Induction of neutralizing antibodies against bovine leukosis virus in rabbits by vaccination with recombinant vaccinia virus expressing bovine leukosis virus envelope glycoprotein

Three kinds of recombinant vaccinia virus (RVV)--mO-HA/ATI, LO1-HA/ATI and mO-HA/7.5kD--expressing bovine leukosis virus (BLV) envelope glycoprotein (gp60) were constructed. The BLV envelope gene of RVV mO-HA/ATI and LO1-HA/ATI or of RVV mO-HA/7.5kD was expressed under control of the promoter of A-type inclusion body (ATI) protein gene of cow-pox virus or vaccinia virus 7.5-kD protein gene, respectively. The vaccinia virus strain, LC16mO, was used as vector for RVV mO-HA/ATI and mO-HA/7.5kD, and strain LO-1 was used for RVV LO1-HA/ATI. Strains LC16mO and LO-1 are attenuated vaccine virus strains originating from the Lister original vaccinia virus. All 3 kinds of constructed RVV expressed gp60 in cultured rabbit kidney cells after infection; mO-HA/ATI expressed more antigen than did mO-HA/7.5kD. Rabbits vaccinated with RVV produced considerable antibody capable of inhibiting syncytium formation, as well as antibody with virion-binding ability. The RVV that used ATI promoter induced higher antibody titer than did the RVV that used 7.5-kD promoter. Results indicate that BLV gp60 is responsible for induction of neutralizing antibodies that suppress in vitro formation of syncytia among BLV-infected cells. Applicability of RVV, especially those using ATI promoter, was evaluated in a vaccine against bovine leukosis.     

Comparison of slot blot nucleic acid hybridization, immunofluorescence, and virus isolation techniques to detect bluetongue virus in blood mononuclear cells from cattle with experimentally induced infection.

A slot blot hybridization technique was applied for detection of bluetongue virus (BTV) in blood mononuclear cells (BMNC) obtained from cattle with experimentally induced infection. This technique lacked sensitivity to detect the viral nucleic acid directly in clinical specimens. When aliquots of mononuclear cells from these cattle were cultivated in vitro for 10 days to amplify virus titer, only 33.3% of the samples collected during viremia gave a positive signal in the slot blot hybridization format. By contrast, results for 34.3% of noncultured and 63.3% of cultured mononuclear cell samples collected during viremia were positive by immunofluorescence. The average number of infected cells, as detected by immunofluorescence in the noncultured mononuclear cell samples, was 1 to 5/300,000, and was usually > 10/300,000 in the cultured cell samples. Virus was isolated from all postinoculation blood samples obtained from 4 heifers that were seronegative at the time of inoculation, but was not isolated from any of the preinoculation samples, or from any of the postinoculation samples obtained from 2 heifers that were seropositive at the time of inoculation. When virus isolation was attempted from separated mononuclear cells in 2 heifers, 43.7% of the noncultured and 87.5% of the cultured samples had positive results.     

Foot-and-mouth disease virus subtyping by sequencing VP1 genes

In order to use nucleotide sequencing for foot-and-mouth disease virus (FMDV) diagnostic subtyping, it is necessary to shorten the time required for preparation of suitable templates. The time required for analysis was reduced by use of the viral RNA present in the total RNA extract of tissue from infected cattle as a template in the Sanger sequencing reaction. Results are now available within 3 days. The sequences determined encode capsid protein VP1 and therefore major neutralization epitopes. Such a sequence of FMDV O1Kaufbeuren, cultured in the animal, was compared with those of tissue-cultured viruses. They did not differ. It was concluded that a change of virus culture conditions does not necessarily account for antigenic variation.     

