West Nile Virus Isolates Research Articles

An outbreak of West Nile Virus infection in the region of Monastir, Tunisia, 2003

Background:A West Nile (WN) fever epidemic occurred in the region of Monastir, Tunisia, between August and October 2003.Aim of the study:We attempt to describe the epidemiology, clinical presentation, and outcome of patients with confirmed West Nile virus (WNV) infection.Methods:Three groups of specimens were prepared. One was made up of serum only (n = 43), the other of cerebrospinal fluid (CSF) only (n = 30), and the third group was made up of both (n = 40). These specimens were obtained from 113 patients. A serological diagnosis and evidence of WNV genome by nested reverse-transcriptase polymerase chain reaction (nRT-PCR) and TaqMan reverse transcription-polymerase chain reaction (RT-PCR) were carried out.Results:Thirty-eight cases (33.6%) were serologically positive. Results of nRT-PCR showed a total of 10 positive cases of WNV (8.8%) detected in group 1 (n = 1/43), group 2 (n = 5/30), and group 3 (n = 4/40) whereas the PCR TaqMan showed 18 positive samples (15.9%) found in group 1 (n = 3/43), group 2 (n = 9/30), and group 3 (n = 6/40). All TaqMan PCR positive cases were nRT-PCR positive. In addition, four serologically probable cases were confirmed by TaqMan PCR. The attempts to isolate WNV by cell culture were unsuccessful. Considering the results of TaqMan assay and the serological diagnosis, WNV infection was confirmed in a total of 42 patients. The main clinical presentations were meningoencephalitis (40%), febrile disease (95%), and meningitis (36%). Eight patients (19%) died. The highest case-fatality rates occurred among patients aged ≧55 years. The phylogenetic analysis revealed that isolates of WNV were closely related to the Tunisian strain 1997 (PAH001) and the Israeli one (Is-98).Conclusions:West Nile virus is a reemerging global pathogen that remains an important public health challenge in the next decade.

Experimental infection of house sparrows (Passer domesticus) with West Nile virus isolates of Euro-Mediterranean and North American origins

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle involving wild birds as reservoir hosts. The virus has recently emerged in North America and re-emerged in Europe. North American WNV outbreaks are often accompanied by high mortality in wild birds, a feature that is uncommon in Europe. The reason for this difference is unknown, but the intrinsic virulence of the viruses circulating in each continent and/or the susceptibility to the disease of Palearctic as opposed to Nearctic wild bird species could play a role. To assess this question, experimental inoculations with four lineage 1 WNV strains, three from southern Europe (Italy/2008, Italy/2009 and Spain/2007) and one from North America (NY99) were performed on house sparrows (Passer domesticus), a wild passerine common in both continents. Non-significant differences which ranged from 0% to 25% were observed in mortality for the different WNV strains. Viremias lasted from 1 to 5–6 days post-inoculation (dpi) in all cases; individuals inoculated with NY99 had significantly higher titres than those inoculated with any of the Euro-Mediterranean strains. Remarkably, host competence was found to be higher for NY99 than for the other strains. Consequently, albeit being pathogenic for house sparrows, some Euro-Mediterranean strains had reduced capacity for replication in -and transmission from- this host, as compared to the NY99 strain. If applicable also to other wild bird host species, this relatively reduced transmission capacity of the Euro-Mediterranean strains could explain the lower incidence of this disease in wild birds in the Euro-Mediterranean area.

Vector competence of Culex pipiens quinquefasciatus (Diptera: Culicidae) for West Nile virus isolates from Florida.

