- Hello, everyone.
Thank you for joining us in this unprecedented time.
We started this JoVE series of protocols
at about the same time we began updating the WHO laboratory
techniques volumes.
And our rationale at that time was ideally,
in the future that all of, or at least
most of or many of the protocols in the WHO laboratory
techniques book would eventually become visual experiments
to assist with not only reading, but actual operational details.
So our objectives today during this pandemic
are to try and provide a glimpse into some of those for you,
one, because of the limitations in travel,
and the decrease in the opportunities
for actual wet laboratories.
Our second objective is for each of our presenters
to update any of the techniques that they
have presented in the JoVE format special rabies series.
And thirdly, to place in perspective
where their particular presentation fits in
regards to rabies prevention, control, research, et cetera,
as we recognize that we're already well into, in 2021,
the ZBT, the Zero by 30, the elimination of human rabies
caused by dogs and how these protocols may
assist in that endeavor.
But not only in regards to canine rabies, which
is our major burden throughout the world,
particularly in Asia and Africa, but we
recognize that also threatens the progress in the Americas,
particularly in Latin America, which
serves as one of the greatest regional demonstrations
that canine rabies can be eliminated.
Obviously, that has been impacted by the COVID pandemic
because surveillance is down.
As surveillance is down, we know that the evaluation of programs
falter.
But not only surveillance, but also sometimes vaccination
programs are also on hold.
So that when we consider the impacts
of the pandemic upon both surveillance as well as dog
vaccination, cases are going to rise, as we see from the news,
for example, in Arequipa, Peru, and in other parts
of Latin America.
And in those parts of the world that have already well achieved
the elimination of canine rabies,
we've had the epidemiological luxury
of wildlife rabies discovery.
And some of the opportunities that we'll
have to talk about today are directly
focused upon enhanced surveillance, which
could be applied to canine rabies,
but are of obvious importance to finding, detecting, preventing,
and controlling wildlife rabies.
And not the least of which are generated
by bats, which are some of the global reservoirs
for lyssaviruses.
So I'm calling in from the Atlanta area.
I wanted to say a happy equinox, whether you're
in the north or south temperate zones.
We'll have each of our presenters
speak for about 10 minutes, introducing their topic,
talking about any of the salient details,
giving any operational updates.
If you have questions, we have a couple
of ways to resolve those.
One, by raising your hand.
You can type into the text box.
And the presenter has the option of either answering
the question if convenient when it comes up,
or at the end of their session.
We will have a short break at about halfway
through the program.
After that, we'll resume and questions either directly
during or after each presentation,
or if we have time, if our presenters, all
being good professionals, stay on time,
at the end for any wrap-up discussions, any questions that
didn't get answered, or any questions
the participants may have of their colleagues.
We're happy to go ahead and start
with our first presentation from Dr. Wei-Cheng Hsu, coming in
from the Animal Health Research Institute,
the Epidemiology Research Department in Taipei,
and will be sharing with us issues of great importance,
particularly in the Asian area, for the discovery
of lyssaviruses associated with bats.
Dr. Hsu.
You have the floor.
I'm going to mute myself.
And we don't hear--
[AUDIO OUT]
Perhaps try unmuting your microphone.
Hello.
Hi, that's-- Hi.
We can hear you.
Thank you.
OK, thank you.
Hello.
I am Wei-Cheng Hsu from Taiwan.
My talk is about standard operating procedure
for lyssavirus surveillance of the bat population in Taiwan.
And this is my outline.
And the introduction.
Lyssaviruses do not take pathogens.
And now we know we have in [INAUDIBLE]
there are 16 species of lyssavirus
and 3 more new lyssaviruses have been found recently.
And this is the map of lyssavirus distribution.
You can see the gray color.
The gray color represent there are no surveillance data.
And in Taiwan, we have 35 species of bats.
And bat diversity is very high in Taiwan.
And the SOP of lyssavirus is [INAUDIBLE] in Taiwan,
including first our target animal,
our dead bats, or bats dying of weakness,
and this case will be submitted to our laboratory,
and we will perform the necropsy and the sample collection,
and we will do an FAT and RT-PCR at the same time,
and we will run the Fujirebio and the Millipore 5100.
These two conjugates.
In RT-PCR, we will run two set of primers.
If either FAT or RT-PCR show the positive, we follow up,
we will do a virus isolation, and phylogenetic analysis.
In any fresh specimen, we will do
a histopathology examination.
In Taiwan we are very lucky, because in Taiwan we
have 22 animal disease control center,
and we also have four bird conservation NGO,
non-government organizations.
So 22 ADCC and four NGO will send some bat carcass to us.
And about the result, we start our lyssavirus surveillance
in 2008, and in the first year we
capture the health bat and euthanasia,
and all case is negative.
And after the first year, we just
consulted with Dr. [INAUDIBLE],, and he gave us
a very good suggestion.
He think we need to start to do these kind of surveillance,
so we change.
We just collect dead bat.
So we stop to do the live bat surveillance.
In 2008 to 2015, we do the lyssavirus surveillance only
by FAT.
And after eight years of surveillance,
we change our SOP.
We do the FAT and the RT-PCR at the same time.
And after we change our SOP, we start
to find new novel lyssaviruses.
There are final novel lyssaviruses.
Belongs to two species of bat in Taiwan.
During 2016 to 2020, the positive rate
of lyssavirus of the Japanese Pipistrelle is about 1%,
and the positive rate of lyssaviruses
in Mountain Noctule is about 100%,
because this species we only got one sample,
and this sample is positive.
And you can see a blue color character and the red color
character.
The blue means the Japanese Pipistrelle.
You can see four cases.
And in all case, the location is different.
And we also find one positive case of Mountain Noctule,
and it's in a red color.
And the phylogenetic analysis show, in Japanese Pipistrelle,
we just named it.
It's a novel-- it's a new lyssavirus,
and we named it as Taiwan bat lyssavirus type I.
In the Mountain Noctule, after a phylogenetic analysis,
it also new lyssaviruses.
We named it Taiwan bat lyssavirus type II,
and this is unpublished data.
You can see the genetic tree.
Taiwan bat lyssavirus type I and type II are clustered together,
and both belong to the [INAUDIBLE] group 1.
And as I mentioned before, we do RT-PCR,
and the type I and type II Taiwan bat lyssavirus
can work well with two primer.
And this is our primer sets.
But we found something stranger in Taiwan bat lyssavirus type
I. Because if we do [INAUDIBLE] and if we scan
we saw Fujirebio conjugate.
We got a negative result. But if we do the Millipore conjugate,
we can find a positive signal.
But type II bat lyssavirus, both conjugate works well.
In virus isolation, we found if we use the mouse neuroblastoma
cell, MNA cell, it's almost impossible
to isolate the Taiwan bat lyssavirus type II.
So in Taiwan lyssavirus type II, we do a mouse inoculation.
We [INAUDIBLE] three times, and after the mouse inoculation,
we use the BHK cell to do the virus isolation.
It still [INAUDIBLE] three times.
So after three times in the mouse and after three times
the BHK cell, we can reach 100% infectivity.
So it is very difficult to isolate the Taiwan bat
lyssavirus type II.
So, discussion.
So during our 10 more years of surveillance,
we found these SOP can work well in Taiwan.
We use this SOP, we found five positive lyssavirus cases.
We [INAUDIBLE] would find every lyssavirus
has different characters.
And a variation of lyssavirus antigen in a sample,
recommend first two different conjugates
should be used in FAT.
Second, more than one primer set in the RT-PCR should be used.
Third, the more detecting tools, the less false negativity.
Nonetheless, cost performance ratio should be considered.
And how to find your own new lyssaviruses?
In my personal point of view, I would say be patient.
Because, in our experience, we found our first lyssavirus
case, we spent more than eight years.
Second, do the right things.
We only collect dead or dying bats,
and with cooperation with the animal disease control
center and the NGO of bat conservation at the same time,
so we can expand our surveillance area
and we employment both FAT and RT-PCR.
And we routinely do virus isolation.
Finally, the most important.
you need to have a good luck.
So that's my presentation.
Thank you.
- Thank you very much, Dr. Hsu.
And we do have a question.
One question is, could it be that,
because you're an Island, that you
have more apparent diversity of lyssaviruses
compared to other locations?
And they wonder why, for example, in the US
there is such a low diversity, which I would disagree with,
but they wanted to know.
Dr. Hsu, do you have any thoughts on their question?
- Yes.
I think it's a very good question,
but I don't have the answer.
And in Taiwan, from 2016 to 2020,
almost every year we test about 100 cases,
and the positive rate is just very close to 1% every year.
So it's very strange.
Very strange, a very high positive rate, but I
don't know why.
Thank you.
- And do you have any human exposures
to these lyssaviruses?
- We do have some bat bite cases.
The bat comes into the house of somebody, and bite the people,
bite human.
And until now, human contact cases
will go to do the [INAUDIBLE],, and we will diagnose
this bat in less than 24 hours.
So until now we don't have human cases.
- Excellent.
Thank you very much for that presentation,
and I hope that you won't have any human cases.
You can stop sharing your screen,
and we'll have our next presenter.
Thank you.
Our next presenter will be Dr. Ye Feng.
Dr. Feng is an associate professor and epidemiologist
at the National Reference Laboratory
for Animal Rabies [INAUDIBLE].
Research interests focus on rabies
epidemiology, diagnostic surveillance, and also
responsible for training of individuals
in China and other countries.
Dr. Feng will be talking with us today
about the evaluation of the universal nested reverse
transcription polymerase chain reaction of the detection
of lyssaviruses.
Dr. Feng?
- Hello, chairs.
Thank you, and I'm afraid I can't [INAUDIBLE]
because someone has flagged their [INAUDIBLE]..
- Can you share your screen?
- I'm afraid someone is still share--
- Ah.
Dr. Hsu, have you stopped sharing your screen?
Dr. Hsu, can you hear me?
Have you stopped sharing your screen?
- OK.
- Ah, now we'll try.
Thank you, Dr. Feng.
The floor is yours.
- I'm Ye Feng, work site OIE reference laboratory in China.
It's my pleasure to be given the opportunity to join you here
for the [INAUDIBLE].
The purpose of my presentation is
to give you a shot into real [INAUDIBLE] universal RT-nested
PCR for the detection of lyssavirus.
To detect rabies virus other member species opportunist
lyssavirus, we have developed a [INAUDIBLE] lyssavirus nested
RT-PCR in 2007.
The principle of this method is the reverse transcription
of [INAUDIBLE] RNA into to CPA by using only [INAUDIBLE]..
[INAUDIBLE]
The CDA [INAUDIBLE] two rounds of PCR.
The two rounds of PCR significantly
increased the sensitivity of PCR.
The CDA undergoes goes the first round PCR
without primers to amplify on 940 [INAUDIBLE] BP fragment.
Then the second round PCR used a first round PCR product
as a template to amplify a 371 BP fragment [INAUDIBLE]
the primers.
The primers of the first round and second round PCR
was designed based on the [INAUDIBLE] region
of the [? engine ?] of seven major lyssavirus species,
because only seven lyssavirus species was found in 2007.
This method was patented in 2009.
The lyssavirus species was [INAUDIBLE] increase
in recent--
[AUDIO OUT] --lyssavirus species was approved by the ICTV.
In order to detect the sensitivity and the specificity
of these 18 lyssavirus species, the other 18 [INAUDIBLE]
for [INAUDIBLE] of each lyssavirus for PCR.
Result are listed RT-PCR to detect an 18 lyssavirus
[INAUDIBLE] on the left.
We can see all 18 lyssavirus putting the same two
[INAUDIBLE],, indicating that the nested RT-PCR detect all 18
lyssavirus.
16 of classmates [INAUDIBLE] application
in two rounds of PCR.
It is also [INAUDIBLE] virus in [INAUDIBLE] was amplified,
unlike in the second round PCR.
And [? coronavirus ?] [INAUDIBLE]
was applied only in the first round PCR.
The sensitivity of the measure will
show in the table on the right.
The sensitivity was varied out in the detection
of [INAUDIBLE] values ranging from 2.24 to 22,400 [INAUDIBLE]
per [INAUDIBLE],, and as shown in the table on the right.
To [INAUDIBLE] the [INAUDIBLE] the cause of this discrepancy,
I think with cooperation of all 18 lyssavirus
with full primer regions who contact.
With the result showing that [INAUDIBLE] and nucleotide
T are autoprimers.
And it to die in the first round PCR.
And the second nucleotide T at three [INAUDIBLE] in the primer
and 371F in the second round PCR was not
identical to the responding position of the Aravan virus
and Ikoma virus.
They have tried to change the [INAUDIBLE] of the primer to T
of [INAUDIBLE] and change the G of the primer to A--
to C, sorry, of the coronavirus.
Both virus was in the PCR.
This result shows that the critical roles
of the three [INAUDIBLE] and nucleotides
of the primary [INAUDIBLE] success
following application of the [INAUDIBLE] region.
The result was unsure.
It means this difficulty can be attributed
to the dismatch between the primers and templates
due to the [INAUDIBLE] sequence that was attained.
To test the validation of the RT nested PCR,
we use 9,624 branches of eight domestic animal species
in 10 years of clinical, [INAUDIBLE] technologies
and surveillance in China.
But the result shows that RT nested
PCR has 100 sensitivity and over 90 [INAUDIBLE]
in corporation with the direct fluorescent antibody test.
300 decayed practical samples will test the positive,
but RT-PCR was up by nested PCR [INAUDIBLE] at FAT.
This result indicated that the nested RTP [INAUDIBLE]
is most of the two, that FAT in detection
of [INAUDIBLE] samples.
In addition, 10 other [INAUDIBLE]
laboratory was invited to [INAUDIBLE]
the test to further write [INAUDIBLE] the nested RT-PCR.
All 10 laboratories are [INAUDIBLE] nested RT-PCR
with 100% in constant with FAT with no false positive or false
negative, indicating that this nested RT-PCR has
the high price of [INAUDIBLE].
Our nested RT-PCR has been approved
as the national standards of previous technologies in China
in 2018.
This method was published in the Journal of [INAUDIBLE]
Experiment in 2018.
Finally, I would like to end my presentation with each
produce how my laboratory.
My director is Professor of [INAUDIBLE],,
[INAUDIBLE] Laboratory, rabies classical [INAUDIBLE],,
and [INAUDIBLE] this group.
My laboratory would like to cooperate with all of you
to make rabies history.
Thank you.
- Thank you for that very nice presentation, Doctor Feng.
And I thought I saw one question.
The person wants to how easy is it
to adjust your primers when looking
for different lyssaviruses?
For example, do you think you ever might have a case where
you think there might be a lyssavirus but cannot find it?
So I think they're speaking, for example,
of having some disparate ones as we go beyond filo group three
on our clock.
