Prevalence and molecular epidemiology of West Nile and Usutu virus infections in Croatia in the ‘One health’ context, 2018

In 2018, Croatia reported the largest outbreak of West Nile virus (WNV) infections as well as the re‐occurrence of human Usutu virus (USUV) infections. For the first time, fatal WNV and USUV infections were detected in wild birds. We analysed epidemiological characteristics and molecular epidemiology of WNV and USUV infections detected during 2018 transmission season. From April to November, 178 patients with neuroinvasive disease and 68 patients with febrile disease were tested for WNV and USUV. Viral RNA was detected in cerebrospinal fluid (CSF) and urine samples using a real‐time RT‐PCR. Positive samples were tested by nested RT‐PCR and nucleotide sequencing. IgM/IgG antibodies were detected in serum/CSF samples using ELISA with confirmation of cross‐reactive samples by virus neutralization test (VNT). WNV neuroinvasive disease was confirmed in 54 and WNV fever in seven patients from 10 continental Croatian counties. Areas affected in 2018 were those in which cases occurred in previous seasons, while in three areas human cases were reported for the first time. Phylogenetic analysis of six strains from patients residing in different geographic areas showed circulation of WNV lineage 2. In three patients, neuroinvasive USUV infection was confirmed by RT‐PCR or VNT. Sequence analysis of one detected strain revealed USUV Europe 2 lineage. During the same period, a total of 2,574 horse and 1,069 poultry serum samples were tested for WNV antibodies using ELISA. Acute asymptomatic WNV infection (IgM antibodies) was documented in 20/0.7% horses. WNV IgG antibodies were found in 307/11.9% horses and in 125/12.7% poultry. WNV RNA was detected in two goshawks and USUV RNA was detected in one blackbird from north‐western Croatia. In the Zagreb area, 3,670 female mosquitoes were collected. One Culex pipiens pool collected in July tested positive for USUV RNA. Our results highlight the importance of continuous multidisciplinary ‘One health’ surveillance of these emerging arboviruses.     

Are tree squirrels involved in the circulation of flaviviruses in Italy?

West Nile virus (WNV ), Usutu virus (USUV ) and tick‐borne encephalitis virus (TBEV ) are emerging zoonotic flaviviruses (family Flaviviridae ), which have circulated in Europe in the past decade. A cross‐sectional study was conducted to assess exposure to these antigenically related flaviviruses in eastern grey squirrels (Sciurus carolinensis ) in Italy. Seventeen out of 158 (10.8%; CI _95%: 5.9‐15.6) squirrels’ sera tested through bELISA had antibodies against flaviviruses. Specific neutralizing antibodies to WNV , USUV and TBEV were detected by virus neutralization tests. Our results indicate that tree squirrels are exposed to Culex and tick‐borne zoonotic flaviviruses in Italy. Moreover, this study shows for the first time USUV and TBEV exposure in grey squirrels, broadening the host range reported for these viruses. Even though further studies are needed to define the real role of tree squirrels in the epidemiology of flaviviruses in Europe, this study highlights that serology could be an effective approach for future investigations aimed at broadening our knowledge about the species exposed to these zoonotic infections.     

A Serological Protein Microarray for Detection of Multiple Cross‐Reactive Flavivirus Infections in Horses for Veterinary and Public Health Surveillance

The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito‐borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu‐like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick‐borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection with WNV and JEV can lead to severe neurological disease and death, while USUV, SLEV and TBEV infections are mainly asymptomatic, however, and induce antibody responses. Horses often serve as sentinels to monitor active virus circulation in serological surveillance programmes specifically for WNV, USUV and JEV. Here, we developed and validated a NS1‐antigen protein microarray for the serological differential diagnosis of flavivirus infections in horses using sera of experimentally and naturally infected symptomatic as well as asymptomatic horses. Using samples from experimentally infected horses, an IgG and IgM specificity of 100% and a sensitivity of 95% for WNV and 100% for JEV was achieved with a cut‐off titre of 1 : 20 based on ROC calculation. In field settings, the microarray identified 93–100% of IgG‐positive horses with recent WNV infections and 87% of TBEV IgG‐positive horses. WNV IgM sensitivity was 80%. Differentiation between closely related flaviviruses by the NS1‐antigen protein microarray is possible, even though we identified some instances of cross‐reactivity among antibodies. However, the assay is not able to differentiate between naturally infected horses and animals vaccinated with an inactivated WNV whole‐virus vaccine. We showed that the NS1‐microarray can potentially be used for diagnosing and distinguishing flavivirus infections in horses and for public health purposes within a surveillance setting. This allows for fast, cheap, syndrome‐based laboratory testing for multiple viruses simultaneously for veterinary and public health purposes.     

