Evaluation of NS4A, NS4B, NS5 and 3'UTR Genetic Determinants of WNV Lineage 1 Virulence in Birds and Mammals.

West Nile virus (WNV) is amplified in an enzootic cycle involving birds as amplifying hosts. Because they do not develop high levels of viremia, humans and horses are considered to be dead-end hosts. Mosquitoes, especially from the Culex genus, are vectors responsible for transmission between hosts. Consequently, understanding WNV epidemiology and infection requires comparative and integrated analyses in bird, mammalian, and insect hosts. So far, markers of WNV virulence have mainly been determined in mammalian model organisms (essentially mice), while data in avian models are still missing. WNV Israel 1998 (IS98) is a highly virulent strain that is closely genetically related to the strain introduced into North America in 1999, NY99 (genomic sequence homology > 99%). The latter probably entered the continent at New York City, generating the most impactful WNV outbreak ever documented in wild birds, horses, and humans. In contrast, the WNV Italy 2008 strain (IT08) induced only limited mortality in birds and mammals in Europe during the summer of 2008. To test whether genetic polymorphism between IS98 and IT08 could account for differences in disease spread and burden, we generated chimeric viruses between IS98 and IT08, focusing on the 3' end of the genome (NS4A, NS4B, NS5, and 3'UTR regions) where most of the non-synonymous mutations were detected. In vitro and in vivo comparative analyses of parental and chimeric viruses demonstrated a role for NS4A/NS4B/5'NS5 in the decreased virulence of IT08 in SPF chickens, possibly due to the NS4B-E249D mutation. Additionally, significant differences between the highly virulent strain IS98 and the other three viruses were observed in mice, implying the existence of additional molecular determinants of virulence in mammals, such as the amino acid changes NS5-V258A, NS5-N280K, NS5-A372V, and NS5-R422K. As previously shown, our work also suggests that genetic determinants of WNV virulence can be host-dependent.

Detection of West Nile Virus Lineage 2 in Eastern Romania and First Identification of Sindbis Virus RNA in Mosquitoes Analyzed using High-Throughput Microfluidic Real-Time PCR.

The impact of mosquito-borne diseases on human and veterinary health is being exacerbated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeastern Romania, we have used a combination of sampling methods in natural, urban and peri-urban sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365 pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of theCentral-Southeast European clade, has a wider circulation in Romania than previously known.

Serological evidence of equine infectious anaemia, West Nile fever, surra and equine piroplasmosis in a herd of horses in northern Argentina.

Northern Argentina hosts equine populations living under preserved natural areas and extensive breeding conditions, with limited access to veterinary care. Horses can be in contact with i) wildlife considered to be a potential reservoir of horse pathogens (e.g. capybara, coatis and pampas deer) and/or ii) potential disease vectors such as ticks, horse flies, Culicidae and vampire bats. In this context, the aim of this study was to assess the exposure of horses from a herd in northern Argentina to different vector-borne pathogens. Serum samples were collected from 20 horses on a farm in Chaco province. Most of these horses were in good health, but a few showed clinical signs such as fever, neurological signs or emaciation. Potential vectors (ticks, horse flies and Culicidae) were present and a fresh bite of a vampire bat (Desmodus rotundus) was observed on one horse. This serological survey revealed that 100% (20/20) were positive for equine infectious anaemia (EIA), 100% (18/18) for West Nile fever (WNF), 53% (10/19) for surra and 45% (9/20) for equine piroplasmosis (Babesia equi). Among these horses, four were found seropositive for all four infections. On the other hand, all the tested horses were seronegative for equine viral arteritis (EVA), Eastern equine encephalomyelitis (EEE), Venezuelan equine encephalitis (VEE), Western equine encephalomyelitis (WEE) and glanders. The data from this survey conducted on a small number of animals illustrate the need for an effective application of surveillance programmes and control measures for equine diseases in northern Argentina and constitute, to our knowledge, the first report of horses simultaneously seropositive for EIA, WNF, surra and equine piroplasmosis.

Contrasted Epidemiological Patterns of West Nile Virus Lineages 1 and 2 Infections in France from 2015 to 2019.

