Serological evidence for Japanese encephalitis and West Nile virus infections in domestic birds in Cambodia

Mosquito-borne flaviviruses with an enzootic transmission cycle like Japanese encephalitis virus (JEV) and West Nile virus (WNV) are a major public health concern. The circulation of JEV in Southeast Asia is well-documented, and the important role of pigs as amplification hosts for the virus is long known. The influence of other domestic animals especially poultry that lives in high abundance and close proximity to humans is not intensively analyzed. Another understudied field in Asia is the presence of the closely related WNV. Such analyses are difficult to perform due to the intense antigenic cross-reactivity between these viruses and the lack of suitable standardized serological assays. The main objective of this study was to assess the prevalence of JEV and WNV flaviviruses in domestic birds, detailed in chickens and ducks, in three different Cambodian provinces. We determined the flavivirus seroprevalence using an hemagglutination inhibition assay (HIA). Additionally, we investigated in positive samples the presence of JEV and WNV neutralizing antibodies (nAb) using foci reduction neutralization test (FRNT). We found 29% (180/620) of the investigated birds positive for flavivirus antibodies with an age-depended increase of the seroprevalence (OR = 1.04) and a higher prevalence in ducks compared to chicken (OR = 3.01). Within the flavivirus-positive birds, we found 43% (28/65) with nAb against JEV. We also observed the expected cross-reactivity between JEV and WNV, by identifying 18.5% double-positive birds that had higher titers of nAb than single-positive birds. Additionally, seven domestic birds (10.7%) showed only nAb against WNV and no nAb against JEV. Our study provides evidence for an intense JEV circulation in domestic birds in Cambodia, and the first serological evidence for WNV presence in Southeast Asia since decades. These findings mark the need for a re-definition of areas at risk for JEV and WNV transmission, and the need for further and intensified surveillance of mosquito-transmitted diseases in domestic animals

Evidence for West Nile Virus and Usutu Virus Infections in Wild and Resident Birds in Germany, 2017 and 2018

Wild birds play an important role as reservoir hosts and vectors for zoonotic arboviruses and foster their spread. Usutu virus (USUV) has been circulating endemically in Germany since 2011, while West Nile virus (WNV) was first diagnosed in several bird species and horses in 2018. In 2017 and 2018, we screened 1709 live wild and zoo birds with real-time polymerase chain reaction and serological assays. Moreover, organ samples from bird carcasses submitted in 2017 were investigated. Overall, 57 blood samples of the live birds (2017 and 2018), and 100 organ samples of dead birds (2017) were positive for USUV-RNA, while no WNV-RNA-positive sample was found. Phylogenetic analysis revealed the first detection of USUV lineage Europe 2 in Germany and the spread of USUV lineages Europe 3 and Africa 3 towards Northern Germany. USUV antibody prevalence rates were high in Eastern Germany in both years. On the contrary, in Northern Germany, high seroprevalence rates were first detected in 2018, with the first emergence of USUV in this region. Interestingly, high WNV-specific neutralizing antibody titers were observed in resident and short-distance migratory birds in Eastern Germany in 2018, indicating the first signs of a local WNV circulation

A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis

The structural flexibility or 'breathing' of the envelope (E) protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F) of the West Nile virus (WNV) E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV), but not Zika virus (ZIKV), E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing

West Nile virus transmission. results from the integrated surveillance system in Italy, 2008 to 2015

IIn Italy a national Plan for the surveillance of imported and autochthonous human vector-borne diseases (chikungunya, dengue, Zika virus disease and West Nile virus (WNV) disease) that integrates human and veterinary (animals and vectors) surveillance, is issued and revised annually according with the observed epidemiological changes. Here we describe results of the WNV integrated veterinary and human surveillance systems in Italy from 2008 to 2015. A real time data exchange protocol is in place between the surveillance systems to rapidly identify occurrence of human and animal cases and to define and update the map of affected areas i.e. provinces during the vector activity period from June to October. WNV continues to cause severe illnesses in Italy during every transmission season, albeit cases are sporadic and the epidemiology varies by virus lineage and geographic area. The integration of surveillance activities and a multidisciplinary approach made it possible and have been fundamental in supporting implementation of and/or strengthening preventive measures aimed at reducing the risk of transmission of WNV trough blood, tissues and organ donation and to implementing further measures for vector control

Outbreak of West Nile virus causing severe neurological involvement in children, Nuba Mountains, Sudan, 2002.

