Andriyan Grinev

The Global Ecology and Epidemiology of West Nile Virus

Since its initial isolation in Uganda in 1937 through the present, West Nile virus (WNV) has become an important cause of human and animal disease worldwide. WNV, an enveloped virus of the genus Flavivirus, is naturally maintained in an enzootic cycle between birds and mosquitoes, with occasional epizootic spillover causing disease in humans and horses. The mosquito vectors for WNV are widely distributed worldwide, and the known geographic range of WNV transmission and disease has continued to increase over the past 77 years. While most human infections with WNV are asymptomatic, severe neurological disease may develop resulting in long-term sequelae or death. Surveillance and preventive measures are an ongoing need to reduce the public health impact of WNV in areas with the potential for transmission.

Monocytes-macrophages are a potential target in human infection with West Nile virus through blood transfusion

BACKGROUND:  West Nile virus (WNV) transmission by transfusion was documented in 2002. Approximately 80 percent of WNV infections are asymptomatic and 1 percent develop severe neurological illness. In animals, Langerhans‐dendritic cells support initial viral replication, followed by replication in lymphoid tissues and dissemination to organs and possibly to the CNS. The cellular tropism of WNV infection after transfusion and the particular human blood cells that sustain viral replication remain largely unknown. Whether primary monocyte‐derived macrophages (MDMs) support WNV infection‐replication and produce infectious virions, with an in vitro system, was investigated.

Evolutionary dynamics of West Nile virus in the United States, 1999-2011: phylogeny, selection pressure and evolutionary time-scale analysis.

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.

Genetic Variability of West Nile Virus in US Blood Donors, 2002–2005

This virus is diverging from precursor isolates as its geographic distribution expands.

Anti-West Nile virus activity of in vitro expanded human primary natural killer cells

BackgroundNatural 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.ResultsCo-culture of PBMC with irradiated K562D2 cells expanded the NK cell number by 2-3 logs in 2-3 weeks, with more than 90% purity; upregulated NK cell surface activation receptors; downregulated inhibitory receptors; and boosted interferon gamma (IFN-γ) production by ~33 fold. The expanded NK (D2NK) cell has strong natural killing activity against both K562 and Vero cells, and killed the WNV infected Vero cells through antibody-dependent cellular cytotoxicity (ADCC). The D2NK cell culture supernatants inhibited both WNV replication and WNV induced cytopathic effect (CPE) in Vero cells when added before or after infection. Anti-IFN-γ neutralizing antibody blocked the NK supernatant-mediated anti-WNV effect, demonstrating a noncytolytic activity mediated through IFN-γ.ConclusionsCo-culture of PBMC with K562D2 stimulatory cells is an efficient technique to prepare large quantities of pure and active NK cells, and these expanded NK cells inhibited WNV infection of Vero cells through both cytolytic and noncytolytic activities, which may imply a potential role of NK cells in combating WNV infection.

Microarray-based assay for the detection of genetic variations of structural genes of West Nile virus.

Adaptation through fixation of spontaneous mutations in the viral genome is considered to be one of the important factors that enable recurrent West Nile virus (WNV) outbreaks in the U.S. Genetic variations can alter viral phenotype and virulence, and degrade the performance of diagnostic and screening assays, vaccines, and potential therapeutic agents. A microarray assay was developed and optimized for the simultaneous detection of any nucleotide mutations in the entire structural region of WNV in order to facilitate public health surveillance of genetic variation of WNV. The DNA microarray consists of 263 oligonucleotide probes overlapping at half of their lengths which have been immobilized on an amine-binding glass slide. The assay was validated using 23 WNV isolates from the 2002-2005 U.S. epidemics. Oligonucleotide-based WNV arrays detected unambiguously all mutations in the structural region of each one of the isolates identified previously by sequencing analysis, serving as a rapid and effective approach for the identification of mutations in the WNV genome.

Dengue virus and other arboviruses: a global view of risks

Arboviruses (arthropod‐borne viruses) are an ecological group of viruses from different families (e.g. Bunyaviridae, Flaviviridae and Togaviridae) that use arthropods such as mosquitoes, flies and ticks as vectors for transmission between different hosts. The superb plasticity of these viruses allows propagation to different host systems including both invertebrates and vertebrates. More than 500 species of arbo‐virus have been described and are listed in the International Catalogue of arbo‐virus (http://www.cdc.gov/nczved/divisions/dvbid/arbovirus.html), many of which are of medical importance.

Genetic Variability of West Nile Virus in U.S. Blood Donors from the 2012 Epidemic Season.

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.

Application of a full-genome microarray-based assay for the study of genetic variability of West Nile virus.

Viral adaptation through fixation of spontaneous mutations is an important factor potentially associated with reoccurrence of West Nile virus (WNV) outbreaks in the New World. The emergence of new genetic variants of WNV represents an important public health issue because it may affect the sensitivity of WNV screening and diagnostic assays, as well as the development and efficacy of WNV vaccines and anti-viral drugs. A microarray assay was developed and optimized to enable simple monitoring of WNV genetic variability and rapid detection of any nucleotide mutations within the entire viral genome. The assay was validated using 11 WNV isolates from the 2007 and 2009 U.S. epidemics. The developed microarray system can potentially serve as a high throughput, rapid, and effective approach for the detection of circulating WNV genetic variants.

Development of a microarray-based assay for rapid monitoring of genetic variants of West Nile virus circulating in the United States.

West Nile virus (WNV) has become endemic in the Western Hemisphere since its first introduction in the United States in 1999. An important factor associated with annual reoccurrence of WNV outbreaks in the U.S. is viral adaptation to domestic mosquitoes and birds through accumulation of spontaneous mutations in the WNV genome. Newly emerged mutations in the viral genome can potentially negatively affect the performance of existing diagnostic and screening assays and future vaccines. Therefore, the genetic monitoring of the WNV viral population during annual outbreaks is extremely important for public health and can only be achieved by application of efficient sample preparation methods followed by high throughput genetic analysis. In this study, we developed and evaluated a method for specific isolation of WNV genomic RNA from plasma samples without cultivation of the virus in cells. In combination with the microarray-based genetic analysis of the isolated WNV genomic RNA, this approach is suitable for fast, high throughput genotyping of circulating WNV genetic variants. The methods were evaluated using WNV isolates from the 1999-2012U.S. epidemics.