To assess vector competence (infection, dissemination and transmission) of Culex pipiens quinquefasciatus for Florida (FL) West Nile virus (WNV) isolates. West Nile virus isolates (WN-FL-03: NY99 genotype; WN-FL-05-558, WN-FL-05-2186, WN-FL-05-510: WN02 genotype) collected from different regions of FL were used for vector competence experiments in Cx. p. quinquefasciatus from Alachua County and Indian River County in FL. Mosquitoes from both colonies were fed blood containing 7.9 ± 0.2 log10 plaque-forming units WNV/ml ± SE and incubated at 28 °C for 14 days. Vector competence, including rates of infection, dissemination, and transmission, was compared between colonies for WN-FL-03 using chi-squared. Virus titres in bodies, legs and saliva were compared using anova. Daily measurements of in vitro replication of WNV isolates were evaluated in Vero cells so that a standardised virus dose for each isolate could be delivered to mosquitoes. Infection and dissemination rates were high (≥ 95%) and not affected by isolate or colony (infection, P = 0.679; dissemination, P = 0.799). Transmission rates were low (≤ 20%), detected in one colony and affected by isolate (P = 0.008). Body and leg titres differed between isolates (body titre, P = 0.031; leg titre, P = 0.044) and colonies (body titre, P = 0.001; leg titre, P = 0.013) while saliva titre did not differ between isolates (P = 0.462). Variation in vector competence of mosquito populations may be attributed, in part, to exposures to WNV with genetic differences leading to different rates of replication in mosquitoes. Evaluation of vector competence for different WNV isolates may help us understand vector-virus interactions and, hence, the role of vectors in complex virus transmission cycles in nature.

Co-circulation of West Nile Virus Variants, Arizona, USA, 2010

Molecular analysis of West Nile virus (WNV) isolates obtained during a 2010 outbreak in Maricopa County, Arizona, USA, demonstrated co-circulation of 3 distinct genetic variants, including strains with novel envelope protein mutations. These results highlight the continuing evolution of WNV in North America and the current complexity of WNV dispersal and transmission.

Molecular evolution of West Nile virus

Phylogenetic analysis and estimation of the rate of evolution of West Nile virus (WNV) were conducted. Sixty-eight nucleotide sequences of WNV E protein were used for the analysis. The rate of nucleotide substitution accumulation was 2.5 × 10−4 substitutions per site per year. Phylogenetic analysis and estimation of WNV evolution time using molecular-clock methodology demonstrated that the WNV genotypes 1, 2, and 4 with an estimated time of divergence from the common precursor of approximately 2360, 2800, and 5950 years, respectively, circulate on the territory of the European part of Russia. The ratio of frequencies of nonsynonymous substitutions (dN) to synonymous substitutions (dS) can vary within 0.022–0.275 for certain WNV strains grouped according to geographical and/or phylogenetic traits. The highest values of dN/dS ratio were found for modern WNV isolates in Russia and in North America, which appeared in new natural biocenoses of these regions in the last 14 years. dN/dS estimation for WNV species shows that indices of intraspecific dN/dS variability can be used for detecting the presence of accelerated evolution of new WNV isolates. All this confirms the hypothesis that favorable conditions exist for wide distribution and rapid evolution of different WNV genotypes (that arose 2000–6000 years ago) in modern natural and climatic conditions.

West Nile Virus Transmission in Winter: The 2013 Great Salt Lake Bald Eagle and Eared Grebes Mortality Event

West Nile Virus (WNV) infection has been reported in over 300 species of birds and mammals. Raptors such as eagles, hawks and falcons are remarkably susceptible, but reports of WNV infection in Bald Eagles (Haliaeetus leucocephalus) are rare and reports of WNV infection in grebes (Podicipediformes) even rarer. We report an unusually large wild bird mortality event involving between 15,000-20,000 Eared Grebes (Podiceps nigricollis) and over 40 Bald Eagles around the Great Salt Lake, Utah, in November-December 2013. Mortality in grebes was first reported in early November during a period when the area was unseasonably warm and the grebes were beginning to gather and stage prior to migration. Ten out of ten Eared Grebes collected during this period were WNV RT-PCR and/or isolation positive. This is the first report of WNV infection in Eared Grebes and the associated mortality event is matched in scale only by the combined outbreaks in American White Pelican (Pelecanus erythrorhynchos) colonies in the north central states in 2002-2003. We cannot be sure that all of the grebes were infected by mosquito transmission; some may have become infected through contact with WNV shed orally or cloacally from other infected grebes. Beginning in early December, Bald Eagles in the Great Salt Lake area were observed to display neurological signs such as body tremors, limb paralysis and lethargy. At least 43 Bald Eagles had died by the end of the month. Nine of nine Bald Eagles examined were infected with WNV. To the best of our knowledge, this is the largest single raptor mortality event since WNV became endemic in the USA. Because the majority of the eagles affected were found after onset of below-freezing temperatures, we suggest at least some of the Bald Eagles were infected with WNV via consumption of infected Eared Grebes or horizontal transmission at roost sites.

