Generation of recombinant bovine interferon tau in the human embryonic kidney cell line and its biological activity

The objective of this study was to generate recombinant bovine interferon tau (rbIFNT) in mammalian hosts. The complementary DNA encoding bovine IFNT2 was cloned for the construction of pRcRSV‐bIFNT2 expression vector. The expression vector was transfected to 293 cells. Transfected cells harboring expression vector were selected with G418. Highly expressing clonal line was adapted to serum‐free suspension culture in a spinner flask. The recombinant protein had 24 kDa apparent molecular mass, suggesting being expressed as a glycoprotein, and was purified from serum‐free conditioned medium by the combination of Diethylaminoethanol Sepharose ion exchange and Sephacryl S‐200 HR gel filtration. A total of 7.3 mg rbIFNT was obtained from 13.5 L conditioned medium. Generated rbIFNT was biologically active in terms of antiviral activity measured by the plaque inhibition assay with Madin‐Darby bovine kidney cells and the vesicular stomatitis virus. The recombinant protein was also utilized for immunization to raise antibodies in the rabbit. The generated antibody was capable of use in both Western blotting and the binding assay. The results in the present study suggest that a certain amount of rbIFNT is raised in mammalian hosts by using conventional plasmid vector and its antibody provides useful tools for studies in the biology of bovine IFNT.     

Role of membrane phospholipids and glycolipids in cell-to-cell fusion by VSV

To identify membrane components of CER cells interacting with vesicular stomatitis virus (VSV) during fusion at acidic pH (fusion from without, FFWO) two different approaches have been used, i.e. (i) treating the whole cells with enzymes and (ii) testing the ability of isolated membrane molecules to interfere with FFWO. Phospholipase A₂ and C digestion of cells greatly reduced syncytia formation, pointing towards the involvement of lipid structures as target sites for VSV. Cell susceptibility to FFWO was also reduced after neuraminidase, β-galactosidase or periodate treatment, suggesting that carbohydrate residues may participate in a complex receptor structure required for virus fusion. When membrane molecules were examined separately for their ability to inhibit viral FFWO, phosphatidylserine, phosphatidylinositol, sphingomyelin, cholesterol and GM3 ganglioside were found to be active, confirming the role of membrane lipid moiety in the cell surface structures involved in the early phases of VSV infection.     

Evaluation of cytotoxicity and antiviral activity of recombinant human interferon alfa-2a and recombinant human interferon alfa-B/D hybrid against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, and vesicular stomatitis virus in vitro

OBJECTIVE: To evaluate cytotoxicity and antiviral activity of recombinant human interferon alfa-2a and recombinant human interferon alfa-B/D hybrid against cytopathic and noncytopathic bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), and vesicular stomatitis virus (VSV) in vitro. SAMPLE POPULATION: Primary bovine testicular cells and Mardin Darby bovine kidney cells. PROCEDURES: To evaluate cytotoxicity, cells were added to serial dilutions of each interferon. To evaluate antiviral activity of each interferon, interferons were serially diluted 1:10, and tissue culture cells were added; virus was then added at 3 time points. Prevention of viral infection by interferon was defined as failure to induce cytopathologic effect for VSV, IBRV, and cytopathic BVDV and failure to detect virus immunohistochemically for cytopathic and noncytopathic BVDV. RESULTS: No evidence of cytotoxicity in either cell line was detected after incubation with interferon alfa-2a or interferon alfa-B/D. However, reduced growth rates of tissue culture cells were detected for each interferon when undiluted interferon was tested. Comparable and profound antiviral activities against cytopathic and noncytopathic BVDV were evident for each interferon. Interferon alfa-2a and interferon a-B/D had comparable antiviral activities against VSV. Neither interferon had antiviral activity against IBRV. CONCLUSIONS AND CLINICAL RELEVANCE: The safety and marked in vitro antiviral activity against noncytopathic BVDV, cytopathic BVDV, and VSV suggest that interferons alfa-2a and alfa-B/D may be useful for treatment of natural disease after infection with these viruses.     

Glycoprotein M of bovine herpesvirus 1 (BHV-1) is nonessential for replication in cell culture and is involved in inhibition of bovine respiratory syncytial virus F protein induced syncytium formation in recombinant BHV-1 infected cells

Cell cultures infected with BHV-1/F(syn), a recombinant bovine herpesvirus 1 (BHV-1) which expresses a synthetic open reading frame encoding the fusion (F) protein of the bovine respiratory syncytial virus (BRSV), showed a cytopathic effect (CPE) indistinguishable from that induced by wildtype BHV-1 although transient transfection experiments demonstrated that expression of the F protein leads to formation of large syncytia. Since it has been shown that glycoprotein M (gM) of pseudorabies virus inhibits BRSV F-induced syncytium formation in transient plasmid transfection experiments [Pseudorbies virus glycoprotein M inhibits membrane fusion. J. Virol. 74 (2000) 6760], the gM ORF of wtBHV-1 and BHV-1/F(syn) was interrupted. Infection of cell cultures with the resulting gM(-) mutant of BHV-1/F(syn) led to formation of syncytia, whereas the CPE in gM(-)BHV-1 infected cells was comparable to the CPE in wtBHV-1 infected cultures. Our results demonstrate that gM is not essential for BHV-1 replication in cell culture and that gM is involved in inhibition of the cell fusion activity of the BHV-1 expressed BRSV F protein.     