An atypical outbreak of West Nile virus (WNV) occurred in Ngorban County, South Kordophan, Sudan, from May to August 2002. We investigated the epidemic and conducted a case-control study in the village of Limon. Blood samples were obtained for cases and controls. Patients with obvious sequelae underwent cerebrospinal fluid (CSF) sampling as well. We used enzyme-linked immunosorbent assay (ELISA) and neutralization tests for laboratory diagnosis and identified 31 cases with encephalitis, four of whom died. Median age was 36 months. Bivariate analysis did not reveal any significant association with the risk factors investigated. Laboratory analysis confirmed presence of IgM antibodies caused by WNV in eight of 13 cases, indicative of recent viral infection. The unique aspects of the WNW outbreak in Sudan, i.e. disease occurrence solely among children and the clinical domination of encephalitis, involving severe neurological sequelae, demonstrate the continuing evolution of WNV virulence. The spread of such a virus to other countries or continents cannot be excluded

Physico-chemical requirements and kinetics of membrane fusion of flavivirus-like particles.

Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus pre-membrane and envelope glycoprotein genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examined the physico-chemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37 °C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on a timescale of 7-20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process

Human case of West Nile neuroinvasive disease in Portugal, summer 2015

A case of West Nile virus (WNV) infection was reported in the Algarve region, Portugal, in the first week of September 2015. WNV is known to circulate in Portugal, with occasional reports in horses and birds (2004 to 2011) and very sporadically human cases (in 2004 and in 2010). Here we present the clinical and laboratory aspects related to the first human case of West Nile neuroinvasive disease reported in Portugal

Assessing the impact of climate change on vector-borne viruses in the EU through the elicitation of expert opinion

Expert opinion was elicited to undertake a qualitative risk assessment to estimate the current and future risks to the European Union (EU) from five vector-borne viruses listed by the World Organization for Animal Health. It was predicted that climate change will increase the risk of incursions of African horse sickness virus (AHSV), Crimean-Congo haemorrhagic fever virus (CCHFV) and Rift Valley fever virus (RVFV) into the EU from other parts of the world, with African swine fever virus (ASFV) and West Nile virus (WNV) being less affected. Currently the predicted risks of incursion were lowest for RVFV and highest for ASFV. Risks of incursion were considered for six routes of entry (namely vectors, livestock, meat products, wildlife, pets and people). Climate change was predicted to increase the risk of incursion from entry of vectors for all five viruses to some degree, the strongest effects being predicted for AHSV, CCHFV and WNV. This work will facilitate identification of appropriate risk management options in relation to adaptations to climate change

Determinants of the population growth of the West Nile virus mosquito vector Culex pipiens in a repeatedly affected area in Italy

Background The recent spread of West Nile Virus in temperate countries has raised concern. Predicting the likelihood of transmission is crucial to ascertain the threat to Public and Veterinary Health. However, accurate models of West Nile Virus (WNV) expansion in Europe may be hampered by limited understanding of the population dynamics of their primary mosquito vectors and their response to environmental changes.<p></p> Methods We used data collected in north-eastern Italy (2009–2011) to analyze the determinants of the population growth rate of the primary WNV vector Culex pipiens. A series of alternative growth models were fitted to longitudinal data on mosquito abundance to evaluate the strength of evidence for regulation by intrinsic density-dependent and/or extrinsic environmental factors. Model-averaging algorithms were then used to estimate the relative importance of intrinsic and extrinsic variables in describing the variations of per-capita growth rates.<p></p> Results Results indicate a much greater contribution of density-dependence in regulating vector population growth rates than of any environmental factor on its own. Analysis of an average model of Cx. pipiens growth revealed that the most significant predictors of their population dynamics was the length of daylight, estimated population size and temperature conditions in the 15 day period prior to sampling. Other extrinsic variables (including measures of precipitation, number of rainy days, and humidity) had only a minor influence on Cx. pipiens growth rates.<p></p> Conclusions These results indicate the need to incorporate density dependence in combination with key environmental factors for robust prediction of Cx. pipiens population expansion and WNV transmission risk. We hypothesize that detailed analysis of the determinants of mosquito vector growth rate as conducted here can help identify when and where an increase in vector population size and associated WNV transmission risk should be expected.<p></p&gt