Practice English Speaking&Listening with: Rebecca Christofferson (LSU) 1: Mosquito-Borne Arboviruses

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Hi. My name is Rebecca Christofferson,

and I'm an Assistant Professor at Louisiana State University

School of Veterinary Medicine,

and I study arbovirology.

Arbovirology is the most fun virology,

or that's at least what we say.

So, what is arbovirology?

Arbovirology is the study of arboviruses.

The term arboviruses comes from

arthropod borne-viruses.

Arthropods are, of course,

things like insects or ticks.

And they are vectors of these arthropod-borne viruses,

and that means that they transmit them.

Now, some examples of arboviruses that you may be familiar with are

yellow fever virus, West Nile virus, dengue virus,

Zika virus, and those are all flaviviruses.

Or alphaviruses like chikungunya or Mayaro virus.

And then there's another set of viruses that I study,

which are bunyaviruses,

like Rift Valley fever virus, La Crosse virus,

Crimean-Congo hemorrhagic fever virus,

or the ones that we are particularly interested in,

Bunyamwera, Batai and Ngari viruses.

Particularly in my lab, we study arbovirus transmission.

And arbovirus transmission is made up of

a milieu of questions that encompass

the virus, the vertebrate, and the vector.

So, again when we're talking about arboviro... arbovirology,

it's this whole interconnectedness of this system

that goes into transmission and how transmission shapes up.

We also have the added component of the environment,

and I'll explain later in this talk how the environment

acts on this interaction of vector, vertebrate, and virus

to sort of shape how an epidemic might look.

When I talk about epidemic shapes,

what I'm looking are... what I'm looking at is just that,

the shape.

So, here are three randomly generated epidemics.

And you can have one, like this blue,

that starts out way over here and has a really long burn in,

and then kind of turns into this epidemic

and then has a really long tail this way.

Or you have something like the yellow, which starts about right here,

and it has a shorter burn in but a higher peak,

and it disappears... also a little shorter.

And then you have something like explosive outbreaks,

like we've seen in the past few decades with chikungunya and Zika viruses

in the Americas,

that start out and they just come in,

and they go all the way to the top,

and then they burn out.

And so, those are the different shapes of an epidemic.

And in my lab, what we're interested in is how are these dynamics of transmission

determined by the interactions of the transmission system,

which are the mosquito-virus interactions,

the vertebrate-virus interactions,

and then the environment-virus-mosquito interactions?

What is a vector?

Well, I've already told you a vector is something that transmits these pathogens,

in my case viruses.

So, there are many types of... or, many species that can act as vectors,

the main ones being, for me, mosquitoes,

which... I'll tell you a lot about mosquitoes;

ticks; we have here sand flies,

which are implicated in, like, leishmaniasis;

and then we have biting midges that are responsible for things like

bluetongue virus.

Ticks, as we all know, can transmit several viruses

but also bacterial agents.

But I'm gonna focus on mosquitoes

because I love mosquitoes.

So, some trivia for the next time you're at a party

that you will love to spout off

is that there are about 3,500 different species of mosquitoes

and 132 species of Aedes mosquitoes.

I primarily study Aedes mosquitoes

and their associated arboviruses.

Here's an interesting tidbit.

It's that only the ladies bite you.

Mosquitoes, as a food source, actually drink nectar,

so they're pollinators.

When the female bites you,

it's not because she's hungry.

It's because she needs your protein, in your blood,

to then make eggs and make little baby mosquitoes.

And so, when you see a mosquito biting you,

it's going to be a female.

Some more trivia, because this is always interesting, is that males

are really, really fuzzy.

So, here you see their antennae --

really, really fuzzy.

And their proboscis tends to have sort of like little decorations on the end,

so they'll have like this fleur-de-lis thing goin' on.

The females... they tend to be a little bit bigger.

And their proboscis, which is this... this thing right here

-- it's their little sippy straw,

the thing that they suck your blood with --

it's... it's just straight.

And their antennae are a lot less fuzzy.

And so, if you really want to hang around and learn how to sex a mosquito,

be my guest.

But if you see it on you and it's biting you,

it's a girl.

So, one of the major things we study in vector-borne, or mosquito-borne, particularly,

arbovirus transmission is vector competence.

Vector competence is the intrinsic ability

of a specific vector to transmit a specific pathogen.

And it's often mathematically represented

as the number that trans...

the number of mosquitoes that transmit

divided by the total number that you are exposed to.

And so, what I'm gonna talk about today

is how we determine a good way

to look at vector competence,

but also I'm gonna tell you physically how we do it in the lab.

