Hi. Me again, again.
I'm Rebecca Christofferson at Louisiana State University,
And previously, I talked about an overview of arbovirology
and then all about mosquitoes.
So, now, I'm gonna go a little bit different and talk about,
what do we do when we have understudied arboviruses?
And the... from top to finish,
how do we characterize these arboviruses?
So, I'm gonna talk about the case of orthobunyaviruses in Rwanda.
So, first, this is a problem of One Health.
But what is One Health?
And why do we care?
So, One Health is the idea that you have
environmental, animal, and people health,
and that you cannot optimize one without optimizing the other.
So, it's just a concept of...
let's all take care of the planet
so that we all have a planet to live on.
And so, vector-borne diseases,
which is, you know, a broader term for more than arboviruses,
but any pathogen transmitted by a vector,
is the perfect example of One Health,
because most often vector-borne diseases
are influenced by the environment,
there is a subset of them that are of public health importance to people,
but a lot of the times they are of veterinary or wildlife
or zoonotic importance.
And so, we really have already been working in this space
of this triad of environment, animal, and human...
and human health.
So, we're gonna talk about One Health in Rwanda.
Cows are culturally and economically important in Rwanda.
And these right here are some native cows to Rwanda,
and they're beautiful.
But they live really close to people...
not just these, but a bunch of different types of cows.
And they're part of a program called
One Cow Per Poor Family.
And this is a program that the government of Rwanda instituted
to combat child growth stunting.
And what they did was they provided every family with a cow,
which was a source of calcium
and other vitamins for children,
to combat child stunting.
And they've seen success with it.
But what that means is that cows, again,
live really close to people.
What they also have in Rwanda,
and in general in Sub-Saharan Africa,
is Rift Valley fever.
And Rift Valley fever virus is a phlebovirus,
which is a type of bunyavirus of animal and human health importance in Rwanda.
This virus is cyclical -- it's seasonal --
and it causes outbreaks in cattle
that manifest as abortions or as hemorrhagic fever.
It's also been called the disease of abattoirs,
which is like a butcher, or veterinarians.
And that's because Rift Valley fever
causes kind of a big mess from infected cattle.
So, if you think about hemorrhaging,
that's... that's a lot of fluids everywhere.
And if you think about abortions or miscarriages in cows,
that's also a lot of fluids.
And so, what that means is that there's a lot of virus
contaminating the environment.
And when a butcher goes to butcher a cow
that is infected -- either symptomatically or not --
then that is a chance for that butcher to become infected.
And the same thing when a veterinarian goes to treat --
there's just a lot of virus around,
and then you have transmission to people.
And so, Rift Valley fever is a problem in Sub-Saharan Africa
and in Rwanda.
Now, these other bunyaviruses of interest
-- Bunyamwera, which is... Bunyamwera, Batai, and Ngari --
have also been sporadically detected or suspected in Sub-Saharan Africa.
These are different kinds of bunyaviruses;
they're of the orthobunyavirus genus.
And they have not really...
they have not been isolated specifically in Rwanda before.
Now, an overview of these orth...
of these three orthobunyaviruses
showed that the distribution of them is somewhat sporadic.
So, you see the red dots pertain to Batai.
So, you see Batai is sort of associated
with European transmission,
and it's maintained in a bird-mosquito cycle,
as far as we know.
And so, Batai, here, again, is that...
one isolation, way back in the '50s or '60s
from Uganda, and then primarily in Europe.
Whereas Bunyamwera and Ngari
kind of stayed in this... this sort of African distribution.
Now, the interesting thing about these three viruses in particular
is that Bunyamwera and Batai
are "parental" viruses for Ngari.
The genetic structure of bunyaviruses
is that they have three discrete genome segments.
And when they have a...
when you have a co-infection of Bunyamwera and Batai, for example,
then you have an opportunity for those segments
to sort of rearrange themselves.
And we have several examples of reassortments
through the bunyavirus family,
but in particular Ngari is thought to be...
is the putative child of Bunyamwera and Batai.
It shares two gene segments of Bunyamwera and one of Batai.
And so, we were really interested in characterizing these viruses,
because they haven't been really well characterized.
Bunyamwera has been more characterized
than the other two.
But also, we kind of wanted to compare,
across several different properties,
the child virus with its parents.
And so, that's the basis of the talk
that I'm going to give today.
So, the first thing you do when you characterize an undercharacterized virus
is you throw it in cell culture.
Let's see what it... how it grows.
So, we put it in Vero cells
because that's what most people have laying around,
and we really didn't know what it was going to do, comparatively.
So, the different colors are doses,
all the way from one virion per milliliter of supernatant
all the way up to six... logs... six logs.
So, we have really no difference in the growth.
So, in Vero cells, there was not a whole lot of difference
in the growth of these three viruses.