How easy is it, do you think, to find new lyssaviruses
based upon your primer design?
- Thank you.
My thought was our primer was designed based on the seven
lyssavirus species.
And after that, we [INAUDIBLE] how 18 lyssavirus species now,
so we just test a classmate to test whether our primer works
now or not.
- Thank you.
- OK, thank you.
- And do you think there's a greater diversity
of bat lyssaviruses in Asia given the results that you've
found so far That.
Haven't been discovered yet?
- Maybe because they [INAUDIBLE] enough surveillance in China
and other Asian countries.
And if we operate more rabies technologies,
maybe we can find more lyssavirus maybe, I think.
- Yes.
And do people who are exposed to bats in China,
do they submit the bats for diagnosis
or receive prophylaxis?
Or do you think because our major burden is with dogs
that the education is focused more upon rabies
in dogs as it should be?
Do people get exposed to bats in China
and receive prophylaxis after a bite?
- Yes, of course.
In China, our work is [INAUDIBLE] dog rabies
because dog [INAUDIBLE] is a major transfer source in China.
It's over 99 human cases was caused by dogs,
so we have lots of dog rabies.
And I don't think they have enough money and enough people
to operate [? with ?] this rabies research.
Maybe in the future we can operate more research on that.
- Yes.
And you probably saw some recent papers from our colleagues
in Europe talking about the diversity of lyssaviruses
in Eurasia as well as in Africa.
For example, the finding of a west Caucasian bat
virus in a cat in Italy.
And I'm wondering, considering beyond [INAUDIBLE]
group one, such as the African and some of the Eurasian
lyssaviruses, do you believe we need
to have new vaccines for the future to cover the broader
spectrum of these lyssaviruses?
- I think they have three groups lyssavirus species.
And our vaccine can cover group one and group two.
Maybe in the future we can operate--
vaccine can cover all the lyssavirus.
But in China, and especially in other countries in Asia,
and we just how rabies virus and work with lyssavirus
and Thailand bat lyssavirus, I think.
So the vaccine ratio is enough for rabies control
and [INAUDIBLE].
- Yes.
And I don't see any other questions,
so I thank you for your time and your presentation.
And if you'd be so kind to stop screen sharing,
we'll go on with our next participant.
Thank you very much.
We're going to stay on the theme about lyssaviruses
and their detection, and in particular,
in regards to molecular detection,
our next participant will be Megan Golding speaking to us
about pan- lyssavirus, a real-time PCR for rabies
diagnosis, recognizing that rabies is caused by lyssavirus
or lyssaviruses cause rabies.
And Megan is coming to us from the Animal and Plant Health
Inspection Agency Rabies and Viral Zoonoses
Group from the UK.
Megan, you have the floor.
- Thank you.
Hi, so I'm Megan.
I work for the Rabies and Viral Zoonoses Group
at the Animal Plant Health Agency
and I'm going to be talking about the real-time PCR
that we use primarily in rabies diagnosis
and comparing it to the other [INAUDIBLE] that we also use.
There we go.
Just a little bit about our group.
First of all, we're a UK and international reference
laboratory for rabies and we carry out
a range of functions and research and diagnostics
by serology and molecular and today I'll
be focusing on our molecular and comparing the assays
we use in lyssavirus diagnostics.
There are a range of ways that we use PCR and our diagnostics
and it's become a really valuable tool
in our infectious disease.
We use it in suspect cases to confirm
disease in an animal or human exhibiting symptoms
of that disease.
We use it primarily in our passive surveillance scheme.
So we have a passive bat surveillance
scheme where we test at bat submitted to us by the public.
This allows us to possibly monitor current lyssaviruses
in the UK, like European bat lyssavirus
too, as well as monitor when you want to merge,
like European bat lyssavirus one,
which we detected for the first time in 2018.
And we also use it in targeted surveillance.
We haven't done this in a long time,
but we can do swabs of bats and test those
by PCR of targeted species or in areas where we've
had a lot of positive cases.
And we also then send our positive PCR products
for sequencing to confirm the species
and to investigate phylogeny.
So the widely accepted PCR as a primary diagnostic tool
for the first time in 2018, recognizing
its advantages in that there's no requirement
for a live virus.
And it's particularly useful in cases
where the sample is sub-optimal.
Those are the three assays that we use listed there
and the one highlighted in green is our primary go-to assay
for initial screening.
And the other two are mainly used as confirmatory assays.
All of our assets are based on the N gene
region of the lyssavirus genome because it
is the most conserved.
And also we do tend to use the molecular assays in combination
with serology, like F80.
So just to break down the assay that we use initially.
First of all, of our assays have an RT step
because the lyssavirus genome is RNA
and the [? san ?] material PCR cDNA.
They're also all one step because this
makes them simpler and more convenient to carry out.
It also reduces how much manipulation
is involved to reduce the chance of contamination.
It is a real time assay.
So the assay works by a intercalataing dye called SYBR
green that binds to double stranded DNA.
And then once found, it emits fluorescence,
which is measured by the real time machine at each cycle.
So it detects and quantifies target DNA or RNA in real time.
And also gives us a CT value, which
is the number which the PCR product crosses
the threshold of detection.
A lower CT would mean that you have a viable nucleic acid
load.
The assay is also pan-lyssavirus,
so we've developed primers that are
successful at detecting all known lyssaviruses,
making this assay great for general lyssavirus
surveillance monitoring current and emerging disease.
However, it isn't capable of differentiating
between the species it detects, so you would need to carry out
further analysis to do that.
This is the thermal profile we use.
The key bit to know is that the assay is rapid.
And [INAUDIBLE] the reverse transcription step is step one.
To make it a one step assay, we amplify for 40 cycles
and we carry out with dissociation curve analysis.
This is the kind of output that we would get.
So on the left, you've got the amplification plot
where you can see which of your samples is amplified
and what their CT is.
So just for example, the blue curve
would have the lower CT value, approximately 20.
But that would also mean it has the highest viral load.
And on the right, you've got the dissociation curve.
Because the dye bind is non-specific,
we analyze this curve to check the melting temperature
is specific to lyssavirus, which we know to be
78 to 79.5 degrees Celsius.
And amplification isn't due to non
specific binding like the formation of primer timers.
So just to summarize, it is a pan-lyssavirus assay,
so it can detect all lyssavirus species
but can't differentiate.
You'd need to carry out further analysis.
The project length is a bit short,
120 base pairs, so it's not ideal for sanger sequencing.
So you'd likely need to carry on another PCR after this.
It comes in real time, so you get CT values.
And it's within a closed tube system,
so there's a minimal risk of contamination.
It's rapid.
And you can use the dissociation curve to confirm specificity.
So I'll just compare that to a conventional hemi-nested PCR.
Because it's a conventional PCR, you
need to carry out post PCR analysis, like gel
electrophoresis, in order to determine
what it is you detected.
It also makes it an open tube system,
so it's much more vulnerable to contamination than our SYBR.
Like our SYBR, it's pan-lyssavirus,
but you still can't differentiate between species.
But the product is much larger at 600 base pairs, which
is ideal for sanger sequencing.
So this assay is one we would typically carry out
if we've had a positive SYBR result
and we want to send the product for sequencing.
It's also a nested PCR, so we carry out
two rounds and with the same forward primer
in the second round but a slightly different reverse
primer to amplify the round one product to confirm specificity
of the first round and improve sensitivity.
It's also much slower than a SYBR
and it takes over five hours to run both
of the rounds and the analysis.
And then to compare that to our other real time
assay, which is our TaqMan.
However, instead of dye, it uses highly specific probes
to detect target sequences.
So we use three probes.
These are EBLV-1 and EBLV-2 because these
are the two lyssavirus species in the UK currently.
And they only concern in certain bat species,
so this assay is great if we have
a preliminary positive result in one of our bat species
and we want to quickly confirm it's the expected lyssavirus.
We also use a RABV probe mainly as an indication
of contamination, because it's extremely unlikely to get
RABV in our bats in the UK.
So it's a differential PCR so you can see what
lyssavirus you have amplified.
You also still get CT values.
And it occurs within a closed system,
so there's a minimized risk of contamination.
It uses the same primers as our SYBR PCR,
so it's the same product length, too small for sanger
sequencing, but this is less needed
than it is with SYBR because you have the probes.
And it's not as fast as SYBR, but still a lot faster
than a hemi-nested.
Controls are a critical part of any PCR,
particularly in diagnostics.
We typically use three in our assays.
Firstly, we have the positive PCR control, which is CVS.
Prepared from infected mouse brain.
Validate and calibrate it to be within a predetermined range.
We track the CT of this control across all of our PCRs
to show good inter-comparability and provide
assurance of the robustness and reproducibility of the assay.
When the control CT deviates from what we would expect,
this would be investigated in the PCR and repeated if needed.
We also have a PCR negative control, like water,
to confirm negative samples and show there's no contamination.
We also use beta-actin, which is a housekeeping gene,
as an internal control system in our samples to show
that extraction was successful.
Any samples with a negative beta-actin
would be investigated and repeated,
or re-extracted as needed.
The other key thing to consider is cost.
Real-time machines-- real-time assays,
sorry, require optic consumables so the machine
can measure through the plastic, which
can be more expensive than what would
be fine with the conventional.
The real-time machines are also much more expensive
than their conventional counterparts.
And just a general consideration of reagents, primers,
and probes.
Especially probes, if you want to design a lot
and you're carrying out low throughput PCR,
the costs can begin to add up.
And also just consider the cost of resources like staff time.
While real-time machines are more expensive,
the assay is much faster.
And nowadays, you can also remote access software
so you could report results from home.
Whereas with conventional, you're a lot more restricted.
So my take home point would be that if you're
needing to choose an assay, make sure you
choose one is best according to your needs, what
you're using it for.
Is it general surveillance or targeted?
Is it high or low throughput?
All of these have implications on cost.
And always carry out local validation
of your essay to make sure it's doing what you need it to do.
Be aware of the impact of false results.
Rabies is obviously incredibly fatal disease,
so getting a wrong diagnostic result
can have serious implications.
Carry follow up investigations of inconclusive or positive
results to confirm them.
And while the real-time PCR does have a reduced
risk of contamination compared to the conventional,
it can still happen.
And always, quality is essential to being
able to rely on your assay and the results produced.
Monitoring [INAUDIBLE] controls, watch
for trends and signs of degradation or contamination.
Always follow good [INAUDIBLE] practice,
like ISO17025 is the international standard for test
accreditation and one that YE requires
assets that are accredited to.
Also continuously validate your assays
to show that it's the one that's most appropriate for you.
All this works towards minimizing contamination
and getting the right result.
Thank you for listening.
You can check out our blog release on World Rabies
Day every year for more info.
- Great.
Thank you, Megan, for that very concise, and yet engrossing,
presentation.
And we have two questions so far.
One, obviously you can apply your SOP
to post-mortem samples.
Suppose you had a suspect human suspected of rabies in the UK.
Would you apply this particular protocol
for antimortem diagnostics?
- We can.
I don't-- just trying to think.
We haven't.
Our suspects usually-- it's post-mortem usually.
But I think if you have tissue that we can extract, yeah,
I don't know.
Because it's normally brain tissue
that it would be done on.
So I don't know if it would be the most appropriate one.
- So suppose you did have a suspect human case
and they were submitting saliva to your laboratory.
Would you be using this or a different protocol
for diagnoses?
- Yeah.
Sorry, yeah.
Yeah, we could extract the saliva
and analyze that with this assay.
Yeah.
- Very good.
We have a secondary follow up question as well.
Do you ever come across submitted specimens
that might have more than one lyssavirus in the sample?
- So we haven't.
So we only have to lyssaviruses in UK and the
also only occur in two bat groups.
And they're very specific.
So EBLV-2 two we've only ever detected in [INAUDIBLE] bats.
And we've only ever detected EBLV-1 in our [INAUDIBLE] bats.
And I think hypothetically it's definitely possible,
so we always test the both.
But no, we haven't yet
- Thank you.
And simpler question, which I think you
may have already answered.
Do you think a person could be infected with more than one
lyssavirus at the same time?
- Yeah.
Yeah.
Again, I think definitely possible
and we would always test all of them.
But nothing we've encountered.
- Yes.
I don't think in any of our collective experiences
on this call from the panelists and their colleagues,
I don't think we've ever detected
more than a single variant in a person
at the same time, which is going to be rare unto itself.
A related question.
What's-- do you know what the sensitivity of your test is
in regards to antemortem samples such as saliva?
- Unfortunately, off the top of my head, I don't.
But it is a really sensitive assay.
So I think it is good, because we have tested saliva before.
So it's definitely still a good assay to choose in most cases.
- Sure.
And do you have any experience with degraded samples,
for example, from animals that may have been dead for a while?
- Oh yeah, definitely.
The bats that we get in are often
ones that have been left to the elements for weeks.
Yet we've had some really- ones in really bad shape.
There's no brain left, in which case serology isn't possible.
Again, it's is still a really good assay.
And we still pick up [INAUDIBLE] in these samples.
It's still the strong sensitive assay.
- In which element would they have been exposed to?
Hydrogen or helium?
- Oh sorry, I mean just they've been left in the environment.
So just left in the rain and the wind for weeks on end.
- Sorry, that was just a subtle attempt at humor
from this side of the--
- Oh, sorry.
[LAUGH]
- --pond in regards to Americana.
Do any of our panelists have any additional questions?
Otherwise, we thank you very much.
And if you'd be so kind to stop your screen sharing
and stick around, if you would be so kind, so
that we could have a more engaging discussion at the end.
Otherwise, thank you very much Megan.
- Thank you.
- Our next presentation will be by Dr. Luka Zaeck coming
from us to us from Europe from the Institute of Molecular
Virology and Cell Biology from our teutonic colleagues
at the Frederick Loeffler Institute in the Isle of Rehm's
in Germany.
And Dr. Luka will be speaking with us today
in regards to a high resolution 3D imaging of rabies virus
infection insolvent cleared brain.
Dr. Zaeck, you have the floor.
- OK now, can you hear me?
Thank you very much for the introduction.
As mentioned, my name is Luka Zaeck
and I am coming from a slightly different angle
in the previous talks because I'm coming more
from the basic research angle on this matter
because I want to talk to you about high resolution
3D imaging of rabies virus infection
in solvent-cleared tissue.
So the question there often is when
trying to understand rabies pathogenesis
is that we have different models.
For example, with in vitro models
using, for example, ex vivo cultures, primary neurons,
primary astrocytes, or looking at material
from infected animals, we have various caveats
that we have to keep in mind when
looking at primary cultures.
We are leaving out other populations
of the central nervous system cell populations.
And we look at conventional immunohistochemistry
and 2-D IHC.
