Sunday, 16 September 2012

Virus evolution: hitting the bottle(neck) in mosquitoes
When a virus population transmits between hosts, regardless of how this is achieved, not all of the progeny viruses make it, most likely the majority are left behind to contemplate a life of non-existence and the impeding destruction. At the animal level this is fairly straightforward; an animal becomes infected, produces virus, some of which will infect a new animal, which then becomes infected etc. etc. But transmission events impose bottlenecks. Imagine a mosquito taking a bloodmeal from a human. The virus population within that person will, more or less, be spread throughout the body. An adult human contains approximately 5 litres of blood; a mosquito will only take a few microlitres, i.e. a few millionths, of the blood, and therefore only a tiny fraction of the virus in the body. Taken to a more subtle level, of the virus which makes it to a new host, only a fraction of that virus will infect cells within the new host. In the case of the mosquito, there are many 'barriers' which are familiar to virologists studying arboviruses. The first step towards infecting a mosquito (or other biting insect such as midges) is to infect the midgut. At this point there's another bottleneck, known as the midgut-infection barrier; only a few of the viruses which were taken up in the blood-meal will be able to infect the gut cells. In the case of bluetongue virus in midges there are other described barriers, including a midgut escape barrier, a disemmination barrier, and a salivary gland infection barrier. All of these must be overcome in order to allow transmission to another mammalian host.

An array of barriers must be overcome by a virus in order to continue the virus lifecycle. Image from Black et al, 2002. 

The significance of this becomes more apparent when the virus population itself is considered. Viral RNA polymerases are notoriously error prone, resulting in a population of viruses with differences in their genomes, in other words the so-called quasispecies (itself something worthy of discussion). What happens to the population structure as the transmission cycle progresses through the various stages and bottlenecks?

An interesting talk at the SGM in Dublin this year has now been published in PLoS Pathogens. In this case, the authors made an artificial virus population of Venezuelan equine encephalitis virus (VEEV) using reverse genetics. This then allowed particular clones to be followed through the various steps of infection as the virus passes from a mouse containing the clones through to how many are injected into a fresh mouse at the end of the mosquito steps of the virus lifecycle.
If they allowed mosquitoes to feed on a mouse containing the different 'clones' of VEEV, and then looked to see how many they could find in the mosquito, they found between 6 and 8 of the clones (i.e. ~70% of all clones). After a few days however, once the infection process is under way, the number of clones had decreased, implying that not all of the clones had made it through the midgut infection process.

By the time the virus has disseminated to the legs/wings, and salivary glands, there were only 1-4 of the clones present, suggesting that yet more had been lost along the way. In the saliva, which represents the population of virus that will be passed on to the new host, they only found on average 1-3 of the clones. This contrasts to the number of clones found in the legs/wings, showing that only some of the clones from the haemocoel managed to successfully infect the salivary glands.

Interestingly, there didn't appear to be a strong bottleneck in transmission to an uninfected mouse; the same clones that were in the saliva were the same clones which infected the mice, although considering the low numbers of clones in the saliva there wasn't a massive choice.

All of this leads to the puzzling question of how does the virus survive? If at each point viruses are removed, then by chance the surviving population may be less fit. Looking for marked viruses as this study has done is an elegant way of doing it, but it is limited to signatures within the genome. A further study using deep sequencing to look at the whole population more comprehensively may offer even more detail about what's going on. Perhaps this all relates to the observation of purifying selection being popular among arboviruses, time will tell, but to begin with this study has shown that such barriers exist. How the severity of the barriers vary, and how the virus deals with these barriers, will be interesting to see.

William C. Black IVa, , , Kristine E. Bennetta, Norma Gorrochótegui-Escalantea, Carolina V. Barillas-Murya, Ildefonso Fernández-Salasb, Marı́a de Lourdes Muñozc, José A. Farfán-Aléd, Ken E. Olsona, Barry J. Beaty (2002). Flavivirus Susceptibility in Aedes aegypti Archives of Medical Research DOI: 10.1016/S0188-4409(02)00373-9

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