Seroepidemiology of Selected Alphaviruses and Flaviviruses in Bats in Trinidad

A serosurvey of antibodies against selected flaviviruses and alphaviruses in 384 bats (representing 10 genera and 14 species) was conducted in the Caribbean island of Trinidad. Sera were analysed using epitope‐blocking enzyme‐linked immunosorbent assays (ELISAs) specific for antibodies against West Nile virus (WNV), Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), all of which are zoonotic viruses of public health significance in the region. Overall, the ELISAs resulted in the detection of VEEV‐specific antibodies in 11 (2.9%) of 384 bats. Antibodies to WNV and EEEV were not detected in any sera. Of the 384 sera, 308 were also screened using hemagglutination inhibition assay (HIA) for antibodies to the aforementioned viruses as well as St. Louis encephalitis virus (SLEV; which also causes epidemic disease in humans), Rio Bravo virus (RBV), Tamana bat virus (TABV) and western equine encephalitis virus (WEEV). Using this approach, antibodies to TABV and RBV were detected in 47 (15.3%) and 3 (1.0%) bats, respectively. HIA results also suggest the presence of antibodies to an undetermined flavivirus(es) in 8 (2.6%) bats. Seropositivity for TABV was significantly (P < 0.05; χ²) associated with bat species, location and feeding preference, and for VEEV with roost type and location. Differences in prevalence rates between urban and rural locations were statistically significant (P < 0.05; χ²) for TABV only. None of the aforementioned factors was significantly associated with RBV seropositivity rates.     

Detection of Antibodies to Alphaviruses and Discrimination between Antibodies to Eastern and Western Equine Encephalitis Viruses in Rabbit Sera using a Recombinant Antigen and Virus‐specific Monoclonal Antibodies

Three arthropod‐borne alphaviruses, western equine encephalitis viruses (WEEV), eastern equine encephalitis viruses (EEEV) and Venezuelan equine encephalitis viruses are the aetiological agents of a sometimes severe encephalomyelitis in equines and humans in the New World. With regard to the different ecology and epidemiology of these viruses, a method applied in serological screening should be able to distinguish between them as well as other related members of the genus Alphavirus in the American continent. However, this has been hampered in the past by (a) the close antigenic relationship between alphaviruses in traditional serological assays, especially in the routinely used haemagglutination‐inhibition, and (b) the need of biosafety level 3 facilities to grow the viral antigens. An epitope blocking assay using an EEEV glycoprotein E1‐expressing recombinant Sindbis virus and virus‐specific monoclonal antibodies (mAbs) binding to the E1 of EEEV (strain NJ/60) and the E1 of Sindbis virus was established using automated flow cytometry. The test was evaluated using sera of infected and vaccinated rabbits. A cut‐off value of 30% inhibition for antigenic complex‐specific seroconversion was found to be sufficient for the detection of the respective infection. By using three different mAbs in parallel, we were able to detect alphavirus genus‐, EEEV‐ and WEEV‐complex‐specific serum antibodies. As this test is based on the inhibition of binding of virus‐specific mAbs, sera of every origin other than mouse can be tested. Thus, this assay may prove useful in the serological screening of a variety of animal species during an outbreak investigation.     

The challenges posed by equine arboviruses

Equine populations worldwide are at increasing risk of infection by viruses transmitted by biting arthropods, including mosquitoes, biting midges (Culicoides), sandflies and ticks. These include the flaviviruses (Japanese encephalitis, West Nile and Murray Valley encephalitis), alphaviruses (eastern, western and Venezuelan encephalitis) and the orbiviruses (African horse sickness and equine encephalosis). This review provides an overview of the challenges faced in the surveillance, prevention and control of the major equine arboviruses, particularly in the context of these viruses emerging in new regions of the world.     

