马动脉炎病毒进化分析及DNA条形码的确定

从GenBank数据库下载马动脉炎病毒所有基因组全序列和同属的其他病毒基因组序列,对其进行多重比对,绘制系统进化树,并根据序列比对筛选保守序列作为马动脉炎病毒的DNA条形码。结界发现马动脉炎病毒分为两个分枝,即欧洲型和美洲型,两者同源性在85%以上,而与同属其他病毒同源性低于40%,亲缘关系较远;对马动脉炎病毒基因组序列进行分析,筛选出9条基因序列作为马动脉炎病毒的DNA条形码,可用于马动脉炎病毒的鉴别诊断。     

Molecular epizootiology of equine arteritis virus isolates from Poland

Phylogenetic analysis was performed on the sequences of 44 Polish isolates of equine arteritis virus that were isolated from the semen of stallions from national and private studs, collected during 2001--2005. These sequences were also compared with 41 reference strains previously described and commonly used in phylogenesis. On the basis of the nucleotide sequence analysis of the ORF5 gene, encoding the glycoprotein GP5, it was demonstrated that the Polish EAV isolates belonged to two subgroups and showed the closest relationship to the European strains. Similar results were obtained using the nucleotide sequences of the ORF7 gene. The nucleotide identity between the ORF5 and ORF7 sequences of all Polish isolates was in the range of 80.1-99.0% and 93.6-100%, respectively. The analysis of genetic diversity within the ORF5 sequences enabled a retrospective epizootic investigation. This study suggested that some of the EAV shedding stallions were probably infected before they were moved to Poland.     

Epizotiology and phylogeny of equine arteritis virus in hucul horses

The aim of the study was to determine the situation of equine arteritis virus (EAV) infections in hucul horses. A total of 176 horses (154 mares and 22 stallions) from the biggest hucul horse stud in Poland were tested. Antibodies against EAV were detected in 97 (55.1%) horses. The EAV seroprevalence among mares was 53.2% while in stallions - 68.2%. The percentage of positive mares increased with their age, thus amongst the mares of less than 2 years of age the percentage was 32.5%, while in the group of 3-5 years old increased to 59.4% and in the mares in the age of 6-10 years and older than 10 years 89.5% and 95% were seropositive, respectively. Among 11 seropositive stallions five were supposed to be shedders of EAV with their semen. It is likely that those persistently infected stallions were the reservoirs of the virus in the stud. Genetic studies using of ORF5 gene showed high homology between the viruses detected in the semen of those stallions what suggested lateral transmission between the stallions sharing the same stable. Persistent infection in an immature stallion, which has not yet been used for breeding, was established as a result of infection via respiratory route. Phylogenetic analysis confirmed that all hucul viruses shared the same ancestor and as most of EAV strains dominating in Polish horse population belonged to the European origin EAV subgroup (EU-1).     

Effective removal of equine arteritis virus from stallion semen

REASONS FOR PERFORMING STUDY: A method of removing equine arteritis virus (EAV) from equine semen used for artificial insemination is urgently needed. Recent medical studies suggest that a double semen processing technique of density gradient centrifugation followed by a 'swim-up' can provide virus-free sperm preparations for assisted reproduction. OBJECTIVES: To investigate the use of the double semen processing technique to obtain virus-free sperm preparations from stallion semen containing EAV. METHODS: Aliquots of an ejaculate from an uninfected stallion were spiked with virus and processed by the double processing technique. The sperm preparations were tested by PCR for the presence of EAV. The procedure was repeated using an ejaculate from a known shedding stallion, testing processed and unprocessed aliquots by PCR and virus isolation. RESULTS: Virus-free sperm preparations were obtained using the double sperm processing technique. The 'swim-up' step is apparently required to ensure complete virus removal. CONCLUSIONS: The double semen processing technique is potentially a useful and simple tool for the removal of EAV from the semen of shedding stallions. POTENTIAL RELEVANCE: The inclusion of density gradient centrifugation and 'swim-up' in protocols for the processing of semen for artificial insemination could help prevent the transmission of viral diseases carried in semen, such as EAV.     

Pathologic features of horses given avirulent equine arteritis virus intramuscularly

Twenty horses that were seronegative for equine arteritis virus antibodies were inoculated IM with live equine arteritis virus vaccine. The inoculation did not cause clinical signs of disease. A mild, transient febrile reaction developed in 6 horses, 3 of which were in poor condition before inoculation. Six horses, 2 of which were in poor condition before inoculation, experienced mild lymphopenia. Necropsy revealed mild lesions in the lymph nodes of 6 horses (3 of which were in poor condition before inoculation). Maximum concentrations of virus were detected in the lymph nodes and were consistently present from postvaccination day 3 through 8. Lesser concentrations of virus were detected in the spleen of 5 horses, liver and kidney of 4, abdominal fluid of 3, pleural fluid of 2, and lungs and urine of 1, between postvaccination days 3 and 7. Virus was not detected in the brain, nasal tract, or serum of any of the horses.     

