To evaluate transmission dynamics, we exposed 25 bird species to West Nile virus (WNV) by infectious mosquito bite. We monitored viremia titers, clinical outcome, WNV shedding (cloacal and oral), seroconversion, virus persistence in organs, and susceptibility to oral and contact transmission. Passeriform and charadriiform birds were more reservoir competent (a derivation of viremia data) than other species tested. The five most competent species were passerines: Blue Jay (Cyanocitta cristata), Common Grackle (Quiscalus quiscula), House Finch (Carpodacus mexicanus), American Crow (Corvus brachyrhynchos), and House Sparrow (Passer domesticus). Death occurred in eight species. Cloacal shedding of WNV was observed in 17 of 24 species, and oral shedding in 12 of 14 species. We observed contact transmission among four species and oral in five species. Persistent WNV infections were found in tissues of 16 surviving birds. Our observations shed light on transmission ecology of WNV and will benefit surveillance and control programs.

West Nile virus (WNV), first recognized in North America in 1999, has been responsible for the largest arboviral epiornitic and epidemic of human encephalitis in recorded history. Despite the welldescribed epidemiological patterns of WNV in North America, the basis for the emergence of WNVassociated avian pathology, particularly in the American crow (AMCR) sentinel species, and the large scale of the North American epidemic and epiornitic is uncertain. We report here that the introduction of a T249P amino acid substitution in the NS3 helicase (found in North American WNV) in a low-virulence strain was sufficient to generate a phenotype highly virulent to AMCRs. Furthermore, comparative sequence analyses of full-length WNV genomes demonstrated that the same site (NS3-249) was subject to adaptive evolution. These phenotypic and evolutionary results provide compelling evidence for the positive selection of a mutation encoding increased viremia potential and virulence in the AMCR sentinel bird species.

In February 2019, following the annual taxon ratification vote, the order Bunyavirales was amended by creation of two new families, four new subfamilies, 11 new genera and 77 new species, merging of two species, and deletion of one species. This article presents the updated taxonomy of the order Bunyavirales now accepted by the International Committee on Taxonomy of Viruses (ICTV).

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In the past decade there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. We recently described the isolation of a novel flavivirus (tentatively designated “Nhumirim virus”; NHUV) (Pauvolid-Correa et al., in review) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. We characterized the in vitro growth potential, 3’ untranslated region (UTR) sequence homology with alternative flaviviruses, and evaluated the virus’s capacity to suppress replication of representative Culex spp. vectored pathogenic flaviviruses in mosquito cells. Only mosquito cell lines were found to support NHUV replication, further reinforcing the insect-specific phenotype of this virus. Analysis of the sequence and predicted RNA secondary structures of the 3’ UTR indicate NHUV to be most similar to viruses within the yellow fever serogroup, Japanese encephalitis serogroup, and viruses in the tick-borne flavivirus clade. NHUV was found to share the fewest conserved sequence elements when compared to traditional insect-specific flaviviruses. This suggests that, despite being apparently insect-specific, this virus likely diverged from an ancestral mosquito-borne flavivirus. Co-infection experiments indicated that prior or concurrent infection of mosquito cells with NHUV resulted in significant reduction in viral production of West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Japanese encephalitis virus. The inhibitory effect was most effective against WNV and SLEV with over a million-fold and 10,000-fold reduction in peak titers, respectively.

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To begin to define parameters that might explain the different virulence phenotypes between these two viruses, temperature-sensitivity assays were performed for both viruses at the high temperatures experienced in viraemic AMCRs. Growth curves of the two WNV strains were performed in African green monkey kidney (Vero; 37-42 6C) and duck embryonic fibroblast (DEF; 37-45 6C) cells cultured at temperatures that were tolerated by the cell line. Unlike the NY99 virus, marked decreases in KEN-3829 viral titres were detected between 36 and 120 h post-infection (p.i.) at temperatures above 43 6C. Replication of KEN-3829 viral RNA was reduced 6500-fold at 72 h p.i. in DEF cells incubated at 44 6C relative to levels of intracellular virus-specific RNA measured at 37 6C. In contrast, replication of virus derived from the NY99 infectious cDNA at 44 6C demonstrated only a 17-fold reduction in RNA level. These results indicated that the ability of WNV NY99 to replicate at the high temperatures measured in infected AMCRs could be an important factor leading to the increased avian virulence and emergence of this strain of WNV. INTRODUCTIONIn North America, West Nile virus (WNV; family Flaviviridae, genus Flavivirus) has become the leading cause of arboviral encephalitis in humans and equines (O'Leary et al., 2004) and is associated with mortality in >200 avian species (Hayes et al., 2005). The North American WNV strain, NY99, is highly virulent for American crows (AMCRs; Corvus brachyrhynchos) (Komar et al., 2003;McLean et al., 2001). AMCRs inoculated with this virus develop peak viraemic titres in excess of 10 log 10 p.f.u. ml 21 and suffer 100 % mortality within 6 days post-infection (p.i.) (Brault et al., 2004;Komar et al., 2003).Mortality in migratory storks and domesticated geese was identified in Israel between 1997(Bin et al., 2001 Malkinson et al., 2002), where a strain almost identical to the NY99 genotype has circulated (Lanciotti et al., 1999). Other studies have demonstrated that WNV strains from Africa and Australia cause reduced viraemia and mortality in AMCRs compared with the NY99 strain (Brault et al., 2004). These experimental avian infection data, coupled with the genetic relatedness of avian virulent strains, indicate that viral genetic determinants are responsible for the emergence of WNV-associated avian mortality.The WNV genome is a single-stranded, positive-sense RNA of approximately 11 kb. The single open reading frame has 59-and 39-terminal non-coding regions (NCRs) and encodes a polyprotein, which is co-and post-translationally cleaved by viral and host proteases to yield three structural et al., 2005), as well as the construction of an infectious clone of the African KEN-3829 strain. We report phenotypic characterization of viruses generated from these constructs in temperature-sensitivity experiments in vitro and in the in vivo AMCR model. METHODSCells and viruses. Duck embryonic fibroblast (DEF; ATCC CCL-141) and African green monkey kidney (Vero) cells were utilized for temperature-sensitivity s...

In October 2018, the order Bunyavirales was amended by inclusion of the family Arenaviridae, abolishment of three families, creation of three new families, 19 new genera, and 14 new species, and renaming of three genera and 22 species. This article presents the updated taxonomy of the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).