Specific detection and differentiation of tick‐borne encephalitis and West Nile virus induced IgG antibodies in humans and horses

Tick‐borne encephalitis virus (TBEV) and West Nile virus (WNV) are important arthropod‐borne zoonotic flaviviruses. Due to the emergence of WNV in TBEV‐endemic regions co‐circulation of both viruses is increasing. Flaviviruses are structurally highly similar, which leads to cross‐reacting antibodies upon infection. Currently available serological assays for TBEV and WNV infections are therefore compromised by false‐positive results, especially in IgG measurements. In order to discriminate both infections novel diagnostic methods are needed. We describe an ELISA to measure IgG antibodies specific for TBEV and WNV, applicable to human and horse sera. Mutant envelope proteins were generated, that lack conserved parts of the fusion loop domain, a predominant target for cross‐reacting antibodies. These were incubated with equine and human sera with known TBEV, WNV or other flavivirus infections. For WNV IgG, specificities and sensitivities were 100% and 87.9%, respectively, for horse sera, and 94.4% and 92.5%, respectively, for human sera. TBEV IgG was detected with specificities and sensitivities of 95% and 96.7%, respectively, in horses, and 98.9% and 100%, respectively, in humans. Specificities increased to 100% by comparing individual samples on both antigens. The antigens could form the basis for serological TBEV‐ and WNV‐assays with improved specificities.     

Development of a Double‐Antigen Microsphere Immunoassay for Simultaneous Group and Serotype Detection of Bluetongue Virus Antibodies

Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme‐linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time‐consuming and require BSL3 facilities. VP2 protein is considered the major serotype‐defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV‐8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti‐VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti‐VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti‐VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously.     

Exposure to West Nile virus and tick‐borne encephalitis virus in dogs in Spain

In the past decade, the spread of emerging zoonotic flaviviruses (genus Flavivirus , family Flaviviridae ) has been reported in many regions worldwide, representing a threat to both human and animal health. A serosurvey was carried out to assess exposure and risk factors associated with antigenically related flaviviruses, particularly West Nile virus (WNV ), Usutu virus (USUV ) and tick‐borne encephalitis virus (TBEV ), in dogs in Spain. Flavivirus antibodies were detected in 39 of 815 dogs (4.8%; 95% CI : 3.3–6.3) by bELISA . Significantly higher seropositivity was observed in hunting dogs compared to pet dogs. Virus neutralization tests confirmed WNV ‐specific and TBEV ‐specific antibodies in 11 and 14 bELISA ‐positive dogs, respectively. This is the first serosurvey of WNV and TBEV in dogs in Spain and the first report of TBEV circulation in this country. The seropositivity obtained indicates widespread, but not homogeneous, distribution of WNV and TBEV in dogs in Spain. In 2013 and 2015, WNV ‐seropositive dogs were detected in those areas of Andalusia where the highest number of WNV outbreaks were reported in both horses and humans. Antibodies against TBEV have been found in dogs sampled in two different periods and regions in Spain. Serosurveillance in dogs could be a complementary way of monitoring the activity of emerging flaviviruses in Spain.     