The immune response to equine arteritis virus: potential lessons for other arteriviruses

The members of the family Arteriviridae, genus Arterivirus, include equine arteritis virus (EAV), porcine reproductive and respiratory syndrome virus (PRRSV), lactate dehydrogenase-elevating virus (LDV) of mice, and simian hemorrhagic fever virus (SHFV). PRRSV is the newest member of the family (first isolated in North America and Europe in the early 1990s), whereas the other three viruses were recognized earlier (EAV in 1953, LDV in 1960, and SHFV in 1964). Although arterivirus infections are strictly species-specific, the causative agents share many biological and molecular properties, including their virion morphology, replication strategy, unique properties of their structural proteins, and their ability to establish distinctive persistent infections in their natural hosts. The arteriviruses are each antigenically distinct and cause different disease syndromes in their natural hosts. Similarly, the mechanism(s) responsible for the prolonged and/or persistent infections that characterize infections with each arterivirus in their natural hosts are remarkably different. The objective of this review is to compare and contrast the immune response to EAV with that to the other three arteriviruses, and emphasize the potential relevance of apparent similarities and differences in the neutralization characteristics of each virus.     

The relationship between equine and human West Nile virus disease occurrence

Cases of human and equine West Nile virus (WNV) disease reported in Texas in 2002 were analyzed to assess their temporal relationship. For each human case with a known residential location, the closest equine case (within a 5 km radius) was selected. A total of 80 human–equine case pairs were identified, 51 (64%) of which were located in urban areas. Dates-of-onset of human and equine cases were positively correlated (r_SP = 0.494, P < 0.001). Although overall there was no significant (P = 0.207) difference between the dates-of-onset of human and equine cases, in urban areas of Texas equine cases were reported significantly (P = 0.011) earlier (August 7) than corresponding human cases (August 19). Monitoring equine populations that are susceptible to WNV disease within close proximity to urban human populations might be useful for predicting disease risk in human populations.     

Lateral transmission of equine arteritis virus among Lipizzaner stallions in South Africa

REASONS FOR PERFORMING STUDY: A serological study conducted in 1995 revealed that 7 stallions at the Lipizzaner Centre, Gauteng, South Africa, were seropositive for antibody to equine arteritis virus (EAV). A Lipizzaner stallion imported into South Africa from Yugoslavia in 1981 had previously (1988) been confirmed to be an EAV carrier. Despite being placed under life-long breeding quarantine, EAV had been transmitted between stallions at the Lipizzaner Centre. OBJECTIVES: To investigate the phylogenetic relationships between the strain of EAV shed in the semen of the original carrier stallion and strains recovered from the semen of 5 other stallions; and to investigate the means whereby lateral transmission of EAV occurred among 7 in-contact, nonbreeding stallions at the Centre. METHODS: EAV was isolated from semen collected from the seropositive stallions using RK-13 cells. Viral RNA was reverse transcribed and amplified by polymerase chain reaction using ORF 5-specific primers, subjected to sequence and phylogenetic analysis. RESULTS: Phylogenetic analysis of strains of EAV recovered from the semen of 6 persistently infected stallions confirmed that all viruses were closely related and probably derived from a common ancestor, i.e. the stallion imported from Yugoslavia. Lateral transmission subsequently occurred among 7 in-contact, nonbreeding stallions at the Centre. It is speculated that these stallions may have been exposed to virus from bedding or fomites contaminated with semen. CONCLUSIONS: These data confirm that lateral transmission of EAV can occur from shedding stallions to susceptible, in-contact horses, including other stallions, which may become persistently infected with the virus. POTENTIAL RELEVANCE: The findings are consistent with lateral spread of a single, unique strain of EAV among a group; and suggest that transmission of EAV may be initiated by infection of one or more stallions with virus on bedding or other fomites contaminated with EAV- infected semen.     

抗马动脉炎病毒N蛋白单克隆抗体的制备

为制备抗马动脉炎病毒(EAV)衣壳蛋白(N)的单克隆抗体(MAb),本研究通过原核表达重组N蛋白,纯化后免疫6周龄雌性BALB/c小鼠,细胞融合后经间接ELISA筛选获得两株能够稳定分泌抗EAV N蛋白的杂交瘤细胞株,MAbs亚型鉴定为IgG1,轻链为κ链。Western blot结果显示,这两株杂交瘤细胞分泌的MAb均能够识别EAV。EAV N蛋白MAb的制备,为EAV血清学检测方法的建立奠定了基础。