:11.690 --> 02:57:14.980 So continuing that work, especially 02:57:14.980 --> 02:57:16.930 in some species that are understudied, 02:57:16.930 --> 02:57:18.940 and in particular Puerto Rico where 02:57:18.940 --> 02:57:22.360 they are a rabies reservoir that interact 02:57:22.360 --> 02:57:26.920 with domestic animals, which can spread that to humans which 02:57:26.920 --> 02:57:29.560 happened in 2015, which is the first documented 02:57:29.560 --> 02:57:32.920 case of a directly mongoose mediated case of rabies 02:57:32.920 --> 02:57:35.260 on a humans on the island. 02:57:35.260 --> 02:57:39.550 We now have through this advent of these additional vaccines, 02:57:39.550 --> 02:57:43.390 the ability to address that issue in the wildlife 02:57:43.390 --> 02:57:47.270 reservoir of domestic animal vaccination outstanding. 02:57:47.270 --> 02:57:51.760 That's another issue in Puerto Rico that will certainly 02:57:51.760 --> 02:57:56.700 be critical in my opinion to eradicating mongoose spreading 02:57:56.700 --> 02:57:58.260 rabies from the island. 02:57:58.260 --> 02:58:03.180 But we have now a vaccine that shows a lot of promise 02:58:03.180 --> 02:58:07.140 whereas not too many years ago, that was not yet available. 02:58:07.140 --> 02:58:09.420 And that's thanks to the continued diligence 02:58:09.420 --> 02:58:12.780 and research of scientists both in the field and laboratory 02:58:12.780 --> 02:58:14.490 in my opinion. 02:58:14.490 --> 02:58:16.680 Thank you, Are, except for using one 02:58:16.680 --> 02:58:21.060 of my most unfavorite, impossible words in 02:58:21.060 --> 02:58:22.950 regards to rabies, and that's eradicate. 02:58:22.950 --> 02:58:25.500 Prevention control, selectively eliminate, 02:58:25.500 --> 02:58:28.260 but we aren't going to eradicate this baby. 02:58:28.260 --> 02:58:33.360 I'm wondering in regards to my colleagues from Asia, 02:58:33.360 --> 02:58:35.100 if you're still there. 02:58:35.100 --> 02:58:38.520 Dr. Tsu, Dr. Zhie, what do you think 02:58:38.520 --> 02:58:42.660 about the utility for new diagnostics or new vaccines. 02:58:42.660 --> 02:58:46.050 Is there a need is there utility for the near future 02:58:46.050 --> 02:58:52.600 from the Asian, local China perspective, what do you think? 02:58:52.600 --> 02:58:54.010 Do we need new tools? 02:58:54.010 --> 02:58:56.337 Are the tools we have now adequate? 02:58:56.337 --> 02:59:00.320 02:59:00.320 --> 02:59:02.600 - There is a question of Dr. Feng 02:59:02.600 --> 02:59:07.630 Ye on the chat about the zero vaccine 02:59:07.630 --> 02:59:10.490 and how the zero vaccine is supporting the stomach acid 02:59:10.490 --> 02:59:10.990 attack. 02:59:10.990 --> 02:59:13.700 02:59:13.700 --> 02:59:14.918 Dr. Feng Ye? 02:59:14.918 --> 02:59:18.790 02:59:18.790 --> 02:59:21.790 There is also a remark from someone from PAHO, 02:59:21.790 --> 02:59:24.200 saying that even in Latin America at the moment 02:59:24.200 --> 02:59:27.010 there is not enough RIG, I'm just reading. 02:59:27.010 --> 02:59:32.160 02:59:32.160 --> 02:59:32.670 - Yes, I-- 02:59:32.670 --> 02:59:34.110 - Hi, hello. 02:59:34.110 --> 02:59:37.790 - Yes, Dr. Feng, please go ahead. 02:59:37.790 --> 02:59:41.580 - Yeah our laboratory we have a vaccine too 02:59:41.580 --> 02:59:44.836 and there is a student and who've 02:59:44.836 --> 02:59:49.710 worked at how to produce a vaccine. 