Whole genome phylogenetic investigation of a West Nile virus strain isolated from a tick sampled from livestock in north eastern Kenya

West Nile virus (WNV) has a wide geographical distribution and has been associated to cause neurological disease in humans and horses. Mosquitoes are the traditional vectors for WNV; however, the virus has also been isolated from tick species in North Africa and Europe which could be a means of introduction and spread of the virus over long distances through migratory birds. Although WNV has been isolated in mosquitoes in Kenya, paucity of genetic and pathogenicity data exists. We previously reported the isolation of WNV from ticks collected from livestock and wildlife in Ijara District of Kenya, a hotspot for arbovirus activity. Here we report the full genome sequence and phylogenetic investigation of their origin and relation to strains from other regions. A total of 10,488 ticks were sampled from animal hosts, classified to species and processed in pools of up to eight ticks per pool. Virus screening was performed by cell culture, RT-PCR and sequencing. Phylogenetic analysis was carried out to determine the evolutionary relationships of our isolate. Among other viruses, WNV was isolated from a pool of Rhipicephalus pulchellus sampled from cattle, sequenced and submitted to GenBank (Accession number: KC243146). Comparative analysis with 27 different strains revealed that our isolate belongs to lineage 1 and clustered relatively closely to isolates from North Africa and Europe, Russia and the United States. Overall, Bayesian analysis based on nucleotide sequences showed that lineage 1 strains including the Kenyan strain had diverged 200 years ago from lineage 2 strains of southern Africa. Ijara strain collected from a tick sampled on livestock was closest to another Kenyan strain and had diverged 20 years ago from strains detected in Morocco and Europe and 30 years ago from strains identified in the USA. To our knowledge, this is the first characterized WNV strain isolated from R. pulchellus. The epidemiological role of this tick in WNV transmission and dissemination remains equivocal but presents tick verses mosquito virus transmission has been neglected. Genetic data of this strain suggest that lineage 1 strains from Africa could be dispersed through tick vectors by wild migratory birds to Europe and beyond.

Characterization of West Nile virus (WNV) isolates from Assam, India: Insights into the circulating WNV in northeastern India

West Nile virus (WNV) is a mosquito-borne flavivirus that causes subclinical symptoms, febrile illness with possible kidney infarction and encephalitis. Since WNV was first serologically detected in Assam during 2006, it has become recognized as an important etiological agent that causes acute encephalitis syndrome (AES) in addition to endemic Japanese encephalitis virus (JEV). Therefore, isolating and characterizing the currently circulating strain of WNV is important. The virus was isolated from the cerebrospinal fluid (CSF) of two patients that presented with AES. The genotyping of the isolates HQ246154 (WNIRGC07) and JQ037832 (WNIRTC08) based on the partial sequencing of 921 nucleotides (C-prM-E) of the genome placed them within lineage 5 along with other Indian strains isolated prior to 1982, but the present circulating virus formed a distinct subclade. The derived amino acid sequence alignment indicated substitution in A81T and A84P of the capsid region in HQ246154. A cross-neutralization assay suggested substantial antigenic variation between isolates. The pathogenesis in mice that suggested the circulating WNV was neuroinvasive and comparatively more pathogenic than previous strains from India.

Molecular detection and isolation of West Nile virus from a human case in northern Greece, 2013.

In order to laboratory confirm the first suspected West Nile fever case in 2013 in northern Greece, a combination of serological molecular and culture methods were applied. It was shown that the causative West Nile virus strain belonged to lineage 2, and possessed the amino acid substitution H249P in the NS3 protein, as in previous years. The significance of this specific strain in Europe remains to be elucidated.