First Detection of Co‐circulation of West Nile and Usutu Viruses in Equids in the South‐west of Tunisia

In the last fifteen years, West Nile Virus (WNV) has dramatically expanded its geographic range and is now considered the most widespread arbovirus in the world. In Tunisia, West Nile Fever (WNF) outbreaks were reported in humans in 1997, 2003 and 2012. Usutu Virus (USUV), which is a ‘new’ emerging Flavivirus antigenically close to WNV, has never been reported in Tunisia. A serological investigation in 284 equids was conducted in 2012 in the southern west region of the country to assess the presence and prevalence of the WNV and USUV infection. Of the 284 samples tested by competitive enzyme‐linked immunoassay, 129 were positive. Of these, 120 (42.3%) had WNV‐specific neutralizing antibodies. The prevalence was significantly higher in areas closer to the oasis compared with that of the surrounding arid areas. Antibody titres against USUV were also reported in 10 equids. This was the first evidence of USUV circulation in Tunisia. Data recorded by this study indicate that WNV and USUV have circulated/are circulating in the region and that there is an urgent need to adapt the current surveillance programmes to this new scenario.     

Development and evaluation of swine vesicular disease isotype‐specific antibody ELISAs based on recombinant virus‐like particles

Swine vesicular disease (SVD) is a contagious viral disease of pigs. The clinical signs of SVD are indistinguishable from other vesicular diseases, such as senecavirus A infection (SVA) and foot‐and‐mouth disease (FMD). Rapid and accurate diagnostic tests of SVD are considered essential in countries free of vesicular diseases. Competitive ELISA (cELISA) is the serological test used routinely. However, although cELISA is the standard test for SVD antibody testing, this test produces a small number of false‐positive results, which caused problems in international trade. The current project developed a SVD isotype antibody ELISA using recombinant SVD virus‐like particles (VLP) and an SVD‐specific monoclonal antibody (mAb) to reduce the percentage of false positives. The diagnostic specificities of SVD‐VLP isotype ELISAs were 98.7% and 99.6% for IgM and IgG. The SVD isotype ELISAs were SVD‐specific, without cross‐reactivity to other vesicular diseases. A panel of 16 SVD‐positive reference sera was evaluated using the SVD‐VLP isotype ELISAs. All sera were correctly identified as positive by the two combined SVD‐VLP isotype ELISAs. Comparison of the test results showed a high level of correlation between the SVDV antigen isotype ELISAs and SVD‐VLP isotype ELISAs. 303 sera from animals lacking clinical signs and history of SVDV exposure were identified positive using SVD cELISA. These samples were examined using SVD‐VLP isotype ELISAs. Of the 303 serum samples, five were positive for IgM, and five of 303 were positive for IgG. Comparable to virus neutralization test results, SVD isotype ELISAs significantly reduced the false‐positive samples. Based on above test results, the combined use of cELISA and isotype ELISAs can reduce the number of false‐positive samples and the use of time‐consuming virus neutralization tests, with benefit for international trade in swine and related products.     

Serological Evidence Indicates that Foot‐and‐Mouth Disease Virus Serotype O,C and SAT1 are most Dominant in Eritrea

Foot‐and‐mouth disease (FMD) is endemic in Eritrea and in most parts of Africa. To be able to control FMD using vaccination, information on the occurrence of various foot‐and‐mouth disease serotypes in Eritrea is needed. In this cross‐sectional study, 212 sera samples were collected from FMD infected and recovered animals in Eritrea. These samples were tested for the presence of antibodies against FMD non‐structural proteins (NSP) and neutralizing antibodies against six of the seven (all but SAT 3) serotypes of FMD virus (FMDV). Of these, 67.0% tested positive to non‐structural protein antibodies in the FMD NS ELISA. By virus neutralization, FMDV serotype O antibodies were shown to be the most dominant (approximately 50%). Virus neutralization test results indicate that infection with serotype C and SAT 1 might have occurred, although there are no reports of isolation of these two serotypes. Because the samples were not randomly selected, further random serological surveillance in all age group animals is necessary both to estimate the prevalence of FMD in the country and to confirm the serological results with serotype C and SAT 1.     

Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine

Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti‐IAV antibodies using homologous and heterologous haemagglutination‐inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)‐blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut‐off of S/N ≤ 0.60, the sensitivity and specificity of the NP‐blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post‐inoculation and the interactions between those factors (P < 0.0001). Serum antibodies against NP provide an ideal universal diagnostic screening target and could provide a cost‐effective approach for the detection and surveillance of IAV infections in swine populations.     