Increase of CD163 but not sialoadhesin on cultured peripheral blood monocytes is coordinated with enhanced susceptibility to porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted tropism mainly for porcine alveolar macrophages (PAMs), but not for peripheral blood monocytes (BMo) in vivo. Previous research showed that only a few BMo became susceptible to PRRSV infection after 1 day culture. Porcine sialoadhesin (PoSn) and CD163 are identified to be the two main PRRSV receptors for binding and internalization. Both receptors are not expressed on BMo, or only expressed at low levels, which may explain why PRRSV cannot infect them. The relationship of BMo differentiation/aging, PRRSV receptor level, and susceptibility to PRRS virus infection has not been thoroughly investigated. In this study, BMo were successfully cultured with pig serum plus L929 cell culture supernatant. Our results showed that both the mRNA and protein expression levels of PoSn were significantly increased after 5-day culture. The mRNA level of CD163 was enhanced more than 20-fold after 1-day culture; CD163-positive BMo increased dramatically from about 2% after 2h- culture to about 50% after 96-h culture. Furthermore, cultured BMo became much more permissive to PRRSV infection, and the percentage of PRRSV-infected BMo was at least the same as PAMs, if not higher, when infected with CH-1a, the first PRRSV strain isolated in China, or HV, a highly virulent strain. Three other PRRSV strains including VR2332, and two classical Chinese isolates could also infect cultured BMo as well. Most importantly, PRRS virus was successfully isolated from 14 of 15 antibody-positive serum samples using cultured BMo. These results suggest that the enhanced susceptibility of cultured BMo to PRRS virus is coordinated with increased CD163 expression, but less related to the delayed (day 5) increased expression of PoSn. Thus, cultured BMo could be an alternative choice for PRRS virus isolation and identification.     

In vitro production of antibodies to bovine virus diarrhoea virus by peripheral blood mononuclear cells from cattle immunized by infection

A method for in vitro production of antibodies to bovine virus diarrhoea virus (BVDV) by peripheral blood mononuclear cells (PBMC) was developed. The PBMC were cultured in microtitre plates coated with detergent-solubilized BVDV and the supernatants were tested in an enzyme-linked immunosorbent assay which detects IgG antibodies to BVDV. Following incubation of PBMC with an optimal concentration of pokeweed mitogen for 5 days, antibodies to BVDV were detected in culture supernatants of PBMC from immune cattle, but not in supernatants of PBMC from seronegative cattle, from persistently BVDV-infected cattle or from a 5-day-old calf that received BVDV antibodies via colostrum. This antibody-production assay may therefore be used as a tool to discriminate between passively acquired antibodies and those actively induced.     

猪流行性腹泻病毒分子生物学研究进展

猪流行性腹泻病毒在Vero细胞上培养成功后,病毒全基因组的序列和分子结构特征,结构蛋白和非结构蛋白的生物学特性和功能,病原学、血清学和分子生物学的诊断技术,体液免疫、细胞免疫和局部黏膜免疫的机理,疫苗和病毒的细胞受体等方面得到了广泛的研究。但是,与其他冠状病毒相比,猪流行腹泻病毒的研究还比较滞后,许多研究还没有开展或不够深入,为了能加快对猪流行性腹泻病毒研究进程,对其最近的分子生物学研究进展做了综述,为猪流行性腹泻病毒的深入研究提供参考。     

Testicular changes observed in boars following experimental inoculation with pseudorabies (Aujeszky''s) virus.

Four boars were inoculated intranasally with pseudorabies virus to determine if microscopic testicular changes occurred as a result of infection. Testicular biopsies and semen samples were taken at two, four and six weeks postinoculation and the boars were castrated immediately after the last sample collection. Testicular samples and semen were cultured to determine if the virus was present. Pseudorabies virus was not isolated from the semen or testicular tissue. Virus was isolated from trigeminal ganglia at necropsy and from nasal swabs taken one day after castration. Consequently, a time of high risk for shed of the virus from clinically normal carrier animals is immediately following castration. Gross changes were not observed in testicular tissues and microscopic changes in the testicles were the result of biopsy. Lesions consistent with pseudorabies virus infection were observed in the central nervous system of all inoculated boars. Temporary lowered fertility may result from the effects of elevated body temperature on spermatogenesis during acute clinical disease. However, it appears that the strain of pseudorabies virus used, lacked the ability to infect and/or replicate in the boars' reproductive tracts.     

