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.     

Equine infectious anemia in mules: virus isolation and pathogenicity studies

There appears to be a lack of information concerning responses of mules to natural infection or experimental inoculation with equine infectious anemia virus (EIAV). In the present study EIAV was isolated from mules, for the first time, and its pathogenicity in naturally infected and experimentally inoculated animals was investigated. Two naturally infected (A and B) and three EIAV free mules (C, D and E) were used for this purpose. Mule A developed clinical signs, whereas mule B remained asymptomatic until the end of the study. Mules C and D were each inoculated with 10ml of blood from mule A and developed signs of the disease; they were euthanatized or died at day 22 and 25 post-inoculation, respectively. Mule E served as a negative control. The virus was isolated from the plasma samples of mules with clinical signs of the disease (A, C and D), but not from the asymptomatic mule B. Both proviral DNA and viral RNA were amplified from blood and tissues of the infected animals by nested polymerase chain reaction (nPCR). Antibodies were not detected in the two experimentally infected mules until their natural death or euthanasia. Clinicopathological and laboratory findings showed that, in mules, EIAV produced clinical signs similar to those observed in horses and ponies. Nested PCR proved to be a rapid, sensitive and specific diagnostic method for the detection of EIAV, regardless of the disease stage.     

应用ViewRNA技术特异性检测感染细胞中的不同马传染性贫血病毒株

为了对体外培养细胞感染的不同马传染性贫血病毒(EIAV)株进行特异的鉴别检测,本研究基于一种新的RNA原位杂交-ViewRNA技术,通过设计合成针对EIAVFDDV13和EIAVUK3病毒株基因组RNA的特异性探针,并结合信号放大技术和荧光标记技术,同时通过激光共聚焦显微镜进行观察,建立了可时两种不同株的EIAV基因组RNA进行细胞内原位检测的方法.研究结果表明,两组探针能够特异地与感染细胞中EIAVFDD13和EIAVUK3的病毒RNA相结合,可在两种不同EIAV株共感染的宿主细胞中实现对两种毒株的有效鉴别和定位检测.该方法为进一步研究EIAV与宿主细胞间相互作用提供了有效手段.     

Effects of equine infectious anemia virus on hematopoietic progenitors in vitro.

Direct effects of equine infectious anemia virus (EIAV) on hematopoiesis in vitro were studied. Bone marrow mononuclear cells from clinically normal horses were incubated with 100 TCID50 of EIAV/10(7) cells. These cells were cultured to assay for colonies derived from erythroid progenitors, granulocyte/monocyte progenitors, and fibroblastic progenitors. The EIAV had a selective suppressive effect on the erythroid progenitors. Colony-forming units-erythroid were suppressed to 80% of that for medium controls (P = 0.011). Burst-forming units-erythroid were suppressed to 70% of that for medium controls (P = 0.003). Significant effect was not apparent on colony-forming units-granulocyte/macrophage or on colony-forming units-fibroblastic.     

马传染性贫血病病毒弱毒疫苗株感染性分子克隆的构建

采用ＰＣＲ方法分三段扩增出马传染性贫血病毒驴白细胞弱毒疫苗株（ＥＬＡＶ　 ＤＬＡ）的前病毒ＤＮＡ,这三个片段覆盖马传染性贫血病毒的全部基因组,ＰＣＲ产物经克隆后顺次连接,获得一个含有ＥＬＡＶ全基因（８．０Ｋｂ）的重组质粒,将其命名为ｐ８．０。将此８．０Ｋｂ ＥＩＡＶ全基因再亚克隆到含有一完整 ＥＩＡＶ ＤＬＡ株长末端重复序列的质粒中,获得一含有 ＥＩＡＶ驴白细胞弱毒前病毒全基因的重组质粒,将其命名为ｐ８．２,经核苷酸序列分析,证明ｐ８．２含有ＥＩＡＶ前病毒的全基因。用ｐ８．２转染驴白细胞,将其作为种毒进行传代,于感染该克隆毒的细胞培养上清中检测出了反转录酶,说明在驴白细胞中由ｐ８．２衍生出了ＥＩＡ病毒。驴白细胞经该克隆毒感染后,第４天出现病变,经透射电镜可观察到典型的马传染性贫血病毒粒子,进一步证明ｐ８．２具有感染性,我们获得了马传染性贫血病毒驴白细胞弱毒疫苗株的感染性分子克隆,为进一步在分子水平上阐明我国ＥＩＡＶ疫苗株的减毒机理和免疫保护机制奠定了基础。     