So in animal samples, while we can perfectly
see whether or not there is infection,
we were having trouble asserting this kind of infection
to particular cell types because of limited multicolor
or multiplexing capabilities, as well
as we aren't really able to say anything about the larger
morphology of cells because IHC, convention HC,
deals with small slices of about five to 10 micrometers.
So how do we get a broader context of infection?
How are we able to look at more of the infection
than just a small layer?
We can do that by taking bigger samples.
Which comes with the problem that a bigger sample,
as you can see here in the image and the photo,
that the light from microscopy cannot go into it anymore
and it doesn't come out anymore.
In order to alleviate this problem,
we can use a protocol called tissue optical clearing, which
in the end, makes our tissue transparent,
or see through, as I hope you can appreciate
because you can now actually read the word
rabies below our sample.
That works in the basic principle
that the organ, or tissues, generally,
are made up of several biomolecules.
There's water, there's fat, there's proteins.
They all refract light differently
and this different reflection and refraction of the light
is causing tissue opacity, so we can't image deeply
into the sample.
By using this specialized protocol,
we can draw those factors out of the sample.
For example, we can draw water and lipids
so that we are left with a primarily proteinacious sample,
which has a high refractive index.
If we immerse that sample in an organic solvent that
also has a higher refractive index,
we are left with a so-called see-through, or transparent,
organ, which is what you just saw in the picture before.
And our workflow basically consists out of that.
We take organs, for example, from experimentally infected
animals.
But equally feasible, as for example,
human clinical samples, which are fixed.
We dissect them to a millimeter thickness, which
is about 50 to 100-fold thicker than this conventional
immunohistochemistry that I mentioned,
we apply our protocol, which consists of several steps,
of treatment steps, of the immunostaining and clearing.
And then finally, we can do confocal laser scanning
microscopy.
So high resolution images.
This allows us to actually get deep informations
on the infection context because we are looking
at a three dimensional volume.
We can assess relations between cell populations.
We can go do multicolor assays to actually pinpoint
the infection of cells to specific cell types.
Because we're at a confocal level here,
we can actually go at a sub-cellular resolution
where we can look at the cells at this mentioned sub-celluar
resolution.
So we can go from a broad overview
to an in-depth analysis on a single cell level.
And of course, the protocol the images
I showed you before were in mouse samples,
experimental animals.
It's equally applicable to basically any kind of tissue.
Here as an example.
There is a RABV infected ferret.
And as you can see, if we take our samples
from different sides of the brain,
we find a different abundance of rabies virus infection
in different areas of the brain.
This is not a real surprise yet.
The strong suit of this way, for example, is also true.
If we look at sub figure d here, we
can find rare and very localized events
because we were looking at such a high volume
that we can miss it by only looking at the small micrometer
thick section and conventional IHC
so that we are able to find those two single infected cells
in this large volume here.
Again much more visual for us to see
if we look at three dimensional reconstructions and renderings
of those volumes because it is allowing us to actually grasp
the spatial morphological distribution, and also just
the neuromorphology of those infected cells.
And as mentioned, the protocol is fully multiplexable
so we can use multiple standings.
Apart from the rabies virus antigens of course,
we can stain for marker proteins like GFAP,
which is a marker for [INAUDIBLE]
in the central nervous system, in this case, astrocytes.
Or we can look at neurons by staining
for neuronal markers like MAP2 and then pinpoint
our location, our infection, to specific cellular
subpopulations.
So to sum this experimental approach up
that we have explained in this protocol that
was used this special issue, it works on the basic principle
that we use refractive index matching
to yield optically clear tissue, which is allowing us to use it
for high volume microscopy.
This is very important because when
we're looking at rabies virus that
is affecting neuronal tissues, we
need to understand the distribution in it's
complex morphology of the neuron with projecting axons
in all directions, not only to be
able to actually understand pathogenesis
at a level of basic research.
This gives us this large spectrum of logical context
so we can actually understand how the infection is
distributed in space.
And it's also an unbiased approach
to detect rare and localized events because we're not
restricting ourselves to several layers
but we're looking at an entire volume.
The question then arises is OK, this is a basic protocol.
What can we actually do with this in a real world scenario?
And I would like to show you two applications of this protocol
in a real world basic research scenario
as well as how we have advanced and further optimized
these kind of protocols.
So first I would like to talk about how
we can use this to in-depth quantify different cells.
It comes as no surprise that rabies virus is
infecting neurons in the brain.
But what about other cell types in the central nervous system?
When we look at pathogenic viruses
with this lab adapted viruses, so basically
strain-dependent differences, we can actually
see that here on the right hand side, all of the viruses
are more or less equally able to infect neurons
while only the pathogenic [INAUDIBLE]
viruses, the wild viruses so to speak,
are able to cause a non-abortive infection of astrocytes.
While lab-adapter strains, or vaccine strains like ERA,
are not able to infect astrocytes
in the brains of IM-inoculated mice non-abortively.
This the same picture for intracranial inoculation.
I'm not showing this year for time reasons.
But by using those large volumes,
we are able to quantify the full volume, giving us
a high and robust quantification to really say
that there is no non-abortive infection within lab adapted
virus restraints in IM-infected mice.
The other application is that besides being
able to use the high volume for qualification purposes,
that we use this high volume for visualization purposes.
And here we optimize our protocols in several ways.
First of all, we take larger tissue samples.
But second of all, we implemented
an additional technical platform that
allows us to purchase this lychee fluorescence
microscope, which in comparison to the confocal that
has a high resolution capacity, this
is a high volumetric capacity allowing us to acquire not just
a region of interest for our high volume sample
but the full high volume sample as in total.
And here we can see what this looks
like for central nervous tissues for the brain.
Again, no surprise.
The brain is positively full of rabies virus infection
as you can see here in the red color.
This is equally true for other CNS parts like the spinal cord.
Again you can here see the spinal cord
inside the spinal column still, which
is positively full of rabies virus antigen,
also visible in detail shots.
But the interesting thing that we can see here
is that in an intramuscularly-inoculated
animal, we can also visualize the nerve strands
from the periphery that allow the virus to actually
infiltrate the central nervous system.
And this arises the question, or bears the question,
what is the [INAUDIBLE] for rabies virus in the periphery?
Besides peripheral nerves of course,
because it has to somehow get to the central nervous system.
But are there other cell types in the periphery that are
being affected by the virus?
So we looked at the inoculation site but looked at hind legs.
This is actually a slice, or a section of a hind leg.
The circular center here is the bone.
And again, we can see the peripheral nerves
that the rabies virus uses to infiltrate
the central nervous system.
And if we go into closer detail here,
we can actually see that in green, you can see neurons.
In Anorectic and rabies virus, we can see infected neurons.
But we can also see that the virus is often
associated with the RABV antigens often associated
around neurons.
In a tomography, again shown here, for example,
that the antigen surrounded around the neurons,
showing us that it doesn't seem to be affecting neurons
exclusively in the periphery.
If we stained for an additional marker, MBP,
myelin basic protein, we can see that it does coalocalize
with this marker, showing us that the virus seems
to be infecting cells in the periphery surrounding neurons,
expressing myelin sheaths.
What kind of cells do that?
The insulators of the periphery, so-called Schwann cells.
So using this high volumetric approach because we
aren't restricted to small volumes ,
we're able to screen a high volume of tissue,
allowing the identification of a new target subpopulation
of virus in peripheral tissues.
In this case, Schwann cells.
So as a global summary of what this basic research--
of course it's a basic research method--
but of course, this three dimensional tissue imaging
can yield is that it can help in elucidating global virus
tropism by, for example, allowing us to gain insights
into strain-dependent differences
in the central nervous system.
By identifying new target cell populations like Schwann
cells in the periphery with pathogenic [INAUDIBLE] rabies
viruses.
Or also by giving additional evidence
for anterograde transport of rabies virus,
which I haven't shown here.
But just to quickly give an insight,
we can also see those infected Schwann cells
after intracranial-inoculated animals giving us
this supporting evidence.
Not just for the exclusive retrograde,
but also for anterograde transport.
This I would like to close my talk.
I am part of the [INAUDIBLE] Group,
[INAUDIBLE] Group at the [INAUDIBLE]
Institute in Germany.
And I would like to thank all of the people
in our rabies group that have been involved with this,
particularly would like to highlight
Madlin Portratz, who's done a lot of the imaging work.
And I'd love to answer any questions there are.
Thank you very much.
- Thank you, Luka.
And I'm still trying to get over my amazement
from your kaleidoscopic investigations, which
have really helped to revolutionize
our understanding of pathobiology
from these techniques.
Very nice job.
Thank you.
We do have some questions, one from our colleague Dr. Tudo who
wanted to know that given that there are
previous articles in the literature
that not only street rabies viruses
but other lyssaviruses may have the capability
to infect astrocytes.
Were you able to compare different species
of lyssaviruses besides rabies virus?
- We haven't done that yet.
So the studies that I've shown you
are exclusively done on RTBV.
But of course, different strains,
pathogenic versus non-pathogenic or field work.
[INAUDIBLE] lab adapted or vaccine viruses.
We're actually working on looking
at astrocyte tropism in the lyssavirus spectrum as a whole.
So we are hopefully able to give an insight
on that in the future.
But concerning astrocyte tropism that has been shown for lab
adapted viruses, that has actually mostly
been done either in vitro cultures
so that we do not have this additional layer
of the entire organism.
We can also see with another nice publication
that those lab adapted viruses seem
to infect astrocytes, but abortively,
because the infection is cleared.
- And do you have any opportunities to look
at species beyond rodents?
- As I showed of course, it is possible to apply the protocol
the variety of tissues.
We are an animal health facility.
Of course, we are mostly working with animal tissue,
not likely human clinical, samples of course.
That would be an extremely interesting endeavor
to also look at those particular questions
in human clinical samples but also with naturally
infected animals, like for example,
bats would be a very interesting endeavor to do.
- Yes.
And one of our participants asks beyond neural cells,
astrocytes, et cetera, what about muscle
cells in the periphery?
Aren't they infected also?
- Literature tells us yes.
We haven't particularly put our focus to that yet.
Also, very interesting question to look at this amplification
stage of what has always been described as an amplification
stage in muscle cells.
But we haven't specifically looked at that yet
and first focused on whether we can
find additional cells of the peripheral nervous system that
are infected by the virus.
- Very good.
And we know that one of our late German colleagues Dr. Leuchter
Schneider did some very nice comparative pathobiological
studies.
And we have one participant ask how based on your techniques
is rabies virus transferred from the periphery
to the central nervous system?
- I don't know if it's possible to ask for clarification here.
What does the person mean by how?
So what we can see from our images
is that we infect the mice at peripheral sites.
So for example, the hind leg muscles, in all case
those experimental mice.
And we see the nerves from the infection site being infected
up until the spinal cord where it then
enters into the central nervous system.
- Excellent.
I hope that answers the question.
- Yes.
And our colleague Dr. Thomas Muller
mentions that recent studies show
that bat-associated lyssaviruses are also
able to infect astrocytes.
We do have a question from one of our colleagues
that I believe this is from Dr. Satoshi Inoway,
perhaps from Japan.
What about the salivary glands?
- Also very interesting to look at salivary glands in terms
of transmission of the virus.
I mean we have to, of course, focus here, or stage here,
that we are dealing with mice.
And I personally am not so sure on the role of mice
with the upkeep of rabies virus transmission cycles.
I would assume they have a rather minor role.
Well we have found some rabies [INAUDIBLE] antigen
in salivary glands.
We are still working on this.
It didn't seem as much as we expected to be there.
- Yes.
And I think you hit that right on the head
that this has been one of our pet peeves for many of us
that no, you're absolutely correct.
Rodents are not a reservoir.
However, whenever we have larger bodied rodents
in contact with, for example, carnivores, in North America,
we have spillover infection from carnivore rabies virus
to large body rodents like beavers,
or groundhogs, woodchucks.
So perhaps some of our erstwhile investigators in the field
might be able to send some of these naturally
infected animals, preserved appropriately, to you
and we might be able to get some answers to questions
if they're differential impacts of these viruses and carnivores
or non-reservoir species.
- Thank you for your very excellent presentation.
I see that we are now in time for a very short break.
For some of us who have been hydrating because we've
been glued to our screens, we don't
want to take any time away from listening to these
so I know I'm going to take a quick bathroom break,
and we will be back in 3 minutes.
Please stay with us until that time.
Be back shortly.
[MUSIC - TALKING HEADS, "ONCE IN A LIFETIME"]
You may find yourself living in a shotgun shack.
You may find yourself in another part of the world.
And you may find yourself behind the wheel
of a large automobile.
And you may find yourself in a beautiful house
with a beautiful wife.
And you may ask yourself, well, how did I get here?
Letting the days go by, let the water hold me down.
Letting the days go by, water flowing underground.
Into the blue again after the money's gone.
Once in a lifetime, water flowing underground.
You may ask yourself, how do I work this?
And you may ask yourself, where is that large automobile?
And you may tell yourself, this is not my beautiful house.
And you may tell yourself, this is not my beautiful wife.
Letting the days go by, let the water hold me down.
Letting the days go by, water flowing underground.
Into the blue again after the money's gone.
Once in a lifetime, water flowing underground.
Same as is it ever was, same as it ever was, same
as it ever was, same as it ever was, same as it ever was, same
as it ever was, same as it ever was, same as it ever was.
Letting the days go by, let the water hold me down.
Letting the days go by, water flowing underground.
Into the blue again after the moneys gone.
Once in a lifetime, water flowing underground.
You may ask yourself, what is that beautiful house?
You may ask yourself, where does that highway go to?
And you may ask yourself, am I right, am I wrong?
And you may say to yourself, my God, what have I done?
Letting the days go by, let the water hold me down.
Letting the days go by, water flowing underground.
Into the blue again, into the silent water.
Under the rocks and stone, there is water underground.
Letting the days go by, let the water hold me down.
Letting the days go by, water flowing underground.
Into the blue again after the money's gone.
Once in a lifetime, water flowing under--
- Hello, everyone, and welcome back from our short break.
You are at the JoVE rabies webinar.
We're going to be rejoining with our next presenter, Dr. Laurent
Dacheux, who is one of the deputy directors
at the National Reference Center for Rabies at the Institut
Pasteur.
Some of the things he does is to supervise the activities
of diagnosis in humans and other animals
in a very high quality assurance environment.
And he's very much invested in the development and validation
of diagnostic techniques related to rabies, particularly
in regards to their WHO collaborating
center activities.
He's a very active researcher, collaborator, and publisher.
And he's going to be sharing with us today
some of his experience in the field
post-mortem rabies rapid immunochromatographic
diagnostic test for resource-limited settings
with further molecular applications.
Dr. Dacheux, the floor is yours.
- Sorry.
Excuse me.
My microphone went off.
So it's OK now?
Yeah, OK.
