Serologic survey for equine infectious anemia virus in Louisiana.

In 1975, a survey was conducted in East Baton Rouge Parish, Louisiana, to determine the prevalence of equine infectious anemia. Using the agar gel immunodiffusion test, 94 of 1,398 horses (6.7%) were found to be infected. Infection rates were especially high in areas where clinical cases of equine infectious anemia had been diagnosed. Clinical signs compatible with the disease were noted in 1 of the 94 seropositive horses. The sample set of 1,398 horses represented 22% of the census population obtained during the 1971 Venezuelan equine encephalomyelitis vaccination campaign.     

马传染性贫血病毒驴白细胞弱毒疫苗株致弱过程中不同代次病毒LTR的进化分析

为揭示马传染性贫血病毒(EIAV)弱毒疫苗的减毒机理,本研究对EIAV弱毒疫苗株在体外驴白细胞传代过程中不同代次毒株的长末端重复序列(LTR)进行扩增和分析。结果显示:随着病毒在体外传代次数的增加,各病毒株遗传多样性逐渐增加,并与致弱前亲本株EIAVDV117的遗传距离逐渐增大;EIAV在体外传代过程中LTR的变异主要集中在U3区和R区的转录起始位点,但随着传代次数的增加,在负调节区丢失了GATA结合位点,并在增强子区出现了E-box基序。此外,传代初期低代次病毒株与后期的高代次弱毒株在负调节区的AP-1结合位点和转录起始位点以及TAR的起始位点存在明显差异。     

Inactivation of equine infectious anemia virus by chemical disinfectants.

Twelve chemicals and commercial disinfectants were tested for inactivation of equine infectious anemia virus. In the presence of 10% bovine serum, all chemicals inactivated 4 log10 (based on 0.1 ml) of the virus within 5 minutes at 23 C. A reduction of at least 4 log10 was observed when the virus was exposed for 1 minute to substituted phenolic disinfectants (3 commercial preparations and sodium orthophenylphenate), halogen derivatives (iodophor and sodium hypochlorite), chlorhexidine, and 70% ethanol. Sodium hydroxide (5%), 2% formalin, and 2% glutaraldehyde were slower to inactivate the virus, but achieved 4 log10 reduction in titer by 5 minutes' contact time. The susceptibility of the equine infectious anemia virus to chemical disinfectants is similar to that of other enveloped viruses.     

Studies on equine infectious anemia virus transmission by insects

There are several factors involved in the mechanical transmission of equine infectious anemia (EIA) virus by insects. Large hematophagous insects, especially tabanids, which feed from extravascular sites (ie, pool feeding) appear to be the most efficient vectors. The biology of the host-seeking and blood-feeding behavior of the vectors are important variables that have been overlooked in the mechanical transmission of pathogens like EIA virus. The biology, population levels, and diversity of the vectors, in addition to the clinical status and proximity of EIA virus-infected horses maintained with susceptible animals are all important variables that contribute to EIA virus transmission in nature.     

带有组氨酸标签的马传染性贫血病毒感染性分子克隆的构建

马传染性贫血(EIA)弱毒疫苗的广泛使用存在野毒和疫苗毒鉴别困难的问题.本研究以已构建的马传染性贫血驴白细胞弱毒疫苗株的感染性分子克隆(pOK8266)为基础,在其S2基因内引入NspV酶切位点,将人工合成的编码6个组氨酸的寡核苷酸插入NspV位点,获得带有组氨酸标签的重组质粒pOK8266-HIS.将pOK8266-HIS转染驴白细胞,将驴白细胞转染产物传至第6代时,在电镜下观察到了典型的马传染性贫血病毒粒子.提取pOK8266-HIS衍生病毒的前病毒基因组DNA,通过PCR扩增和测序表明,衍生病毒基因组中引入了6个组氨酸标签,从而获得了带有分子标志的马传染性贫血弱毒疫苗株,为野毒株和疫苗病毒的鉴别诊断奠定了基础.本研究还证明了S2基因中的插入突变并不影响马传染性贫血病毒的体外复制.     