马传贫驴白细胞弱毒疫苗株跨膜蛋白主要免疫决定区基因的克隆与表达

从感染驴白细胞的马传贫驴白细胞弱毒疫苗株前病毒DNA中克隆了编码跨膜蛋白主要免疫决定区(TMIR)的基因，并在大肠杆菌中进行了表达。所表达的融合蛋白有一部分是可溶的，其氨基端带有6个组氨酸的标签，因此可以用固定化金属离子亲和层析法在非变性条件下进行纯化。在间接酶联免疫吸附试验(ELISA)和免疫印迹试验中，重组的TMIR蛋白可与马传贫阳性血清样品发生反应，而与健康马血清无任何反应。这表明该重组蛋白具有良好的抗原性和特异性，可用于马传贫弱毒疫苗株在体内外复制、接种马体内免疫应答及马传贫诊断的研究。     

Use of western blot and radioimmunoprecipitation for diagnosis of feline leukemia and feline immunodeficiency virus infections.

The uses and limitations of the western blot (WB) and radioimmunoprecipitation assay (RIPA) techniques for study of feline immunodeficiency virus (FIV) and FeLV were evaluated. Western blot analysis was used to detect antigenic relatedness between the 2 lentiviruses. Using a rabbit serum directed against p26 of the equine infectious anemia virus (EIAV) and anti-EIAV horse serum obtained from an infected horse, cross-reactivity with p24 of FIV was revealed. Cat sera obtained late after experimentally induced FIV infection recognized p26 of EIAV, which indicates reciprocal cross-reactivity. For RIPA, FIV was metabolically labeled, and virus-coded proteins were identified, using immunoprecipitation. Polypeptides with apparent molecular mass of about 15, 24, 43, 50, 120, and 160 kilodaltons were detected. An additional polypeptide of 10 kilodaltons was found only by use of WB analysis.     

Differential responses of Equus caballus and Equus asinus to infection with two pathogenic strains of equine infectious anemia virus

Most in vivo studies with equine infectious anemia virus (EIAV) have been performed in horses and ponies (Equus caballus) with little published information available detailing the clinical responses of donkeys (Equus asinus) to infection with this virus. Consequently, donkeys were inoculated with two strains of EIAV (EIAV(PV) and EIAV(WY)) which have been documented to produce disease in E. caballus. Four ponies, 561, 562, 564 and 567 and two donkeys, 3 and 5 were infected with EIAV(PV) and one horse (94-10) and one donkey (4) were infected with EIAV(WY). Although the horse and ponies all experienced clinical signs of disease, which in some cases were severe, the donkeys remained asymptomatic throughout a 365-day observation period, except for mild transient reductions in platelet counts. The results from serological assays, virus isolation from plasma and detection of plasma-associated viral RNA by RT-PCR, indicated that initial replication of EIAV(PV) and EIAV(WY) was lower in donkeys than in horses and ponies. This conclusion was confirmed using competitive RT-PCR, in which viral RNA levels in the plasma of EIAV(PV)-infected ponies was up to 100,000-fold higher than in infected donkeys during the first 20 days post-infection (dpi). Similar results were obtained in the EIAV(WY)-infected animals, in which viral RNA burdens in the donkey at 20 dpi were 1000-fold less than in the horse. However, infection of donkey and horse monocyte-derived macrophage cultures with EIAV(PV) demonstrated that these cells in vitro were equally susceptible to virus-induced cytopathic effects and yielded similar levels of progeny virus. This result suggests that factors other than host cell permissiveness mediate the clinical differences observed between horses and donkeys infected with EIAV(PV) or EIAV(WY).     

