通过暂时免疫抑制研究跨膜蛋白C末端缺失对EIAV疫苗株EIAV_(FDDV12)体内复制的影响

马传染性贫血病毒(EIAV)减毒疫苗是世界首例慢病毒疫苗,但其作用机理尚不明了。我们的前期研究发现EIAV疫苗株EIAVFDDV12编码跨膜蛋白gp45的基因在马体内发生高频率G2230A点突变形成终止子,使该蛋白C末端出现154个氨基酸的截短。为了探讨该截短蛋白对EIAV疫苗株生物学特性的作用,本研究以EIAV弱毒疫苗株感染性克隆基础上,构建了gp45截短型感染性克隆,脂质体转染细胞,增殖传代,获得病毒株EIAVFDDVTM-36,接种到马体内。在接种后167 d内未观察到临床症状,连续注射10 d地塞米松以暂时抑制免疫系统,并用迟发性超敏反应和淋巴细胞增殖实验评价免疫抑制效果。结果显示,截短型跨膜蛋白疫苗株EIAVFDDVTM-36接种组免疫抑制前后病毒载量和中和抗体效价差异不显著,而完整型跨膜蛋白疫苗株感染性克隆EIAVFDDVTM-45接种组4匹马中3匹免疫抑制后病毒载量显著上升,同时组内中和抗体效价明显提高。以上结果表明,马体特异性免疫压力对EIAVFDDVTM-36复制无影响,而对亲本株EIAVFDDVTM-45复制有抑制,显示跨膜蛋白截短型EIAV作为疫苗更为安全。但免疫抑制造成的gp45跨膜蛋白未截短型疫苗株感染性克隆体内病毒载量升高,可以促使机体产生更高的中和抗体效价,提示该疫苗的安全性和有效性在一定程度内呈负相关(相互制约)。因此,安全性和有效性的平衡是慢病毒减毒疫苗研发需注意的重要因素。     

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.     

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.     

A propagating epizootic of equine infectious anemia on a horse farm

An epizootic of equine infectious anemia (EIA) involved 35 horses on a farm in south Georgia. During a 126-day period, 21 of these horses became seropositive for EIA. After the initial diagnosis in July, the horses were tested every 7 to 10 days. At least one additional horse was found to be seropositive on each testing day. As soon as they were determined to be seropositive, the horses were removed from the herd and sent to slaughter. The removal of the seropositive horses, however, did not stop the epizootic. We believe the initial infection was from a 7-year-old stallion that recently had been purchased or from 1 of 2 mares that were seropositive for EIA on the first test. None of the horses had been tested for EIA at the time of purchase or within 60 days before the epizootic.     

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.     

Virulence and in vitro growth of a cell-adapted strain of equine infectious anemia virus after serial passage in ponies

Five serial passages of a cell-adapted strain of equine infectious anemia (EIA) virus were conducted in Shetland ponies. The 13 recipient ponies became agar-gel immunodiffusion test-positive by 25 days after they were inoculated. The virulence of the cell-adapted strain of EIA virus markedly increased through 3 serial passages, although individual variation within passages was high. The 1st serial-passage recipient remained afebrile through 200 days, whereas a febrile episode occurred about every 185, 44, 35, and 33 days in the 2nd, 3rd, 4th, and 5th serial-passage recipients, respectively. Severe clinical signs of EIA were noted in the ponies at each serial passage, but the mean virulence rating of each passage, graded on frequency of febrile episodes and number of clinical signs evident within 200 days after ponies were inoculated, increased from 0 through 4, 21, 24, and 29 for the 1st through 5th serial passages, respectively. Isolates of EIA virus, made in fetal equine kidney cells, were obtained from plasma of 75% of the samples of blood collected during febrile episodes and from 45% of the samples collected during afebrile periods, indicating that the cell culture growth capacity of this strain of EIA virus may be relatively stable through 5 serial passages in Shetland ponies.     

Role of insects in the transmission of bovine leukosis virus: potential for transmission by stable flies, horn flies, and tabanids

The ability of stable flies (Stomoxys calcitrans), horn flies (Haematobia irritans), and tabanids (Diptera: Tabanidae) to transmit bovine leukosis virus (BLV) was investigated. Stable flies and horn flies were fed on blood collected from an infected cow, and the flies' mouthparts were immediately removed, placed in RPMI-1640 medium, ground, and inoculated into sheep and calves. Infection of sheep occurred with mouthparts from as few as 25 stable flies or 25 horn flies. However, sheep were not infected when removal of stable fly mouthparts was delayed greater than or equal to 1 hour after blood feeding. Infection of calves occurred after inoculation of mouthparts removed immediately after feeding from as few as 50 stable flies or 100 horn flies. Infected blood, applied by capillary action to the mouthparts (labella) of 15 deer flies (Chrysops sp) and a single horse fly (Tabanus atratus) caused infection in each of 2 sheep. Infection did not occur in 2 calves inoculated daily for 5 days with mouthparts from 50 horn flies collected after feeding on a BLV-infected steer. Four calves receiving bites from 75 stable flies interrupted from feeding on a BLV-positive cow also were not infected. Seronegative cattle held for 1 to 4 months in a screened enclosure with positive cattle in the presence of biting flies were not infected with BLV. The feeding behavior of each insect is discussed to assess their potential as vectors of BLV.     