Continued Evolution of West Nile Virus, Houston, Texas, USA, 2002–2012

We investigated the genetics and evolution of West Nile virus (WNV) since initial detection in the United States in 1999 on the basis of continual surveillance studies in the Houston, Texas, USA, metropolitan area (Harris County) as a surrogate model for WNV evolution on a national scale. Full-length genomic sequencing of 14 novel 2010–2012 WNV isolates collected from resident birds in Harris County demonstrates emergence of 4 independent genetic groups distinct from historical strains circulating in the greater Houston region since 2002. Phylogenetic and geospatial analyses of the 2012 WNV isolates indicate closer genetic relationship with 2003–2006 Harris County isolates than more recent 2007–2011 isolates. Inferred monophyletic relationships of these groups with several 2006–2009 northeastern US isolates supports potential introduction of a novel WNV strain in Texas since 2010. These results emphasize the need to maintain WNV surveillance activities to better understand WNV transmission dynamics in the United States.

Resurgence of West Nile Virus in 2012: Lack of Evidence for Viral Genetic Determinants

Since the introduction into North America in 1999 of West Nile virus (WNV) and its subsequent dramatic spread across the United States and Canada, human cases of WNV infection declined from their 2003 peak of 9,862 to only 712 cases and 43 fatalities confirmed in 2011 (http://www.cdc .gov/ncidod/dvbid/westnile/index.htm). The reasons for this decline remain unclear because avian amplification host and human herd immunity remains low in most regions and WNV continues to circulate in most areas of the United States. However, last year an unexpected resurgence in West Nile fever and encephalitis cases occurred; a total of 5,674 human cases was detected in the United States, with 286 deaths, making 2012 the deadliest year yet. The epidemic was focused in the central part of the United States, with Texas reporting 1,739 human infections and 89 deaths, or about 33% of the national total and more than three times the number of any other state. Within Texas, Dallas County was the epicenter, with 398 human infections and 20 deaths. The reasons for this dramatic resurgence in WNV infections are not clear; however, several factors could be involved ranging from increased enzootic bird-mosquito-bird circulation, resulting in more human spillover infections, to increased human virulence. In this issue, Duggal and others report complete genomic sequences of 17 WNV isolates from several regions of Texas and phylogenetic studies to determine if viral genetic changes might have been responsible for the resurgence in human cases. Two distinct WNV genotypes were identified, and both circulated in the Dallas and Houston regions where disease incidence differed markedly (about four times higher in Dallas). Importantly, no signs of adaptive evolution that could have resulted in more efficient infection of mosquitoes or birds were identified in any of the recent WNV strains. These results, along with epidemiological analyses, suggest that the 2012 resurgence in the Dallas area resulted from an increase in transmission by mosquitoes from birds to humans (spillover infections), rather than an increase in WNV neuroinvasiveness. Unusually high summer temperatures in the Dallas area, perhaps combined with ecological changes affecting mosquito or avian populations, probably played important roles in driving the 2012 outbreak. These critical findings presented by Duggal and others 1 and the magnitude of the 2012 West Nile encephalitis epidemic underscore the importance of a better understanding of how WNV, other arboviruses, and vector-borne diseases in general will respond to climate and anthropogenic ecological changes that now appear inevitable. Rising temperatures exert a wide variety of complex effects on mosquito survival, reproduction, and behavior. Because temperature also influences vector infection, dissemination, and transmission of arboviruses, often in unpredictable ways, we remain unable to anticipate the effects of global warming on arboviral disease. Droughts, which are predicted to increase in some regions of the United States and elsewhere, can also have dramatic impacts on mosquito populations, especially on West Nile mosquito vectors because of their affinity for standing, highly polluted water and to irrigated agricultural lands. Drought conditions can also concentrate avian amplifying hosts in wetter locations amenable to mosquito transmission of WNV. Thus, rigorous experimental and modeling studies are needed to improve the ability of our public health systems to anticipate and respond to the effects of climate change and its influences on vector-borne pathogens such as WNV.