So thank you, Dr. Rupprecht, For this nice introduction.
So as you indicated, I would like
to share with you some results and experience about the field
post-mortem diagnosis with a RIDT or Rapid
Immunochromatographic Diagnosis Test,
which was published in the special edition of JoVE.
So the context.
We know that, unfortunately, rabies,
and especially canine rabies underreporting, especially
in enzootic countries and low- and middle-income countries.
And this fact is that the this impact
impairs disease surveillance and control
due to the lack of reliable data.
We know also that to have some reliable data,
we need to confirm rabies cases, especially animal cases,
by biological diagnosis tests which are done
on post-mortem brain tissue.
And the references methods are provided by WHO and OIE--
of course, the classical one with a direct fluorescence
antibody test, but also with a Direct Rapid
Immunohistochemistry Test, DRIT, and also
the molecular technique such as the one
you will see in this webinar, like qPCR and RT-qPCR.
However, these techniques, these reference techniques,
have some limitations.
Because there is a fact that many--
[CLEARS THROAT] excuse me-- they are mainly
restricted to central veterinary laboratories.
Indeed, they have some different technical and material
constraints, like it should be done in equipped laboratories
with a trained staff with power suppliers
or with the [INAUDIBLE] maintenance, and so on.
So what are the needs?
What we are looking in, for example, in Africa and Asia,
is to have a rapid and reliable diagnostic tests and protocols
also [INAUDIBLE] low technical expertise.
And these diagnosis methods should be accessible
to decentralized laboratories, especially
in the rural area for example, so even in the field,
like a Point-Of-Care Test or POCT.
And we know that several diagnosis protocols were
recently developed and tested.
And the one I would like to focus on
is Rapid Immunochromatographic Diagnosis Test, or RIDT,
which also is referred as Lateral Flow Device, LFD,
or LFA, Lateral Flow Immunoassay.
So this is the aim of this short presentation,
is to present you an application of RIDT in an African context,
for example, from the brain sample
collection to the analysis of the results,
and also further, to the medical diagnosis based on this RIDT.
Different rapid immunochromatographic tests
are available on the market for the detection of rabies.
But my purpose of my talk will be
focused on only one which has been
tested in this purpose, the rapid rabies antigen from
[INAUDIBLE].
And you can see here the content of the kit,
where have all inside which can be used on the file.
What you need to have access to have a tube
to preserve brain samples.
So rapidly, what is the principle of the lateral flow
device?
So it's based on the immunochromatographic
techniques using gold-conjugated detector antibodies.
So you have here the device with a sample pad,
the conjugate pad, the nitrocellulose membrane,
the test line of T-line, the control line, C-line,
and the absorbent pad.
And what are very important here are the antibodies.
We have here the gold-labeled IgG
to detect the antibodies which are called detector antibodies.
Here on the test line you have the sensor antibodies,
IgG sensor antibodies.
And the control line, you have the antibodies,
again the IgG antibodies.
And so the principle is quite-- they are very simple,
like a pregnancy test.
So you have the brain samples, crushed brain sample,
as I specified before.
You put it on the pad, and then you
have a mixture between the antigens in red here
and the detector antibodies.
If you have antigens, you have an immune complex
which are formed.
And then we [INAUDIBLE] in addition
with a free IgG detector.
And then if you have the immune complex,
they will be fixed at the T-line by the sensor antibody.
And the three IgG detector antibodies [INAUDIBLE]
and will be kept fixed under control line
by the anti-IgG antibodies.
For this test, one key point is the collection
of the brain samples.
In the lab, you can make an autopsy of the total brain
and the head of the animals.
But in the field, it's not very practical.
So what way [INAUDIBLE] need to have access
directly to the brain stem.
You can see here, we have the occipital lobe.
And you will see a presentation of that,
I think, later on in the webinar.
It is compatible with the field application.
And these are to be done quite freshly on fresh samples.
And you can see here the foramen magnum and the access
to the brain stem.
Here we can see that.
And what is very important here, as a key point,
is to have the good sample, because we
know that brain stem reference samples for the diagnosis,
which is quite rich in terms presence of antigens
and viruses, that the teams or the staff
have to be trained for this step, which
is highly recommended.
And after all, here you can see that on the field
we can collect the brain stem with different disposal things,
like a plastic pipette, a drinking straw
or plastic drinking straw, clamps,
even with a disposable droppers, provided in the kits.
And for the protocol of the [INAUDIBLE],,
the initial protocol provided by the [INAUDIBLE]
for the manufacturer, you have the brain samples.
You put that in--
you put that in the PBS, with a dilution of 10%.
You mix it with a swab provided in the kit.
PBS are not provided in the kit, so you
have to carry it with you, as well as a tube.
And then after, you put this dilution
on the buffer contained in the kit.
You mix with a swab provided in the kit,
take some drops here, and put on the pad
and wait for the [INAUDIBLE].
[INAUDIBLE] That's quite simple.
But the problem is that you have to put some PBS,
and that also you have to [? resolve ?] a clear PBS
and tube.
So in our protocol, it was very simple,
we just skipped these dilution steps to have
a more rapid diagnosis method.
And in fact, we also demonstrated
that this technique was able to increase the level
of sensitivity and specificity.
And here are just the [INAUDIBLE] results.
I mean, here we have a positive result
with a tester line and a control line.
So two [INAUDIBLE],, this is positive.
One [INAUDIBLE] is the control line, it's negative.
And here if you have no [INAUDIBLE],,
no migrations, for only the test,
like no control [INAUDIBLE],, that
means that you have invalid results,
and the test has to be repeated.
And here is an example of a representative result
we had from our experiments.
We performed that in five laboratories.
And what was interesting, it [INAUDIBLE]
being done in three laboratories in the field in Africa.
And here we performed that in only more than 250 samples.
And you can see, the results--
their sensitivity and specificity are not so bad.
For the sensitivity we have nearly 98%,
and for the specificity 95%.
Another interesting point of these kits
is that if you have the device here,
so you already use it for the detection of antigens,
you can also use it for the detection
of RNA, viral RNA, which are fixed
in storage under the membrane.
So you have to open, when it is finished, the device carefully.
You take the membrane, just get [INAUDIBLE]
the part where you put the samples,
and perform to an extraction with different protocol.
Here we use a protocol based on TRIzol extraction.
And you can use whatever techniques, validity technique,
for the detection of viral RNA--
RT-qPCR or [INAUDIBLE] PCR.
And this could be also done for the Sanger sequencing
by genotyping.
And here we have some examples which
were obtained in a study conducted in Chad in 2016.
And you see here, we have the samples
which have been tested directly in the lab or samples which
have been collected from the field
and sent to the lab at room temperature.
And here are the combined results.
And we were able to have a positive detection by PCR
when combining of nearly 96%.
Of course it was lower for the samples from the field
than in the lab.
And from these positive samples, which are in total 18,
we are able to make the sequencing and typing for 14.
These results have-- interesting results have been confirmed,
or at least observed, in several other studies.
Here I just put some example, which is
the first one here published.
This one is interesting, because it demonstrated also
that it's working for other lyssaviruses
and rabies viruses.
This is the one from 2016--
sorry, it's a mistake--
conducted in Chad.
This one was one conducted on interlaboratory trial
with RIDT.
And this one is the last one published by Kimitsuki in 2020.
And they're interesting, because it's
comparing other kits like this one from ADTEC, Bionote,
and even Elabscience.
They demonstrated a good sensitivity and specificity--
a specificity [INAUDIBLE] and specificity of 88%,
for example, for ADTEC and 96% for Bionote.
And what is interesting is that they also
use this skipping the dilution step
and increase that sensitivity.
There were some other studies, two studies,
that demonstrated also not good results.
For example, for the two studies here, that
evaluated some different commercial tests
and demonstrated that they have very great variation
between the kits, the manufacturers,
and it was [INAUDIBLE] in 2020.
So we've got just some key messages here for this test.
It's quite simple, rapid, and quite low-cost,
even though it depends how you manage to drive
the price of this test.
It's simple to perform and interpret.
And as you can see here, so it's suitable for field surveillance
applications.
You can use it in different various areas,
especially in Africa, for example, or even in Asia,
where we need careful validation of these methods,
because as [INAUDIBLE] indicated,
that we can add some potential batch-to-batch variation.
Also, depending on the manufacturers,
you can [INAUDIBLE] low quality for the test.
Today, it's not recommended by WHO and OIE
for working diagnosis, and this is very [INAUDIBLE],,
but from our experience, it's very interesting,
because it improves the motivation of people working
and who are [INAUDIBLE] viral area to be involved
in the fight of rabies.
And also, you can have some information from the field
to the laboratory.
And it could be done, also, to complete e reference
steps which have been--
which are done in a centralized laboratory, for example.
Thank you for your attention.
I will open for questions.
- Thank you, Laurent, for a very clear presentation.
I had a quick question in regards to your figure.
You're not suggesting that they should
be collecting the samples without gloves
on that hand in terms of biosafety?
When we go ahead and look at the diagram--
there, the diagram-- yes.
- Oh, yeah.
[LAUGHTER]
- Sorry.
- You're right.
- I couldn't resist.
- Yeah.
Yeah, sure, sure, sure.
Of course.
Even if you're vaccinated, sure.
- And we do have another question.
One of the participants asks how to avoid contamination
when working with PBS.
- In fact, we just keep-- we don't use PBS.
That's why in this protocol, we skip this step.
So we take directly, the sample directly,
in the tube here of the buffer.
And after, when it is finished, you
can get a closer [INAUDIBLE].
So from here, we don't have any contamination.
- Excellent.
Another quick question just came in.
One-- this is from Andre [INAUDIBLE],,
asking, seeing such a large variation in the diagnostic,
let's say, results between different brands
or manufacturers, is it feasible to ever
have this test approved by the OIE,
or would a specific brand have to be
approved if all the manufacturers are not
up to standard?
- That's a good question.
That's a good point.
Currently we are making a study of that in partners
in Asia and Africa to investigate that.
We are focusing on this Bionote kit.
But maybe other people have demonstrated that [INAUDIBLE]
it would be good.
And colleagues from FLI demonstrated
that some are not so good.
But I think some are not so bad.
For Bionote in our hands--
[INAUDIBLE] you were there--
if we confirm that this manufacturer or the quality
of this device are not so bad if you [INAUDIBLE]
some control for each batch, for example.
I don't know why they cannot be used or validated.
It's the same for, for example, for conjugated,
when you have a antibody against rabies,
you have to look at the [INAUDIBLE] check.
And you have to [INAUDIBLE] some batch.
But yeah, I think it's like a classical diagnosis method.
And if you make the same things, it's the same control.
So yeah, for sure, you have to validate them.
If you validated them before, and you are checking the time,
like a coefficiency [INAUDIBLE] and a comparison with FAT.
- Exactly.
And I think this is the whole issue with both OIE and WHO,
that it has to do with the science behind the technique as
opposed to a particular producer of--
a manufacturer of a conjugate, a kit, et cetera.
And it's just as you said.
It really comes down to quality control, and licensure issues,
and the regulatory authorities, which, unfortunately, are often
lacking.
It seems that this has really started
a whole suite of questions.
What's your recommendations for saliva as opposed to brain?
- Oh, saliva.
For the moment, some manufacturers
said it's good for saliva.
We test just-- you can see in the paper, other [INAUDIBLE]..
So it's very, very highly concentrated and very low
in quality, because you have very few antigens.
So we at all don't recommend for saliva.
- And how do you recommend storing
the kits with the samples for sending them
to a lab for later testing?
Room temperature, refrigeration?
- We test that [INAUDIBLE].
And it was taking a lot of time to come.
And we are still OK.
But for sure, like RNA, the more time you spend at room
temperature-- especially in Africa where it's quite hot--
you can degrade your RNA.
If you can keep that in a freezer not a [INAUDIBLE]..
One key point, also--
I think it's the same thing on the FDA paper, you know,
[INAUDIBLE].
The problem is that you can have some RNA.
If you store them at room temperature for a long time,
probably the RNA will degrade, so you will
have a little piece of RNA.
So it's good for qPCR, for example.
But if you want to make some genotyping,
this could be more difficult. This is what we experienced.
- And given the findings that you've
had about the elimination of the dilution step,
do you anticipate that the Bionote manufacturers will
change their recommendations in the directions for the kit?
- I don't know.
- I think that's probably the best answer.
- Yeah.
so maybe-- that depends on the data.
Maybe if we have some robust and reliable data on that, maybe.
But probably, they will not change
[INAUDIBLE] their quality control and test again.
Probably they will wait for other people to do that.
No.
- Very good.
Thank you very much for your presentation.
If you'd be so kind to stop screen sharing,
we'll move on to our next presenter who's
a relative novitiate in the lyssavirus group-- no,
I'm just kidding.
This is another colleague from Institute Pasteur
who'll be joining us.
It's Dr. Noel Tordo from the antiviral unit.
And also, maybe joining us today from the Pasteur Institute
in Guinea in Conakry.
Dr. Tordo, you have the floor.
- Thank you very much, Chuck.
And hello to everybody there.
Can you hear me?
- Yes, I think you can do your screen share, too,
to make the slides bigger.
So when you go to Presentation mode at the bottom right-hand
screen--
- I did.
- Ah, good.
- That's good.
That's good, right?
- That's good, thanks.
- OK.
So I will talk about an in-vitro ELISA test
to evaluate rabies vaccine potency.
This job has been done essentially
by Corinne Jallet, a technician in my lab in Paris,
which Antiviral Strategy Unit.
And as you said, Chuck, I'm now in Guinea,
in a real rabies country, leading the Pasteur Institute
that I'm building there.
So the purpose of the test is that animal welfare globally
is encouraging manufacturers and national control
laboratories to follow the 3Rs strategy for replacement,
reduction, and refinement of the laboratory animal testing.
And this consists to try to avoid using animals
and replace them by in-vitro, and WHO
and the regulatory authorities require the development
of these in-vitro approaches as alternatives to the NIH
for validating rabies vaccine potency.
So as you know, the lyssavirus glycoprotein,
so the rabies glycoprotein, is a trimer.
And you can see [INAUDIBLE] here,
the trimer form of the glycoprotein which
is on the surface of the virus.
And indeed, this is the more immunogenic form
to which more of the antibodies are going.
And G protein is the main viral antigen. Triangles of G
are the most immunogenic form.
And G induces neutralizing antibodies,
and G carries most of the antigenic sides.
This is a monomer of this trimer that you can see here.
And you can see in brown, then site II is located here, and--
site III, sorry, and in pink site II.
I will talk about these two sites later.
So we are talking about one antibody, which name is D1-25.
I will say D1 in the following lines.
And D1 has been obtained by Balb-C mice immunized
with inactivated rabies vaccine PV strain.
[INAUDIBLE] IgG 1 isotype, which is recognizing the site III.