Since 2015, annual West Nile virus (WNV) outbreaks of varying intensities have been reported in France. Recent intensification of enzootic WNV circulation was observed in the South of France with most horse cases detected in 2015 (n = 49), 2018 (n = 13), and 2019 (n = 13). A WNV lineage 1 strain was isolated from a horse suffering from West Nile neuro-invasive disease (WNND) during the 2015 episode in the Camargue area. A breaking point in WNV epidemiology was achieved in 2018, when WNV lineage 2 emerged in Southeastern areas. This virus most probably originated from WNV spread from Northern Italy and caused WNND in humans and the death of diurnal raptors. WNV lineage 2 emergence was associated with the most important human WNV epidemics identified so far in France (n = 26, including seven WNND cases and two infections in blood and organ donors). Two other major findings were the detection of WNV in areas with no or limited history of WNV circulation (Alpes-Maritimes in 2018, Corsica in 2018-2019, and Var in 2019) and distinct spatial distribution of human and horse WNV cases. These new data reinforce the necessity to enhance French WNV surveillance to better anticipate future WNV epidemics and epizootics and to improve the safety of blood and organ donations.

Serological evidence of infection with dengue and Zika viruses in horses on French Pacific Islands.

New Caledonia and French Polynesia are areas in which arboviruses circulate extensively. A large serological survey among horses from New Caledonia and French Polynesia was carried out to investigate the seroprevalence of flaviviruses in the horse population. Here, 293 equine sera samples were screened for flaviviruses using a competitive enzyme-linked immunosorbent assay (cELISA). The positive sera were then confirmed using a flavivirus-specific microsphere immunoassay (MIA) and seroneutralization tests. This serosurvey showed that 16.6% (27/163) and 30.8% (40/130) of horses were positive for cELISA tests in New Caledonia and French Polynesia, respectively, but the MIA technique, targeting only flaviviruses causing neuro-invasive infections in humans and horses (i.e. West Nile virus [WNV], Japanese encephalitis virus [JEV] and tick-borne encephalitis virus [TBEV]), showed negative results for more than 85% (57/67) of the cELISA-positive animals. Seroneutralization tests with the main flaviviruses circulating in the South Pacific revealed that 6.1% (10/163; confidence interval [95% CI] 3.0%-11.0%) of sera in New Caledonia and 7.7% (10/130; 95% CI 3.8%-13.7%) in French Polynesia were positive for dengue virus serotype 1 (DENV1) and 4.3% (7/163; 95% CI 1.7%-8.6%) in New Caledonia and 15.4% (20/130, 95% CI 9.7%-22.8%) in French Polynesia were found positive for Zika virus (ZIKV). Seroprevalence of the JEV and WNV flaviviruses on the 293 samples from both island groups were comparatively much lower (less than 2%). This seroprevalence study in the horse population shows that horses can be infected with dengue and Zika viruses and that these infections lead to seroconversions in horses. The consequences of these infections in horses and their role in ZIKV and DENV epidemiological cycles are two issues that deserve further investigation.

Improved reliability of serological tools for the diagnosis of West Nile fever in horses within Europe.

West Nile Fever is a zoonotic disease caused by a mosquito-borne flavivirus, WNV. By its clinical sensitivity to the disease, the horse is a useful sentinel of infection. Because of the virus' low-level, short-term viraemia in horses, the primary tools used to diagnose WNV are serological tests. Inter-laboratory proficiency tests (ILPTs) were held in 2010 and 2013 to evaluate WNV serological diagnostic tools suited for the European network of National Reference Laboratories (NRLs) for equine diseases. These ILPTs were designed to evaluate the laboratories' and methods' performances in detecting WNV infection in horses through serology. The detection of WNV immunoglobulin G (IgG) antibodies by ELISA is widely used in Europe, with 17 NRLs in 2010 and 20 NRLs in 2013 using IgG WNV assays. Thanks to the development of new commercial IgM capture kits, WNV IgM capture ELISAs were rapidly implemented in NRLs between 2010 (4 NRLs) and 2013 (13 NRLs). The use of kits allowed the quick standardisation of WNV IgG and IgM detection assays in NRLs with more than 95% (20/21) and 100% (13/13) of satisfactory results respectively in 2013. Conversely, virus neutralisation tests (VNTs) were implemented in 33% (7/21) of NRLs in 2013 and their low sensitivity was evidenced in 29% (2/7) of NRLs during this ILPT. A comparison of serological diagnostic methods highlighted the higher sensitivity of IgG ELISAs compared to WNV VNTs. They also revealed that the low specificity of IgG ELISA kits meant that it could detect animals infected with other flaviviruses. In contrast VNT and IgM ELISA assays were highly specific and did not detect antibodies against related flaviviruses. These results argue in favour of the need for and development of new, specific serological diagnostic assays that could be easily transferred to partner laboratories.

Spatio-temporal trends and risk factors affecting West Nile virus and related flavivirus exposure in Spanish wild ruminants.