So, here we see no sort of advantage
of the child virus over the parents.
But we did notice something interesting when we did this.
And that was at 30 days post infection,
we still had a lot of detectable RNA.
So, when we go and we look at these growth curves,
what we do is we usually test for RNA
using something called quantitative real-time PCR.
And we noticed that there was a lot of RNA,
and so we wanted to see,
does that RNA equal infectious virus?
So, to do that, we took RNA from...
I'm sorry, we took the virus
and we just put it in a tube
with media, cell-free media,
and we stuck it in the incubator for 30 days.
After 30 days, we took that tube...
we took what was in it -- the tube-supernatant combination --
and we put it on new cells,
and we tested it over a week,
and then we looked for viral growth.
So, the ide... the concept is if you get more out than you put in,
then there was replication.
And for all three viruses, we saw that we did actually
get more out than we put in.
So, this tells us that these three viruses
are stable in extracellular conditions
for up to 30 days post infection.
And why that's important will become clearer later on.
Just remember that in your head for now.
But don't worry.
We also figured out how to kill it.
So, we took a commonly used detergent,
used in things like ELISAs, and...
it's called Triton X-100,
and we saw that it inactivated all three viruses.
So, this is a representative of all three,
but they all three look the same,
but this is [Bunyamwera].
This is cell culture at... negative control,
so you see there's not a whole lot of disruption of the monolayer.
Cells are... they look okay.
This is Bunyamwera at four days post infection.
So, you see these kind of divots and pivots and whatnot...
that's just Bunyamwera doin' its thing.
It's killin' all the cells.
But if you look at the inactivation of Bunyamwera...
this is after one hour of incubation with the detergent,
and we... four days post inoculation onto Vero cells,
you see that it looks more like the negative control,
which has no virus,
than it does the infectious treatment.
So, here we are.
We've shown that we can inactivate the virus.
Even if it's environmentally stable,
we know how to kill it.
So, that was part of what we did in vitro.
Then we moved on to in vivo.
So, a lot of studies...
not a lot of studies, but some studies
have looked at one or two of these viruses in a mouse model.
But we wanted to put in all three and compare, again.
And so, the first thing we did was put it into
a competent model, a mouse model
that was not immunocompromised in any kind of way.
It's just a regular old mouse.
And what we found was that this mouse
did not get sick.
So, here we have Batai and Bunyamwera,
and what we see is this...
this detection of virus at day 1
is probably the same virus we put in.
It's not replicating, it's not progeny, it's just... it was still there.
We call this process clearance.
So, the virus was cleared out of the...
out of these mice -- and there was only five per group --
really, really quickly.
Ngari showed a little bit more variability
than the parental strains.
And it was an interesting find
that may or may not have significance,
but we kind of harken back to the reports of Ngari
having more of a...
more of a severe manifestation in human populations
than has been reported with the other two.
But again, that's based on limited reports from human populations
and limited study of these viruses.
So, we don't really want to say that this is necessarily a thing,
but it was interesting to see.
So, what do we find out from this infection?
Well, we did some other things with it,
but the main goal is that this mouse
is not a good candidate for infection studies.
Because sometimes in science you get negative results.
And the first thing that most students want to do is cry,
until I remind them that negative results
are still results.
So, we got data, and it may not be the data that people are looking for,
but we did determine that this mouse
is not a good candidate for infection studies.
And what did that lead us to?
Well, it led us to a knockout mouse.
So, with some other studies that I have done for dengue and Zika...
and Zika virus,
we've used a knockout mouse.
Because these... particularly flaviviruses are...
they don't infect mice very well.
So, we used what's called
an interferon regulatory factor 3 and 7 double knockout mouse.
And what this mouse model is deficient in is
these two factors, down here,
that directly go into the pathway
that makes interferon type I.
And interferon type I, which is interferon alpha and beta,
is one of the first innate responses...
immune responses that's antiviral in an infection.
And so, we did not ablate it -- it's not completely gone --
but we did downregulate this antiviral response
in this particular mouse.
I say we; the mouse already came like that.
But the mouse does have a deficient antiviral response.
And so, in dengue and in Zika,
this means that they're more susceptible
than the immunocompetent mice
like the ones we used before.
So, when we put Bunyamwera into this mouse model,
what did we see?
We saw that 100% percent of the mice
developed hunched posture and lethargy,
which is a common clinical symptom for...
or clinical sign for mice just not feeling well.
We also noticed that 62.5% of them
presented with facial swelling.
And this was very marked, and it was posthumously noted,
but facial edema is seen often with another bunyavirus infection in humans,
which is Lassa fever.
Now, I'm not saying that Bunyamwera is Lassa fever,
because, remember, this is in mice,
and it's in a messed up mouse.
This is not directly comparable to what we see in the real world.
But it is an interesting finding.
And I'll tell you what we could possibly use this model for in a minute.