02:59:49.710 --> 02:59:54.450 And it's her question and where she 02:59:54.450 --> 03:00:00.690 tried to right overall vaccine to animal 03:00:00.690 --> 03:00:06.120 and he found it was not very effective. 03:00:06.120 --> 03:00:12.240 So her question is it's true at all for a vaccine 03:00:12.240 --> 03:00:17.550 to add more to overcome the stomach, like act attack. 03:00:17.550 --> 03:00:23.230 03:00:23.230 --> 03:00:24.850 - Very good, thank you. 03:00:24.850 --> 03:00:27.340 Do you think we need new diagnostics? 03:00:27.340 --> 03:00:29.980 I'll also open that up to Nuro? 03:00:29.980 --> 03:00:34.991 03:00:34.991 --> 03:00:40.950 - I'm afraid they don't have the method on how 03:00:40.950 --> 03:00:45.890 to produce the ultra vaccine. 03:00:45.890 --> 03:00:49.370 - Yes, I know there's been a very recent paper 03:00:49.370 --> 03:00:54.200 about the serum system that was done 03:00:54.200 --> 03:00:56.930 conducted in China recently. 03:00:56.930 --> 03:00:59.360 And unfortunately, although, China 03:00:59.360 --> 03:01:04.730 has made huge progress in human rabies case decrease 03:01:04.730 --> 03:01:08.990 primarily from post exposure prophylaxis, 03:01:08.990 --> 03:01:16.700 from the standpoint of the ZBT and on a score from 0 to 5, 03:01:16.700 --> 03:01:20.930 nationwide it looks like China is only about a 1.5 03:01:20.930 --> 03:01:23.420 in terms of preparedness. 03:01:23.420 --> 03:01:26.900 And we only have nine years to go. 03:01:26.900 --> 03:01:29.180 What do your colleagues in China think? 03:01:29.180 --> 03:01:32.570 Is China going to be able to eliminate 03:01:32.570 --> 03:01:35.710 canine rabies in this decade? 03:01:35.710 --> 03:01:38.270 03:01:38.270 --> 03:01:39.080 - Yes. 03:01:39.080 --> 03:01:44.360 I had published this article with other colleagues. 03:01:44.360 --> 03:01:48.290 And I know we have serious problems 03:01:48.290 --> 03:01:54.860 in animal rabies in China and we still work on it. 03:01:54.860 --> 03:02:03.110 And we have a very serious problem of dog rabies. 03:02:03.110 --> 03:02:09.470 But we have a decrease of human rabies. 03:02:09.470 --> 03:02:13.750 I know we still have less than 10 years 03:02:13.750 --> 03:02:21.130 but all the people want China to make rabies history in China. 03:02:21.130 --> 03:02:22.190 - Yes, thank you. 03:02:22.190 --> 03:02:27.100 And we look forward to your continued efforts. 03:02:27.100 --> 03:02:29.800 And I know our colleagues at pass two 03:02:29.800 --> 03:02:34.030 have very recently published a very interesting paper 03:02:34.030 --> 03:02:40.310 about the treatment of animals with illness. 03:02:40.310 --> 03:02:41.540 Laurent, what do you think? 03:02:41.540 --> 03:02:44.960 Is their utility to using these and other modalities 03:02:44.960 --> 03:02:48.835 not just for prophylaxis but also for treatment? 03:02:48.835 --> 03:02:53.200 03:02:53.200 --> 03:02:55.120 - In fact I was not involved in that. 03:02:55.120 --> 03:02:59.056 But though was it compromising, yes, to do that? 03:02:59.056 --> 03:02:59.750 - Yes. 03:02:59.750 --> 03:03:02.770 - The main problem is that for the moment 03:03:02.770 --> 03:03:07.100 that you have to inject this directly into the brain. 03:03:07.100 --> 03:03:12.970 So we have to make efforts to wonder of this access 03:03:12.970 --> 03:03:18.220 more easily because to date it's a good example of what 03:03:18.220 --> 03:03:22.400 we can do, is that pave the way for future experiments. 