Detection and Early Warning of West Nile Virus Circulation in Central Macedonia, Greece, Using Sentinel Chickens and Mosquitoes

Following the West Nile Virus (WNV) epidemic in 2010 in Central Macedonia, Greece, which resulted in 197 human neuroinvasive disease cases, we determined the seasonal appearance and prevalence of the virus in 2011 by testing weekly for WNV genomic RNA in mosquitoes collected in carbon dioxide-baited traps, and for anti-WNV antibodies in sentinel chickens. Preliminary findings of the surveillance program regarding the circulation of "Nea Santa-Greece-2010" in sentinel chickens were rapidly communicated to public health authorities. In the present article, the full 2011 data produced by this surveillance program are presented. We detected enzootic circulation of WNV in chickens 1 month prior to the onset of the first human cases in 2011. Culex pipiens and Cx. modestus were abundant throughout the sampling period and at all sites of increased transmission. Molecular identification and phylogenetic analysis of WNV isolates from two chickens and one Cx. pipiens mosquito pool suggested that: (1) the virulent "Nea Santa-Greece-2010" WNV lineage 2 strain responsible for the 2010 epidemic was actively circulating in 2011, and (2) all Greek isolates belong to a distinct recent evolutionary clade. In Europe, where numerous strains of different virulence coexist, sequencing information for WNV is important for phylogeography and identification of virulent strains for human health risk assessment.

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.

The Culex pipiens Complex in Continental Portugal: Distribution and Genetic Structure

Portugal is a southern European country that displays favorable ecological conditions for the establishment of West Nile virus (WNV) transmission cycles. Competent mosquito vector species are present throughout the country. Among the species with reported cases of WNV isolation in Portugal, Culex pipiens is the most ubiquitous and abundant mosquito. This species exhibits two biological forms with differences in host preferences. The molestus form has a greater tendency to feed upon humans and other mammals whereas the pipiens form prefers avian hosts. In northern latitudes, both forms are physically separated, with molestus occupying underground habitats and pipiens being found aboveground. However, the warmer climatic conditions of southern regions such as Portugal may favor the sympatric occurrence of both forms hence promoting interform hybridization. Genetic introgression between molestus and pipiens forms may result in a higher propensity for admixed populations to serve as bridge-vectors of WNV between humans and birds. Here we revise our present knowledge on the distribution, role in WNV transmission and genetic structure of the Cx. pipiens complex in continental Portugal. We focus on recent findings of sympatric molestus and pipiens populations that display considerable levels of hybridization and discuss the epidemiological repercussions of this occurrence.

Molecular evolution of lineage 2 West Nile virus

Since the 1990s West Nile virus (WNV) has become an increasingly important public health problem and the cause of outbreaks of neurological disease. Genetic analyses have identified multiple lineages with many studies focusing on lineage 1 due to its emergence in New York in 1999 and its neuroinvasive phenotype. Until recently, viruses in lineage 2 were not thought to be of public health importance due to few outbreaks of disease being associated with viruses in this lineage. However, recent epidemics of lineage 2 in Europe (Greece and Italy) and Russia have shown the increasing importance of this lineage. There are very few genetic studies examining isolates belonging to lineage 2. We have sequenced the full-length genomes of four older lineage 2 WNV isolates, compared them to 12 previously published genomic sequences and examined the evolution of this lineage. Our studies show that this lineage has evolved over the past 300-400 years and appears to correlate with a change from mouse attenuated to virulent phenotype based on previous studies by our group. This evolution mirrors that which is seen in lineage 1 isolates, which have also evolved to a virulent phenotype over the same period of time.

Dynamic transmission of West Nile virus across the United States–Mexican border

Confirmed clinical and veterinary cases of West Nile virus (WNV) infection in Mexico remain restricted to northern Mexico, supporting a unidirectional transmission model from the US into Mexico. Full-length genomic sequencing of nine WNV isolates obtained from Culex spp. mosquito pools in El Paso, Texas (n=7) and Cuidad Juarez, Mexico (n=2) from 2005 to 2010 demonstrates the co-circulation of three independent genetic groups, two of which belong to the southwestern (SW/WN03) genotype and the other to the North American (NA/WN02) genotype. These results indicate ongoing dynamic circulation of WNV between the United States and Mexico.