National Seroprevalence and Risk Factors for Eastern Equine Encephalitis and Venezuelan Equine Encephalitis in Costa Rica

Eastern equine encephalitis and Venezuelan equine encephalitis are endemic neglected tropical diseases in the Americas, causing encephalitis in both horses and humans. In 2013, a cross-sectional study was performed in 243 horses located in the highlands and lowlands throughout Costa Rica. Serum samples were analyzed with an IgG ELISA and confirmed by the plaque-reduction neutralization test (PRNT80). Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) overall seroprevalences by the PRNT80 were 36% (95% confidence interval [CI]: 29.9–42.5; 78/217 horses) and 3% (95% CI: 1.3–5.9; 6/217 horses), respectively. Both the viruses occurred in the lowlands and highlands. Rainfall and altitude were associated with VEEV seropositivity in the univariate analysis, but only altitude <100 meters above sea level was considered a risk factor in the multivariate analysis. No risk factors could be identified for the EEEV in the multivariate analysis. This is the first study that estimates the seroprevalence of the EEEV and VEEV in Costa Rican horses. The VEEV is widely distributed, whereas the EEEV occurs at a much lower frequency and only in specific areas. Clinical cases and occasional outbreaks of both viruses are to be expected.     

Encephalitic alphaviruses

This review will cover zoonotic, encephalitic alphaviruses in the family Togaviridae. Encephalitic alphaviruses, i.e. Western- (WEEV), Eastern- (EEEV), Venezuelan equine encephalitis virus (VEEV) and, more rarely, Ross River virus, Chikungunya virus and Highlands J virus (HJV), are neuroinvasive and may cause neurological symptoms ranging from mild (e.g., febrile illness) to severe (e.g., encephalitis) in humans and equines. Among the naturally occurring alphaviruses, WEEV, EEEV and VEEV have widespread distributions in North, Central and South America. WEEV has found spanning the U.S. from the mid-West (Michigan and Illinois) to the West coast and extending to Canada with human cases reported in 21 states. EEEV is found along the Gulf (Texas to Florida) and Atlantic Coast (Georgia to New Hampshire), as well as in the mid-West (Wisconsin, Illinois and Michigan) and in Canada, with human cases reported in 19 states. In contrast, transmission of VEEV occurs predominantly in Central and South America. As with their geographical distribution, equine encephalitis viruses differ in their main mosquito vector species and their zoonotic potential.     

Detection of antibodies to alphaviruses and discrimination between antibodies to eastern and western equine encephalitis viruses in rabbit sera using a recombinant antigen and virus-specific monoclonal antibodies

Three arthropod-borne alphaviruses, western equine encephalitis viruses (WEEV), eastern equine encephalitis viruses (EEEV) and Venezuelan equine encephalitis viruses are the aetiological agents of a sometimes severe encephalomyelitis in equines and humans in the New World. With regard to the different ecology and epidemiology of these viruses, a method applied in serological screening should be able to distinguish between them as well as other related members of the genus Alphavirus in the American continent. However, this has been hampered in the past by (a) the close antigenic relationship between alphaviruses in traditional serological assays, especially in the routinely used haemagglutination-inhibition, and (b) the need of biosafety level 3 facilities to grow the viral antigens. An epitope blocking assay using an EEEV glycoprotein E1-expressing recombinant Sindbis virus and virus-specific monoclonal antibodies (mAbs) binding to the E1 of EEEV (strain NJ/60) and the E1 of Sindbis virus was established using automated flow cytometry. The test was evaluated using sera of infected and vaccinated rabbits. A cut-off value of 30% inhibition for antigenic complex-specific seroconversion was found to be sufficient for the detection of the respective infection. By using three different mAbs in parallel, we were able to detect alphavirus genus-, EEEV- and WEEV-complex-specific serum antibodies. As this test is based on the inhibition of binding of virus-specific mAbs, sera of every origin other than mouse can be tested. Thus, this assay may prove useful in the serological screening of a variety of animal species during an outbreak investigation.     