03:03:22.400 --> 03:03:27.700 But if you look back in the context of real life, what 03:03:27.700 --> 03:03:31.930 we are far from giving this ZBT to lower middle income 03:03:31.930 --> 03:03:34.180 countries you know we're going to have a real problem. 03:03:34.180 --> 03:03:36.250 So that's promising but I think we 03:03:36.250 --> 03:03:44.050 have work to do not only on the biological products themselves 03:03:44.050 --> 03:03:48.550 but on the way to deliver them in the target organs, which 03:03:48.550 --> 03:03:50.470 is the brain. 03:03:50.470 --> 03:03:54.050 - Yes, excellent point. 03:03:54.050 --> 03:03:56.060 So we've covered the spectrum today 03:03:56.060 --> 03:04:02.560 from diagnostics, to therapy, to prevention, 03:04:02.560 --> 03:04:06.550 to vaccination of free ranging animals, 03:04:06.550 --> 03:04:10.420 to the utility of such techniques for potency 03:04:10.420 --> 03:04:14.500 of the biologics we use in humans and other animals. 03:04:14.500 --> 03:04:16.960 And how to go ahead and gauge the utility 03:04:16.960 --> 03:04:19.710 in free ranging animals. 03:04:19.710 --> 03:04:23.730 We certainly have many challenges ahead of us. 03:04:23.730 --> 03:04:25.540 Nature abhors a vacuum. 03:04:25.540 --> 03:04:28.830 I know there's a new lyssaviruses to be found. 03:04:28.830 --> 03:04:31.950 We hope that it's never going to come anything 03:04:31.950 --> 03:04:35.040 close to what this past year has shown us 03:04:35.040 --> 03:04:38.820 in regards to coronavirus but you never say never. 03:04:38.820 --> 03:04:41.250 And I think that's the challenge for some of us 03:04:41.250 --> 03:04:44.880 who want to increase the cross reactivity 03:04:44.880 --> 03:04:48.030 of our current biologics to the Filo Group two, 03:04:48.030 --> 03:04:52.260 three and the new lyssaviruses to come. 03:04:52.260 --> 03:04:56.340 Any closing thoughts by any of our panelists 03:04:56.340 --> 03:04:57.390 since we're over time? 03:04:57.390 --> 03:05:00.900 03:05:00.900 --> 03:05:04.290 Not hearing any, I want to thank everyone 03:05:04.290 --> 03:05:06.960 from Asia and in the middle of the night 03:05:06.960 --> 03:05:08.740 to our European colleagues. 03:05:08.740 --> 03:05:11.820 Next time we'll have African colleagues to boot. 03:05:11.820 --> 03:05:14.520 To those on this side of the pond 03:05:14.520 --> 03:05:18.060 wishing you a very good night, good afternoon, good day, 03:05:18.060 --> 03:05:20.310 and thank you very much for your participation 03:05:20.310 --> 03:05:23.280 in this very wonderful webinar. 03:05:23.280 --> 03:05:25.950 And please stay healthy, get vaccinated out there. 03:05:25.950 --> 03:05:27.300 Bye bye. 03:05:27.300 --> 03:05:29.414 - Bye bye, ciao, ciao 03:05:29.414 --> 03:05:30.990 - OK, thanks, everyone. 03:05:30.990 --> 03:05:32.610 Thank you, Charles. 03:05:32.610 --> 03:05:35.830 And we have a recording of this webinar. 03:05:35.830 --> 03:05:39.420 So we'll add subtitles to it and we'll 03:05:39.420 --> 03:05:42.010 send it to all the participants later this week. 03:05:42.010 --> 03:05:43.340 - Thank you, sir. 03:05:43.340 --> 03:05:45.935 Thanks for Joe for hosting us, bye bye. 03:05:45.935 --> 03:05:47.310 - No problem, thank you everyone. 03:05:47.310 --> 03:05:48.102 - Thanks, everyone. 03:05:48.102 --> 03:05:49.660 - Bye bye. 03:05:49.660 --> 03:05:50.620 - Thank you. 03:05:50.620 --> 03:05:52.370 - Thank you. 03:05:52.370 --> 03:05:54.000