Application of a radioimmunoassay for detection of the major internal antigen (p24) of bovine leukemia virus from cultured lymphocytes of cattle

A sensitive and specific radioimmunoassay for the major internal antigen of bovine leukemia virus has been applied to detecting this protein in cultured lymphocytes of infected cattle. The specificity inherent in this assay offers obvious advantages over a previously described syncytium induction assay for infectious bovine leukemia virus, because false positive reactions due to other viruses such as bovine syncytial virus are avoided. Investigations of various culture conditions indicated that maximal amounts of antigen had been produced after incubation for 72 hr at 37°C. Lymphocyte concentrations of 10⁶?5×10⁷ cells/ml gave satisfactory results. Tests of cultured lymphocytes from bovine leukemia virus infected or bovine leukemia virus-free cattle indicated a comparable sensitivity between the radioimmunoassay and syncytium induction assay in the detection of bovine leukemia virus infections.     

伪狂犬病病毒gE／gI双基因缺失株的生物学特性

为了解gE和gI双基因缺失株伪狂犬病病毒gE-／gI—PRVSA738和野毒株PRV—SA的生物学特性,对该病毒进行了理化特性和体外增殖曲线的研究。结果显示：该缺失株对氯仿、酸、热及冻融次数敏感,从双基因缺失毒株与野毒株在BHK-21细胞上的增殖曲线来看,在病毒培养50h之前野毒株的毒价均高于缺失株,而在50h以后缺失株的毒价又高于野毒株,并且在36h时2株病毒的毒价均达到最高峰。     

A bovine herpesvirus (BHV-4) as passenger virus in ethmoidal tumours in Indian cattle

A herpesvirus was isolated from tumours of the ethmoidal mucosa in two of three head of cattle in the State of Kerala, India. The virus designated M40 was cytopathic for a variety of cultured bovine and porcine cells and it did not kill suckling mice or chicken embryos. Sera from tumour-bearing cattle and goats reacted with the M40 virus. Immunofluorescence tests with FITC-conjugated IgG from a bovine monospecific antiserum to bovine herpesvirus 4 (BHV-4) stained the M40 virus specific antigen in infected cells. Experimental infection of goats with the M40 virus did not result in development of tumours. This virus is therefore considered to represent a "passenger" virus. A great similarity was found between restriction patterns of DNAs extracted from M40 virus and the strain 66-P-347, a reference strain of the BHV-4 group.     

Feline panleukopaenia virus and mink enteritis virus. A serological study.

The serological relationship of Danish feline panleukopaenia virus and mink enteritis virus and strains from Great Britain, USA, Germany and Canada was examined in neutralization tests using a direct immunofluorescence technique. Vaccine strains of the virus were used representing virus strains from the different countries. It was found that all Danish feline panleukopaenia virus strains and the mink enteritis strain belong to the same serotype and further that they are of similar antigenicity as feline panleukopaenia virus strains and mink enteritis strains isolated in other countries.     

Experimental vesicular stomatitis virus infection of swine: Extent of infection and immunological response

Swine, a natural host species for infection by vesicular stomatitis virus (VSV), were infected with VSV-New Jersey (VSV-NJ) serotype virus obtained from a recent field isolate. Tissues collected from the infected pigs were examined for the presence of infective virus, for viral antigens, and/or for viral nucleic acid. Infective virus could be recovered from tissues near the site of infection for as long as 6 days after the primary infection with VSV. However, no infective virus was recovered following hypothermia induced 11 weeks after infection, or following a secondary challenge with virus 22 weeks after initial infection. Immunofluorescence tests for viral antigens and nucleic acid hybridization assays failed to detect viral antigens or nucleic acids in tissues from which no infective virus could be recovered. Titers of serum-neutralizing antibody peaked 3–5 weeks after infection and then fell slightly until the secondary infection which caused a rapid anamnestic response. Peripheral blood mononuclear cells (PBM) tested 3, 5, 8 or 18 weeks after primary infection all produced readily detectable antigen-specific proliferative responses when cultured with VSV. Thus, although direct tests failed to demonstrate persistence of virus after infection, the humoral and cellular immune response remained elevated for months. Infective VSV was not required to stimulate the proliferative response since UV-inactivated VSV was immunogenic in these in vitro tests. Following primary infection, antigen-specific proliferative responses could be stimulated by several strains of VSV-NJ, but not by VSV-Indiana (VSV-Ind) serotype virus. Secondary infection had relatively little effect on the proliferative response to VSV-NJ strains, but it did cause the PBM to gain responsiveness to VSV-Ind.     