So, interestingly,

not all mosquitoes that get exposed to a virus will eventually become infectious.

And so, we kind of have this this whole, like,

filter, which is time, that determines...

given I exposed 100, maybe only 60% of them will actually transmit

given a certain amount of time.

That time component is called the extrinsic incubation period.

It's the time between exposure of the mosquito

to an infectious bloodmeal

to the mosquito is actually transmitting.

And I'll probably say EIP a lot.

That's what I'm talking about:

extrinsic incubation period.

So, again... how do I measure it?

Do I measure it at one time point? At two time points?

Does it really matter?

And then, how, physically, do we do this in the lab?

One of the things I noticed when I was first starting out

on my vector competence journey of life

was that there was sort of a bias in sampling

at 7 and 14 days post exposure.

So, this is from a paper where we looked at

chikungunya vector competence in the literature,

and we found... so, you can see here...

we found that there's a lot of people sampling at 7 days

and then again at 14 days.

And you can see it here...

this is in a separate mosquito species,

but the way that these lines...

these are data points...

how many data points per time point.

And so, you see there's this, really, kind of propensity

to measure at 7 and 14.

Well, why do we do 7 and 14?

Well, just one week and two weeks is kind of easy to talk about,

and it's really sort of a historical sort of artifact.

There's no biological basis for 7 and 14 days.

So, again, how do we measure it?

Well, it's a process... vector competence.

So, what I have been trying to champion

is that we really need to understand

the temporal component of vector competence.

For example, this is some data out of my laboratory

in some Aedes aegypti mosquitos

looking at Zika virus.

And these are the days post exposure.

So, we give the mosquitoes a bloodmeal that has Zika in it,

and then we test them over time.

We test a subset of them over 5 days post exposure

all the way to 23.

And this is the percent that had virus in their saliva.

So, you see we start out with 0.

We get some at 10%.

And then all of a sudden we're at 60%.

But, if I had measured at 7 in 14 days,

then I really would say maybe 10% is the max,

or 10% is vector competence.

But we're really not getting to the 60% there.

So, again, what is a good way to do this?

This is the same data in graphical form.

And so, what I wanted to kind of show you here

is that if we look at 7 and 14 days

-- which I put on this graph as dotted lines --

it doesn't capture the whole story.

But what if I were to say 10 days or 20 days or 15 days?

My point being... any combination of one or two discrete time points

that are somewhat arbitrarily chosen

are really not gonna tell the whole story

of this process of vector competence.

And so, what we do in my laboratory

is we fit a distribution to the data.

And I've been doing vector competence for a long time,

and I've found that the cumulative logistic distribution

-- or, to some people, a sigmoid curve --

fits the... fits the data more often than not.

And you do this by a magic thing called statistics,

which is not as scary or as boring as people make it out to be.

But you find you get this really nice curve.

And then what you can do with this curve

is just basic arithmetic or basic algebra.

And you find the point at which this curve is equal to 50%.

And then that defines the EIP50,

or the extrinsic incubation period - 50.

In this case, the EIP50 is 16.6 days post exposure.

Now, what does this mean?

The EIP50 is the time it takes for

50% of mosquitoes to become infectious.

And it's a similar concept to something like the lethal dose - 50 or LD50,

which is used a lot in toxicology and vertebrate studies,

or lethal concentration - 50,

or tissue culture infectious dose - 50.

These are all measures where you apply some...

a treatment to a system

and then you look for 50% of your required outcome --

infection or lethality or whatever.

And so, it's the same concept,

but it makes sense because it allows us to sort of tailor our experiments

to our particular system.

What does that mean?

Okay, so let's compare.

This is the same data,

but all I did was add 10 days

to the days post exposure.

Because it's easy; 10 is easy.

I can do that.

And I have to fight with sixth-grade math homework,

so I tend to make things easy for myself.

So, EIP50 here is 26.6 days.

It's the same logistic regression...

the same logistic function,

and we solve for 50%.

What does this mean?

So, if we look at the two data -- the red and the purple....

this is our original, and this is the one we added --

the data points here start at 8 and go all the way to 23.

So, that defines our period of sampling

for that particular system.

If we were to start at day 8 for the purple line,

we're actually not gonna see transmission until day 18.

That's a lot of mosquitoes,

and a lot of sampling,

and a lot of time and resources for something that...

you're just gonna get a bunch of zeroes.

So, what you can do is if you're planning

a really big experiment,

you can do a small pilot study

and get an idea of when your virus

starts to sort of follow this upward path,

and tailor when you sample, or the times you sample on this x axis.