恒河猴Tetherin蛋白对马传染性贫血病毒抑制作用的研究

Tetherin是新发现的具有限制多种病毒从细胞膜释放功能的细胞蛋白。本研究采用RT-PCR技术从恒河猴巨噬细胞中扩增Tetherin基因,将其插入真核表达载体pEF4/myc-His B中构建重组质粒pHF-Tetherin。并将其转染293T细胞,通过western blot和激光共聚焦分析Tetherin在293T细胞中的表达及亚细胞定位。将pHF-Tetherin分别与马传染性贫血病毒(EIAV)或猴免疫缺陷病毒(SIV)假病毒粒子感染性质粒共转染293T细胞,分析Tetherin对EIAV及SIV出芽的限制作用。研究结果表明,pHF-Tetherin转染293T细胞48 h后可以检测到Tetherin的表达并定位于细胞膜,而且该蛋白具有抑制EIAV及SIV从转染细胞中出芽的生物学功能。     

感染马传染性贫血病毒的巨噬细胞差异表达蛋白质组分析

为研究马传染性贫血病毒(EIAV)感染其主要靶细胞马巨噬细胞(eMDM)后与细胞蛋白的相互作用,本研究采用EIAV强毒株EIAVDLV34感染48 h后的马外周血单核细胞分化的eMDM,并以未感染eMDM为对照,提取细胞的蛋白质样品,进行双向凝胶电泳(2-DE)分离,并分析凝胶中差异蛋白点。结果共检测出19个表达差异的蛋白点(ratio1.4,p0.05),其中感染组相对于对照组有7个蛋白质上调表达,12个蛋白质呈现下调表达。将差异蛋白进行串联质谱分析进行鉴定,并通过生物信息学方法对这19个差异表达蛋白进行了蛋白互作用分析。此外,对其中5个比较重要蛋白的mRNA水平进行了荧光定量PCR分析,其表达变化与2-DE结果一致。本研究为进一步分析EIAV与其宿主细胞eMDM的相互作用提供了参考。     

Cytotoxic T lymphocytes in protection against equine infectious anemia virus

Cytotoxic T lymphocytes (CTL) are associated with virus control in horses infected with equine infectious anemia virus (EIAV). Early in infection, control of the initial viremia coincides with the appearance of CTL and occurs before the appearance of neutralizing antibody. In carrier horses, treatment with immunosuppressive drugs results in viremia before a change in serum neutralizing antibody occurs. Clearance of initial viremia caused by other lentiviruses, including human immunodeficiency virus-1 and simian immunodeficiency virus, is also associated with CTL and not neutralizing antibody. In addition, depletion of CD8+ cells prior to infection of rhesus monkeys with simian immunodeficiency prevents clearance of virus and the same treatment of persistently infected monkeys results in viremia. Cats given adoptive transfers of lymphocytes from vaccinated cats were protected and the protection was MHC-restricted, occurred in the absence of antiviral humoral immunity, and correlated with the transfer of cells with feline immunodeficiency virus-specific CTL and T-helper lymphocyte activities. Therefore, a lentiviral vaccine, including one for EIAV, needs to induce CTL. Based on initial failures to induce CTL to EIAV proteins by any means other than infection, we attempted to define an experimental system for the evaluation of methods for CTL induction. CTL epitopes restricted by the ELA-A1 haplotype were identified and the MHC class I molecule presenting these peptides was identified. This was done by expressing individual MHC class I molecules from cDNA clones in target cells. The target cells were then pulsed with peptides and used with effector CTL stimulated with the same peptides. In a preliminary experiment, immunization of three ELA-A1 haplotype horses with an Env peptide restricted by this haplotype resulted in CTL in peripheral blood mononuclear cells (PBMC) which recognized the Env peptide and virus-infected cells, but the CTL response was transient. Nevertheless there was significant protection against clinical disease following EIAV challenge of these immunized horses when compared with three control horses given the same virus challenge. These data indicated that responses to peptides in immunized horses needed to be enhanced. Optimal CTL responses require help from CD4+ T lymphocytes, and experiments were done to identify EIAV peptides which stimulated CD4+ T lymphocytes in PBMC from infected horses with different MHC class II types. Two broadly cross-reactive Gag peptides were identified which stimulated only an interferon gamma response by CD4+ T lymphocytes, which indicated a T helper 1 response is needed for CTL stimulation. Such peptides should facilitate CTL responses; however, other problems in inducing protection against lentiviruses remain, the most significant of them being EIAV variants that can escape both CTL and neutralizing antibody. A possible solution to CTL escape variants is the induction of high-avidity CTL to multiple EIAV epitopes.     

Induction of a cell membrane antigen by equine infectious anemia virus.

Equine fibroblasts persistently infected with equine infectious anemia virus acquire a new cell membrane antigen demonstrable by indirect radioimmunoassay, using infected horse serum as an antibody source.     

Scanning and transmission electron microscopic study of equine infectious anemia virus.

Scanning and transmission electron microscopy were used to study in detail the morphogenesis and replication of equine infectious anemia virus (EIAV) in cultured, persistently infected equine fetal kidney fibroblasts. The EIAV was shown by thin-section electron microscopy to resemble morphologically more closely the members of the genus Lenti-virus in the family Retroviridae than other genera. Scanning electron microscopy demonstrated budding virus on only about 5% of the equine fetal kidney fibroblasts; however, the entire surface of these cells was involved in viral replication. Except where virus budding was observed, EIAV-infected cells were smooth and free of the topographic surface alterations characteristic of cells transformed by type C retroviruses. The morphologic relationship of EIAV and pathologic manifestations of EIAV infection to those of other Retroviridae are discussed.     