Foot‐and‐Mouth Disease Seroprevalence in Cattle in Eritrea

Information about seroprevalence of foot‐and‐mouth disease (FMD) and virus serotypes in Eritrea is unavailable, but is very important as it may guide the choice of intervention measures including vaccination to be implemented. We carried out a cross‐sectional study from February to June 2011 in Eritrea with a two‐stage cluster design, sampling cattle in 155 villages with the objective of determining the seroprevalence of FMD in four administrative regions of the country. We analysed cattle sera (n = 2429) for FMD virus antibodies using the non‐structural ELISA (NS ELISA) and virus neutralization test (VNT). The overall seroprevalence was 26% and 30% for the NS ELISA and VNT, respectively. FMD virus serotypes O (14%) and A (11%) were the most prevalent. Gash Barka showed the highest (39%) seroprevalence both in NS ELISA and VNT compared to the other three administrative regions. Strategic FMD virus vaccination with type O and A (matching circulating strains) in combination of zoo‐sanitary measures would be the best control option for Eritrea which could be started in areas where the disease is less endemic.     

Different dynamics of Usutu virus infections in Austria and Hungary, 2017–2018

Usutu virus (USUV), a mosquito‐borne flavivirus closely related to West Nile virus, emerged in Austria in 2001, when it caused a considerable mass‐mortality of Eurasian blackbirds. Cases in birds increased until 2003 and quickly declined thereafter, presumably due to developing herd immunity. Since 2006, no further cases were recorded, until two blackbirds were tested positive in 2016. In Hungary, USUV first appeared in 2005 and has caused only sporadic infections since then. Initially, the only genetic USUV lineage found across both countries was Europe 1. This changed in 2015/2016, when Europe 2 emerged, which has since then become the prevalent lineage. Due to dispersal of these strains and introduction of new genetic lineages, USUV infections are now widespread across Europe. In 2009, the first cases of USUV‐related encephalitis were described in humans, and the virus has been frequently detected in blood donations since 2016. To monitor USUV infections among the Austrian wild bird population in 2017/2018, 86 samples were investigated by RT‐PCR. In 67 of them, USUV nucleic acid was detected (17 in 2017, 50 in 2018). The majority of succumbed birds were blackbirds, found in Vienna and Lower Austria. However, the virus also spread westwards to Upper Austria and southwards to Styria and Carinthia. In Hungary, 253 wild birds were examined, but only six of them were infected with USUV (five in 2017, one in 2018). Thus, in contrast to the considerable increase in USUV‐associated bird mortality in Austria, the number of infections in Hungary declined after a peak in 2016. Except for one case of USUV lineage Africa 3 in Austria in 2017, Europe 2 remains the most prevalent genetic lineage in both countries. Since USUV transmission largely depends on temperature, which affects vector populations, climate change may cause more frequent USUV outbreaks in the future.     

Seroprevalence of selected flaviviruses in free‐living and captive capuchin monkeys in the state of Pernambuco,Brazil

Mosquito‐borne diseases such as dengue, yellow fever and, more recently, Chikungunya virus (CHIKV ) and Zika virus (ZIKV ) have a great impact in the public health. In addition, the presence of such viruses might have an impact on wild animal conservation as well as their possible role as animal reservoir. Here, we performed a serological survey searching for antibodies against a panel of flaviviruses [ZIKV , Dengue virus (DENV ), Yellow Fever virus (YFV ), West Nile virus (WNV ), Saint Louis Encephalitis virus (SLEV ), Ilheus virus (ILHV ) and Rocio virus (ROCV )] using plaque reduction neutralization test (PRNT ₉₀) in both free‐ranging and captive capuchin monkeys (Sapajus flavius and Sapajus libidinosus ). Captive and free‐living monkeys were sampled between June 2015 and January 2016 in the state of Pernambuco, including in the border with State of Paraíba, the epicentre of the ZIKV epidemics in Brazil. We have found neutralizing antibodies for ZIKV , DENV ‐1, DENV ‐2, DENV ‐3, DENV ‐4, YFV , ILHV and SLEV in both S . flavius and S . libidinosus samples. No positives samples were found for ROCV and WNV . Our results suggest that these flaviviruses might be circulating in capuchin monkey in the studied region. The possible presence of these viruses represents a risk for public health, as well as for animal conservation, especially for S . flavius which is a critically endangered species, facing high risk of extinction.     