BACKGROUND: During the last decade, the spread of many flaviviruses (Genus Flavivirus) has been reported, representing an emerging threat for both animal and human health. To further study utility of wild ruminant samples in West Nile virus (WNV) surveillance, we assessed spatio-temporal trends and factors associated with WNV and cross-reacting flaviviruses exposure, particularly Usutu virus (USUV) and Meaban virus (MBV), in wild ruminants in Spain. Serum samples from 4693 wild ruminants, including 3073 free-living red deer (Cervus elaphus), 201 fallow deer (Dama dama), 125 mouflon (Ovis aries musimon), 32 roe deer (Capreolus capreolus) and 1262 farmed red deer collected in 2003-2014, were screened for WNV and antigenically-related flavivirus antibodies using a blocking ELISA (bELISA). Positive samples were tested for neutralizing antibodies against WNV, USUV and MBV by virus micro-neutralization tests. RESULTS: Mean flavivirus seroprevalence according to bELISA was 3.4 ± 0.5 % in red deer, 1.0 ± 1.4 % in fallow deer, 2.4 ± 2.7 % in mouflon and 0 % in roe deer. A multivariate logistic regression model revealed as main risk factors for seropositivity in red deer; year (2011), the specific south-coastal bioregion (bioregion 5) and presence of wetlands. Red deer had neutralizing antibodies against WNV, USUV and MBV. CONCLUSIONS: The results indicate endemic circulation of WNV, USUV and MBV in Spanish red deer, even in areas without known flavivirus outbreaks. WNV antibodies detected in a free-living red deer yearling sampled in 2010, confirmed circulation this year. Co-circulation of WNV and USUV was detected in bioregions 3 and 5, and of WNV and MBV in bioregion 3. Sampling of hunted and farmed wild ruminants, specifically of red deer yearlings, could be a complementary way to national surveillance programs to monitor the activity of emerging flaviviruses.

A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses.

West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using the Drosophila S2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.

A recombinant novirhabdovirus presenting at the surface the E Glycoprotein from West Nile Virus (WNV) is immunogenic and provides partial protection against lethal WNV challenge in BALB/c mice.

West Nile Virus (WNV) is a zoonotic mosquito-transmitted flavivirus that can infect and cause disease in mammals including humans. Our study aimed at developing a WNV vectored vaccine based on a fish Novirhabdovirus, the Viral Hemorrhagic Septicemia virus (VHSV). VHSV replicates at temperatures lower than 20°C and is naturally inactivated at higher temperatures. A reverse genetics system has recently been developed in our laboratory for VHSV allowing the addition of genes in the viral genome and the recovery of the respective recombinant viruses (rVHSV). In this study, we have generated rVHSV vectors bearing the complete WNV envelope gene (EWNV) (rVHSV-EWNV) or fragments encoding E subdomains (either domain III alone or domain III fused to domain II) (rVHSV-DIIIWNV and rVHSV-DII-DIIIWNV, respectively) in the VHSV genome between the N and P cistrons. With the objective to enhance the targeting of the EWNV protein or EWNV-derived domains to the surface of VHSV virions, Novirhadovirus G-derived signal peptide and transmembrane domain (SPG and TMG) were fused to EWNV at its amino and carboxy termini, respectively. By Western-blot analysis, electron microscopy observations or inoculation experiments in mice, we demonstrated that both the EWNV and the DIIIWNV could be expressed at the viral surface of rVHSV upon addition of SPG. Every constructs expressing EWNV fused to SPG protected 40 to 50% of BALB/cJ mice against WNV lethal challenge and specifically rVHSV-SPGEWNV induced a neutralizing antibody response that correlated with protection. Surprisingly, rVHSV expressing EWNV-derived domain III or II and III were unable to protect mice against WNV challenge, although these domains were highly incorporated in the virion and expressed at the viral surface. In this study we demonstrated that a heterologous glycoprotein and non membrane-anchored protein, can be efficiently expressed at the surface of rVHSV making this approach attractive to develop new vaccines against various pathogens.

Comparison of the neuropathology induced by two West Nile virus strains.

Some strains of West Nile virus (WNV) are neuroinvasive and may induce fatal encephalitis/meningitis in a variety of animal species including humans. Whether, however, there is a strain-specific signature in the brain is as yet unknown. Here we investigated the neuropathogenesis induced by two phylogenetically distant WNV strains of lineage 1, WNV(IS98) and WNV(KUN35 911). While four-week old C57Bl/6J mice were susceptible to both strains and succumbed rapidly after intraperitoneal inoculation, differences were observed in virulence and clinical disease. WNV(KUN35 911), the less virulent strain as judged by determination of LD50, induced typical signs of encephalitis. Such signs were not observed in WNV(IS98)-infected mice, although they died more rapidly. Histological examination of brain sections also revealed differences, as the level of apoptosis and inflammation was higher in WNV(KUN35 911)- than WNV(IS98)-infected mice. Moreover, staining for cleaved caspase 3 showed that the two WNV strains induced apoptotic death through different molecular mechanisms in one particular brain area. Finally, the two strains showed similar tropism in cortex, striatum, brainstem, and cerebellum but a different one in hippocampus. In summary, our data show that, upon peripheral administration, WNV(IS98) and WNV(KUN35 911) strains induce partially distinct lesions and tissue tropism in the brain. They suggest that the virulence of a WNV strain is not necessarily correlated with the severity of apoptotic and inflammatory lesions in the brain.