And this antibody is neutralizing.
As you can see in this picture, when
you have a trimer of glycoprotein,
you need to have the transmembrane and the central--
and the [? superplasmid ?] part of the protein to maintain it.
If you cut it, and you have what we name the G ecto,
they are only monomers.
And you can see that D1, which is presented
in the red [INAUDIBLE],, is recognizing
the total G, so the triangles of glycoprotein,
so the more immunogenic form, and not the monomers.
So this ELISA test with a D1 antibody
has been tested since a long time versus NIH.
More than 1,000 vaccine lots have been tested.
There is a good concordance between the two
tests for vaccine potency.
You can see here in blue you have
the results of the ELISA test.
In red you have the NIH test.
And you can see that basically, from one
to the other of the vaccines, we have
approximately the same fluctuation
in the quality of the test.
And the ELISA test with the D1 antibody
is more sensitive, more rapid--
it's only one day-- and less fluctuant.
You can see that in this second part of the test,
the NIH test was done only once.
And you can see that we have a large fluctuation
for each batch, while the blue line is staying more
into the variation.
So rapidly, the different steps of the test
with the monoclonal antibody D1-25.
So the sensitization, passivation, ELISA test assay.
And I will talk about results and analysis of the results
after talking about security precaution, and conclusion
and perspectives first.
The sensitization.
So you can see that we take, of course, a plate, and to put
on the plate the antibody [INAUDIBLE] to fix
on the plate these antibodies.
And this is done by just putting--
diluting the antibody in carbonate buffer
at a concentration of about 1 microgram per mL.
3 hours' incubation in humidity, you
remove the supernatant, and dry with absorbent
paper for 5 minutes.
Then is starting the passivation.
So for the passivation, it consists
to avoid that any vaccine after will
have the nonspecific binding with the plate.
And so you fill up the plate with a passivating proteins,
like BSA for example, that are occupying nonspecifically
the plate.
And this is, again, the same type of experiment,
very simple, 30 minutes at 37 degrees in humidity.
Remove the supernatant, dry.
And this plate can be kept for approximately 3 months
at minus 20 degrees before being used for ELISA.
So the ELISA is done by two different steps.
First, the capture-- so after washing extensively the plate,
we are preparing serial two-fold dilution of the reference
vaccine and two-fold dilution of the tested vaccine.
And then we distribute 200 microliters
in duplicates of this dilution, of the reference
and the tested vaccine, plus, of course, the blank, which
is a diluent buffer.
Incubation for 1 hour at 37 degrees, discard supernatant,
wash.
And then the revelation.
The revelation is done within our hands.
We can change-- I will talk about that later on--
with the same antibody, D1, which
is conjugated with peroxydase.
So we grow this antibody, we incubate, we wash.
And then you put the substrate-chromogen solution,
which after incubation for 30 minutes at room temperature,
allows to go to the stop solution.
And you can read the optical density
at 492 nanometers with a spectrophotometer.
So this is a typical result. So you calculate the mean--
the optical density of each duplicate for reference vaccine
by subtracting the blank, of course.
And you plot the OD value on the vertical axis,
and the G protein trimer concentration
on the horizontal axis.
And you draw the reference curve.
And once you have that, you can estimate the tested vaccine
concentration, essentially trying
to look at the linear places here with your tested vaccine.
And you can estimate it in terms of glycoprotein, trimeric
glycoprotein concentration.
So we are providing a reference vaccine in the test,
which is a purified inactivated rabies virus vaccine,
the lot 09.
Its G protein content is well known.
It has been measured by different tests, BCA
and also by estimation on SDS.
And the result that we got are in the concentration.
And they can be transformed in equivalent international units
by comparison to the WHO International Standard
that we can test in parallel.
So just to say that we have to highlight
some security and precautions.
You have to wear adequate personal protective equipment--
coats, gloves, mask, glasses.
I'm sorry, she's not Corinne.
I didn't-- I wasn't able to capture the picture that we
have on the job article.
It's someone else, but it is basically what we can do.
When this is live virus that you are titrating,
you work on the biosafety cabinet.
And what I said since the beginning is
that we are removing many times supernatant.
And this supernatant, if they are contaminated,
should be put in a bleach solution, 2.5 sodium
hydrochloride, in order to avoid any contamination.
And of course, you do that by using good laboratory practice.
So some conclusions.
This ELISA test is recognizing the trimeric immunogenic form
of the glycoprotein.
And it is a suitable alternative to the NIH test.
And this is the first paper in which we showed that in 2014.
It is also able to discriminate sub-potent vaccine lots.
In this paper, it is shown that you can heat up, for example,
a vaccine for degradation and the level of the response
is going down.
It is used, indeed, by human and veterinary vaccine producers
since more than 20 years, despite the fact that it is not
recognized officially, because they are using it to follow
production before submitting their batch release
to their national authorities.
And this antibody can also be used
in competition for titration of anti-rabies antibodies
with the anti-rabies antibodies in equine and human sera,
for example, if you want to test the quality
of your anti-rabies sera.
Perspectives.
Working with monoclonal antibodies poses all the time
the risk that your vaccine strain
is disappearing because it has some mutated epitopes.
And I would like to come back on the first presentation
of this webinar, where, if I remember correctly,
one of the [? TW ?] BLB1 was not recognized by one of the--
I don't remember what is the conjugate.
And so this is the risk that you have
when you're working with monoclonal antibodies.
So in our case, we are using the same monoclonal antibodies,
the D1, recognizing the site III for both capture
and revelation.
You have seen that.
This method is applicable, of course, with other antibodies.
And this antibody is an antibody of the [? Wistar. ?]
And currently, an ELISA combining
D1 site III for capture and this [? Wistar ?] antibody
for site II for revelation is currently
tested by vaccine companies from many countries
in the world and the National Control Laboratories
under the umbrella of the European Directorate
for Quality and Medicine and Healthcare,
and the Biological Standardization Program.
And of course, the goal of this is
to end up with an ELISA which could be recognized
by the different authorities, national and international
authorities, and to have a sort of ELISA test
that will be proposed in the end to replace the NIH test.
So I have to thank, apart from Corinne Jallet Sylvie
Morgeaux at ANSM--
this is a French authority in Lyon;
Jean-Michel Chapsal from Sanofi Pasteur in Lyon;
Sabrina Kali from my lab in Paris [INAUDIBLE];;
and Pierre Perrin was indeed the guy
who created this 20 years ago, and he's now retired.
And thank you, Pierre.
And thank you for your attention.
- Thank you, Dr. Tordo, for a very clear presentation.
And obviously, these monoclonals in this particular recipe
has great utility for determining
the potency of biologics in the future
and hopefully putting to rest, finally, this NIH test.
I have one question.
What about application towards veterinary vaccines
that might be adjuvanted?
- So we are currently testing that.
One of the difficulties is that when
you are talking about human vaccine, as you know,
there is only a lot of vaccine strains
that are used up to now.
This could increase.
And so most of them are strains that
are recognized by D1 antibody or your antibody, Chuck.
By the way, it is possible that some vaccine strain, even
the human part, could be not recognized by one
or the other of the antibodies.
If we are talking about the veterinary world,
it's more complex.
We know that we have more diversity.
So not only the adjuvant, but also the diversity.
So we are making some tests now with some veterinary companies
in order to enlarge that to the veterinary one.
But the problem that I can see is
that we have to be certain that this
is covering exactly the strain that is of interest, of course.
- Very good.
And do you see any utility to the use
of one versus the two monoclonal antibodies used in the test?
Is there an advantage to just use D1
for both capture and detection?
- The kit that we are-- because we are selling this kit already
on MTA--
- Ah, so you're selling.
It so there's a potential conflict.
[LAUGHS]
- No, no, no.
We are selling since 20 years.
So it's not really a problem, because we are participating
also to this trial of having a test which will combine,
indeed, the two antibodies, the D1 and the [INAUDIBLE]
antibody.
The interest that I saw at the beginning in that one
is that it is recognizing this trimeric form.
And it is clear that this is a form which is
a more, let's say, immunogenic.
So if you propose a vaccine along the line,
you would like to have a vaccine that
is inactivated vaccine, vaccine which is still keeping
the native format, if you want.
And in this way, D1 is interesting.
But maybe-- I don't know if it has ever been looked at.
Maybe also, the other antibodies can specifically
fix on the trimeric form.
So no, I have no problem to me with another antibody.
- Thank you for your very diplomatic answer.
And it's very good to see you again.
Glad you're in good health.
Thanks for your presentation.
- Thank you very much.
- Thank you, sir.
We're going to move to our next presentation now.
We'll be with Jodie Jarvis.
Jodie received her degree in wildlife biology
from the University of Wisconsin.
She's been a member of the New York State Rabies Laboratory
since 2001 and the deputy director since 2019.
The New York state Rabies Lab is one of our premiere ones
here, now that we've gone over the pond
from Asia, from Europe, and now into North America.
New York, Texas, and California are
three of our major laboratories here in the United States
detecting--
having submissions throughout the country of sometimes
in excess of 100,000 samples--
surveillance, surveillance, surveillance.
And Jodie will give us some insights,
as we've talked about, that these tests are marvelous,
as long as we have the proper sample.
And so will be sharing with us some rabies necropsy techniques
in both small as well as mega or large animals,
to ensure that we actually have the proper sample
from the CNS for our tests.
Jodie, the floor is yours.
- Sorry, I had to unmute myself.
Do I have it on the correct screen?
Sorry.
Is that-- am I doing the correct screen?
- Looks good from here.
If you want to move it to Presentation mode
in the bottom right hand, you may.
- Is it on presentation mode?
I'm sorry.
This is my first time doing a Zoom.
Is the [INAUDIBLE] right one?
OK.
There we go.
OK.
Sorry about that.
So I'm Jodie Jarvis.
And as Dr. Rupprecht has said, I've
been working at the Rabies Lab for about 18 years now.
And all of the other authors on this paper
have been here for quite a while, too.
I'm actually one of the newer people.
And I wanted to give you just a little bit of background
on the Wadsworth Lab.
The Wadsworth Center is the main rabies laboratory
for New York State.
New York City has its own laboratory
that tests just human exposure cases within the city.
But we do all of the other testing
for the rest of the state.
We receive over 6,000 specimens per year.
In 2019, the year that this article is published,
the Wadsworth Rabies Lab tested 7,225 samples.
And during our busiest days, we can receive
between 1 and 200 samples.
And anything arriving before 2:00 PM gets same-day results.
So our testing methods follow the guidance
given by the protocol for post-mortem diagnosis of rabies
in animals by direct fluorescence antibody
testing, the minimum standard for rabies diagnosis
in the United States.
Basically, the takeaway from this
is that our laboratory has a lot of experience in necropsy
and doing it in a safe and time-efficient manner.
Every sample package we open is like a surprise.
We receive submissions from veterinarians
to private citizens, all with different capabilities
and resources available to them to perform a decapitation.
And that's why we have several different methods
dependent on the decapitation And just to note,
we do not ask private citizens to perform their decapitations,
but we can't control what they do on their own.
Most of our samples are delivered to us
by mail, UPS, or FedEx, in shipping boxes supplied
by Wadsworth Center.
Our large box is suitable for a large dog head.
But if larger, like most livestock, other methods
are needed to get the submission to the lab.
And that was the initial reason for doing this video--
a source for us to recommend to large animal veterinarian that
need to collect samples in the field.
We added the small animal portion
to show a safer alternative for tissue collection that doesn't
create as much aerosol as saws.
This picture shows the only type of tools
that we use for necropsy--
no saws or power tools of any kind.
From left to right, we have short blunt scissors;
forceps for manipulating smaller items without direct contact;
hammer and chisel for going through bone;
tenaculars for moving the head into position and holding
muscle when cutting with the knife--
and at no time do we directly touch the head
with our gloved hand; a carving knife
to cut through skin and muscle to get to the skull.
These last three items are not added unless we're
using the large animal method.
And they are a long knife ground down to a thinner blade;
a chemistry spoon; and a long-handled tablespoon
with the spoon part ground down to be just a little bit wider
than the handle.
In addition to well-maintained tools,
the technician has safety protocols to follow.
A minimum of eye protection, a mask, and gloves
are worn, as well as being rabies vaccinated.
In the lab, we actually wear scrubs and surgical gowns
as well, but in the field that might not be available.
So I know there are other websites available that
show how to do the brain collection through the foramen,
particularly for transmissible spongiform encephalopathy.
However, when asked by large animal veterinarians for advice
on how to perform it, we preferred
to have this video to [INAUDIBLE]..
Eliminates any confusion if there's
any differences in tissue collected or safety protocols.
The large animal method is best for livestock
with cervical vertebrae removed.
The requirement is that the foramen is large enough
for the tools to pass through.
Using the thin bladed knife, circle
along between the meninges and the brain stem.
When it feels loose, hold onto the brain stem with forceps
and insert the thin long-handled spoon
or the chemistry spoon between the meninges and the brain
stem.
You can then use the spoon to disconnect the brain stem
and cerebellum at the back end of the cylinder shape created
by the knife.
You can then use the forceps and spoon simultaneously
to remove the brain stem in one piece.
As our lab requires pieces of all three
lobes of the cerebellum for testing,
it usually requires going back in with the spoon
to scoop out the cerebellum.
The ventral method is an easy method
when the foramen is too small to do the large animal method,
and there are no vertebrae attached.
With this method, the ventral side of the head is facing up.
With the hammer and chisel, make two cuts,
forming an upside down V around the foramen.
You will need to bend that V down.
I personally use my chisel under the tip of the V
and tap a few times with the hammer.
But it can also be done with tenaculars or forceps.
It mostly depends on the thickness of the skull,
how much force you will need--
or how much force you will need.
With the curved scissors, with the blades closed,
that can be used as a scoop for scooping out
the cerebellum and brain stem.
It's sometimes left with the foramen,
and you can use forceps to pull that out.
The dorsal method is the most labor intensive,
but is most useful if tissues other than brain stem
and cerebellum is needed.
It is also used if the vertebrae are still
attached to the skull, hindering the other methods.
In this method, the dorsal part of the head is facing up.
The muscle needs to be cut away to expose the skull.
That is done with a carving knife using tenaculars
to hold the head in place.
Do not use your hands.
Once the skull is exposed, cut two horizontal cuts
on either side of the skull from the midline down
towards your workspace.
From that line, you want to make cuts
along the sides of the skull towards the back of the skull.
It will look like an upside down U.
Gripping the face of the U with the teeth of one of the--
with the teeth of the tenaculars,
you can use that to pry open the flap of the skull.
And once opened, scissors can be used to scoop out
the cerebellum and brain stem.
Of course, the specimens from the video and article
are examples of good submissions.
But this is the real world, and not
everything comes in perfect shape.
