West Nile virus (WNV), an arbovirus maintained in a bird-mosquito enzootic cycle, can infect other vertebrates including humans. WNV was first reported in the US in 1999 where, to date, three genotypes belonging to WNV lineage I have been described (NY99, WN02, SW/WN03). We report here the WNV sequences obtained from two birds, one mosquito, and 29 selected human samples acquired during the US epidemics from 2006–2011 and our examination of the evolutionary dynamics in the open-reading frame of WNV isolates reported from 1999–2011. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses and selection pressure analyses were conducted with the HyPhy package. Phylogenetic analysis identified human WNV isolates within the main WNV genotypes that have circulated in the US. Within genotype SW/WN03, we have identified a cluster with strains derived from blood donors and birds from Idaho and North Dakota collected during 2006–2007, termed here MW/WN06. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. The mean nucleotide substitution rate for WNV isolates obtained from humans was calculated to be 5.06×10⁻⁴ substitutions/site/year (s/s/y). The Bayesian skyline plot shows that after a period of high genetic variability following the introduction of WNV into the US, the WNV population appears to have reached genetic stability. The establishment of WNV in the US represents a unique opportunity to understand how an arbovirus adapts and evolves in a naïve environment. We describe a novel, well-supported cluster of WNV formed by strains collected from humans and birds from Idaho and North Dakota. Adequate genetic surveillance is essential to public health since new mutants could potentially affect viral pathogenesis, decrease performance of diagnostic assays, and negatively impact the efficacy of vaccines and the development of specific therapies. West Nile Virus (WNV) is a mosquito-borne virus of African origin that is widespread around the world. The WNV life-cycle involves mosquitoes and birds, but humans and other animals can be infected, although they are not considered to be important players in the transmission cycle. Clinically, most WNV infections are unapparent, but the virus can disseminate to the central nervous system causing a potentially fatal neurological disease, especially in susceptible populations including elderly and immunocompromised individuals. West Nile virus can also be transmitted by organ transplant and by transfusion of blood and blood components. Like other arboviruses, WNV has the extraordinary capacity of growing in the different microenvironments represented by the invertebrate vector and the vertebrate hosts. From an evolutionary standpoint, the arrival of WNV in the US in 1999 represents a unique opportunity to explore the processes involved in the adaptation and dissemination of an arbovirus in a naïve environment. From the study of WNV sequences, we can not only learn about the evolutionary mechanisms that govern arboviruses, but also update diagnostic tests that rely on the detection of the viral genome upon the occurrence of mutations and study the existence of genetic markers that may be responsible for increases in clinical cases and their severity.

Natural Killer (NK) cells are a crucial component of the host innate immune system with anti-viral and anti-cancer properties. However, the role of NK cells in West Nile virus (WNV) infection is controversial, with reported effects ranging from active suppression of virus to no effect at all. It was previously shown that K562-mb15-41BBL (K562D2) cells, which express IL-15 and 4-1BBL on the K562 cell surface, were able to expand and activate human primary NK cells of normal peripheral blood mononuclear cells (PBMC). The expanded NK cells were tested for their ability to inhibit WNV infection in vitro.

West Nile virus (WNV) is an arbovirus maintained in nature in a bird-mosquito enzootic cycle which can also infect other vertebrates including humans. WNV is now endemic in the United States (U.S.), causing yearly outbreaks that have resulted in an estimated total of 4–5 million human infections. Over 41,700 cases of West Nile disease, including 18,810 neuroinvasive cases and 1,765 deaths, were reported to the CDC between 1999 and 2014. In 2012, the second largest West Nile outbreak in the U.S. was reported, which caused 5,674 cases and 286 deaths. WNV continues to evolve, and three major WNV lineage I genotypes (NY99, WN02, and SW/WN03) have been described in the U.S. since introduction of the virus in 1999. We report here the WNV sequences obtained from 19 human samples acquired during the 2012 U.S. outbreak and our examination of the evolutionary dynamics in WNV isolates sequenced from 1999–2012. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses. Selection pressure analyses were performed with the HyPhy package using the Datamonkey web-server. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. Thirteen of the 19 completely sequenced isolates from 10 U.S. states were genetically similar, sharing up to 55 nucleotide mutations and 4 amino acid substitutions when compared with the prototype isolate WN-NY99. Overall, these analyses showed that following a brief contraction in 2008–2009, WNV genetic divergence in the U.S. continued to increase in 2012, and that closely related variants were found across a broad geographic range of the U.S., coincident with the second-largest WNV outbreak in U.S. history. West Nile virus (WNV; family Flaviviridae, genus Flavivirus) is a mosquito-borne virus maintained in a bird-mosquito enzootic cycle. WNV can occasionally infect other animals and humans, which are considered dead-end hosts because they produce too little virus in blood to re-infect mosquitoes. Most human infections (~80%) do not cause symptoms, and when symptoms do occur, they may vary from mild flu-like illness to fatal neuroinvasive disease (~1%). WNV can be transmitted by transfusion of blood and blood components and by organ transplantation, posing a risk to the blood supply and public health. There is no specific therapy or vaccine for WNV in humans. WNV now is one of the most widely distributed flaviviruses in the world. Comparative studies of WNV genetic sequences have described two major groupings of WNV, lineages I and II, and up to five newer lineages, which correlate well with the geographical point of isolation. Since 1999, WNV has spread from New York City throughout the U.S. and the Americas including Mexico, Canada, the Caribbean and South America. The emergence of WNV in the U.S. with annual outbreaks represents a unique opportunity to understand how a mosquito-borne virus adapts and evolves in a new environment. Viral adaptation to domestic mosquitoes and birds is considered to have played an important role in the spread of WNV in the U.S. Continuous surveillance of WNV genetic variation is needed to protect public health because the tests used to diagnose infection and screen blood, as well as vaccines and drug therapies currently in development, may not perform as well against newer genetic variants of WNV.

West Nile virus (WNV) appeared in the U.S. in 1999 and has since become endemic, with yearly summer epidemics causing tens of thousands of cases of serious disease over the past 14 years. Analysis of WNV strains isolated during the 2006–2007 epidemic seasons demonstrates that a new genetic variant had emerged coincidentally with an intense outbreak in Idaho during 2006. The isolates belonging to the new variant carry a 13 nt deletion, termed ID-Δ13, located at the variable region of the 3′UTR, and are genetically related. The analysis of deletions and insertions in the 3′UTR of two major lineages of WNV revealed the presence of conserved repeats and two indel motifs in the variable region of the 3′UTR. One human and two bird isolates from the Idaho 2006–2007 outbreaks were sequenced using Illumina technology and within-host variability was analyzed. Continued monitoring of new genetic variants is important for public health as WNV continues to evolve.

We report the complete sequences of two West Nile virus strains (FDA-Hu02 and NY99) used for the formulation of CBER/FDA RNA reference reagents and lot release panels for use with nucleic acid technology testing.