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.     

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.     

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.     

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.     

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.     

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.     

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。     

Diagnoses of infections by the so-called chick anemia agent: anemia and direct transmission electron microscopic detection of virus.

Blood from 48 chicks was examined for anemia (packed cell volume), and plasma was examined for virus particles by direct transmission electron microscopy (DTEM). There was agreement between the occurrence of anemia and the presence of CAA virus particles in plasma from anemic chicks (Kappa = 0.2425, Z = 2.096, P = 0.036). Although DTEM is a method that can be used to diagnose CAA in chicks, more sensitive, economical and less laborious diagnostic assays are needed.     

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。     

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.     

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.     

Structural proteins of equine infectious anemia virus and their antigenic activity

Using purified equine infectious anemia (EIA) virus labeled with 3H-glucosamine or 14C-protein hydrolysate, structural proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, 2 glycoproteins and 10 proteins with molecular weights (mol wt) ranging from 12,000 to 115,000 daltons were demonstrated. Of 12 structural proteins, 3 proteins, namely a glycoprotein with mol wt of 76,000 (gp76) and 2 proteins with mol wt of 25,000 (p25) and 12,000 (p12), respectively, had distinct antigenic activity from one another in immunodiffusion. Development of antibodies against gp76 and p25 was compared in infected horses. The antibody to gp76 appeared earlier and stronger than to p25 in horses infected with the homologous virus strain. The fraction with glycoproteins was found to have hemagglutinating activity which was inhibited by the serum sample from horses infected with equine infectious anemia virus.     

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.     

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.     

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.     

马传染性贫血驴强毒gp90基因的克隆和序列分析

以EIAV驴强毒株D-AmRNA为模板，利用RT-PCR技术，扩增了约1．4kb的gp90基因。将其克隆后进行了测序。测序结果表明所扩增的1338个核苷酸片段含有完整的gp90基因全序列。核苷酸和氨基酸序列比较分析结果表明：D-A EIAV与国内分离株辽系强毒L株差异率仅有1．8％，而与国外毒株(克隆1369，WENVl7、WENVl6、PSPEIAVl9)核苷酸差异率在35．5％～37．2％之间；D-A株与国内分离株L株氨基酸水平差异率在2．9％，而与国外毒株氨基酸水平上的差异率在42．6％—46．0％；D-AEIAV有19个N-连接糖基化位点，L株、WENVl7和WENVl6是18个，克隆1369、WENVl6和WENVl7亲本毒株PSPEIAVl9是12个。     

Scanning electron microscopic study of bovine leukemic cells

Scanning electron microscopic observation of (i) leukemic cells in peripheral blood and tumor tissues of 11 cattle with bovine leukosis (adult form, n = 5; calf form, n = 2; and thymic form, n = 4) and (ii) peripheral lymphocytes from 2 cattle with persistent lymphocytosis and from 3 healthy cattle revealed morphologic differences of cell surface structures among various forms of bovine leukosis. These differences indicated an interrelationship of cell surface morphologic features between peripheral lymphocytes and tumor cells. Leukemic cells from cattle with the thymic and calf forms characteristically had a smooth surface. In cells from cattle with the adult form, the majority of abnormal cells in the peripheral blood, possessed numerous elongated microvilli on the cell surface, whereas tumor cells in the tissue were pleomorphic with a long villous, plicated surface or had stubby projections. Most of the peripheral lymphocytes from cattle with persistent lymphocytosis were characterized by a dense arrangement of elongated microvilli on the cell surface.     

马属动物SLFN11具有促进马传染性贫血病毒复制的能力

SLFN是一类干扰素诱导蛋白,还可以通过干扰素调节因子(IRF3)途径被病原体直接诱导表达。最新研究表明,SLFN11可通过抑制病毒蛋白合成,对慢病毒属的HIV-1具有明显抗病毒作用。为阐明SLFN11对马传染性贫血病毒(EIAV)的作用,本研究采用RT-PCR技术从健康马淋巴细胞中首次扩增获得SLFN11基因,构建了真核表达重组质粒pHA-eSLFN11。将其与EIAV的感染性克隆共转染HEK293T细胞,western blot检测结构蛋白的表达情况,并采用激光共聚焦试验分析SLFN11在亚细胞中的定位。研究结果显示,SLFN11能够促进EIAV结构基因(Gag)的表达,此外马源SLFN11(eSLFN11)与人源SLFN11(hSLFN11)明显定位不同。以上结果提示,eSLFN11与EIAV的作用明显区别于hSLFN11对HIV-1的限制作用,其机制有待进一步研究。