Molecular characterization of the capsid gene of two serotypes of turkey astroviruses

Astrovirus infections mainly cause acute gastroenteritis in children and young animals. Human astroviruses are well characterized antigenically and genetically. However, information on turkey astroviruses is limited. We isolated two astroviruses (TAstV1987 and TAstV2001) from turkeys and classified them as two different serotypes using a virus neutralization test. To elucidate the differences between these two isolates at the molecular level, further genetic characterization and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were carried out. The sequences of the complete capsid protein gene of these two isolates were obtained by cloning and sequencing. The percentage nucleotide and predicted amino acid identities for these two sequences along with those of 16 other capsid protein gene sequences from human and animal astroviruses retrieved from GenBank were calculated using MegAlign. The results showed that TAstV1987 and TAstV2001 had 73.3% nucleotide and 82.8% amino acid identities, respectively. An unrooted Neighbor-joining phylogenetic tree of these sequences was generated using MEGA 3 software with 1000 bootstrap replicates. The results of evolutionary analysis showed that TAstV1987 was closely related genetically to another virus, designated TAstV-2, whereas TAstV2001 was not as close to TAstV-2 as TAstV1987. The analysis of the capsid proteins of the two viruses by SDS-PAGE revealed that they had different band patterns, indicating that their capsid proteins consisted of different viral proteins. The findings in this study revealed the molecular differences in the capsid protein gene of TAstV1987 and TAstV2001, which may provide the molecular basis of the antigenic differences between these two serotypes of turkey astroviruses.     

猪细小病毒VP2蛋白单克隆抗体的制备及抗原捕捉ELISA方法的建立

为制备猪细小病毒VP2蛋白单克隆抗体(McAb),建立检测猪细小病毒的抗原捕捉ELISA方法 (AC-ELISA),本研究以原核表达的重组VP2蛋白作为免疫原,免疫6周龄BALB/c雌鼠,取其脾细胞与骨髓瘤细胞SP2/0进行融合,经间接ELISA方法筛选,成功获得了2株能稳定分泌抗猪细小病毒VP2蛋白的McAb,命名为3C4、5F8。以多克隆抗体作为捕获抗体、单克隆抗体5F8作为检测抗体,通过双抗夹心ELISA各个反应条件的优化,建立检测猪细小病毒抗原捕捉ELISA方法。该方法与日本乙脑病毒(JEV)、猪流行性腹泻病毒(PEDV)、伪狂犬病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)均不发生交叉反应;与RT-PCR相比较,符合率、敏感性和特异性分别为93.6%、90.9%、94.4%。本研究建立的猪细小病毒AC-ELISA有良好的重复性、敏感性和特异性,可应用于猪细小病毒感染的早期诊断。     

Antigenicity of two turkey astrovirus isolates

Astroviruses are positive-sense single-stranded RNA viruses. These viruses cause gastroenteritis in humans and in a variety of animal species, including turkey poults. Only human astroviruses are well characterized antigenically. In the current study, two turkey astrovirus isolates, TAstV1987 and TAstV2001, were antigenically compared using cross-neutralization tests in turkey embryos, as well as cross-reactivity of the two isolates by an enzyme-linked immunosorbent assay (ELISA). The antigenic relatedness values (R) were calculated using the Archetti and Horsfall formula. The R value based on the cross-neutralization tests was 0.56%, which indicates that TAstV1987 and TAstV2001 belong to different serotypes; the R value of the two viruses based on ELISA was 70.7%, which suggests these two viruses share common antigen(s).     

应用单克隆抗体ＡＣ—ＥＬＩＳＡ检测鸡蛋卵黄中鸡毒支原体

采用抗鸡毒支原体阳性血清包被酶标板作为捉抗体，以４株鸡毒支原体特异单克隆抗体作为第二抗体和酶标羊抗鼠ＩgG作为指示体建立了一种检测鸡蛋卵黄中鸡毒支原体的双夹心ＡＣ－ＥＬＩＳＡ。该法具有简便、灵敏、快速等优点，从采样到获得结果６小时内完成，检出率高于分离培养法，且重复性良好，该方法的建立鸡群毒支原体感染的检测和净化提供了一种新的可靠方法。     

Differential detection of Newcastle disease virus strains by degenerate primers based RT-PCR

Degenerate primers based RT-PCR (previously described by [Avian Dis 26 (1997) 837]) has been used for the detection and differentiation of Newcastle disease (ND) viruses. Two sets of primers (A+B and A+C), with common forward primer and distinct reverse degenerate primers, designed from fusion protein gene encoding for cleavage site, could differentiate virulent and avirulent Newcastle disease viruses (NDV). Both sets of primers amplified "F" gene sequence of virulent (velogenic and mesogenic) viruses, whereas in avirulent strains, amplification was only with primer set A+C. Total 10 NDV isolates and two clinical samples including both known and unknown pathotypes, were checked. Based on amplification results 5 viruses were found to be virulent type and 6 as avirulent with one of the two clinical samples, earlier positive by RT-PCR using non-degenerate "F" gene specific primers was found negative in this study. The technique has been found to be a simple and quick for the detection and differentiation of virulent and avirulent NDV, which is important for control of the disease in the events of the outbreaks.     

Characterization of group II avian adenoviruses with a panel of monoclonal antibodies.

