JoVE Methods Collections: Current Research Methods in Rabies Diagnosis, Prevention, Treatment, and Control

Abstract

The following is a transcript of the webinar:

JoVE Methods Collections: Current Research Methods in Rabies Diagnosis, Prevention, Treatment, and Control

Moderated by Dr. Charles E. Rupprecht

Originally hosted by JoVE on March 31, 2021

- 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.

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 ICTV 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 represents 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 surveillance 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 sets 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 centers, and we also have four bird conservation NGO, non-government organizations. So 22 ADCC and four NGO will send some bat carcasses 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. Rupprecht, and he gave us a very good suggestion. He thinks we need to start to do this kind of surveillance, so we change. We just collect dead bats. So we stop to do the live bat surveillance.

In 2008 to 2015, we did 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 final 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 primers. And this is our primer set. But we found something stranger in Taiwan, bat lyssavirus type I. Because if we do the brain impression smear 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 , our passage is three times, and after the mouse inoculation, we use the BHK cell to do the virus isolation. It still passes 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, recommending 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 employ both FAT and RT-PCR. And we routinely do virus isolation.

Finally, the most important. you need to have 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 humans. And until now, human contact cases will go to do the PEP, 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 in Changchun. 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, Charles. Thank you, and I'm afraid I can't because someone has slides their powerpoint

- 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, worksite OIE reference laboratory in China. It's my pleasure to be given the opportunity to join you here for the rabies webinar. The purpose of my presentation is to give you a shot into real [offered] universal RT-nested PCR for the detection of lyssavirus.

To detect rabies virus other member species opportunist lyssavirus, we have developed a pan- lyssavirus nested RT-PCR in 2007. The principle of this method is the reverse transcription of target RNA into cDNA by using only [INAUDIBLE]. the amplification of the cDNA by two rounds of PCR. The two rounds of PCR significantly increased the sensitivity of PCR.

The cDNA undergoes the first round PCR without primers to amplify on 940 final BP fragments. Then the second round PCR used a first round PCR product as a template to amplify a 371 BP fragment using inner primers. The primers of the first round and second round PCR were designed based on the conserved region of the N gene of seven major lyssavirus species, because only seven lyssavirus species were found in 2007. This method was patented in 2009.

The lyssavirus species had a rapid 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 commercial plasmids for N gene of each lyssavirus for PCR.

Results are listed RT-PCR to detect an 18 lyssavirus species on the left. We can see all 18 lyssavirus putting the same two positive controls, indicating that the nested RT-PCR can detect all 18 lyssavirus. 16 of plasmids efficient amplification in two rounds of PCR. It is also [INAUDIBLE] virus in the first round that was amplified, unlike in the second round PCR. And Ikoma virus [INAUDIBLE] was applied only in the first round PCR.

The sensitivity of the method will show in the table on the right. The sensitivity was varied in the detection of different lyssavirus plasmids limit values ranging from 2.24 to 22,400 molecules per microliter, and as shown in the table on the right.

To investigate the cause of this discrepancy, I think, with cooperation of all 18 lyssavirus with full primer regions was conducted. With the result showing that 3' end nucleotide T of outer primers N829 in the first round PCR. And the second nucleotide T at the 3' end of inner primer N371F 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 G of the primer to T of Aravan virus and change the G of the primer to A-- to C, sorry, of the Ikoma virus.

Both viruses were in the PCR. This result shows the critical roles of the three end or near end nucleotides of the primers in the successful amplification of the target region. The result was unsure. It means this difficulty can be attributed to the mismatch between the primers and templates due to the viral sequence that was attained.

To test the validation of the RT nested PCR, we use 9,624 brain tissues of eight domestic animal species in 10 years of clinical, rabies technologies and surveillance in China. The result shows that RT nested PCR has 100 sensitivity and over 99 specificity in comparison with the direct fluorescent antibody test. 300 decayed practical samples were tested positive, but RT-PCR was up by nested PCR but negative by FAT. This result indicated that the nested RT PCR is more sensitive than FAT in detection of highly decayed samples.

In addition, 10 other rabies laboratories were invited to conduct the test to further validate the nested RT-PCR. All 10 laboratories obtained the nested RT-PCR result 100% in consistent with FAT with no false positive or false negative, indicating that this nested RT-PCR has the highest specificity.

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 Visualized Experiments in 2018. Finally, I would like to end my presentation with the people in my laboratory. My director is Professor Changchun Tu, [INAUDIBLE] Laboratory, rabies classical [INAUDIBLE], and bat disease 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 know 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 count 18 lyssavirus species now, so we just test a plasmid 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 of the lack of 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 focused on dog rabies because dogs are a major transfer source in China. Over 99 human cases were caused by dogs, so we have lots of dog rabies. And I don't think they have enough money and enough people to operate bat lyssavirus 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 phylo 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 of lyssavirus species. And our vaccine can cover group one and group two. Maybe in the future we can operate-- vaccines can cover all the lyssavirus. But in China, and especially in other countries in Asia, we just know how rabies virus works with lyssavirus and Thailand bat lyssavirus, I think.

So the vaccine ratio is enough for rabies control until now.

- 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 assays 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 off, all of our assays have an RT step because the lyssavirus genome is RNA and the standard material PCR is 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 an intercalating 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, an hour 38, 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 that 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 the only concern is 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 brains. 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 an 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. Monitor and trace your controls, watch for trends and signs of degradation or contamination. Always follow good lab 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 antemortem 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 two lyssaviruses in the UK and they also only occur in two bat groups. And they're very specific. So EBLV-2 two we've only ever detected in our Dauneton's bats. And we've only ever detected EBLV-1 in our serotine bats. And I think hypothetically it's definitely possible, so we always test the both. But no, we haven't yet

- Thank you. And a 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 is still a really good assay. And we still pick up beta-actin 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 American. 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 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 proteinaceous 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 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 information 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 subcellular resolution where we can look at the cells at this mentioned subcellular 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 neural glial 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 in 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 kinds 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 the 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 field 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 cell tropism 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 stain for an additional marker, MBP, myelin basic protein, we can see that it does colocalize 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 field 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.

Thus I would like to close my talk. I am part of the [? Finke ?] Group, [INAUDIBLE] Group at the Friedrich-Loeffler 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, 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 to a 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 the case of 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 source 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"]

- 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.

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 this impact impairs disease surveillance and control due to the lack of reliable data.

We also know 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 culture maintenance, and so on.

So what are the needs? What we are looking for, for example, in Africa and Asia, is to have rapid and reliable diagnostic tests and protocols that also should require 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 BioNote. And you can see here the content of the kit, which has 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 is 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 is formed.

And then we migrate 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 migrate 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 could be 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 disposable droppers, provided in the kits.

And for the protocol of the kits this is the initial protocol provided by the brand or 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 migrations. That's quite simple. But the problem is that you have to put some PBS, and that also you have to include 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 representative results. I mean, here we have a positive result with a tester line and a control line. So two bands, this is positive. One band is the control line, it's negative. And here if you have no bands, no migrations, only the test line no control band, 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 had been 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 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 conventional 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 examples, 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 an 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 of 1.00 and sensitivity 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 conducted 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, 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 of course indicated, that we can add some potential batch-to-batch variation. Also, depending on the manufacturers, you can have poor low quality for the test.

Today, it's not recommended by WHO and OIE for working diagnosis, and disease surveillance, but from our experience, it's very interesting, because it improves the motivation of people working and who are in the 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.