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.     

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.     

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.     

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.     

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.     

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.     

Serological evidence of widespread exposure of Grenada fruit bats to chikungunya virus

Antibody detection against selected potentially zoonotic vector‐borne alphaviruses and flaviviruses was conducted on sera from bats from all six parishes in Grenada, West Indies. Sera were tested for (i) antibodies to flaviviruses West Nile virus, St. Louis encephalitis virus, Ilhéus virus, Bussuquara virus (BSQV), Rio Bravo virus and all four serotypes of dengue virus (DENV) by plaque reduction neutralization test (PRNT); (ii) antibodies to alphaviruses western equine encephalitis virus, Venezuelan equine encephalitis virus and eastern equine encephalitis virus by epitope‐blocking enzyme‐linked immunosorbent assay (ELISA); and (iii) antibodies to the alphavirus chikungunya (CHIKV) by PRNT. Two species of fruit bats were sampled, Artibeus jamaicensis and Artibeus lituratus, all roosting in or within 1,000 m of human settlements. Fifteen (36%) of the 42 bats tested for neutralizing antibodies to CHIKV were positive. The CHIKV‐seropositive bats lived in localities spanning five of the six parishes. All 43 bats tested for epitope‐blocking ELISA antibody to the other alphaviruses were negative, except one positive for Venezuelan equine encephalitis virus. All 50 bats tested for neutralizing antibody to flaviviruses were negative, except one that had a BSQV PRNT₈₀ titre of 20. The CHIKV serology results indicate that bats living close to and within human settlements were exposed to CHIKV in multiple locations. Importantly, bats for this study were trapped a year after the introduction and peak of the human CHIKV epidemic in Grenada. Thus, our data indicate that bats were exposed to CHIKV possibly during a time of marked decline in human cases.     

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.     

Equine viral encephalitis.

The most important neurotropic viral infections of the horse are the arthropod-borne encephalitides. These include Venezuelan encephalitis (VE), eastern encephalitis (EE) and western encephalitis (WE), which are found in the Americas, and Japanese B encephalitis which occurs in the Far East. All the viruses cause encephalitis in man. Between 1969 and 1972 an epidemic of VE occurred in Central America. In 1971 the disease was reported in Texas, where it was brought under control by the vaccination of susceptible horses with an attenuated live virus vaccine and by the reduction of the mosquito population with insecticides sprayed from aircraft. A high titre viraemia occurs with VE virus in the horse and epidemics are maintained by a mosquito/horse cycle; infection of man and other species is incidental. EE and WE have been recognised as separate diseases since 1933 and in the U.S.A. horses are protected by routine vaccination. Epidemics of these diseases are routine vaccination. Epidemics of these diseases are now uncommon. In contrast with VE, both EE and WE viruses are maintained by a bird/mosquito cycle. The viraemia in the horse is generally considered insufficient to infect mosquito vectors; the horse is a "dead end host". Several species of mosquito can act as vectors of VE, WE and EE. The extension of other arthropod-borne diseases to areas originally outside their geographical distribution (e.g. bluetongue in sheep) serves to illustrate the potential of VE, WE and EE to cause disease on other continents.     

First Report on Molecular Detection of Equine Upper Respiratory Infectious Viruses in Republic of Korea

The prevalence of equine respiratory virus infections among a suspected population of race horses was examined using polymerase chain reaction (PCR). One or more of five equine respiratory viruses were detected in the nasal swabs of 45 of 89 horses (50.6%), and the detection rate of equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), equine herpesvirus type 5 (EHV-5), equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV) were 5.6%, 7.9%, 39.0%, 2.2%, and 6.7%, respectively. Among the 45 infected horses, 7 were co-infected with EHV and/or equine rhinitisvirus (ERV). Equine influenzavirus and equine arteritisvirus were not detected in any samples. Specific antibodies to EHV-1 and/or EHV-4 were detected in 59 of 73 tested sera (80.8%), using a virus neutralization test. This investigation suggests that equine respiratory viruses are endemic at Seoul Race Park and that the impact of viral infections on race horses’ health in Republic of Korea should be evaluated.     

1. Isolation and Characterisation of Equine Rhinopneumontis Virus and Other Equine Herpesviruses from Horses

Equine herpesviruses (EH viruses) were isolated from 9 horses in three separate outbreaks of respiratory disease. The pattern of disease in the three stables is described and evidence is presented that some of the horses were ill, possibly as a result of recurrent infection, and that reactivation of a persistent, latent infection may have occurred. An ulcerative condition of the pharyngeal region was seen in some of the horses with EH virus infection.
The cytopathogenicity for equine foetal kidney cells of the 9 EH viruses varied considerably. One isolate, EH 39 virus, which was recovered from an acute, upper respiratory tract infection, was rapidly cytopathic for equine foetal kidney cell cultures and was shown in neutralisation tests to be identical with, or closely related to equine rhinopneumonitis virus (EH virus type 1) that is associated with acute respiratory disease and abortion in other countries. More slowly cytopathic isolates were recovered from mild to subclinical upper respiratory tract infections. Evidence is presented that the property of slow cytopathogenicity is probably related to the tendency of these viruses to remain cell associated. Slowly cytopathic isolates were recovered from the nasal cavity of horse 89 on two occasions 79 days apart. One of the eight slowly cytopathic isolates, EH 86 virus, was shown to be antigenically distinct from equine rhinopneumonitis virus (EH 39 virus).     

