Explorations in West Nile virus ecology and evolution

2021 Spring.Includes bibliographical references.West Nile virus (WNV) continues to be a major cause of human arboviral neuroinvasive disease. Susceptible non-human vertebrates are particularly diverse, ranging from commonly affected birds and horses to less commonly affected species such as alligators. The literature review in Chapter 1 summarizes the pathology caused by West Nile virus during natural infections of humans and non-human animals. While the most well-known findings in human infection involve the central nervous system, WNV can also cause significant lesions in the heart, kidneys and eyes. Time has also revealed chronic neurologic sequelae related to prior human WNV infection. Similarly, neurologic disease is a prominent manifestation of WNV infection in most non-human non-host animals. However, in some avian species, which serve as the vertebrate host for WNV maintenance in nature, severe systemic disease can occur, with neurologic, cardiac, intestinal and renal injury leading to death. The pathology seen in experimental animal models of WNV infection and knowledge gains on viral pathogenesis derived from these animal models are also briefly discussed. A gap in the current literature exists regarding the relationship between the neurotropic nature of WNV in vertebrates, virus propagation and transmission in nature. This and other knowledge gaps, and future directions for research into WNV pathology, are addressed. In Chapter 2, experimental evolution work is described. For arboviruses, the vertebrate and invertebrate hosts in which they circulate shape viral evolution and can lead to the emergence of new genotypes. Previous work in mosquitoes and birds has identified species-specific effects on viral populations when species were assessed in isolation. We united mosquito and bird species to perform experimental evolution studies which paired Culex (Cx.) pipiens with American crows, Cx. quinquefasciatus with American crows and Cx. quinquefasciatus with American robins. Crow and Cx. pipiens transmission cycles were the most successful and robin and Cx. quinquefasciatus transmission cycles were the least successful at reaching three complete rounds of bird-to-mosquito transmission. These findings suggest that crows may be more important to WNV maintenance in nature over robins. The greater success of crow cycles when paired with Cx. pipiens in comparison to crows paired with Cx. quinquefasciatus may also suggest fitness losses associated with Cx. quinquefasciatus. In multiple rounds of transmission, infection rates (WNV-positive mosquito midgut) and transmission-capability (WNV-positive mosquito saliva) decreased with each subsequent round of transmission, suggesting that pairings in isolation experience fitness losses. Competitive fitness assays of transmission cycles exhibited cyclical increases and decreases in fitness as virus moved through crows and mosquitoes, respectively. That the stronger competitive fitness tended to occur with samples from the avian host while virus from mosquitoes tended to have decreased fitness may be consistent with genetic restriction and strong purifying selection in birds and genetic expansion and weak purifying selection in mosquitoes. Sequencing is needed to assess whether differences in transmission cycle success and competitive fitness can be attributed to genetic changes. In Chapter 3, the avian single cell viral environment is assessed. Error-prone replication of RNA viruses generates the viral diversity required for adaptation to rapidly changing environments. This is crucial for arboviruses whose viral populations exist as mutant swarms maintained between both mosquito and vertebrate hosts. By infecting cells and birds with barcoded WNV stock and sequencing single cells, we demonstrated that the richness and frequency of rare variants in crows far exceeded that found in robins. Moreover, those rare occurring variants were maintained by crows more than they were by robins. We further demonstrated that bird viremia functions as a determinant of multiplicity of infection in peripheral blood mononuclear cells (PBMCs), a significant site of viral replication. We found that increased viremia leads to increased polyinfections of individual PBMCs with maintenance of defective genomes and less prevalent variants, specifically in crows, presumably through complementation. When two pairings of variably-fit viruses were used to co-infect American robins and American crows, we observed increases in replication for one of the less fit viruses when viremia was higher. The ability of the low fitness virus to better replicate at higher viremia is likely a result of polyinfections and complementation at the cellular level. Our findings suggest that weak purifying selection in highly susceptible crows is attributable to higher viremia, polyinfections and complementation while viral divergence and fewer variants rising to fixation in robins is a result of overall lower levels of viremia and fewer polyinfections. In Chapter 4, the potential contributions of American alligators to natural WNV ecology are examined. West Nile virus (WNV) overwintering is poorly understood and likely multifactorial. Interest in alligators as a potential amplifying host arose when it was shown that they develop viremias theoretically sufficient to infect mosquitoes. We examined potential ways in which alligators may contribute to the natural ecology of WNV. We experimentally demonstrated that alligators are capable of WNV amplification with subsequent mosquito infection and transmission capability, that WNV-infected mosquitoes readily infect alligators and that water can serve as a source of infection for alligators but does not easily serve as in intermediate means for transmission between birds and alligators. These findings indicate potential mechanisms for maintenance of WNV outside of the primary bird-mosquito transmission cycle. We performed a diverse array of experiments which utilize novel techniques and technologies to characterize the mechanisms of WNV evolution. We also identified a potential non-avian WNV amplifier host in alligators. This work represents a significant contribution to the West Nile virus literature by working with the unique species which contribute to virus propagation and assessing their effects on viral evolution and ecology