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.