Seroprevalence and Rate of Infection of Equine Influenza Virus (H3N8 and H7N7) and Equine Herpesvirus (1 and 4) in the Horse Population in Israel

Equine influenza and equine rhinopneumonitis are among the Office International des Epizooties or the World Organisation for Animal Health notifiable, contagious respiratory diseases. Although vaccination of horses in Israel against equine influenza virus (EIV) and against equine herpesvirus (EHV) is routinely performed, information regarding the occurrence and the epidemiology of the diseases is lacking. We hereby attempt to determine seroprevalence and rate of infection for EHV-1 and 4 and for EIV in horses distributed throughout Israel and describe demographic and environmental risk factors associated with seroprevalence. Despite the fact that last reported isolation of EIV in Israel occurred in 2007, we found a 26.4% (29/110) (95% confidence interval [CI]: 18.18–34.62) seroprevalence for H3N8, a 16.4% (18/110) (95% CI: 9.49–23.31) for H7N7, and a 6.4% (7/110) (95% CI: 1.83–10.97) rate of seroconversion for H3N8, suggesting current and active circulation of EIV in horses in Israel. Age, housing management type, and type of farm activity were significantly associated with seroprevalence, with activities allowing exposure to new horses positively associated with seroprevalence to EIV and an only pasture housing management negatively associated with seroprevalence. No association was detected between other demographic variables (gender, breed, and color) and environmental factors (climatic regions). Seroprevalence to EHV-1 and 4 were very low (<1%) and very high (>99%), respectively, raising questions regarding the appropriate vaccination guidelines. Our findings of the occurrence of EIV in horses in Israel imply an underdiagnosis of this virus in this country and warrant further investigation as to the strains that circulate in this region and their accordance with the current vaccine strains.     

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.     

Equine viral encephalomyelitis in Canada: a review of known and potential causes

Rabies, equine herpesvirus type I, and eastern and western encephalomyelitis viruses, known causes of equine neurological disease, are reviewed with emphasis on epidemiology, pathogenesis, clinical signs, and diagnosis.

Several arboviruses known to be active in Canada and capable of producing neurological disease in humans (Powassan, St. Louis encephalitis, snowshoe hare, and Jamestown Canyon viruses) are discussed as potential causes of encephalomyelitis in horses.

     

Detection of cattle infected with bovine viral diarrhea virus using nucleic acid hybridization.

A ribonucleic acid (RNA) hybridization assay to identify cattle infected by bovine viral diarrhea virus (BVDV) is described. The RNA probe was derived from the coding region at the 3' end of the genome of the NADL strain of BVDV. Total RNA from infected cell cultures or peripheral blood leukocytes from suspect animals was extracted and applied to nylon membranes with a slot blot apparatus. Peripheral blood leukocytes were tested concurrently for BVDV by virus isolation. The results of hybridization and virus isolation were in agreement for 92% of the cases. When compared with virus isolation, hybridization had a sensitivity of detection of 59.5% and a specificity of 95%. Cross-reactivity to RNA extracts of border disease virus-infected cells was noted. No cross-reactivity was detected to other common bovine viruses (bovine herpesvirus-1, bovine respiratory syncytial virus, parainfluenza-3 virus, and bluetongue virus), to viruses classified in related families (equine arteritis virus and Venezuelan equine encephalitis virus), or to viruses having similar genomic organization (dengue virus type 2 and Japanese encephalitis virus).     

Prevalence of antibodies against Saint Louis encephalitis and Jamestown Canyon viruses in California horses

Jamestown Canyon (JC) and Saint Louis encephalitis (SLE) viruses are mosquito-transmitted viruses that have long been present in California. The objective of this study was to determine the seroprevalence of these two viruses in horses prior to the introduction of West Nile (WN) virus. Approximately 15% of serum samples collected in 1998 from 425 horses on 44 equine operations horses throughout California had serum antibodies to JC virus, whereas antibodies were not detected to SLE virus. The results indicate that horses in California were commonly infected prior to 1998 with mosquito-transmitted Bunyaviruses that are identical or closely related to JC virus, but not with SLE virus. The different seroprevalence of SLE and JC viruses in horses likely reflects the unique ecology of each virus, and it is predicted that WN virus will have a wider distribution in California than closely related SLE virus.     

Mortality supposedly due to intoxication by pyrrolizidine alkaloids from Heliotropium indicum in a horse population in Costa Rica: a case report.

This article describes a case of massive mortality among horses which was probably due to intoxication by pyrrolizidine alkaloids from Heliotropium indicum. Over 4 years more than 75% of a population of about 110 horses on a farm in Costa Rica died after showing nervous neurological symptoms. Two clinical manifestations were encountered, an acute and a chronic one, both with a fatal outcome. Pathological findings in 2 horses coincided with those reported in the literature for intoxication by pyrrolizidine alkaloids and were not specific for VEE. However Venezuelan equine encephalitis (VEE) was the main differential diagnosis and could not completely be excluded because this disease was endemic in the region and VEE titres were found to be high. Taxonomic and toxicological investigations implicated Heliotropium indicum as the most probable principal cause of the intoxication.     

Adsorption of Wild Rabies Virus and Neurotropic-Adapted Viruses by Activated Attapulgite

The adsorption by attapulgite (a naturally occurring clay) of wild rabies virus in naturally rabid dogs was investigated. Attapulgite selectively absorbed rabies virus in the brain tissue of naturally rabid dogs and rejected rabies virus in extracts from the submaxillary salivary glands of the rabid dogs. Attapulgite rejected the neurotropic-adapted viruses lymphocytic choriomeningitis, Eastern equine and Western equine encephalitis, Japanese B encephalitis, and St. Louis encephalitis in the brain tissue of suckling mice.