The animal may have full damage due to trauma, gunshot,
or poor decapitation.
The tissue may not scoop up nicely
because of decomposition.
Foreign objects like quills or packing materials
may cause obstacles.
Sometimes, it's just due to the species of animal--
goats and deer with their horns and antlers
in the way to lay the head on the workspace,
or some breeds of dogs or cats with more rounded skulls that
won't stay in a position, or full-body animals that
require more manipulation.
Those are the times when modifications need
to be made to those methods.
Regardless of which method or what modifications are made,
safety is still top priority.
With the methods demonstrated in this video,
safety is worked into the procedure.
By eliminating the use of saws, the amount
of potentially dangerous aerosols are reduced.
Using the ventral or large animal method
eliminates the need for a knife, reducing the risk
for a cut or puncture wound.
If dorsal method is used, make sure your knife is sharp.
Don't handle the head directly with your gloved hand.
Tenaculars will help to avoid cuts and punctures
from quills bone shards, broken blades embedded
from the decapitation, or other foreign objects.
And never hold the head steady with your hands when cutting.
Proper PPE, and if available the use of a fume hood or BSC
can help protect the technician from splashes.
Proper PPE is also essential for safety from aerosol and shards.
Remember that the safety protocols are not
just for rabies.
These animals could have a different illness,
and it's best to stay protected.
Thank you very much.
- Great.
Thank you, Jodie.
Quick question.
Do you recall, have you ever had a case in which the brain
stem was negative but the forebrain or the hippocampus
was positive?
- With the brain stem negative, you asked?
- Yes.
- No.
I think we've seen--
- Good.
- --where the brain stem is positive and not very much
in the cerebellum.
- Excellent.
That's the ideal answer, thank you.
And a similar question-- have you had situations where--
and oftentimes this is complicated
because of TSEs, mad cow, et cetera, chronic wasting--
where if you don't have a full thickness
cross-section of the brain stem, that a simple may be negative.
- We have seen in a lateral rabies virus in the stem.
They're very strict on having a full cross-section of brain
stem for that reason.
We've seen it in a cow.
And I know there is a picture of it in the protocol
that we followed.
I believe it's from a donkey.
- Yes.
And if we think of the transmission from peripheries
to the CNS, we can think of how that may be possible.
It's not very common, but still it has occurred.
And I also saw that we had a comment from our colleagues
at in Texas that the same as with your experience,
and between--
and which California was on as well--
between New York and Texas, the denominators
are overwhelming in the sense that, yes,
you can have other areas of the brain negative
but the brain stem positive, demonstrating
the anatomic pathologic criticality of having
the brain stem for diagnosis.
So I certainly thank you for pointing that out.
And a very excellent presentation.
I'm looking quickly to see in the Q&A.
I don't see any open questions there or in the chat.
No.
What are your criteria for rejecting a specimen
as not sufficient for doing, for example, DFA--
Direct Fluorescent Antibody testing?
- If we don't have a full cross-section of stem,
we'll still test it but we can't call it negative.
We'll call it [? unsat. ?] Same with if we don't have samples
from all three lobes of the cerebellum.
Decomposition is a tricky one.
- If you have samples that are, say, less than ideal--
if the material looks like ice cream
and it's obviously denatured, decaying, has a bad odor,
will you ever resort to molecular techniques
for diagnostics?
Or if it's not suitable for DFA, then you don't report results?
- So we'll run DFA.
If it's positive, we can still call it that.
If it's negative, we do have it--
do molecular testing on it.
Depending on the history will depend if we
can release the PCR results.
If it's more surveillance we'll do it.
If there's actual treatment or anything involved with a human,
then we prefer to steer on the side of caution,
have them be treated for an [INAUDIBLE]..
- Thank you, Jodie, for your very excellent presentation.
Demonstrating the comparative anatomy
is not only important but critical to rabies diagnostics.
If you don't have the right specimen,
you can't expect to get the correct true result. I'll ask,
if you'd be so kind, to stop screen sharing.
And we'll move on to our next panelist.
Erin Patrick is the Tennessee, Kentucky rabies biologist--
[CHEERING]
- Did I talk at warp speed?
- No, you did a good--
- Oh, I think you're need--
I think, Jodie, I think you need to mute your microphone.
[LAUGHTER]
Erin is the biologist for the United States
Department of Agriculture APHIS Wildlife Services.
She worked for Wildlife Services for 16 years,
including experience in Florida and in Kentucky
as a rabies biologist before moving to Tennessee.
Her current responsibilities include
implementation of the National Rabies Management
Plan in Tennessee and Kentucky to help
stop the westward spread of raccoon rabies virus variant.
She received her degree from the University of Tennessee
and the University of Arkansas.
She lives in Knoxville, where she's
going to be sharing some of her experience
on the direct rapid immunohistochemistry
test, which again demonstrates that you--
it's great to be a trained laboratorian
and with 10,000 hours of laboratory experience,
but the utility of some of these techniques such as we
heard for the lateral flow assay or now for the DRIT,
that even a wildlife biologist can do it.
Erin, you have the floor.
- OK.
Can you hear me OK?
- [INAUDIBLE]
- OK, great.
All right, good.
OK, yes.
So definitely, coming from a little bit
of a different background, not a lab background.
But I think what we do is obviously very important.
And it plays an important role with rabies management
worldwide.
And so it is very important for people without lab backgrounds
to have the ability to kind of help diagnose
and then make plans for rabies management.
And that's really what this presentation is about.
So I want to recognize my co-authors, who--
obviously, most of those, they work
for the National Rabies Management Program
and USDA Wildlife Services.
But-- I'm going to make sure--
OK.
So of course, I'm talking about the DRIT test,
which is the Direct Rabbit Immunohistochemical Test.
And I will refer to it as DRIT for the rest
of this presentation, because it's a lot easier to say.
And obviously, it is an OIE and WHO recognized alternative
to DFA, is becoming a very important component
of our national rabies management program.
If you have not had a chance to watch our video
that we did for JoVE, which kind of takes you
through the DRIT test step by step,
or if you've not had a chance to use it,
just really quickly, it is a relatively straightforward test
that takes just a couple hours from,
you can go from getting the sample in the field--
whether that's picking up a roadkill
or having a suspect animal--
going from the sample in the field
to removing the brain stem--
it's just like she just explained from the New York
Lab--
taking brain stem.
And then we make touch impressions.
Then the touch impressions are put
through a series of slide staining,
and the slide staining is only about an hour.
So start to finish, you can do the entire thing in just
a couple hours and have a result.
So the significance of DRIT, it's
been incredibly significant to us in the field.
But it's got obviously a lot of other significance.
And first of all, the accuracy is
comparable to DFA, which obviously is very important.
The sensitivity and specificity has
proven to be comparable in multiple studies.
One of the big positive things is that it is--
it can be conducted at ambient temperatures, and on benchtop--
or, as we like to say in the field of wildlife biology,
on a tailgate.
We don't necessarily use tailgates that often,
but meaning that we don't need to be at a centralized lab
to be able to conduct this test.
It uses basic laboratory techniques
that can be attainable and easy for people to learn.
We do not need specialized equipment
We can run this test, of course, with just
light microscopy, which is obviously a huge benefit.
And it requires training, yes, but a short time
to gain proficiency.
So we can take people who have a background, maybe,
in biology or natural resources-- of course,
wildlife biology--
and take them through the training in a couple of days,
and then they can go back into their field settings
and quickly have proficiency with this test.
So that's a huge benefit.
And overall, it's inexpensive, which
is because of the simplicity of it, I guess.
The DRIT test does have the ability
to recognize all variants of rabies.
And I do want to point out that we and this is USDA Wildlife
Services, we use this as a complement
to public health surveillance.
This is not for--
we don't use this to, of course, diagnose a rabies
in human health and safety cases or cases
where an animal has exposed a human or domestic animal.
We use this for enhanced rabies surveillance
to get a better, bigger picture of what's going on.
And over the years, the DRIT has really
became a highly critical tool for us
to consult to enhance rabies surveillance.
So it allows us to conduct this enhanced rabies surveillance
to kind of piggyback off the public health surveillance,
and it allows us to have real time over the management
decisions that can be made from this test.
So why do we want to do that?
If you, excuse me, if you're familiar with our work then
you probably understand, but if you're not,
our agency works to prevent the spread of terrestrial rabies
in the United States and to eliminate rabies variants
at local, regional, and national level.
So that's one of our big goals with the National Rabies
Management Program.
To do this, we will distribute oral rabies vaccines
across a very large area.
17 states where last year had oral rabies vaccines
distributed, and over time we've distributed over 9 and 1/2
million rabies vaccines to work on this goal of preventing
and eliminating rabies variants.
So obviously, there's a lot of time, effort, personnel,
and money going into getting these vaccines out
on the landscape.
So it's very important that we get these vaccines out
in the right places.
So the test has allowed us to gain confidence and make
management decisions with confidence over the years.
So DRIT by the numbers.
So how much are we talking about Here?
First of all in 2020, USDA Wildlife Services conducted
over 7,000 tests on samples and about 2.3% of those
were positive.
Since the beginning of when we started doing DRIT,
we have tested over 110,000 samples
of using DRIT and about 2.1% have been positive.
So we've been able to get a lot of data point
over the years that has allowed us to make our management
decisions with confidence.
You know, a lot of times I get questions from people saying,
well that's a lot of negatives, that's
a lot of time and effort, you guys
are spending on the negatives.
And I always tell people that the negatives when you're
doing, of course, surveillance, are just
as important as the positives, so those negatives have allowed
us to help make our management decisions just as
much as the positives have.
Today, we've had 87 Wildlife Services employees that
have been trained to do DRIT.
And again, most all of those are field biologists or field
technicians, and they've been very successful at it.
We do confirm our positives and of course, number
of negatives with the confirmation lab,
and we also do proficiency testing.
And so, it's been a very helpful test to learn,
and it's been one that has been totally doable by the field
biologists including myself.
We have 24 field biologists that are currently
performing the test.
And the other big thing that has been kind of a big deal to us
to the field is that we don't have
to have the centralized testing locations in order
to do the test.
We conduct these tests in 15 different facilities currently.
The majority of those are in office spaces.
So in office spaces that are USDA Wildlife Services offices,
and we're able to basically turn a room of that office
into a DRIT testing location.
Those were great.
We also work with cooperating agencies
at times, where we will work maybe
with a University or a public health lab or something,
and we will do our there.
And we also have a couple of mobile labs.
And those mobile labs kind of take that you
can do this to the field.
The second picture from the top is a picture of one
of our mobile labs, and we have this dropdown table
and you can do the entire test there without any power.
The only time you would need power
is at the end, when you are reading on a flood.
So we have the ability to take either of these mobile labs
into a situation or an area that we
need maybe a lot of testing with a quick turnaround
or just testing in general with a quick turnaround.
And so that has been a pretty big deal and helpful part
of that we do to test.
So I want to provide just an example
of how we are using today.
Literally, today to make these management decisions.
We've recently had a couple of positive cases of rabies
that were detected through our enhanced rabies surveillance
test.
And they were areas that we hadn't had rabies
quite in those areas before.
So we're able to--
first of all, we pull in the tests
that we've done in the past couple of years
to kind of get an idea of what the surveillance is like
and where we need to kind of look.
And so, as a field biologist, that's helpful, first of all,
to pull in all the negatives.
Again, those negatives are really important when
you're looking at rabies and potentially a different area.
But pulling in all of those samples
that we had done over the last year or two,
and then making a plan of where we need to kind of focus
and where we need to try to get some additional samples.
And in this situation, we can potentially
pull one of our mobile labs over,
and we could have people out in the field, which are out
in the field today, looking for sick animals,
trying to get some surveillance numbers up.
And I mean, we could literally have an animal
and have a result within two hours.
So that's an example of how we are
using DRIT to make management decisions,
because this will could potentially
change how we end up putting the vaccine out and on the ground.
So it's definitely a test that we are utilizing every day
both in the field, and then taking
that data back to our planners and the people that
do the modeling, and so we're able to work together
to work towards our goal of elimination.
So I will summarize, there's really
no changes to the standard operating procedure
if you have seen the video or read our document.
Number of changes.
We're still doing things the way that we have been doing them
and they work well, so we're thankful for that,
and we'll hopefully continue that.
The ongoing challenge has been availability and reliability
of the antibodies to the mouse.
And overall, the drug test has been
and is a safe, efficient, effective, and affordable way
to provide rabies testing.
It's allowing field biologists to make science based,
data driven management, which is obviously we're
using this every day, and it's really
became a game changer for effective Wildlife rabies
management.
So with that, we definitely want to thank
we work closely with a lot of state programs
within Wildlife Services for people that are conducting
the DRIT, so we want to obviously acknowledge
them and their hard work that they're
doing not only in the lab but out in the field
to collect the sample.
And then CDC and the Wadsworth, and wistar.
And then we have so many cooperators that are helping us
with samples and have been for quite a while now,
so we always want to acknowledge those people as well.
So with that, I will take any questions.
- Super thank you, Erin.
And we do want to mention that one
participates in proficiency testing for DRIT the same as
for DFA.
And so you participate in national DRIT proficiency
testing, don't you?
- Yes, we do.
We do proficiency testing just like you said, like would be--
I think, twice a year now, we do not get the blood samples
and do that.
And overall, we've been extremely successful with that.
Yeah.
So yeah.
- Great.
- It's been a very successful thing.
- And how do you considering their reportable nature,
how do you share your results from
enhanced surveilling with local or state public health
officials?
- Yeah.
That's a good question.
So when we have positive, a couple of different things.
One is we get them confirmed by a confirmation lab
either Wadsworth or the CDC.
So they're going there for confirmation.
And once we get confirmation from their,
each state works independently, I
guess, with their state health lab in Tennessee, Kentucky.
When I get positive, I send them right on to the state lab.
So we do work in conjunction with our state
public health lab and public health agencies,
so to make sure that everything is matching up.
And so we're definitely in communication with them
and that goes both ways.
So they're communicating with us and us with them.
- And I noticed that these DRIT results are now
officially recognized and appear in
our annual national surveillance report that CDC puts out.
So it--
- Yeah.
- --demonstrates the utility that Wildlife Services
biologists and others have been doing.
And I'm happy to point out that although--
obviously, who wouldn't rather use a lab
when you don't have to use the field,
but the DRIT actually started in parts of Africa,
not only for dogs but a variety of wildlife.
DRIT's been done all over the world,
and similarly it's not so much a kit,
because as you've demonstrated in your protocols
and from your video, that the variety of chemicals,
fixatives like Formalin and washing with Tween PBSC.
Most of the parts of DRIT scalpels for collecting
the correct brain stem sample, biosafety protocols,
most of these things are readily available.