A hamster-derived West Nile virus strain is highly attenuated and induces a differential proinflammatory cytokine response in two murine cell lines

Increasing evidence suggests that West Nile virus (WNV) induces a persistent infection in some humans and animals. Here, we characterized infection of mouse macrophage and kidney epithelial cell lines with a strain of WNV (H8912), cultured from urine of a persistently infected hamster. WNV H8912 had a reduced replication rate, concurrent with a lower interferon (IFN)-β gene expression in both cell types compared to its parent strain – WNV NY99. In WNV H8912-infected macrophages, we observed higher interleukin (IL)-6 and tumor necrosis factor (TNF)-α expression and more nuclear factor kappa B (NF-κB) activation than in cells infected with WNV NY99. In contrast, there were reduced levels of TNF-α and IL-6 expression, as well as less NF-κB activation following WNV H8912 infection in the kidney epithelial cells compared to WNV NY99. Overall, our results demonstrate that the WNV isolate obtained from hamster urine is an attenuated virus and induces a differential proinflammatory cytokine response in mouse macrophage and kidney epithelial cell lines.

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.

Culex Flavivirus and West Nile Virus inCulex quinquefasciatusPopulations in the Southeastern United States

Little is known of the interactions between insect-only flaviviruses and other arboviruses in their mosquito hosts, or the potential public health significance of these associations. The specific aims of this study were to describe the geographic distribution, prevalence, and seasonal infection rates of Culex flavivirus (CxFV) and West Nile virus (WNV) in Culex quinquefasciatus Say in the Southeastern United States, investigate the potential association between CxFV and WNV prevalence in Cx. quinquefasciatus and describe the phylogenetic relationship among CxFV and WNV isolates from the Southeastern United States and around the world. Using ArboNET records, 11 locations were selected across Georgia, Mississippi, and Louisiana that represented a range of WNV human case incidence levels. Cx. quinquefasciatus were trapped weekly throughout the summer of 2009 and pools were screened for flavivirus RNA by reverse transcriptase polymerase chain reaction. Cx. quinquefasciatus from Georgia had significantly higher CxFV infection rates than either Mississippi or Louisiana. CxFV was not detected in Mississippi after July, and no CxFV was detected in Cx. quinquefasciatus in Louisiana. In Georgia, CxFV infection rates were variable between and within counties and over time. WNV infection rates were not significantly different across states or months, and WNV sequences from all three states were identical to each other in the envelope and NS5 gene regions. Phylogenetically, NS5 and E gene sequences from Georgia CxFV isolates clustered with CxFV from Japan, Iowa, and Texas. Multiple CxFV genetic variants were found circulating simultaneously in Georgia. No evidence was found supporting an association between WNV and CxFV infection prevalence in Cx. quinquefasciatus.

Molecular detection and characterization of West Nile virus associated with multifocal retinitis in patients from southern India

In late 2009/early 2010, approximately 2000 people were affected by a mysterious viral outbreak in a southern district of Tamil Nadu; this particularly affected those living in coastal areas. Blood samples from affected patients were sent for clinical analysis to determine the actual cause of the illness, but reports were inconclusive. The present study describes the clinical observations and laboratory investigations involving molecular methods performed on 170 of the 2000 clinically suspected cases. These were patients who were admitted to Aravind Eye Hospital, Madurai, Tamil Nadu with ocular complications. Conventional reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) assays were used to detect West Nile virus (WNV) infection. Further investigation of the genetic diversity of the WNV implicated in ocular complications was undertaken by sequence phylogeny. Out of 170 samples, 25 (15%) were positive for chikungunya IgM antibody, 10 (6%) for chikungunya antigen, and 30 (18%) were positive for dengue IgM antibody. The remaining 105 seronegative samples were further processed for WNV detection by IgM capture ELISA and molecular methods. Out of the 105 samples, 35 (33%) were positive for WNV IgM antibody, 15 (14%) were positive for WNV by RT-PCR, and 27 (26%) were found to be positive for WNV by both real-time RT-PCR and RT-LAMP assays. Comparative evaluation with acute-phase patient serum samples revealed 100% concordance between the real-time RT-PCR and RT-LAMP assays. These assays had an overall higher sensitivity than the conventional RT-PCR as they picked up 12 additional samples with a low copy number of template. Further genotyping through sequence phylogeny revealed that all the WNV isolates were grouped in lineage I. The association of West Nile virus with ocular infection in South India during an epidemic of mysterious fever in the first half of 2010 was clearly established through molecular approaches employing envelope gene-specific real-time RT-PCR and RT-LAMP assays followed by nucleotide sequencing.