Vector-Borne Transmission Imposes a Severe Bottleneck on an RNA Virus Population

Abstract

RNA viruses typically occur in genetically diverse populations due to their error-prone genome replication. Genetic diversity is thought to be important in allowing RNA viruses to explore sequence space, facilitating adaptation to changing environments and hosts. Some arboviruses that infect both a mosquito vector and a mammalian host are known to experience population bottlenecks in their vectors, which may constrain their genetic diversity and could potentially lead to extinction events via Muller's ratchet. To examine this potential challenge of bottlenecks for arbovirus perpetuation, we studied Venezuelan equine encephalitis virus (VEEV) enzootic subtype IE and its natural vector Culex (Melanoconion) taeniopus, as an example of a virus-vector interaction with a long evolutionary history. Using a mixture of marked VEEV clones to infect C. taeniopus and real-time RT-PCR to track these clones during mosquito infection and dissemination, we observed severe bottleneck events that resulted in a significant drop in the number of clones present. At higher initial doses, the midgut was readily infected and there was a severe bottleneck at the midgut escape. Following a lower initial dose, the major bottleneck occurred at initial midgut infection. A second, less severe bottleneck was identified at the salivary gland infection stage following intrathoracic inoculation. Our results suggest that VEEV consistently encounters bottlenecks during infection, dissemination and transmission by its natural enzootic vector. The potential impacts of these bottlenecks on viral fitness and transmission, and the viral mechanisms that prevent genetic drift leading to extinction, deserve further study. The ability of arboviruses to perpetuate in nature given that they must infect two disparate hosts (the mosquito vector and the vertebrate host) remains a mystery. We studied how viral genetic diversity is impacted by the dual host transmission cycle. Our studies of an enzootic cycle using Venezuelan equine encephalitis virus (VEEV) and its natural mosquito, Culex taeniopus, revealed the stages of infection that result in a viral population bottleneck. Using a set of marked VEEV clones and repeated sampling at various time points following C. taeniopus infection, we determined the number of clones in various mosquito tissues culminating in transmission. Bottlenecks were identified but the stage of occurrence was dependent on the dose that initiated infection. Understanding the points at which mosquito-borne viruses are constrained will shed light on the ways in which virus diversity varies, leading to selection of mutants that may result in host range changes or alterations in virulence.