So it's not a kit per se as you would think formerly,
because one of the questions was, well,
how can we get a DRIT kit to X. Or one has to do
is go ahead and read the protocols.
There's a variety of protocols out there.
DRIT has been recognized as a companion
to DFA for antigen testing.
And really, I would say, the most critical part
of the DRIT or the antibodies, and those antibodies
can either be monoclonal.
And most of the WHO collaborating centers,
some of whom are on this call--
CDC, CFIA, our colleagues in Europe.
There are monoclonal's that are available that
have been able to demonstrate their utility in the DRIT,
and our colleagues in Southern Africa
have demonstrated the utility of polyclonal antibodies
and performing the DRIT.
Once you've demonstrated sensitivity and specificity
of your biologic, all the other parts, the slides,
the fixatives, the washes, the immunohistochemical stains,
the microscopes, all of those are commercially available.
So you don't need a kit, you just need the ability to read,
the ability to learn, the ability to watch,
the ability to train, and the ability to do.
So I thank you very much for pointing out your experience
with us today, and I hope you'll stay on for the roundtable
and would you be so kind to stop your screen sharing,
so we'll go on to our next panelist.
Our next panelists will be closing up the presentations
before our discussions, and it demonstrates
that we've come full circle from talking
about the utility of laboratory testing,
to protocols and techniques, for diagnostics
in and out of the lab, in the utility of oral vaccination,
not only for Wildlife, but obviously, for the future.
In real time, there have been some very nice recent papers
about oral vaccinations applied to dogs, for free ranging dogs,
for the utility in the ZBT, to 0 by 30 elimination
of human rabies caused by dog rabies.
And so our next panelist, Harry Bernstein,
a biologist, also with the United States Department
of Agriculture, a National Wildlife Research Center,
working on the rabies project as a Wildlife biologist,
had a lot of time and experience in Puerto Rico, working
on another rabies reservoir, so beyond dogs, raccoons, skunks,
foxes, bats.
We have a non-indigenous imported reservoir vector
in the Caribbean into Puerto Rico, the mongoose.
And Harry will be talking about some of his experience
and thinking about biomarkers that
can be incorporated in debates for oral vaccination
to gain some insight as to the utility of such programs
in reaching those free ranging animals in the field.
Harry?
[NON-ENGLISH SPEECH]
- Thank you very much.
There we go.
See here-- can everybody-- pardon me.
Can everybody see the slide without my notes?
- I can.
- Fantastic.
Good morning, good afternoon, and good evening,
depending on your time zone. .
Thank you very much for including me
in this talk today.
I'd like to start off by acknowledging my co-authors
here from the Hawaii field station, as well as the rabies
project leader of the National Wildlife Research Center, Dr.
M. Gilbert, oour collaborators from Germany,
and in particular, Mr Stephen Volker, who is the chemist, who
refined this method that I'm going to be describing today
on the use of Iophenoxic acid, which I'll refer to
as IPA, not to be confused with the beverage in mongoose serum
using liquid chromatography and mass spectrometer techniques.
For those unfamiliar, Iophenoxic IPA
is a white crystalline powder, and there are multiple--
pardon me, derivatives that are typically
identified by the branch chain.
So there's the methyl, ethyl, propyl, butyl, isobutyl,
and pentyl IPA that are available.
Historically, the use of IPA as a biomarker was relied
on measuring an increase in--
- Excuse me.
- --an idea-- yes.
- I see one of the charts, it said, are you
on PowerPoint mode.
So please put slides on PowerPoint mode
for presentation.
Yeah, there you go.
That's great.
Thank you.
- Is that it?
- Good display settings again, in the PowerPoint,
at the top middle.
- At your top middle, Harry.
- I had this earlier.
- If you press From Beginning--
and then, here, Display Settings.
At the top middle.
- 1 over.
Display Settings.
- I'm unfortunately not seeing that on my screen.
- Oh.
- Why don't I stop sharing and try it again,
is that all right?
- Sure.
- Should be able to go to share screen.
- OK.
Let see here.
Can share screen.
When we go here--
- And then bottom.
Oops.
You've just jumped over again.
Do you see at the top, it says, Show Taskbar Display Settings
or End.
- The taskbar, I have that.
- Do you have Display Settings next to it?
- No, that's what's so odd.
We did earlier.
- Yeah, I don't have control of it.
Central, come in, do you have control of his screen?
- No.
That would be within his computer
that that needs to be changed.
- Yeah, but I don't think he can see that.
So how do we go from this to just presentation mode resume?
- Maybe-- oh, there we go.
- Oh, there you go.
- How's that?
OK.
My note--
[INTERPOSING VOICES]
- --OK.
So just to backtrack a moment, I can only
suggest that IPA, there are multiple derivatives,
and they can be previously, they were used as a biomarker,
and that was dependent on evaluating blood iodine
levels, which required a pre and post
baiting sampling to look for that elevated iodine levels.
In recent years, there have been chromatography
and mass spectrometer methods developed,
where we can actually detect these IPA
derivatives independently in serum,
individually unquantified.
Ethyl IPA is, as you can see on the right side,
it's the most common form and derivative of IPA
that's out there right now.
And in our case, we used ethyl as well as methyl.
And we refined to little the process,
the liquid chromatography mass spectrometer method
for isolating and quantifying the concentration of IPA
derivatives in moongoose serum.
And then we took this into the laboratory in the field
as well, and used it as a biological marker
for oral rabies vaccination using placebo baits.
So again, thanks to Stephen Volker
for providing a layman's version of this extraction procedure,
since I'm not a chemist, provided in language
that I can understand, and hopefully
share with you clearly.
So this is a very selective and highly sensitive
method to quantify multiple Iophenoxic acid
derivatives in moongoose serum.
So an overview of the extraction procedure
was the serum was fortified with an internal standard.
Now in our case, we used propyl-IPA
because it was not something we were going to use in the field.
If you're using an internal standard,
you want to use something that is
separate from what's going to be used in your actual baits.
The serum was fortified and extracted
with an acidifide acetonitrile.
Following that, excess sodium chloride
was added to the sample in order to phase-separate acetonitrile
in the water.
Then a portion of that acetonitrile phase
was removed from the sample, and essentially dried
using a flow of natural gas and clean water bath.
This dry extract was then reconstituted with a 75% to 25%
ratio of water and acetonitrile, and transferred
to the high precision liquid chromatography
vial for analysis using liquid chromatography and mass
spectrometry.
- So to look at the workflow of this procedure--
and this is the final procedure that we ended up
using after several methods to try to refine everything.
We used 50 microliters of mongoose serum.
And then we applied 50 microliters
of our internal standard-- in this case, it was propyl-IPA.
We added 950 microliters of acetonitrile
with 0.5% [? trichloroacetic ?] acid.
The sample was then vortex mixed, and then
that excess of sodium chloride--
150 milligrams-- was added to that sample.
It was mixed again, centrifuged, and then add supernatant
800 microliters of supernatant was transferred again
to another vial.
Following that, the solvents were
removed through that drying process
that I described with the liquid nitrogen in a heated water
bath.
And the sample was reconstituted.
After this entire process, it was
transferred to a vial for the chromatography and mass control
analysis.
So basically, the entire process is
reduced to a tiny vial, which gets
inserted into our quote, unquote, black box machine.
And unfortunately, I don't have my notes in front of me.
I can't tell you the exact model of this.
And it was basically set to run overnight.
We had multiple samples to run, and it was set
to run for a 24-hour period.
This provides a nice example of the chromatogram that
resulted from the analysis.
And the analysis of individual samples
actually took less than 2 and 1/2 minutes to perform.
And on the y-axis, we can see the actual molecule count
at each point in time for each derivative.
So we have our methyl-IPA, and then our ethyl-IPA,
and that our standard or reference standard,
the propyl-IPA at approximately 1 and 1/2 minutes in.
So this provides some really interesting information
from a single sample.
We can differentiate different derivatives
of IPA, which became very useful, which I'll
be describing in a few slides.
The method validation for this particular extraction
and quantification is we have a detection
limit for methyl and ethyl-IPA.
For methyl, it was 0.01 micrograms per milliliter,
and for ethyl, it was approximately 0.14 micrograms
per milliliter.
And that is just the detection limit.
That is a presence or absence.
Following that, we also had a quantitative limit
where we could actually quantify the concentration
of these derivatives in that serum sample.
Quantitation limit for methyl being approximately micrograms
per milliliter.
Ethyl IPA was higher at almost 0.5.
So validating this method, we took clean mongoose serum
that had no known marker in it, and fortified them
with three concentrations or three levels of methyl-IPA
and ethyl-IPA here to run the analysis.
And the accuracy range for this ranged anywhere from 95% up
to 113%.
And that's very common in this type of analysis
for that accuracy range to be below and above 100%
with the mean being ideally around 100%.
And the coefficient of variance, which
is simply the standard deviation above mean was less than 5%.
And that provides and the standardization of variance
regardless of the accuracy.
So it's suffice to say that this process was extremely
accurate in quantifying the concentration of IPA residues
in mongoose serum
We conducted a series of laboratory trials
where we offered captive mongooses,
various concentrations of both ethyl and methyl
iophenoxic acid in a placebo or rabies vaccine bait.
A bait is approximately 5 centimeters long,
weighs about 2 to 3 grams.
It Consists of a foil blister pack,
in which there is a liquid, which in the field
would consist of the vaccine.
And an external bait matrix.
In this case, it's an egg flavored external bait matrix.
So in the laboratory we offered these baits
to captive mongooses and then collected a serum sample
prior to bait offering, as well as on day one following bait
offering.
And then weekly for between four and eight weeks.
And you can see more details about how
these concentrations that are presented in the paper.
You can see how they were explained and calculated.
Looking at the ethyl IPA concentrations
over approximately an eight week period,
as you can see from day 0 there was no concentration,
which is not unusual.
That's what we expected to see.
But what was really unexpected is
looking at the longevity of this marker in serum samples.
Now, this is highly variable across species.
In some species it's metabolized within a few days
and I believe in the dog it can be up to a year.
And we were very surprised to see how long this persisted,
this ethyl IPA marker persisted in mongoose samples.
And look at the trend, there's not
much of a decrease over an eight week period
regardless of the concentration and the method
by which that product was delivered.
Either it was in-cooperated into the external bait matrix
or within the internal blister pattern.
So seeing that longevity, we also
evaluated methyl IPA to see if it was consistent.
And we sampled for four weeks for the methyl IPA.
And you can see from the figure, that there was also very little
degradation over time.
And the curves are pretty flat going across
for that four-week period.
This had a kind of unintended benefits
when we conducted our field operations.
Typically, what we do when we conduct
more rabies vaccination in the field
is collect a blood sample, a serum
sample approximately five to six weeks following vaccination
to look for rabies virus neutralizing antibodies,
as well as a biomarker.
And in this case, we were hoping to use iophenoxic acid
as that biomarker.
We conducted a field application, a field trial
with a placebo bait.
There was no vaccine in this bait
but there was 2.8 milligrams of ethyl IPA in the fall of 2016.
And then we used methyl IPA for a follow up trial
in the spring of 2017.
Baits were distributed in South West Puerto
Rico in a subtropical dry forest habitat at an application
rate of 200 baits per kilometer square.
We gave them mongooses about five days following
bating to find the baits and consume
them, hopefully, consume them.
And that we conducted trapping daily
for 10 consecutive days after that,
taking a blood sample from each animal
that we captured every animal.
Looking at these field results from the fall of 2016,
we sampled 87 mongooses across all our study sites.
55 of those or 63% of those animals that we sampled,
were positive for ethyl iophenoxic acid,
suggesting that they had in fact consumed baits.
In the spring, we captured 123 animals and sampled them.
84 of those were positive for methyl IPA.
It's about 68%.
Now, if we pull these samples and we pull back and look at it
from a larger viewpoint and account for recaptures
between seasons, we sampled a total
of 180 individual animals, unique animals.
And 134 of those across those two seasons
were positive for either one or both
of those iophenoxic acid revenues.
And that's a portion of approximately 74%
bait consumption.
And this is where it's very interesting.
The longevity of that ethyl IPA worked to our advantage.
Because we were able to in the spring of 2017,
capture animals and find them positive
for ethyl iophenoxic acid that we had not
captured in the fall of 2016.
So that helped bolster our sample size for that season.
It also allowed us to determine that there
was a certain proportion of animals
that we captured in the spring that
were positive for both biomarkers,
suggesting they had consumed baits during both time periods.
Looking at the proportion of bait consumption from the study
is very encouraging when we consider
that some of the literature in some species
suggest that a vaccination rate of 60% to 80% to 90%
annually may be required in order to interrupt
virus transmission in certain species.
So this is a new technique for the species, certainly not
a new technique. iophenoxic acid, as I mentioned,
has been used as a biomarker for a variety of reasons
in the literature.
Just to wrap up and to conclude that this study,
we were able to refine our method
to evaluate the IPA concentration
and be able to quantify it in mongoose serum.
And this is also important for use as a biological marker
to estimate bait consumption in particular in areas where there
is a natural rabies virus exposure background not
from rabies virus exposure, where you have naturally
occurring rabies virus neutralizing antibodies making
the evaluation of these antibodies post vaccination
very difficult because it's very difficult to determine
whether those antibodies came from the natural exposure
or from vaccination.
So IPA has proved to be a useful biomarker
to estimate this consumption in free ranging mongooses
in our trials in Puerto Rico.
And with that, I'll open it up to any questions.
I apologize for the technical difficulties
here at the beginning.
- Thank you Are.
One quick question, how easy or difficult
is it to perform this analysis, I'm
thinking in particular for some of our colleagues
around the globe as to cost, experience, technicality, et
cetera?
- The extraction and analysis is fairly straightforward.
The work flow is a fairly standard protein extraction
analysis that's been slightly modified using the salt
and acid additions in order to be able to concentrate
the iophenoxic acid.
It would be costly to farm it out to an external laboratory
but it can be done in-house with a little bit of training
and with the refined technique.
And we've done the iterative process to refine it.
I think it's fairly straightforward.
It's just a little bit time-consuming
to get the samples prepared.
- And based upon the use of biomarkers,
either iophenoxic acid or others together
with say, enhanced surveillance, how would one
go about determining how much vaccine would be needed
per area based upon these insights,
whether it's a mongoose, a fox, a raccoon, or a dog?
- So how much--
if you could rephrase the question to--
- Sure.
So in other words, you find out that 65% of your mongoose
have evidence of consuming a bait based on iophenoxic acid.
Or based upon tetracycline, that we find out
that 70% of our foxes are eating a bait.
Or based upon enhanced surveillance,
we find out that X proportion of raccoons, or skunks, or foxes,
or dogs, are positive.
How would you combine these laboratory techniques
based on biomarkers, serology, enhanced surveillance.
And I'm opening it up to all the participants
based on the question.