马传贫驴白细胞弱毒疫苗株基质蛋白基因的克隆与表达

从感染驴白细胞的马传贫驴白细胞弱毒疫苗株前病毒DNA中克隆了编码基质蛋白（p15)的基因，并在大肠杆菌中进行了表达，所表达的蛋白是一种可溶性的融合蛋白，其氨基端带有6个组氨酸的标签，因此可以用固定化金属离子亲和层析法在非变性条件下进行纯化，在间接ELISA和免疫印迹试验中，重组的基质蛋白可与马传贫阳性血清样品发生反应，而与健康马血清无任何反应，这表明该重组蛋白具有良好的抗原性和特异性，可用于马传贫弱毒疫苗株在体内外复制及在接种马体人免疫应答的研究中。     

利用Real-time PCR对外周血白细胞中EIAV前病毒及血浆中病毒RNA含量的测定

利用Real-time PCR和Real-time RT-PCR方法对马传染性贫血病(EIA)驴白细胞弱毒疫苗(DLA-EIAV)、DLA-EIAV感染性分子克隆衍生毒(vOK8226)、强弱毒嵌合病毒(vOKVltr)以及EIA强毒接种马后不同时期外周血白细胞中前病毒含量及血浆中病毒含量进行了监测,结果发现直接攻击强毒的2匹马及1匹接种vOK8226后再用强毒攻击的马血浆中病毒含量快速升高,伴随着明显的临床反应,最后均以死亡结束.其它免疫接种马在攻击强毒后均获得保护,没有发病,马血浆中有低水平病毒存在,攻毒后3个月血浆中检测不到病毒,说明感染马体内病毒的大量增殖与疾病的进程有着直接的关系.而外周血白细胞中前病毒的含量在各试验马各时期均能检测到,说明EIAV以前病毒的形式潜伏在感染马体内,疫苗的免疫只能控制发病,而不能清除感染的病毒.     

Serosurveillance for equine infectious anaemia in the Ardahan province of Turkey

Equine infectious anaemia is a retroviral infection of horses. All infected horses, including those that are asymptomatic, become carriers and are infectious for life. In this study, blood samples of all equines in the province of Ardahan were collected. The material consisted of 8,947 equines, including 8,769 horses and 178 donkeys, from Ardahan province in northeastern Turkey. Blood was collected from all horses and donkeys and the sera were analysed for the presence of antibodies to equine infectious anaemia virus (EIAV) using an enzyme-linked immunosorbent assay. The results revealed that none of the horses and donkeys were positive for antibodies to EIAV.     

Reduction of CD4+ and CD8+ T lymphocytes during febrile periods in horses experimentally infected with equine infectious anemia virus

Three horses were experimentally infected with equine infectious anemia virus (EIAV). All horses were febrile after inoculation with EIAV and then developed chronic symptoms with intermittent fever. The febrile period was characterized by a rise in body temperature with reduced PBL and erythrocyte counts. Flow cytometric analysis showed that the reduced number of lymphocytes was due to significant decreases in CD4+ and CD8+ T cells in the absence of any change in B cell number. At the end of the febrile period the body temperature began to recover and numbers of CD4+ and CD8+ T cells showed a tendency to increase. For CD8+ T cells, this increase continued for several days after the febrile period. B cell number also significantly increased after the febrile period in two out of three horses. The decrease of CD8+ T cells was greater than that of CD4+ T cells. Although the PBL numbers and the CD4/CD8 ratio returned to the level of the preinoculation period, erythrocyte numbers decreased as the body temperature normalized after each intermittent fever. These results suggest that the recurring cycle of fever accompanied with viremia is caused by a reciprocal relationship between EIAV replication and the host immune response. Furthermore, we demonstrate that the lymphocytic response mitigates fever and viremia in EIAV infection despite the absence of virus neutralizing antibody.     

Leukoencephalitis associated with selective viral replication in the brain of a pony with experimental chronic equine infectious anemia virus infection

Neurologic disease occurs sporadically in horses infected with the equine infectious anemia virus (EIAV). This report describes a case of clinically severe neurologic disease in a pony experimentally infected with EIAV. This pony did not have fever or anemia, which are the characteristic clinical signs of disease. The histopathologic changes were characterized as lymphohistiocytic periventricular leukoencephalitis. Polymerase chain reaction and in situ hybridization data showed that the brain lesions were directly associated with viral replication and that high-level viral replication occurred selectively within the lesion and not in other tissues. These findings suggest that EIAV-associated neurologic disease is the direct result of viral replication.     