Cowpox virus isolate virulent in humans shows attenuated phenotype in mice

We have cultured Cowpox virus (CPXV) from skin lesion material of a human patient from Austria. Phylogenetic comparison of the HA-gene revealed a rather homogeneous cluster with other local isolates from recent years, the A36R-gene was mostly related to elephant derived strains from Germany. Despite causing disease in human, the isolate AT/Carinthia/788/07 surprisingly even at high titers showed a highly reduced virulence in BALB/c mice upon intranasal inoculation as compared to vaccinia virus. This contrasts earlier reports on other CPXV isolates. Using shotgun DNA sequencing several insertions and deletions were found in genes presumably involved in host range, immune regulation as well as established virulence factors. These preliminary data could be an indication that CPXV strains with proven pathogenicity for humans may have reduced virulence in mice and vice versa. Additionally strains with a reduced virulence may have an advantage in persisting in less dense rodent populations.     

鸡痘鹌鹑化弱毒在犊牛睾丸细胞上的适应性试验

鸡痘鹌鹑化弱毒在犊牛睾丸细胞上培养传至7代,各代次均产生程度不同的致细胞病变作用,经测定至第6代病毒收获液在犊牛睾丸细胞(BTC)上的致病变作用(CPE)最明显,细胞产毒效价最高可达105.5 EID50/0.2 mL.另外,对7个代次的病毒液进行的安全试验结果表明,该病毒收获液安全有效,符合制苗标准.此试验为用BTC生产鸡痘活疫苗提供了重要依据.     

3种藏药复方抗ORFV作用研究

将岩青、轮叶棘豆等3种藏药材按一定比例配成复方,采用乙醇提取法进行提取,将浓缩药液用细胞维持液配制成1g／L的药液,并根据对兔肾细睁（PRK）安全浓度的测定结果,稀释成不同梯度浓度,分别与脓疱皮炎病毒（ORFV）加入到已培养24h的单层PRK中,通过M1TI’法测定OD值,分析其对ORFV病毒增殖的影响。经回归分析,复方药液在7．81—62·5mg／mL浓度范围内,药物浓度与活细胞存在线性关系,对病毒增势有显著的抑制作用（P〈0·05）。表明藏药材复方药液具有抗脓疱皮炎病毒感染的作用。     

肾型鸡传染性支气管炎病毒新疆株的生物学特性研究

通过致病力试验和免疫原性检测对肾型鸡传染性支气管炎病毒新疆株(IBVXJ-2株)进行生物学特性的研究。致病力检测结果证实,IBVXJ-2株可以致SPF鸡胚卷缩、僵化,EID50达10-8.3/0.1 mL;鸡胚肾细胞在接毒后96 h出现皱缩、脱落等明显的细胞病变;对20日龄非免疫鸡攻毒后第2天,试验鸡出现甩鼻、伸颈张口呼吸等症状。将IBVXJ-2株抗原经乳化后接种1月龄非免疫鸡,在接种后22 d和44 d用ELISA和HI检测到了IBV特异性抗体。     

Replication of parainfluenza type-3 virus and bovine respiratory syncytial virus in isolated bovine type-II alveolar epithelial cells.

