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West Nile virus (WNV) has become firmly established in northeastern US, reemerging every summer since its introduction into North America in 1999. To determine whether WNV overwinters locally or is reseeded annually, we examined the patterns of viral lineage persistence and replacement in Connecticut over 10 consecutive transmission seasons by phylogenetic analysis. In addition, we compared the full protein coding sequence among WNV isolates to search for evidence of convergent and adaptive evolution. Viruses sampled from Connecticut segregated into a number of well-supported subclades by year of isolation with few clades persisting ≥ 2 years. Similar viral strains were dispersed in different locations across the state and divergent strains appeared within a single location during a single transmission season, implying widespread movement and rapid colonization of virus. Numerous amino acid substitutions arose in the population but only one change, V → A at position 159 of the envelope protein, became permanently fixed. Several instances of parallel evolution were identified in independent lineages, including one amino acid change in the NS4A protein that appears to be positively selected. Our results suggest that annual reemergence of WNV is driven by both reintroduction and local-overwintering of virus. Despite ongoing evolution of WNV, most amino acid variants occurred at low frequencies and were transient in the virus population.

West Nile virus is similar to most other RNA viruses in that it exists in nature as a genetically diverse population. However, the role of this genetic diversity within natural transmission cycles and its importance to virus perpetuation remain poorly understood. Therefore, we determined whether highly genetically diverse populations are more fit compared to less genetically diverse WNV populations. Specifically, we generated three WNV populations that varied in their genetic diversity and evaluated their fitness relative to genetically marked control WNV in vivo in Culex quinquefasciatus mosquitoes and chickens. Our results demonstrate that high genetic diversity leads to fitness gains in vector mosquitoes, but not chickens.

Rabies is a fatal zoonotic disease that requires fast, accurate diagnosis to prevent disease in an exposed individual. The current gold standard for post-mortem diagnosis of human and animal rabies is the direct fluorescent antibody (DFA) test. While the DFA test has proven sensitive and reliable, it requires high quality antibody conjugates, a skilled technician, a fluorescence microscope and diagnostic specimen of sufficient quality. The LN34 pan-lyssavirus real-time RT-PCR assay represents a strong candidate for rabies post-mortem diagnostics due to its ability to detect RNA across the diverse Lyssavirus genus, its high sensitivity, its potential for use with deteriorated tissues, and its simple, easy to implement design. Here, we present data from a multi-site evaluation of the LN34 assay in 14 laboratories. A total of 2,978 samples (1,049 DFA positive) from Africa, the Americas, Asia, Europe, and the Middle East were tested. The LN34 assay exhibited low variability in repeatability and reproducibility studies and was capable of detecting viral RNA in fresh, frozen, archived, deteriorated and formalin-fixed brain tissue. The LN34 assay displayed high diagnostic specificity (99.68%) and sensitivity (99.90%) when compared to the DFA test, and no DFA positive samples were negative by the LN34 assay. The LN34 assay produced definitive findings for 80 samples that were inconclusive or untestable by DFA; 29 were positive. Five samples were inconclusive by the LN34 assay, and only one sample was inconclusive by both tests. Furthermore, use of the LN34 assay led to the identification of one false negative and 11 false positive DFA results. Together, these results demonstrate the reliability and robustness of the LN34 assay and support a role for the LN34 assay in improving rabies diagnostics and surveillance.

A key feature in the recent widespread epidemic of the mosquito-borne alphavirus chikungunya virus (CHIKV) was the important role of Aedes albopictus, formerly regarded as a secondary vector, compared to the presumed primary vector Aedes aegypti. Ae. albopictus, a container-inhabiting mosquito, is an invasive species that occurs over a wide geographic range spanning tropical and temperate latitudes. In this study we examine the effects of a broad range of larval rearing temperatures on CHIKV infection, dissemination, and viral titer in Florida F(1) Ae. albopictus. Adults from larvae reared at 18 degrees C, 24 degrees C, and 32 degrees C differed significantly in size, development time, and CHIKV infection rate. Adult females with the largest body size were produced from the coolest temperature, took the longest to mature, and six times more likely to be infected with CHIKV than females reared at 32 degrees C. There was also a significant effect of rearing temperature on viral dissemination, resulting in an increase in population dissemination at the coolest temperature. This study indicates that climate factors, such as temperature, experienced at the larval stage, can influence the competence of adult females to vector arboviruses.

The increased difficulty and expense of using live animals for delivering infectious blood meals in arthropod-borne virus vector competence experiments has resulted in an increase in the use of artificial feeding systems. Compared to live hosts, artificial systems require higher viral titers to attain mosquito infection, thereby limiting the utility of such systems with low or moderate titer virus stocks. Based on the report that freshly propagated virus is more infectious than previously frozen virus, we determined whether such a preparation would enhance the ability to use artificial feeding systems. Culex quinquefasciatus and Aedes aegypti mosquitoes were offered blood in artificial membrane feeders containing freshly collected or previously frozen St. Louis encephalitis and dengue serotype-2 viruses (family Flaviviridae), respectively. Infection rates and estimates of vector competence were significantly lower (P < 0.05) for mosquitoes feeding on blood meals containing frozen–thawed compared to freshly collected virus. We indicate that the use of freshly propagated virus in artificial feeding systems can be an effective blood delivery method for low-titer viruses and viruses that are otherwise inefficient at infecting vectors in such systems. Fresh viruses used in artificial feeding systems may be a viable alternative to the heavily regulated and expensive use of live animals.