Differential virulence and pathogenesis of West Nile viruses.

West Nile virus (WNV) is a neurotropic flavivirus that cycles between mosquitoes and birds but that can also infect humans, horses, and other vertebrate animals. In most humans, WNV infection remains subclinical. However, 20%-40% of those infected may develop WNV disease, with symptoms ranging from fever to meningoencephalitis. A large variety of WNV strains have been described worldwide. Based on their genetic differences, they have been classified into eight lineages; the pathogenic strains belong to lineages 1 and 2. Ten years ago, Beasley et al. (2002) found that dramatic differences exist in the virulence and neuroinvasion properties of lineage 1 and lineage 2 WNV strains. Further insights on how WNV interacts with its hosts have recently been gained; the virus acts either at the periphery or on the central nervous system (CNS), and these observed differences could help explain the differential virulence and neurovirulence of WNV strains. This review aims to summarize the current state of knowledge on factors that trigger WNV dissemination and CNS invasion as well as on the inflammatory response and CNS damage induced by WNV. Moreover, we will discuss how WNV strains differentially interact with the innate immune system and CNS cells, thus influencing WNV pathogenesis.

Recent circulation of West Nile virus and potentially other closely related flaviviruses in Southern France.

In recent years, the number of West Nile virus (WNV) cases reported in horses and humans has increased dramatically throughout the Mediterranean basin. Furthermore, the emergence of Usutu virus (USUV) in Austria in 2001, and its subsequent expansion to Hungary, Spain, Italy, Switzerland, the United Kingdom, and Germany, has given added cause for concern regarding the impact of the spread of flaviviruses on human and animal health in western Europe. Despite frequent detection of WNV and USUV cases in neighboring countries, no case of WNV has been detected in France since 2006 and USUV has never been reported. However, recent investigations focused on detecting the circulation of flaviviruses in France are lacking. We investigated the circulation of WNV and USUV viruses in wild birds in southern France on the basis of a serological survey conducted on a sentinel species, the magpie (Pica pica), in the Camargue area from November, 2009, to December, 2010. We detected WNV-neutralizing antibodies at a high titer (160) in a second-year bird showing recent exposure to WNV, although no WNV case has been detected in humans or in horses since 2004 in the Camargue. In addition, we observed low titers (10 or 20) of USUV-specific antibodies in six magpies, two of which were also seropositive for WNV. Such low titers do not give grounds for concluding that these birds had been exposed to USUV; cross-reactions at low titers may occur between antigenically closely related flaviviruses. But these results urge for further investigations into the circulation of flaviviruses in southern France. They also emphasize the necessity of undertaking epidemiological studies on a long-term basis, rather than over short periods following public health crises, to gain insight into viral dynamics within natural reservoirs.

IS-98-ST1 West Nile virus derived from an infectious cDNA clone retains neuroinvasiveness and neurovirulence properties of the original virus.

Infectious clones of West Nile virus (WNV) have previously been generated and used to decipher the role of viral proteins in WNV virulence. The majority of molecular clones obtained to date have been derived from North American, Australian, or African isolates. Here, we describe the construction of an infectious cDNA clone of a Mediterranean WNV strain, IS-98-ST1. We characterized the biological properties of the recovered recombinant virus in cell culture and in mice. The growth kinetics of recombinant and parental WNV were similar in Vero cells. Moreover, the phenotype of recombinant and parental WNV was indistinguishable as regards viremia, viral load in the brain, and mortality in susceptible and resistant mice. Finally, the pathobiology of the infectious clone was examined in embryonated chicken eggs. The capacity of different WNV strains to replicate in embryonated chicken eggs closely paralleled their ability to replicate in mice, suggesting that inoculation of embryonated chicken eggs could provide a practical in vivo model for the study of WNV pathogenesis. In conclusion, the IS-98-ST1 infectious clone will allow assessment of the impact of selected mutations and novel genomic changes appearing in emerging European strains pathogenicity and endemic or epidemic potential. This will be invaluable in the context of an increasing number of outbreaks and enhanced severity of infections in the Mediterranean basin and Eastern Europe.