We also saw high viremia.
They got really high viremia -- up at about 6 logs.
Now, we put in 3, so that's a pretty low dose.
We put in 3 logs,
which is about a thousand virions per milliliter,
and we put it in the mouse.
We got about 8 at peak...
7 to 8 logs out.
And then, we also had 100% percent mortality by day 6.
So, these mice are very susceptible,
even at relatively low doses of this virus.
We also looked at the pathology of these mice.
And what we found was that
we had necrotizing oophoritis in the ovary.
But we also had some necrosis in the uterus.
And this is interesting because the symptoms...
or... not the symptoms, but the manifestations
of Bunyamwera in cattle
tend to be abortions or affect the reproductive tract of the...
of the cattle.
And so, finding these sort of reproductive tract-associated lesions
is a potential use for this mouse model.
We also saw skin necrosis;
a lot of mast cell degranulation,
which just means, like, a lot of inflammation in your skin;
and then we found diffuse edema in the eyelid,
so this just means that the eyelid skin
and the space between cells
is just filled with fluid,
and that's probably what contributed to the facial swelling
and the edema of these mice.
So, those were some interesting finds
that we weren't necessarily expecting to find in this mouse.
And so, what did this tell us?
It told us that this mouse model is very susceptible
to Bunyamwera infection...
because we only did Bunyamwera;
we didn't do the other two.
And it does recapitulate some of the aspects of the disease
seen in ruminants, like abortions in cattle.
The other thing is that it's been suggested
that we could use this model as a possible
diffuse hemorrhagic disease model,
a proxy model.
And the reason that this is interesting is because Bunyamwera is a BSL2,
or Biosafety Level 2 virus,
because it's not deemed to be
really, really dangerous for trained laboratory personnel.
And so, if you have the opportunity
to study a diffuse hemor...
hemorrhagic disease at a lower Biosafety Level,
that opens up the study of this type of pathology
to people who don't have access to high biocontainment laboratories.
Because, again, we're not saying Bunyamwera equals Lassa fever;
we're saying Bunyamwera in this messed-up mouse
gives us interesting things that may or may not
look like Lassa fever.
And so, some of those... those are some of...
always the caveats of studying mouse models,
or studying vertebrate models,
and trying to extrapolate back to what we see
in the real world.
And that's just part of... part of science and what we have to do.
So, we've done in vivo.
We've done in vitro.
We've kind of had some ideas about characterizing these viruses.
And what we had originally planned to do
was we had originally planned to go to Rwanda,
because my student, the newly-minted Dr. Fausta Dutuze,
is from Rwanda.
And she wanted to see, does...
is there evidence that these viruses are circulating in Rwanda?
Because Rift Valley fever is there,
and sometimes these viruses
have been attributed to a Rift Valley fever-like disease,
so... are they there, and we just haven't found it yet?
So, originally, we were gonna go do a serosurvey.
And what that means is we were gonna bleed a bunch of cows
and check their serum for antibodies against these viruses.
That was what we originally planned.
Well, it turns out... plans change.
There was a giant outbreak in 2018
of Rift Valley fever in Rwanda.
And at that point, we were able to partner
with the Rwandan Agricultural Board,
and their very, very smart and wonderful scientists there,
to sort of test Rift Valley fever suspected cases to see,
are these viruses contributing to Rift Valley fever-like disease?
So, we had 157 blood samples from cattle,
all over the country,
that had suspected Rift Valley fever.
And the inclusion criteria where
they had an abortion within 5 days,
they were having symptoms of hemorrhagic fever,
or they shared a farm with a death case of Rift Valley fever suspicion
within the last 5 days.
So, we found that
70% of the Rift Valley fever suspected cases
were actually negative,
and only 30% were positive.
And we determined positivity by regular old PCR.
All cases of hemorrhagic fever were indeed Rift Valley fever.
So, here you see a cow who's got some hemorrhaging...
hemorrhaging from his nose, and then over here.
So... very obvious hemorrhaging.
And then we decided to test the negatives
for our orthobunyaviruses of interest.
And we found two of our orthobunyaviruses.
So, even though Ngari is a reassortant of Bunyamwera and Batai,
the way that we designed our diagnostics,
we were able to distinguish between
Ngari and the other two viruses.
And so, what we... this is representative of Batai
-- this is the M and L segments that we looked at --
and we found that we had two infections of Bunyamwera,
three infections of co-infection
of Bunyamwera and Batai,
and two infections of just Bun... Batai infection.
And so, we had seven total cattle
that showed that this...
these viruses are, in fact, circulating.
What we also found was that
we had some combinations of all of these gene segments
in ten cows that were infected with Rift Valley fever
but seemed to be co-infected with something else.