The determination of in vivo envelope-specific cell-mediated immune responses in equine infectious anemia virus-infected ponies

Distinct from human lentivirus infection, equine infectious anemia virus (EIAV)-infected horses will eventually enter an inapparent carrier state in which virus replication is apparently controlled by adaptive immune responses. Although recrudescence of disease can occur after immune suppression, the actual immune correlate associated with protection has yet to be determined. Therefore, EIAV provides a model for investigating immune-mediated protective mechanisms against lentivirus infection. Here, we have developed a method to monitor EIAV-envelope specific cellular immunity in vivo. An EIA carrier horse with no clinical signs infected 7 years ago and 4 related experimental ponies infected 6 months previously were used in this study. Forty-four 20-mer peptides, representing the entire surface unit protein (gp90) of EIAV, were combined into 14 peptide pools and intradermally injected into the neck of EIAV-infected horses. An identical volume of saline alone was injected into a fifteenth site as a negative control. After 48h, those sites with palpable infiltrations were measured prior to the collection of 2mm and 4mm punch biopsies. Total RNA was extracted from each 2mm biopsy for determination of CD3 and interferon-γ (IFN-γ) mRNA expression by real-time PCR. The 4mm skin biopsies were formalin-fixed and paraffin-embedded for immunohistochemistry (IHC) staining for CD3, CD20, CD25 and MAC387 (macrophage marker). Peripheral blood mononuclear cells (PBMC) were obtained prior to the injection and tested for in vitro reactivity against the same peptides. Histological examination showed that some of the envelope peptides elicited a lymphocytic cellular infiltration at the injection site, as evidenced by positive staining for CD3. Gp90 peptide-specific increases in CD3 and IFN-γ gene expression were also detected in the injection sites. Furthermore, differences were found between in vivo and in vitro responses to gp90 specific peptides. These results demonstrate a novel method for detecting in vivo cell-mediated immune responses to EIAV-specific peptides that is readily applicable to other host/pathogen systems.     

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.     

An investigation of equine infectious anaemia infection in the central Anatolia region of Turkey

In this study, 162 horses, 80 donkeys and 51 mule serum samples were collected in Konya city. Additionally, 64 horse serum samples from Ankara and 49 samples from Kayseri city were included in the study. A total of 406 serum samples were examined by agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for antibody to equine infectious anaemia virus (EIAV) and no positive result was detected.     

Virus-like particles in buffy coat cells of normal goats and goats infected with progressive pneumonia virus.

By electron microscopy, virus-like particles (VLP) were seen in neutrophils and lymphocytes from buffy coats prepared from 5 goats inoculated with progressive pneumonia virus (PPV) and 3 noninoculated goats. The VLP were 80 to 120 nm in diameter, limited by a unit membrane, and resembled PPV, visna virus, and other members of the oncornavirus family. Some VLP seemed to have electron-dense nucleoids and external spikes. In neutrophils, VLP were observed budding into vacuoles; rarely, intravacuolar clusters were seen. In lymphocytes, VLP were seen in vacuoles. Free VLP and budding of VLP from external cell surfaces were not seen. In vitro incubation of buffy coat cells with mitogens did not affect the frequency of VLP. Although the VLP are similar to PPV, the exact nature of the particles and their relationship to PPV is not clearly understood.     

Real-time quantitative RT-PCR and PCR assays for a novel European field isolate of equine infectious anaemia virus based on sequence determination of the gag gene

In 2006, an outbreak of equine infectious anaemia (EIA) occurred in Ireland. The initial source of the outbreak is believed to have been contaminated plasma imported from Italy. This paper presents the nucleotide sequence of the gag gene of the virus identified in Ireland (EIAV(Ire)), the first for a European strain of EIAV. Comparison of the gag gene with North American and Asian strains of the virus showed that the gag gene is less well conserved than previously believed, and that EIAV strains can have similar phenotypes despite considerable variations in genotype. On the basis of the deduced sequence of the EIAV(Ire) gag gene, highly sensitive, specific and quantitative RT-PCR and PCR assays were developed, and used to quantify the EIAV nucleic acid in postmortem tissues, plasma and secretions of infected horses. This is the first report of the detection and quantification of EIAV in nasal, buccal and genital swabs by RT-PCR.     

H3N8亚型马流感病毒单克隆抗体的制备及鉴定

以灭活马流感病毒(EIV)A/Equine/Jilin/1/1989(H3N8)为免疫原,免疫Balb/c小鼠,经常规细胞融合后,用血凝抑制试验(H1)和间接ELISA方法筛选获得3株(3C2、5G10和5A10)能稳定分泌H3N8亚型马流感病毒单克隆抗体(mAb)的杂交瘤细胞株.其中3C2和5G10为IgG2α,5A...