Review of diagnostic plaque reduction neutralization tests for flavivirus infection

Flavivirus infections (including Japanese encephalitis, West Nile encephalitis and dengue fever/severe dengue) present a worldwide public health problem. Recent climate change may affect the geographical distribution of the arthropod vectors for these viruses and so the risk of flavivirus epidemics may increase. Many methods have been developed for the serological diagnosis of flavivirus infections, such as haemagglutination inhibition assay, enzyme-linked immunosorbent assay, and immunofluorescence in staining. However, the specificity of these assays varies.The plaque reduction neutralizing test (PRNT) using live viruses is currently the ‘gold standard’ for the differential serodiagnosis of flaviviruses. The specificity of results obtained with PRNT is better than that for other protocols and many laboratories apply the PRNT protocol to the differential serodiagnosis of flaviviruses. Here, recent refinements to the PRNT protocols with genetically modified recombinant viruses or reporter-harbouring virus-like particles are reviewed. Further, the problems associated with the differential serodiagnosis of flaviviruses using PRNT are discussed.     

Serological Investigations of Red Foxes (Vulpes vulpes L.) for Determination of the Spread of Tick‐borne Encephalitis in Northrhine‐Westphalia

Serum samples from 786 red foxes shot between January 1995 and August 1996 in the southern half of Northrhine‐Westphalia, located in western Germany, were tested for the presence of antibodies against tick‐borne encephalitis (TBE) virus using the Immunozym FSME IgG All Species‐ELISA^® (Immuno, Heidelberg, Germany) as a screening test: 759 sera were negative, 23 (2.9 %) were borderline, and four (0.5 %) were positive. Nine of the 27 ELISA reactive sera were confirmed by the TBE Western‐Blot (Immuno, Heidelberg, Germany). Furthermore these 27 sera were tested for neutralizing antibodies by means of a plaque reduction neutralization test (PRNT) against TBE and West Nile viruses. Only one single serum was found to have a neutralization titre (+ 1 : 800 PRNT₈₀) against TBE virus. All other 26 sera were negative for neutralizing antibodies against TBE or West Nile virus. Since the titre of the single serum is low, it can be interpreted that if TBE virus is present, its prevalence is extremely low. Northrhine‐Westphalia is not classified as a TBE‐endemic area. Further calculated serological testing of game and virological investigation of collected ticks in the affected area seem to be meaningful and necessary.     

5种脑炎人兽共患病病毒多重RT-PCR检测方法的建立

为建立同时检测流行性乙型脑炎病毒(JEV)、森林脑炎病毒(TBEV)、东方马脑炎病毒(EEEV)、西方马脑炎病毒(WEEV)和基孔肯雅病毒(CHIKV)5种人兽共患脑炎病病毒的多重RT-PCR方法,本研究根据GenBank登录的相关病毒基因序列设计特异引物,通过优化引物组合及PCR反应条件,建立可同时检测5种病毒的方法,扩增片段长度分别为411 bp(JEV)、945 bp(TBEV)、193 bp(EEEV)、545 bp(WEEV)和769 bp(CHIKV);该方法具有良好的特异性,对病毒核酸最低检测拷贝数分别为7.1×103、3.6×103、2.2×103、5.6×103和5.1×103.该方法具有特异性强、灵敏度高、操作简便等优点,为以上5种人兽共患脑炎病病毒提供快速检测手段.     

Phage display identifies an Eastern equine encephalitis virus glycoprotein E2-specific B cell epitope

The present study identified a linear B-cell epitope in the Eastern equine encephalitis virus (EEEV) E2 glycoprotein by screening a phage-displayed random 12-mer peptide library using an EEEV E2 specific monoclonal antibody (mAb) 7C11 and defined L/F-E/R-Y-T-W-G/R-N-H/W-P as the consensus binding motif. A sequence ((321)EGLEYTWGNHPP(332)) encompassing this consensus motif was found in the EEEV E2 glycoprotein and synthesized for further epitope confirmation. Meanwhile, the corresponding epitope peptides in E2 protein of associated alphaviruses were synthesized for specificity identification. Results showed the mAb 7C11 and murine antisera all reacted strongly against the synthesized polypeptide of EEEV antigen complex, but no reaction with Western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) was detected. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against EEEV.     