So, we did... we weren't able to determine what exactly,
but there are other orthobunyaviruses
-- like Germiston, like Ilesha,
possibly others that are circulating in the region --
and so perhaps they are co-infected
with Rift Valley fever and these other orthobunyaviruses.
So, putting it all together,
all of the cattle that were infected with Bunyamwera
So, this harkens back to our mouse model,
where we saw some involvement of the ovaries and the uterus.
And perhaps there's something there that
we could look into further in this mouse model
to sort of help us study the pathogenesis of these viruses
and how it leads to this reproductive tract involvement
But going back to the extracellular...
cellular stability of these three viruses,
an abortion in cows results in
not only a fetus that is just covered in goo
but an environment that's also covered in goo.
And so, if you remember, I talked about Rift Valley fever
and the transmission to people being more
a function of the environmental goo,
or the blood products, or the fluids from these hemorrhagic or abortion cases.
We show that the stability that's seen
in extracellular media
may contribute to that also being a risk for these three orthobunyaviruses,
or at least for Bunyamwera and Batai that we actually found in this region.
So, again, we now can tie back to our in vitro cell culture experiments
to sort of make an estimated guess
about what we think might be a risk in the field.
And so, again, we go all the way from in vitro
through the field to really characterize
what's going on with these... with these orthobunyaviruses.
So, what did we learn?
We learned that these viruses have similar kinetics in vitro.
And so, at first glance, that looks unimportant.
But in following experiments,
you usually want to do what we call matched titer.
And that means you want to make sure that
every group gets the same amount of virus
across all three viruses.
And so, when we know the growth kinetics of all these viruses,
that helps us determine,
well, I have to shoot the virus five days before my mouse experiment
so I can harvest on peak day,
and actually, in Bunyamwera it's day four...
so it helps you kind of plan your experiments,
so it increases efficiency in how your lab runs
for future experiments.
We also learned that the stability of extracellular...
in extracellular conditions...
but we can kill it, so that's good.
This harkens back to biosafety,
both in the field and in the lab.
And also, it does identify the utility of that Triton X-100
to deactivate the virus.
We haven't done anything with mosquitos yet,
because we're waiting to do that.
But that is something that we really need to do to finish characterizing these viruses.
And then we learned that the C57 mouse
is not a good infection model.
So, I've done this so that you don't have to.
And then, we have determined that the knockout mouse
and that there may be utility of this mouse to study pathogenesis
in the reproductive tract.
And then, maybe it can also be used as a proxy model
for diffuse hemorrhagic fever... disease.
And then, finally, our fieldwork.
These viruses are in Rwanda.
That was our... that was our hypothesis,
and that's what we showed.
So, it's interesting because we now have
sort of filled in a little gap in the known distribution of these viruses.
They weren't previously published in...
that they were in Rwanda.
So, now we can say that they are.
That's not particularly surprising,
except for the case of Batai.
So, again, Batai, if you remember the distribution,
was mainly a virus isolated and detected in Europe.
Well, now, we have evidence that it's circulating in Rwanda.
The other interesting thing is that it's circulating in cattle.
It's infecting cattle, causing abortions.
So, this bird virus apparently can...
can cause damage in more than one vertebrate host,
and that's very interesting.
The other interesting thing is that...
the co-infection of cattle.
So, if you remember, I talked about Ngari.
We didn't find it in Rwanda.
But I talked about it being the child of
Bunyamwera and Batai.
The reassortment to make a new virus
has to happen when co-infection occurs.
And so, generally we think about this happening in the vector,
just because there's more opportunity.
More vectors generally tend to get...
tend to get infected,
and then the infection last longer in vectors.
So, once a vector is infected,
it doesn't generally clear it.
So, there's more opportunity for co-infection,
more opportunity for this reassortment to occur.
But, now we've shown that co-infection
can happen in cattle,
so maybe we need to start looking at cattle
as maybe a vessel for potential reassortment
of these orthobunyaviruses.
And finally, the most important conclusion is that
we never have all the answers.
So, even though I seem like I've given you a lot of information here
that sound conclusive,
all I've really done is answer more que...
is raised more questions.
So, if the viruses... especially with Batai...
if it's in... if it's in Rwanda,
but it's only in Europe...
there's a lot of space between Rwanda and Europe,
so where else is it?
Those are the kinds of questions that we still need to ask,
and those are the kinds of questions that come up.
When you do research,
you never have all the answers.
So, finally, I want to thank my lab members.
This is Fausta Dutuze.
She is... this is right after she defended
and was minted a newly... PhD from my lab.
I'm very proud of her.
She's back in Rwanda.
This is Handly in a box;
she thinks she's part cat, clearly.
And this is Chrissy, who is a PhD and my student,
who's demonstrating how a spillover event might happen.
Just kidding -- this is her pet squirrel, Rocky.
And finally, I have to, again,
acknowledge my funding,
and the support of HHMI and iBiology.
And... that's it.