The interaction between a panel of ten monoclonal antibodies and hemorrhagic enteritis virus, a group II avian adenovirus, was determined. The monoclonal antibodies reacted with all nine isolates of group II avian adenoviruses, but not with any of five types of group I avian adenoviruses. All ten monoclonal antibodies recognized antigenic determinants on the hexon protein of hemorrhagic enteritis virus when analyzed by immunoprecipitation and immunoblotting. They reacted only with the native hexon protein and not with protein denatured by sodium dodecyl sulfate or guanidine-HCl/urea treatment combined with reduction and carboxymethylation. Based on the results of competitive binding assays, the panel of monoclonal antibodies could be subdivided into two groups, which recognized different antigenic domains of the hemorrhagic enteritis virus hexon protein. The monoclonal antibodies in group 1 neutralized hemorrhagic enteritis virus infectivity while the monoclonal antibodies of group 2 did not. Group 1 consisted of eight monoclonal antibodies which could be further subdivided into subgroups 1A, 1B, 1C and 1D. The subdivision of the monoclonal antibodies was based on the degree of blocking in the competitive binding assays and differences in their ability to induce enhancement. In general, the monoclonal antibodies had a higher avidity for the virulent isolate of hemorrhagic enteritis virus than for the avirulent hemorrhagic enteritis virus isolate.     

单克隆抗体捕捉猪瘟病毒抗原ELISA方法的建立

分别用原核表达的猪瘟病毒（CSFV）主要抗原E2蛋白和猪瘟基因疫苗免疫BALB/c小鼠,通过细胞融合与克隆筛选出5株稳定分泌CSFV抗体的杂交瘤细胞株1E2、1G7、3A2、3B7和4B6.间接免疫荧光和Western-blotting试验结果表明,5株单抗均与CSFV E2蛋白和全病毒抗原反应.将筛选的CSFV特异性单抗1E2、3B7和4B6纯化后等量混合后包被酶标板（捕捉抗体）,与兔抗CSFV IgG（检测抗体）联合应用,建立起CSFV抗原捕捉ELISA（AC-ELISA）方法.随后采用方阵滴定法确定了单抗与多抗的最适工作浓度及判定检测结果的OD450临界值.最后以建立的AC-ELISA检测CSFV细胞培养物、CSFV攻毒死亡猪的病料和临床猪瘟组织样品,结果表明,该方法敏感、特异、重复性好,与病毒分离和RT-PCR方法符合率分别为86.2%和90.3%.     

牛病毒性腹泻病毒(BVDV)抗原捕获ELISA检测方法的建立

用牛病毒性腹泻病毒(BVDV)单克隆抗体包被酶标板,以兔多克隆抗体作为夹心抗体,建立BVDV抗原捕获ELISA(AC-ELISA)检测方法,优化反应条件并对该方法的稳定性等指标进行了测试和评价。结果表明,单抗最佳包被质量浓度为5μg/mL,兔多抗血清最佳质量浓度为10μg/mL;单抗在4℃包被12~24 h,多抗在37℃作用1 h为双抗体的最佳反应条件;酶标抗体最适稀释度1∶10000,最适作用时间为1 h;采用1%BSA和1%明胶分别在抗体包被后和加入待检抗原反应后进行两次封闭效果好。用AC-ELISA方法检测临床采集的11份牛腹泻病料和12份健康牛组织样品,同时以病毒分离和RT-PCR检测方法做对比,3种方法符合率很高。研究表明AC-ELISA方法稳定性好,可用于BVDV的临床快速检测。     

Polymerase chain reaction tests for the identification of Ross River, Kunjin and Murray Valley encephalitis virus infections in horses

OBJECTIVE: To develop and validate specific, sensitive and rapid diagnostic tests using RT-PCR for the detection of Ross River virus (RRV), Kunjin virus (KV) and Murray Valley encephalitis virus (MVEV) infections in horses. METHODS: Primer sets based on nucleotide sequence encoding the envelope glycoprotein E2 of RRV and on the nonstructural protein 5 (NS5) of KV and MVEV were designed and used in single round PCRs to test for the respective viruses in infected cell cultures and, in the case of RRV, in samples of horse blood and synovial fluid. RESULTS: The primer pairs designed for each of the three viruses amplified a product of expected size from prototype viruses that were grown in cell culture. The identity of each of the products was confirmed by nucleotide sequencing indicating that in the context used the RT-PCRs were specific. RRV was detected in serums from 8 horses for which there were clinical signs consistent with RRV infection such that an acute-phase serum sample was taken and submitted for RRV serology testing. The RRV RT-PCR was analytically sensitive in that it was estimated to detect as little as 50 TCID50 of RRV per mL of serum and was specific in that the primer pairs did not amplify other products from the 8 serum samples. The RRV primers also detected virus in three independent mosquito pools known to contain RRV by virus isolation in cell culture. Samples from horses suspected to be infected with KV and MVEV were not available. CONCLUSION: Despite much anecdotal and serological evidence for infection of horses with RRV actual infection and associated clinical disease are infrequently confirmed. The availability of a specific and analytically sensitive RT-PCR for the detection of RRV provides additional opportunities to confirm the presence of this virus in clinical samples. The RT-PCR primers for the diagnosis of KV and MVEV infections were shown to be specific for cell culture grown viruses but the further validation of these tests requires the availability of appropriate clinical samples from infected horses.