Most RNA viruses exist in their hosts as a heterogeneous population of related variants. Due to error prone replication, mutants are constantly generated which may differ in individual fitness from the population as a whole. Here we characterize three WNV isolates that contain, along with full-length genomes, mutants with large internal deletions to structural and nonstructural protein-coding regions. The isolates were all obtained from lorikeets that died from WNV at the Rio Grande Zoo in Albuquerque, NM between 2005 and 2007. The deletions are approximately 2 kb, in frame, and result in the elimination of the complete envelope, and portions of the prM and NS-1 proteins. In Vero cell culture, these internally deleted WNV genomes function as defective interfering particles, reducing the production of full-length virus when introduced at high multiplicities of infection. In mosquitoes, the shortened WNV genomes reduced infection and dissemination rates, and virus titers overall, and were not detected in legs or salivary secretions at 14 or 21 days post-infection. In mice, inoculation with internally deleted genomes did not attenuate pathogenesis relative to full-length or infectious clone derived virus, and shortened genomes were not detected in mice at the time of death. These observations provide evidence that large deletions may occur within flavivirus populations more frequently than has generally been appreciated and suggest that they impact population phenotype minimally. Additionally, our findings suggest that highly similar mutants may frequently occur in particular vertebrate hosts.

West Nile virus (WNV) (Flaviviridae: Flavivirus) is transmitted from mosquitoes to birds, but can cause fatal encephalitis in infected humans. Since its introduction into North America in New York in 1999, it has spread throughout the western hemisphere. Multiple outbreaks have also occurred in Europe over the last 20 years. This review highlights recent efforts to understand how host pressures impact viral population genetics, genotypic and phenotypic changes which have occurred in the WNV genome as it adapts to this novel environment, and molecular epidemiology of WNV worldwide. Future research directions are also discussed.

Due to error-prone replication, RNA viruses exist within hosts as a heterogeneous population of non-identical, but related viral variants. These populations may undergo bottlenecks during transmission that stochastically reduce variability leading to fitness declines. Such bottlenecks have been documented for several single-host RNA viruses, but their role in the population biology of obligate two-host viruses such as arthropod-borne viruses (arboviruses) in vivo is unclear, but of central importance in understanding arbovirus persistence and emergence. Therefore, we tracked the composition of West Nile virus (WNV; Flaviviridae, Flavivirus) populations during infection of the vector mosquito, Culex pipiens quinquefasciatus to determine whether WNV populations undergo bottlenecks during transmission by this host. Quantitative, qualitative and phylogenetic analyses of WNV sequences in mosquito midguts, hemolymph and saliva failed to document reductions in genetic diversity during mosquito infection. Further, migration analysis of individual viral variants revealed that while there was some evidence of compartmentalization, anatomical barriers do not impose genetic bottlenecks on WNV populations. Together, these data suggest that the complexity of WNV populations are not significantly diminished during the extrinsic incubation period of mosquitoes.

West Nile virus has spread rapidly throughout the United States since its introduction in 1999, into some areas that are also endemic for St. Louis encephalitis virus (SLEV). These viruses are in the same antigenic complex within the genus Flavivirus, family Flaviviridae. Further, both viruses are transmitted primarily by Culex spp. mosquitoes and use birds as amplifying hosts. These viruses could contemporaneously coinfect individual vectors wherein changes in mosquito immune responses might occur. To explore this possibility, we evaluated the effect of sequential infection with both West Nile virus and SLEV on the infection and dissemination rates of these viruses in the vector mosquito, Culex quinquefasciatus. Prior exposure to either virus lowered susceptibility to infection with the second virus, and lower dissemination rates of the second virus, compared to controls. Exposure to one virus followed by a second virus resulted in similar infection rates for the first virus to those of controls, but higher SLEV dissemination rates when exposed first to SLEV than in singly SLEV infected controls. While some mosquitoes became infected with both viruses, only one of those viruses disseminated from the midgut into the legs, indicating a midgut infection barrier to secondary infection. Lower infection rates in mosquitoes exposed to both viruses could change transmission patterns when these viruses are present at epizootic levels.

Chikungunya virus (CHIKV) is an arbovirus (genus Alphavirus, family Togaviridae) that has recently caused disease outbreaks in the Indian Ocean basin and southern Europe. These outbreaks could be associated with a possible shift in primary vector from Aedes aegypti to Ae. albopictus. To evaluate vector competence differences in possible CHIKV vectors, we evaluated the dose-dependant susceptibility of Florida strains of Ae. albopictus and Ae. aegypti for infection with a La Reunion island strain of CHIKV. Pledget and water-jacketed membrane feeding systems were also evaluated. We show that both Aedes spp. were susceptible to the highest CHIKV doses, whereas only Ae. albopictus developed disseminated infections after exposure to the two lowest doses. Infection rates for both mosquito species were significantly affected by the bloodmeal delivery method used. This information is important in assessing risk of an outbreak of imported CHIKV in the United States, in determining differences in vectorial capacity of these two vector species, and in evaluating arbovirus delivery methods in the laboratory.