That is a really good way to save,

again, time and resources.

But the EIP50...

the EIP50 lets you compare regardless of if your x axis,

or your tampling sime points... tamp...

sampling time points, match.

So, why is this useful?

Well, let's say for example that you have a student

who is working really, really hard,

and has this really big experiment coming up or in process.

And they're doing all the work and doing a really good job,

and then an epic flood hits and you can't get to work.

True story.

So, that last time point that involves hundreds and hundreds of mosquitoes,

lots of tears, lots of blood, sweat, and tears...

I can say blood because we feed them blood...

but now we don't have to scratch the whole experiment.

Because we can use this to sort of say,

well, even though we're off on that one day on the x axis,

the EIP50 still standardizes

how we can compare all this data.

So, physically, how do we get the virus into the mosquito?

Do we use very tiny spoons?

That would be a no.

We tried... we didn't really try.

So, we use something called a Hemotek.

This is the best invention ever.

It's from a lab... Discovery Labs in the United Kingdom.

And it's basically an artificial membrane feeding system,

where we put blood into a little disk,

and we put a membrane on the bottom to hold the blood in,

and it heats it up to 37 degrees Celsius,

which is approximately body temperature for people,

and then we stick it on top of a carton full of mosquitoes...

screen, bloodmeal...

mosquitoes come up, they poke...

probe through the screen to the bloodmeal,

and then they get big and fat and juicy and red.

At this point, we take the mosquitoes

that are big and fat and juicy and red,

and we put those into a new carton,

because we know these mosquitoes have been exposed.

They've gotten the bloodmeal that has

whatever in it that we want them to have.

And so, now, we can look at things like vector competence.

How do we get it back out?

Well, we take the mosquitoes,

we flash freeze them in a freezer,

and then we take their legs and their wings off.

And then we stick their bodies onto tape with the sticky side up,

and then we a really thin pipette

and we thread their proboscis through this pipette.

Now, this solution right here has ATP in it,

which is adenosine triphosphate.

And that's one of the energy building blocks of respiration in our body

and everything.

That is actually what stimulates salivation in the mosquito.

So, ATP in this solution right here

is making that mosquito spit.

So, that's how we get it out.

And then we test it using a vary...

a varied repertoire of methods to see if there's RNA from our viruses

or if there's actually infectious viruses.

So, now I'm gonna switch a little bit and talk about zoonoses.

Zoonoses are important to arboviruses

because most arboviruses are zoonoses.

It's estimated that vector-borne diseases

and zoonotic diseases

make up 70-75% of emerging pathogens of public health importance

in the... in the future.

And so, like I said, most arboviruses are zoonoses.

A zoonotic disease is one where

humans are not the primary reservoir...

primary vertebrate host.

A non-human vertebrate is the primary host.

And it's often associated with, like, a natural sort of habitat.

So, some examples are West Nile,

where the primary vertebrate host is birds;

yellow fever, and the primary vertebrate host

-- or the reservoir host, we call it --

is a non-human primate;

Usutu virus, which is a...

like West Nile in Europe

is also trans... is also...

the reservoir host is birds;

and then tick-borne encephalitis is...

the reservoir is rodents;

and then in dengue, Zika, and chikungunya,

it's probably primates.

Although some zoonotic diseases

have come out of the natural or sylvan cycle,

which I'll explain in a minute,

and have come into urban transmission,

and are maintained primarily there,

without a whole lot... not a whole lot of input from the sylvatic cycle.

Dengue is one of those.

It exists happily in an urban cycle

without the input from that sylvatic cycle.

So, what does all that mean?

Well, to explain, I'm gonna tell you about the case of yellow fever.

Yellow fever is maintained in the sylvatic cycle.

Sylvatic comes from a word meaning jungle,

and it's basically just that.

It's mostly found in non-human primates

and their associated jungle mosquitoes of... in South America.

It's the Haemagogus genera.

And so, it's maintained happily.

It just keeps going.

It doesn't generally cause high mortality

in the non-human primates.

We call the non-human primates, again,

the reservoir host.

And we call the associated sylvan vector

the enzootic vector.

So, it's maintained in this big old sylva...

sylvatic cycle.

So, what happens... how does it get into a human outbreak?

Because we've had several yellow fever outbreaks

of pretty big magnitude in the last couple of decades.

So, what happens is, generally,

we have something called a spillover event.

And that is when usually something

we refer to as a "bridge vector"

comes from the sylvatic cycle

into an urban area and infects,

you know, patient zero in the urban cycle.