Detection of Zaire ebolavirus in swine: Assay development and optimization

Ebolaviruses (family Filoviridae , order Mononegavirales ) cause often fatal, haemorrhagic fever in primates including humans. Pigs have been identified as a species susceptible to Reston ebolavirus (RESTV) infection, with indicated transmission to humans in the Philippines; however, their role during Ebola outbreaks in Africa needs to be clarified. To perform surveillance studies, detection of ebolavirus requires a prerequisite validation of viral RNA and antibody detection methods in swine samples. These diagnostic tests also need to be suitable for deployment to low‐level containment laboratories. In this study, we developed a set of tests for detection of antibodies against Zaire ebolavirus (EBOV ) in swine. Recombinant EBOV nucleoprotein was produced using a baculovirus expression system for indirect ELISA development. Evaluation of this assay was performed using laboratory and field samples, achieving a diagnostic specificity of 99%. Importantly, the indirect ELISA was able to detect antibodies to EBOV at 7 dpi, 3 days earlier than virus neutralization tests (VNT ). The format of the VNT in this work was modified to a microtitre plaque reduction neutralization assay (miPRNT ) complemented with immunostaining to provide a more rapid and highly specific assay. Finally, a confirmatory immunoblot assay was generated to supplement the indirect ELISA results.     

Circulation of a Simbu Serogroup Virus,Causing Schmallenberg Virus‐Like Clinical Signs in Northern Jordan

Schmallenberg virus (SBV)‐like clinical cases of abortions in northern Jordan in early 2013, together with the emergence of SBV in Europe in 2011, its rapid spread within the following years and the detection of this virus in Turkey, raised questions about the distribution of SBV or related orthobunyaviruses. To evaluate the occurrence of SBV or related members of the Simbu serogroup of orthobunyaviruses in Jordan, bulk milk (cattle) and serum samples (cattle, sheep and goat) collected in northern Jordan in 2013 were first tested by commercially available SBV antibody ELISAs. Indeed, 3 of 47 bulk milk samples and 57 of 115 serum samples provided positive results, but SBV specificity of the ELISA results could not be confirmed by virus neutralization assays. Instead, subsequent cross‐neutralization tests were able to further investigate the specificity of these antibodies. Here, a significant inhibition of Aino virus was observed. Thus, the causative agent was most likely a Simbu serogroup virus closely related to Aino virus. Consequently, these results confirm that members of this group of virus are not only present in Europe, Africa or Australia, but also in the Middle East.     

Natural Immunity of Sheep and Lambs Against the Schmallenberg Virus Infection

Since the first reports of the Schmallenberg disease (SBD) outbreaks in late 2011, the disease has spread across Europe, affecting cattle and sheep farms. While Schmallenberg virus (SBV) causes a mild clinical disease in adults, infection of pregnant females may lead to the production of typical congenital malformations (CMFs) in their offspring. It is speculated that the immunity acquired after a SBV infection is effective in preventing further infections. However, this has not been proven in naturally infected sheep, especially if they are pregnant when reinfected. The aim of this study was to monitor the natural immunity in SBV‐infected sheep. Twenty‐four ewes from the only Spanish farm with a SBV OIE‐notified outbreak were sampled. Subsequently, nine pregnant ewes were inoculated with SBV infectious plasma under controlled conditions. Six of them were euthanized before delivery, and their fetuses were inspected for lesions indicative for the SBV infection. The three remaining ewes were allowed to deliver one lamb each. Inoculation of the lambs was scheduled at approx. 3 months after birth. All samples were analyzed for viral RNA by RT‐PCR, and for antibodies by an indirect ELISA and a virus neutralization test (VNT). The majority of the 24 ewes showed a serological reaction against SBV. The three ewes that were allowed to lamb down demonstrated variable degrees of seroconversion which corresponded to the levels of immune reaction observed in their lambs. Moreover, no viral RNA was detected, no lesions were observed in the fetuses, and no clinical signs were detected in the inoculated animals. These findings suggest that the immunity acquired by sheep following a natural SBV infection could be sufficient to stop SBV reinfection. However, vaccination could be a valuable tool to control SBV infections and associated economic losses as it affords a more uniform and predictable protection at the flock/herd level.     