Serological investigations of red foxes (Vulpes vulpes L.) for determination of the spread of tick-borne encephalitis in Northrhine-Westphalia

Serum samples from 786 red foxes shot between January 1995 and August 1996 in the southern half of Northrhine-Westphalia, located in western Germany, were tested for the presence of antibodies against tick-borne encephalitis (TBE) virus using the Immunozym FSME IgG All Species-ELISA (Immuno, Heidelberg, Germany) as a screening test: 759 sera were negative, 23 (2.9%) were borderline, and four (0.5%) were positive. Nine of the 27 ELISA reactive sera were confirmed by the TBE Western-Blot (Immuno, Heidelberg, Germany). Furthermore these 27 sera were tested for neutralizing antibodies by means of a plaque reduction neutralization test (PRNT) against TBE and West Nile viruses. Only one single serum was found to have a neutralization titre (+1:800 PRNT80) against TBE virus. All other 26 sera were negative for neutralizing antibodies against TBE or West Nile virus. Since the titre of the single serum is low, it can be interpreted that if TBE virus is present, its prevalence is extremely low. Northrhine-Westphalia is not classified as a TBE-endemic area. Further calculated serological testing of game and virological investigation of collected ticks in the affected area seem to be meaningful and necessary.     

Combining anti‐IgM and IgG immunoassays for comprehensive chikungunya virus diagnostic testing

Chikungunya virus (CHIKV) is a mosquito‐borne pathogen that causes CHIKV fever. Definitive diagnosis is crucial for patients experiencing symptoms similar to other arboviral diseases because they can vary in clinical consequences. An increasing number of patients experience long‐term rheumatic effects of CHIKV infection, but these cases may not be optimally detected by molecular assays and anti‐CHIKV IgM ELISAs (M‐ELISAs) used for confirmation and screening, respectively. The subsequent confirmatory serological test, the plaque reduction neutralization test (PRNT), is laborious and time‐consuming. In this study, we evaluated a new diagnostic algorithm in which the M‐ELISA is conducted in parallel with an anti‐CHIKV IgG ELISA (G‐ELISA) and observed that the Euroimmun M‐ELISA combined with the Euroimmun G‐ELISA or the Abcam G‐ELISA exhibited excellent sensitivity and specificity for CHIKV. The combinations demonstrated perfect and near perfect inter‐rater agreement with the PRNT, respectively, suggesting their potential to be used as alternatives to the confirmatory serological PRNT assay for CHIKV.     

Arbovirus infections in several Ontario mammals, 1975-1980.

Serological studies for arboviruses were conducted on 725 animal sera collected in 22 Ontario townships between 1975 and 1980 including 44 coyote (Canis latrans), 277 red fox (Vulpes vulpes), 192 raccoon (Procyon lotor) and 212 striped skunk (Mephitis mephitis). Hemagglutination inhibition antibodies to two flaviviruses, namely St. Louis encephalitis and Powassan were found in 50% of coyote, 47% of skunk, 26% of fox and 10% of raccoon sera. Similarly, hemagglutination inhibition antibodies to a California serogroup virus, snowshoe hare, were found in 12% of fox, 7% of skunk, 7% of raccoon and 5% of coyote sera. No antibodies were detected to two alphavirus, namely eastern equine encephalitis and western equine encephalitis, antigens. This study affirms the endemic presence of Powassan and snowshoe hare virus and further delineates the scope of St. Louis encephalitis activity in Ontario.     

Detection of antibodies to West Nile virus in equine sera using microsphere immunoassay.