Detection of equine infectious anemia virus in horse leukocyte cultures derived from horses in various stages of equine infectious anemia viral infection

The enzyme-linked immunosorbent assay (ELISA) antigen-positive and agar-gel immunodiffusion test (AGID)-negative horses do not have infective equine infectious anemia (EIA) virus. The ELISA testing of horse leukocyte culture (HLC) supernatants did detect EIA virus in a HLC that was infected with the Wyoming strain of EIA virus and in HLC derived from horses in febrile, acute, or subacute stages of EIA infection. In supernatants of HLC derived from chronic and inapparent carrier horses, EIA virus was not detected with ELISA. Direct fluorescent antibody tests detected EIA virus in HLC infected with 10(6)TCID50 of the Wyoming strain of EIA virus and in 50% of the HLC from febrile acute or subacute horses. The direct fluorescent antibody testing of HLC derived from chronic and inapparent carrier horses did not detect cell-associated EIA virus. The pony inoculation test proved to be the most reliable and accurate method for detecting infective EIA virus in horses in various stages of EIA infection and accurately correlated with the AGID test.     

Prospective study of progeny of inapparent equine carriers of equine infectious anemia virus

Progeny of a band of horses, positive by the agar-gel immunodiffusion (AGID) test for equine infectious anemia (EIA) antibody, were observed through their weaning over a 4-year period. Sentinels (AGID test-negative) were allowed to mingle with EIA-infected mares and their foals in pasture situations in an area with high populations of potential vectors. Of 27 adult sentinels, 8 (30%) seroconverted in annual rates ranging from 0% to 75%. In contrast, only 2 of 31 (6%) foals weaned became infected. Difference in infection rates between adult sentinels and foals was significant (chi 2, P less than 0.05). Possible explanations for differences included protective value of colostral immunity and differences in attractiveness to blood feeding vectors. Detectable colostral immunity to EIA virus in the AGID test persisted for 25 to 195 days, with a mean of 124 days.     

Failure to propagate equine infectious anemia virus in mosquitoes and Culicoides variipennis.

Laboratory-colonized mosquitoes, Culex tarsalis, aedes aegypti, Culiseta inornata, and Anopheles free-borni, and the biting gnat, Culicoides variipennis, were exposed to equine infectious anemia virus. Exposure to the virus was by intrathoracic inoculation for mosquitoes and by oral ingestion of an infective blood meal through a membrane for C variipennis. After various intervals, groups of 15 to 20 insects were homogenized and inoculated into susceptible ponies. Positive immunodiffusion test results were used as criterion for equine infectious anemia infection in ponies. Virus was not detected in the 4 species of mosquitoes at 3, 6, 12, and 18 days after inoculation, or in C variipennis at 6, 8, 12, 14, 21, and 26 days after oral exposure to the virus.     

Indirect hemagglutination test in equine infectious anemia.

An indirect hemagglutination was developed for the diagnosis of equine infectious anemia using sheep red blood cells coated with group specific virus antigen which had been highly purified by affinity chromatography. The presence of indirect hemagglutination antibodies was demonstrated in horses with equine infectious anemia since the cells were specifically agglutinated by all the serum samples obtained from experimentally infected horses. Antibodies appeared within 35 days after inoculation, and development of which coincided well with that of precipitating and complement fixing antibodies. Titer of indirect hemagglutination antibodies were ten to 320 times greater than those of precipitating antibodies. Test results could be read more clearly by the indirect hemagglutination test especially in weakly positive cases. Ninety-six samples from suspected field cases collected from every region of Japan which were positive on the immunodiffusion test were also positive on indirect hemagglutination test. Serum samples from 420 horses in one race track were examined by both the indirect hemagglutination and immunodiffusion tests to determine the reliability of the indirect hemagglutination test for diagnosis of equine infectious anemia. The same result was obtained on both tests. Based on this evidence, the indirect hemagglutination test can be employed as a very sensitive serological test for the diagnosis of equine infectious anemia.     

Demonstration of equine infectious anemia virus in primary leukocyte cultures by electron microscopy.

Electron microscopy was used to demonstrate the presence of viral particles in primary cultures of leukocytes taken from a horse after SC inoculation with the Wyoming strain of equine infectious anemia virus. Unlike previous studies, the exposure virus was not passaged through cell culture prior to horse inoculation. Cultures were begun approximately 1 week before and 1 week after the 1st pyrexic period after inoculation. In both samples, viral particles and cytoplasmic alterations were observed resembling those previously reported in equine infectious anemia virus and other retravirus-infected cells.