How would you use these laboratory techniques
to help determine what your bait density might be for a given
species in a given area?
- Right.
So one of the ways we determined the actual application rate
was to look at the population density of the target species.
And that helps drive as well as our RV
in raccoon, that helps drive the application rate looking
at that initial ecological information.
This is particularly useful, iophenoxic acid
is particularly useful in areas, somewhere like Puerto Rico
where there is a naive population.
But there is a level of background exposure,
so you would have to conduct a free vaccination
survey to look at what that background is just like you
would do in ORV application in the lower 48 states
as well as a follow up sampling to look at the serum conversion
rate.
But you also would look for that biomarker
to help distinguish animals that may have been previously
exposed, either through previous vaccination in the lower
48 or through natural virus exposure in naive places
like Puerto Rico where there is a background level of serology,
is that help?
- Sure.
So based on your experience to date, what do you think
might be a reasonable density of baits for distribution
in the prevention and control of rabies
and mongoose in Puerto Rico or elsewhere in the Caribbean?
- Right now we're starting with 200 baits per square kilometer.
That proved to be at this particular study site, a very
good application rate.
We do have plans to conduct a follow up trials
in slightly different habitats.
And that, excuse me, that bait application rate
may have to vary depending on the habitat type, which
can drive mongoose population densities.
So in areas where you have very high densities
you may have to increase that application
rate because the non-target species that may consume them.
In Puerto Rico those tend to be predominantly
nocturnal rodents i.e. rats and/or mice
that may consume those bats.
That has to be taken into consideration.
So it may vary by habitat.
But at least to start with our research
suggests so far that 200 baits per square
kilometer maybe an adequate application
rate for the species.
- Any guesstimates on when oral vaccination
studies, trials with vaccine may actually begin in Puerto Rico?
- We are planning on conducting a pilot trial
in a limited area in North Central Puerto Rico most likely
in the spring of 2022.
We had hoped that we might be able to pursue it this fall,
but given the COVID pandemic and a lot of other circumstances,
it may be delayed.
But we do have plans to conduct that trial in a limited area
in next spring using iophenoxic acid as a biomarker
to again, distinguish between that natural exposure
and vaccine uptake.
So we're very excited about it.
It's something that hasn't yet been done
and I'm very proud to have been part of this team conducting
this research.
- Great, and I'll broadly open this up
to all of our participants beyond free ranging
carnivores from dogs, foxes, skunks, raccoons, raccoon dogs,
ferret badgers, et cetera.
And you probably have seen the literature, her discussions,
what do you think about the concept
of oral vaccination of bats?
- And Are or Erin or Noel or anybody
is free to take that up.
You look like you have some thoughts,
but you don't want to say them.
- I do.
Do you remember--
- Go ahead, Noel.
- Do you remember that we published a paper on that let's
say 20 years ago.
It was with vampire bats and together with Alvaro in Mexico.
- Yes.
- Yeah it was--
it was done in the lab and we captured bats.
We gave them, so I think it was the Merial vaccine
if I remember correctly.
And--
- I think it--
yes, it was vaccinia recombinant, yes.
- Yeah, it was the vaccinia recombinant.
And they got some positive antibodies and that has
been published and that's it.
I remember to have given a talk on this
just for joking, a talk of this in class, America.
And in front of me was a specialist
of a bat rabies from Argentina.
And he was looking at me saying that you are totally crazy.
We know to do with a bat rabies or vampire
rabid in South America.
We are just vaccinating cows around
and we are just staying the same like that.
And after a while there is a certain proportion
of the cave, which is dying.
And the rest is no more rabid for 10 years.
This is what he replied like a sort
of self vaccination of bats.
So this is where we stopped indeed.
We didn't go ahead.
But it was money given by the European community.
- Yes.
It seems how what's old is new.
And if any of our colleagues from Europe or Latin America
are on, particularly one of our colleagues Daniel Straker,
this has now become novel again to try to think
about vaccines for vampires.
But not only as we did, meaning the passive transferable,
but now they're thinking about transmissible vaccines.
And so trying to find unique viruses only hopefully,
secluded in vampires and putting in your genes
and infecting the vampires and having infectious clones
go from vampire to vampire to vampire.
What do we think about transmissible vaccines
for bats?
- Who is thinking?
[LAUGHTER]
- Erin, Are what do you think?
Any of the other panelists?
I mean, if we're going to think about transmissible vaccines
for vampires or other bats for rabies,
lyssas, other diseases, why aren't we
thinking about transmissible vaccines, viruses
for carnivores?
It would certainly make it easier
than the 200 baits per square kilometer for a mongoose?
- One thing that's interesting about this
is, and I'm not a bat guy, so I'm going
to have to punt on that one.
I'm very, very new to anything bat related.
One thing that is interesting that we found
is with this background exposure in mongooses
there is a certain level of having
positive animals in some areas and that can also help
guide that vaccination process.
Do you decide that areas that have a high background serology
are a low priority for vaccination
because there may be some natural immunity,
or is it a higher priority to kind of bump up
what's already there in order to cross
that threshold required to interrupt a virus transmission?
And those are some of the questions
that we're still working with answering.
- Yes.
And I think this issue of transmissibles
is worth days, if not weeks of more discussion.
And I am wondering what either any of our other panel members
may think about that topic or do they have any questions for any
of the presenters today.
It's open to our colleagues from Asia.
I know it's probably getting kind of late for you.
If you have any questions or comments or New York,
or Luka, or--
yes, Noel.
- If I remember correctly, behind this story
of vampire bats that we tried to vaccinate orally,
the idea behind was to sort of take
a certain number of those bats and to put them
on the body, the vaccine, I mean the recombinant vaccine
in order to send them back in the cave for grooming
each other.
And so in the sense of what you were mentioning before,
allowing them to inter vaccinate, let's say,
to transmit the vaccination.
So it was the basis of the idea at the time also.
Maybe we have to rethink.
If I can remember the things you knew two years ago when
we were using a vaccine for humans,
a recombinant vaccine the same way
that we are using now in COVID didn't exist.
Some cases were existing in vaccinating
for example for rabies, dogs with DNA vaccine,
but apart from that this new technology didn't exist.
And we were staying at the inactivated or attenuated
vaccine.
And you have seen how a pandemic and before a pandemic, how
Ebola for example as the proper use of VSV
for human use or other new vaccine for human use.
So maybe as you said, yes, it was old things.
But we have to see old things with new eyes that sometimes
help us to go ahead more rapidly.
- So given that point and given how incredibly rapidly
the world responded to the current pandemic
with new vaccine applications, although some of us
are not crazy about having to store them
at minus 20 degrees, which of the techniques, technologies,
both from the New vaccines as well as the ones to come,
do you have any particular excitement as to how they
might be applied for rabies?
- Move along the audience.
- Go ahead.
You have the com, what do you think?
Or do we really need them?
I mean, given what we've already been able to accomplish.
All developed countries have eliminated
dog rabies, developing countries, if we look
at the Americas look at the case of Mexico, have eliminated
not only human rabies transmitted by dogs but dog
to dog transmission.
So it is obviously attainable in not just the ultra wealthy
countries.
And I'm saying this for any of our participants
who are still on.
I know we had quite a few from the Indian subcontinent,
from Pakistan, from Africa, et cetera.
Do we need new vaccines for rabies
given what we've accomplished so far in either humans
or domestic animals?
Look at the new WHO recommendations.
We don't need to have daily inoculations intracutaneously
with nerve tissue based vaccines.
We can use modern cell cultured dose bearing
and do post exposure in a week.
Do we really need new vaccine-- or is this just an excuse?
Is this an excuse for us to drag our feet or can
we accomplish the tools with what we have now?
- In my opinion there are two things.
Never stop thinking, dreaming, proposing new things.
So this is one point and we are scientists
and we should do that.
The second is that, I'm in Africa now, I'm in Guinea.
In Guinea we did the sort of overview of what
was available, not available.
The vaccine of course, it was it was
a limited amount of vaccine, we are trying to improve that.
I'm talking only about human vaccine for the moment,
I'm not talking about dog vaccine.
If we are looking to the WHO recommendation,
WHO recommendation is 95% of the case.
This is a bleeding bite, so you have
to make immunoglobulin and a vaccine.
There is not a single drop or immunoglobulin in Guinea.
When someone from the embassy recently was bitten,
I was forced to go to Morocco to find some
in order to give them.
So we are two sets, two ways.
One the scientists are dreaming new, this is our job.
And after there is a recommendation, and sometimes
we have the impression that the recommendation are followed,
while in many countries.
They are not followed simply because the I mean
monoclonal antibodies you remember,
we were saying that 10 years of looking at monoclonal antibody
to replace RIG or eRIG.
There are still waiting on let's say developed country.
But a lot of developing countries
have not reached this level.
So we have both to complete what is already done
and what is recommended and to dream for future.
- Yes.
And on that topic of human and the dilemma
of post exposure in RIGs or monoclonals,
we have that disparity between a small fraction of the world
uses RIG, the majority of the world uses vaccine only.
And so we've either been incredibly fortunate and lucky
or incredibly blurred in our surveillance.
Because we can't explain.
We don't have massive post exposure failures
even though the majority of our patients
out there are only receiving vaccines.
And although I highly agree with you never stop dreaming,
look where we've gone from since past Doris time.
I also wonder from this new wave of both diagnostics
and vaccines because of COVID, will we
become even more neglected and not
have the ability of even use the proven tools that we
have because of supply chain interruptions.
Because of issues of stay at home.
Because of problems of not having any money
left over for everyday things once this particular pandemic
is curtailed.
This is one of my concerns.
Go ahead, please.
- Sorry speaking to the do we have the tools available,
I think, yes.
But I also think that continuing to find more efficient tools--
and since the mongoose has been the primary area of my work
over the last several years, previous attempts
at vaccinating mongoose using the currently available
and licensed vaccines at the time, at least of the doses
in which they were delivered, were
found not to be immunogenic in that species.
And with the advent of these newer vaccines,
we have found them to be immunogenic
at least in laboratory trials.
So continuing that work, especially
in some species that are understudied,
and in particular Puerto Rico where
they are a rabies reservoir that interact
with domestic animals, which can spread that to humans which
happened in 2015, which is the first documented
case of a directly mongoose mediated case of rabies
on a humans on the island.
We now have through this advent of these additional vaccines,
the ability to address that issue in the wildlife
reservoir of domestic animal vaccination outstanding.
That's another issue in Puerto Rico that will certainly
be critical in my opinion to eradicating mongoose spreading
rabies from the island.
But we have now a vaccine that shows a lot of promise
whereas not too many years ago, that was not yet available.
And that's thanks to the continued diligence
and research of scientists both in the field and laboratory
in my opinion.
Thank you, Are, except for using one
of my most unfavorite, impossible words in
regards to rabies, and that's eradicate.
Prevention control, selectively eliminate,
but we aren't going to eradicate this baby.
I'm wondering in regards to my colleagues from Asia,
if you're still there.
Dr. Tsu, Dr. Zhie, what do you think
about the utility for new diagnostics or new vaccines.
Is there a need is there utility for the near future
from the Asian, local China perspective, what do you think?
Do we need new tools?
Are the tools we have now adequate?
- There is a question of Dr. Feng
Ye on the chat about the zero vaccine
and how the zero vaccine is supporting the stomach acid
attack.
Dr. Feng Ye?
There is also a remark from someone from PAHO,
saying that even in Latin America at the moment
there is not enough RIG, I'm just reading.
- Yes, I--
- Hi, hello.
- Yes, Dr. Feng, please go ahead.
- Yeah our laboratory we have a vaccine too
and there is a student and who've
worked at how to produce a vaccine.
And it's her question and where she
tried to right overall vaccine to animal
and he found it was not very effective.
So her question is it's true at all for a vaccine
to add more to overcome the stomach, like act attack.
- Very good, thank you.
Do you think we need new diagnostics?
I'll also open that up to Nuro?
- I'm afraid they don't have the method on how
to produce the ultra vaccine.
- Yes, I know there's been a very recent paper
about the serum system that was done
conducted in China recently.
And unfortunately, although, China
has made huge progress in human rabies case decrease
primarily from post exposure prophylaxis,
from the standpoint of the ZBT and on a score from 0 to 5,
nationwide it looks like China is only about a 1.5
in terms of preparedness.
And we only have nine years to go.
What do your colleagues in China think?
Is China going to be able to eliminate
canine rabies in this decade?
- Yes.
I had published this article with other colleagues.
And I know we have serious problems
in animal rabies in China and we still work on it.
And we have a very serious problem of dog rabies.
But we have a decrease of human rabies.
I know we still have less than 10 years
but all the people want China to make rabies history in China.
- Yes, thank you.
And we look forward to your continued efforts.
And I know our colleagues at pass two
have very recently published a very interesting paper
about the treatment of animals with illness.
Laurent, what do you think?
Is their utility to using these and other modalities
not just for prophylaxis but also for treatment?
- In fact I was not involved in that.
But though was it compromising, yes, to do that?
- Yes.
- The main problem is that for the moment
that you have to inject this directly into the brain.
So we have to make efforts to wonder of this access
more easily because to date it's a good example of what
we can do, is that pave the way for future experiments.
But if you look back in the context of real life, what
we are far from giving this ZBT to lower middle income
countries you know we're going to have a real problem.
So that's promising but I think we
have work to do not only on the biological products themselves
but on the way to deliver them in the target organs, which
is the brain.
- Yes, excellent point.
So we've covered the spectrum today
from diagnostics, to therapy, to prevention,
to vaccination of free ranging animals,
to the utility of such techniques for potency
of the biologics we use in humans and other animals.
And how to go ahead and gauge the utility
in free ranging animals.
We certainly have many challenges ahead of us.
Nature abhors a vacuum.
I know there's a new lyssaviruses to be found.
We hope that it's never going to come anything
close to what this past year has shown us
in regards to coronavirus but you never say never.
And I think that's the challenge for some of us
who want to increase the cross reactivity
of our current biologics to the Filo Group two,
three and the new lyssaviruses to come.
Any closing thoughts by any of our panelists
since we're over time?
Not hearing any, I want to thank everyone
from Asia and in the middle of the night
to our European colleagues.
Next time we'll have African colleagues to boot.
To those on this side of the pond
wishing you a very good night, good afternoon, good day,
and thank you very much for your participation
in this very wonderful webinar.
And please stay healthy, get vaccinated out there.
Bye bye.
- Bye bye, ciao, ciao
- OK, thanks, everyone.
Thank you, Charles.
And we have a recording of this webinar.
So we'll add subtitles to it and we'll
send it to all the participants later this week.
- Thank you, sir.
Thanks for Joe for hosting us, bye bye.
- No problem, thank you everyone.
- Thanks, everyone.
- Bye bye.
- Thank you.