One hundred and ninety-one sera from horses that recently were exposed to West Nile virus (WNV) by either vaccination or natural infection or that were not vaccinated and remained free of infection were used to evaluate fluorescent microsphere immunoassays (MIAs) incorporating recombinant WNV envelope protein (rE) and recombinant nonstructural proteins (rNS1, rNS3, and rNS5) for detection of equine antibodies to WNV. The rE MIA had a diagnostic sensitivity and specificity, respectively, of 99.3% and 97.4% for detection of WNV antibodies in the serum of horses that were recently vaccinated or naturally infected with WNV, as compared to the plaque reduction neutralization test (PRNT). The positive rE MIA results were assumed to be WNV-specific because of the close agreement between this assay and the PRNT and the fact that unvaccinated control horses included in this study were confirmed to be free of exposure to the related St Louis encephalitis virus. The NS protein-based MIA were all less sensitive than either the rE MIA or PRNT (sensitivity 0-48.0), although the rNSI MIA distinguished horses vaccinated with the recombinant WNV vaccine from those that were immunized with the inactivated WNV vaccine (P < 0.0001) or naturally infected with WNV (P < 0.0001). The rE MIA would appear to provide a rapid, convenient, inexpensive, and accurate test for the screening of equine sera for the presence of antibodies to WNV.     

Molecular epidemiological studies of veterinary arboviral encephalitides

Recent studies using molecular genetic approaches have made important contributions to our understanding of the epidemiology of veterinary arboviral encephalitides. Viruses utilizing avian enzootic hosts, such as Western equine encephalitis virus (WEEV) and North American Eastern equine encephalitis virus (EEEV), evolve as relatively few, highly conserved genotypes that extend over wide geographic regions; viruses utilizing mammalian hosts with more limited dispersal evolve within multiple genotypes, each geographically restricted. Similar findings have been reported for Australian alphaviruses. This difference may be related to vertebrate host relationships and the relative mobility of mammals and avians. Whereas EEEV and Venezualan equine encephalitis virus (VEEV) utilize small mammalian hosts in the tropics, most WEEV genotypes probably utilize avian hosts in both North and South America. The ability of mobile, infected avian hosts to disperse alphaviruses may result in continual mixing of virus populations, and thus limit diversification. This high degree of genetic conservation is also exhibited by EEE and Highlands J viruses in North America, where passerine birds serve as amplifying hosts in enzootic transmission foci. Most equine arboviral pathogens, including EEEV, WEEV and Japanese encephalitis virus (JEV), occur in a naturally virulent enzootic state and require only appropriate ecological conditions to cause epizootics and epidemics. However, VEE epizootics apparently require genetic changes to convert avirulent enzootic strains into distinct epizootic serotypes. All of these arboviruses have the potential to cause severe disease of veterinary and human health importance, and further molecular epidemiological studies will undoubtedly improve our ability to understand and control future emergence.     

Health evaluation of free-ranging and captive blue-fronted Amazon parrots (Amazona aestiva) in the Gran chaco, Bolivia.

Bolivia has a total of 47 species of Psittacidae, seven of which have been identified in our study site, the semiarid Gran Chaco of the Isoso. One species, the blue-fronted parrot (Amazona aestiva), is frequently captured by local Isose?o Guaraní Indians for exploitation on the national and international market. These birds are often temporarily housed in small villages under unhygienic conditions with poultry and other domestic species. On occasion, these parrots escape back to the wild. Additionally, many of these birds are kept as pets or are used to lure wild. parrots within slingshot range for subsequent capture. In this study, we evaluated the health status, including the level of exposure to selected infectious agents, in the wild-caught captive birds and free-ranging birds. Physical examinations were performed, and blood was collected, from 54 live birds (20 captive and 34 free-ranging). Feces were collected from 15 birds (seven captive and eight free-ranging). Necropsies were also performed on four recently dead wild-caught birds. On serologic testing, no birds were found to have antibodies to avian influenza virus, Chlamydophila psittaci, infectious bronchitis virus, infectious bursal disease virus, infectious laryngotracheitis virus, Marek's disease virus, paramyxovirus-1, paramyxovirus-2, paramyxovirus-3, polyomavirus, eastern equine encephalitis virus, western equine encephalitis virus, or Venezuelan equine encephalitis virus. Positive antibody titers were found for psittacine herpesvirus (8/44, 18.2%), Aspergillus spp. (3/51, 5.9%), and Salmonella pullorum (33/49, 67.3%). All three of the birds that tested antibody positive for Aspergillus spp. were captive, whereas six of the eight and 15 of the 33 birds that tested positive for psittacine herpesvirus and S. pullorum, respectively, were wild.     