At that point, then, yellow fever

establishes a sort of independent urban cycle,

where the bridge vector

may or may not be the same vector here,

but it is not the same vector as in here.

So, these are two discrete cycles

that are bridged by a bridge vector.

Now, that being said, yellow fever in Brazil,

in this last big outbreak,

was found to have more transmission

associated with the sylvatic vector

than with what we think is the urban vector.

And so, that indicates that there are multiple spillover events

that have... that have contributed to this outbreak.

And so, spillover events and emergence

from the sylvan to the urban cycle

is never a one-to-one or a single event.

It's always a spectrum.

Some of the other things about arbovirology

that makes it really interesting

are the impacts of environment.

And since we're dealing with the constant sort of plague of climate change

and development,

we really have to think about...

what is the environment...

how are the environmental changes that we're expecting

impacting what we see with arbovirus transmission?

So, we do this through laboratory studies,

but also there are some field observations

that kind of make the point.

So, the laboratory studies we have been conducting

are the... Zika and temperature.

And a lot of other labs have been looking at this.

And generally, what we know is that

an increase in temperature increases vector competence.

It means that more mosquitoes will become infectious

and the time it takes for that to happen generally is shorter.

But that's not the whole picture,

because temperature also affects the life traits of the mosquito.

So, what we've been looking at is two temperatures:

one is 24 degrees, which is relatively mild,

and 28 degrees, which is pretty optimal

for Zika and Aedes mosquitoes.

And if we expose these two sets of mosquitoes

at the same time -- 3 days post emergence,

which is a 3-day-old adult --

and we then put them at two different temperatures,

what we find is that we have to look at transmission

and the mortality rate, or survival time,

of the mosquito in order to really understand

vector competence in its totality.

So, we found the minimum time to transmission

for this system was 27 days,

which is pretty long.

The average lifespan of the mosquito at this point is 32.4 days.

At 28 degrees, we had

a 14-day minimum time to transmission,

and we had a lifespan of 28.5 days.

So, what does this mean?

So, again, this is not the EIP50 that I've been talking about;

this is minimum time to transmission.

So, what we have, now, is a difference in the maximum potential days of transmission

that is attributable to temperature only,

because these are the same mosquitoes and the same virus.

We have 5.5 days of maximum potential transmission

at lower temperatures,

while we have 14.5 days of maximum potential transmission

at what we call the optimal temperature.

Now, if we up the temperature

and the mosquitoes start dying too fast,

then we... then we have to look at the trade-off

between that decreased EIP

and the faster time to death for the mosquitoes.

And generally, there comes a point

where the mortality rate of the mosquitoes will win out,

and that advantage that you get from the viral standpoint

of higher temperature equals more virus out quicker

is lost,

because the mosquitoes just don't live long enough.

So, the case of West Nile and the drought.

This was something that was interesting in 2012.

There was a really big drought...

especially, we were looking in Dallas.

There was a big, big drought,

and then there was also a really big increase of West Nile cases in humans.

And this was puzzling for a couple of reasons.

One is that we know -- because I've told you --

that the West Nile is a zoonotic disease,

and it's maintained in this bird-mosquito cycle.

So, birds and Culex mosquitoes

that live both in really urban centers

but also in periurban or suburban areas.

Culex mosquitoes like to breed in water.

They like stand... they like puddle water, dirty water, sewage water.

Just... water in the ground...

as long as it's still and it's wet, they will lay their eggs.

And so, when we had this drought

and we had this increase in West Nile cases,

we sort of were thinking,

well, there's less water,

so there should be less mosquitoes,

because there's not a lot of places for them to breed.

However, what we ended up looking at...

or ended up sort of, as a community, coming to consensus

is that we had what's called water source crowding.

And what that meant was that the birds and the mosquitoes

moved into town

because the most reliable source of water was man-made sources.

And we had, anecdotally,

Culex mosquitoes breeding in places they don't usually breed,

which are the at the...

at the edges of manmade water...

water features or water fountains.

And so, what that did is it brought this cycle of intense transmission

between birds and mosquitoes

closer to humans.

And so, humans just become an incidental

result of being close to that transmission cycle.

And so, that was one way that the environmental effects

really drove the dynamics of West Nile virus

in people that year.

So, what did we learn?

We learned that [arbovirology]

is the study of arthropod-transmitted viruses.

We learned that vector competence is a measure

of transmission capability for the vector.

And it's a combination of vector, vertebrate, virus,

and the environment.

And what else did we learn?

We learned that arbovirology

is virology at its most fun.

Thank you.

The Description of Rebecca Christofferson (LSU) 1: Mosquito-Borne Arboviruses