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 a horse with an equivocal agar gel immunodiffusion test reaction

A horse whose serum reacted equivocally in the agar gel immunodiffusion (AGID) test for equine infectious anemia was studied over a 3-year period. The horse remained afebrile and virus was detected in only 1 of 6 horse inoculation tests. The intensity of AGID test reactions increased temporarily following this evidence for virus. Although the AGID test reaction was equivocal and 5 of the 6 transmission attempts failed, the 1 successful transmission proved the horse was infected.     

Blood-feeding patterns of horse flies in the French Pyrenees

Horse flies can mechanically transmit Besnoitia besnoiti, the agent of bovine besnoitiosis. Although previously limited to enzootic areas, especially the French Pyrenees Mountains, bovine besnoitiosis is now considered a re-emerging disease in western Europe. To improve understanding of the role of horse flies as mechanical vectors, this study investigated their blood-feeding ecology in the eastern French Pyrenees, in two high-altitude summer pastures whose main domestic ungulates were cattle, and in a wildlife park with native fauna. Species-specific PCR assays were conducted to identify the sources of blood meals: wild boar, horse, cattle (or bison), sheep (or mouflon), goat, red deer, roe deer and izard (or Pyrenean chamois). In La Mouline pasture, tabanids (N = 20) fed on red deer (70%) and cattle (30%). In Mantet pasture, tabanids (N = 24) fed on cattle (52%), red deer (20%), wild boar (16%), horse (8%) and sheep (4%). In the wildlife park, Tabanus bromius (N = 32), the most abundant species collected, fed on red deer (85%), bison (9%) and wild boar (6%). Despite relatively high densities in both the pastures and in the wildlife park, small wild ungulates (izard, mouflon and roe deer) were not detected as a source of blood meals. Only two mixed blood meals were identified in two specimens of T. bromius: cattle/horse for the specimen collected in the pastures, and bison/wild boar for the specimen collected in the wildlife park. Our findings showed that tabanids display a level of opportunistic feeding behaviour, in addition to a preference for red deer, the latter being particularly true for Philipomyia aprica, the most abundant species collected in the pastures.     

Immune-mediated hemolytic anemia in a horse

An 18-year-old Quarter Horse gelding was determined to have immune-mediated hemolytic anemia after detection of autoagglutination of RBC spherocytosis as well as a positive direct Coombs test result. A lack of response to treatment with corticosteroids necessiated the administration of cyclophosphamide and azathioprine. The anemia resolved after treatment with chemotherapeutic drugs.     

Acute hemolytic anemia after oral administration of L-tryptophan in ponies

The hematologic and pathologic effects of orally administered L-tryptophan and indoleactic acid and of L-tryptophan administered IV were studied in ponies. Sixteen adult Shetland ponies were allotted into 4 experimental groups. Group 1 consisted of 5 ponies (1-5) given 0.6 g of tryptophan/kg of body weight in a water slurry via stomach tube. Group 2 included 4 ponies (6-9) given 0.35 g of tryptophan/kg orally. Group-3 ponies (10-13) were given 0.35 g of indoleacetic acid/kg orally. Group 4 consisted of 3 ponies (14-16) given a single 4-hour IV infusion of 0.1 g of tryptophan/kg. Restlessness, increased respiratory rate, hemolysis, and hemoglobinuria were detected in 4 of the 5 group-1 ponies. Only pony 7 in group 2 developed hemolysis, hemoglobinuria, and a significant increase in respiratory rate. Renal pathologic lesions, consistent with hemoglobinuric nephrosis, were seen in ponies 2, 4, 5, and 7. Bronchiolar degeneration was evident in 4 of 9 ponies given tryptophan orally. The importance of these respiratory lesions was unknown. Clinical or pathologic abnormalities were not noticed in the ponies of groups 3 and 4. Mean plasma tryptophan values increased significantly in groups 1 and 2 at 6 hours after dosing. A second peak of tryptophan was detected in both groups at 12 hours. Values returned to predose values by 48 hours. Plasma indole and 3-methylindole concentrations were detectable in only 2 ponies (4 and 7). In vitro incubations of cecal fluid from ponies 6, 8, and 9 yielded a percentage conversion of tryptophan to indole of 16.75%, 5.84%, and 7.96%, respectively. 3-Methylindole was not produced. These results suggested that indole was the major metabolite of orally administered tryptophan in these ponies.