Serosurvey Reveals Exposure to West Nile Virus in Asymptomatic Horse Populations in Central Spain Prior to Recent Disease Foci

West Nile fever/encephalitis (WNF) is an infectious disease affecting horses, birds and humans, with a cycle involving birds as natural reservoirs and mosquitoes as transmission vectors. It is a notifiable disease, re‐emerging in Europe. In Spain, it first appeared in horses in the south (Andalusia) in 2010, where outbreaks occur every year since. However, in 2014, an outbreak was declared in horses in central Spain, approximately 200 km away from the closest foci in Andalusia. Before that, evidence of West Nile virus (WNV) circulation in central Spain had been obtained only from wildlife, but never in horses. The purpose of this work was to perform a serosurvey to retrospectively detect West Nile virus infections in asymptomatic horses in central Spain from 2011 to 2013, that is before the occurrence of the first outbreaks in the area. For that, serum samples from 369 horses, collected between September 2011 and November 2013 in central Spain, were analysed by ELISA (blocking and IgM) and confirmed by virus neutralization, proving its specificity using parallel titration with another flavivirus (Usutu virus). As a result, 10 of 369 horse serum samples analysed gave positive results by competitive ELISA, 5 of which were confirmed as positive to WNV by virus neutralization (seropositivity rate: 1.35%). One of these WNV seropositive samples was IgM‐positive. Chronologically, the first positive samples, including the IgM‐positive, corresponded to sera collected in 2012 in Madrid province. From these results, we concluded that WNV circulated in asymptomatic equine populations of central Spain at least since 2012, before the first disease outbreak reported in this area.     

First isolation of West Nile virus in Zambia from mosquitoes

Mosquito surveillance studies to identify mosquito‐borne flaviviruses have identified West Nile Virus (WNV ) for the first time in Zambia. The Zambian WNV isolate from Culex quinquefasciatus mosquitoes collected in the Western Province was closely related genetically to WNV lineage 2 South African strains which have been previously shown to be highly neuroinvasive. These data provide the first evidence of the circulation of WNV in Zambia and suggest there should be an increased awareness of possible associated human and animal diseases in that country.     

Mapping the Serological Prevalence Rate of West Nile fever in Equids,Tunisia

West Nile fever (WNF) is a viral disease of wild birds transmitted by mosquitoes. Humans and equids can also be affected and suffer from meningoencephalitis. In Tunisia, two outbreaks of WNF occurred in humans in 1997 and 2003; sporadic cases were reported on several other years. Small‐scale serological surveys revealed the presence of antibodies against WN virus (WNV) in equid sera. However, clinical cases were never reported in equids, although their population is abundant in Tunisia. This study was achieved to characterize the nationwide serological status of WNV in Tunisian equids. In total, 1189 sera were collected in 2009 during a cross‐sectional survey. Sera were tested for IgG antibodies, using ELISA and microneutralization tests. The estimated overall seroprevalence rate was 28%, 95% confidence interval [22; 34]. The highest rates were observed (i) in the north‐eastern governorates (Jendouba, 74%), (ii) on the eastern coast (Monastir, 64%) and (iii) in the lowlands of Chott El Jerid and Chott el Gharsa (Kebili, 58%; Tozeur, 52%). Environmental risk factors were assessed, including various indicators of wetlands, wild avifauna, night temperature and chlorophyllous activity (normalized difference vegetation index: NDVI). Multimodel inference showed that lower distance to ornithological sites and wetlands, lower night‐time temperature, and higher NDVI in late spring and late fall were associated with higher serological prevalence rate. The model‐predicted nationwide map of WNF seroprevalence rate in Tunisian equids highlighted different areas with high seroprevalence probability. These findings are discussed in the perspective of implementing a better WNF surveillance system in Tunisia. This system might rely on (i) a longitudinal survey of sentinel birds in high‐risk areas and time periods for WNV transmission, (ii) investigations of bird die‐offs and (iii) syndromic surveillance of equine meningoencephalitis.