The objectives of our research were to determine whether bovine pulmonary type-II alveolar epithelial cells could be isolated from bovine lung and maintained in tissue culture and to determine whether isolated bovine type-II alveolar epithelial cells would support productive viral replication of bovine parainfluenza type-3 virus and bovine respiratory syncytial virus. Type-II alveolar epithelial cells were isolated from lungs of 4- to 7-day-old male Holstein calves by enzymatic dissociation of pulmonary tissue with trypsin and by separation of cells with the use of filtration and centrifugation on continuous Percoll gradients. Cells were further separated by panning on IgG-coated plastic plates and by lectin binding. Isolated type-II alveolar cells were maintained on basement membrane-coated tissue cultured plates. In culture, type-II cells formed alveolar structures and maintained other cytologic features of type-II cells, including osmiophilic lamellar inclusions. Cell cultures were inoculated with and supported productive replication of bovine parainfluenza type-3 virus and bovine respiratory syncytial virus. This was determined by recovery of infectious viruses from inoculated cell cultures and by identification of viral structures in type-II alveolar epithelial cells by transmission electron microscopy.     

Serologic survey in a colony of captive common marmosets (Callithrix jacchus) after infection with herpes simplex type 1-like virus.

An outbreak of herpesvirus caused the death of four of five common marmosets (Callithrix jacchus) in a private colony. Gross lesions included acute ulcerative gingivitis, glossitis, and enlarged mandibular lymph nodes. Histologically, all fatal cases showed meningoencephalitis and eosinophilia with intranuclear inclusion bodies in neurons and glial cells. A herpes simplex-like virus was cultured from the brain and was identified as herpes simplex type 1 virus or a closely related virus by immunofluorescence. Serologic testing (complement fixation test) indicated that the surviving adult female was serologically positive for more than 4 yr and that the offspring she produced was seronegative. The most likely source of the outbreak was the owner who mouth fed hand-raised offspring.     

Border disease in sheep caused by transmission of virus from cattle persistently infected with bovine virus diarrhoea virus.

Two outbreaks of border disease occurred on farms with sheep flocks and breeding cattle. The infection of the pregnant sheep was probably caused by transmission of virus from calves persistently infected with non-cytopathic bovine virus diarrhoea virus (BVDV) which were kept in close confinement with the ewes during mid-pregnancy. Border disease was also induced experimentally in eight lambs by exposing their dams at 38 to 78 days of gestation to a heifer persistently infected with BVDV. Both the natural and the experimental infections were characterised by typical signs such as 'hairy-shaker' lambs and high lamb mortality. The diagnosis was confirmed by virus isolations from live-born lambs, seroconversion and pathology. The study supports the assertion that cattle persistently infected with BVDV and in close contact with pregnant sheep, are an important source of strains of virus capable of causing border disease.     

Chronic leukopenia associated with feline immunodeficiency virus infection in a cat

Leukopenia attributable to lymphopenia and neutropenia was detected over a 28-week period in a 12-year-old domestic cat infected with feline immunodeficiency virus (FIV). Mild normocytic, normochronic anemia also was evident. Platelet counts were normal, and serum biochemical values were unremarkable. Antibodies to FIV were detected in serum by use of immunofluorescence and immunoblot electrophoresis assays. Cytologic evaluation of bone marrow aspirates revealed normal cellular morphologic features, maturation, and myeloid-to-erythroid ratio. Normal marrow cellularity was determined histologically. There was, however, a significant (P less than 0.01) inhibition of colony-forming unit granulocyte/macrophage-derived progenitors when marrow cells were cultured in the presence of autologous serum, compared with that when marrow cells were cultured in the presence of serum obtained from clinically normal cats, thus suggesting the presence of a humoral inhibitory substance directed specifically at the granulocyte/macrophage lineage. These cell culture results were consistent with those reported for human beings with acquired immunodeficiency syndrome and neutropenia. Thus, FIV infection may be an excellent animal model in which to study human immunodeficiency virus and should be considered in the differential diagnosis of cats with chronic leukopenia.