Serological surveillance for Tahyna virus (California encephalitis orthobunyavirus,Peribunyaviridae) neutralizing antibodies in wild ungulates in Austria,Hungary and Romania

A serosurvey for Tahyna virus (TAHV), a mosquito‐borne California encephalitis orthobunyavirus (Peribunyaviridae) endemic to Europe, was performed to estimate the activity of TAHV on a broad geographic scale. Sera from wild boar (Sus scrofa), roe deer (Capreolus capreolus) and red deer (Cervus elaphus) were collected from Austria, Hungary and Romania. Samples were tested for neutralizing antibodies against TAHV using a virus microneutralization assay. The results demonstrate that TAHV transmission to mammals is widespread in Europe, particularly in the wild boar population where the mean rate of seroconversion is 15.2%.     

Genetic characterization of rabies viruses isolated from frugivorous bat (Artibeus spp.) in Brazil

In Latin America, rabies cases related to frugivorous bats have been reported since 1930's. Recently, two viruses isolated from Artibeus lituratus were proved to be vampire bat variants by monoclonal antibodies panels [2], but their genetic information is not well known. In this report, four rabies viruses were isolated from frugivorous bats (Artibeus spp.) in Brazil and their nucleoprotein gene sequences were determined. These isolates were found to be genotype 1 of lyssavirus and showed the maximum nucleotide sequence homology of 97.6-99.4% with vampire bat-related viruses in Brazil [6]. These results indicate that the Brazilian frugivorous bat rabies viruses in this study are closely related to vampire bat-related viruses that play a main role in rabies virus transmission to livestock in Brazil.     

Serological evidence for Japanese encephalitis and West Nile viruses in domestic animals of Nepal

A regional survey was conducted in Nepal for antibody to Japanese encephalitis virus (JEV) in domestic animals. Sera from pigs, and limited numbers of ducks and horses were collected from 16 districts in 2002-2003 and subjected to three serological tests. Of 270 porcine sera tested by C-ELISA, 55% were found positive for the presence of antibodies against Japanese encephalitis virus. Additional testing for IgM antibody to JEV revealed less than 2% of C-ELISA positive sera had evidence of recent JEV infection. Plaque reduction neutralisation tests (PRNT) using JEV, Murray Valley encephalitis (MVEV) and Kunjin (KUNV) viruses implicated JEV as the flavivirus associated with the observed antibody response in most sero-positive pigs. However, eight porcine sera with predominant neutralising antibody for KUNV (an Australasian subtype of West Nile Virus) provided evidence for the circulation of West Nile virus in Nepal.     

Serological evidence of West Nile virus infection in human populations and domestic birds in the Northwest of Morocco

West Nile virus (WNV) was recently detected in Culex pipiens mosquitoes in Morocco. The aim of this study was to evaluate the seroprevalence of WNV in humans and in domestic birds in two regions of Morocco by the detection of IgG antibodies. Blood samples were obtained from 91 human patients and 92 domestic birds from September to December 2019. All study samples were tested using competitive enzyme-linked immunosorbent assay (cELISA) and WNV neutralization tests (VNT) were performed on positive sera. Of all samples, 4 (4.39 %) humans and 4 (4.34 %) birds were found to be seropositive for flaviviruses by the cELISA test. The VNT revealed that three of the four human samples detected positive by cELISA contained neutralizing antibodies against WNV. Two bird samples were confirmed positive by VNT. These results show a significant seroprevalence of anti-WNV antibodies and therefore suggest the active circulation and exposure of human and bird populations in the northwest of Morocco.