The interferon sensitivity of selected porcine viruses.

The objective of this study was to compare the sensitivity of 11 porcine viruses to the antiviral effects of porcine interferon-alpha in serum from piglets which had been infected 19 h previously with transmissible gastroenteritis virus, and of porcine interferon-beta prepared in PK-15 cells by induction with polyinosinic:polycytidylic acid, in yield reduction assays in pig kidney cells which were treated with interferon before virus challenge, and both before and after virus challenge. The most sensitive virus to both types of interferon was vesicular stomatitis. A porcine isolate of bovine herpesvirus type 1, hemagglutinating encephalomyelitis virus and porcine enterovirus types 1 and 2 were also highly sensitive to interferon-alpha. There was little reduction in the yield of porcine parvovirus or porcine rotavirus, while swinepox, swine influenza and transmissible gastroenteritis viruses were intermediate in their sensitivity to interferon-alpha. In addition to vesicular stomatitis virus, porcine adenovirus type 3, swine influenza, hemagglutinating encephalomyelitis and porcine rotavirus were highly sensitive to interferon-beta, while swinepox, bovine herpesvirus type 1, porcine parvovirus, transmissible gastroenteritis and porcine enteroviruses were less sensitive than the above viruses to interferon-beta, although all showed significant reductions in virus yield.     

Cellular response to rabies virus infection

The effects of rabies virus on host cells were studied and compared to those obtained with another rhabdovirus, vesicular stomatitis virus [J. Virol. 34, 777-781 (1980)]. We show here: (1) that rabies infection has no effect on cell morphology, while infection with vesicular stomatitis virus caused cell retraction. Thus, only vesicular stomatitis virus induced a depolymerization of the microfilaments; and (2) that microtubules and microfilaments do not play a major role in rabies virus production, as it is suggested by results obtained with several effectors (colcemid, colchicine and cytochalasin-B) which directly or indirectly affect cytoskeleton organization. The same properties were observed with directly or indirectly affect cytoskeleton organization. The same properties were observed with vesicular stomatitis virus. Furthermore, the use of cytochalasin-B shows that an inhibition of glycosylation of the virion spike protein occurs only in rabies infected cells. As vesicular stomatitis viral glycosylation is normal in cytochalasin-B treated cells, results obtained indicate that two types of interactions can occur between a virion and the host-cell depending on the rhabdovirus type.     

Effect of human interferon on vesicular stomatitis virus released from bovine embryonic kidney cells

Human lymphoblastoid interferon (IFN) had an antiviral activity in bovine embryonic kidney cells that resulted in the release of vesicular stomatitis virus (VSV) particles with decreased infectivity. The inhibition was dose dependent and the cells were highly sensitive to human IFN. Examination of the proteins of VSV released from bovine cells after IFN treatment showed a reduction in the glycoprotein. Electron microscopic studies revealed a large number of VSV particles with characteristic spike-like surface projections released from nontreated cells. There was a reduction in the number of mature virions produced in IFN-treated cells and the virions lacked the characteristic surface projections.     

Preliminary characterization of a serum viral inhibitor in goats

A serum viral inhibitor (SVI) was isolated from goats and partially characterized. The inhibitor prevented the cytopathic effects of vesicular stomatitis virus, encephalomyocarditis virus, and a caprine herpesvirus, indicating broad antiviral activity. The SVI was distinct from interferon because SVI did not induce an antiviral state in cells (ie, lack of protection of SVI-treated cells from virus challenge). The SVI had activity on heterologous cells, including human, bovine, and ovine cells. The lack of antiviral activity in mouse cells indicated that SVI was not an antibody. Like fibroblast interferon, however, SVI was sensitive to trypsin, was acid stable at pH 2 and 4 C for 4 days, was heat stable at 56 C for 1 hour, and could not be sedimented by centrifugation at 100,000 X g for 4 hours.     

Microarray-based detection of viruses causing vesicular or vesicular-like lesions in livestock animals

Definitive diagnosis of vesicular or vesicular-like lesions in livestock animals presents challenges both for veterinary clinicians and diagnostic laboratories. It is often impossible to diagnose the causative disease agent on a clinical basis alone and difficult to collect ample vesicular epithelium samples. Due to restrictions of time and sample size, once laboratory tests have ruled out foot-and-mouth disease, vesicular stomatitis and swine vesicular disease a definitive diagnosis may remain elusive. With the ability to test a small quantity of sample for a large number of pathogens simultaneously, DNA microarrays represent a potential solution to this problem. This study describes the application of a long oligonucleotide microarray assay to the identification of viruses known to cause vesicular or vesicular-like lesions in livestock animals. Eighteen virus isolates from cell culture were successfully identified to genus level, including representatives of each foot-and-mouth disease virus serotype, two species of vesicular stomatitis virus (VSV), swine vesicular disease virus, vesicular exanthema of swine virus (VESV), bovine herpesvirus 1, orf virus, pseudocowpox virus, bluetongue virus serotype 1 and bovine viral diarrhoea virus 1. VSV and VESV were also identified in vesicular epithelium samples, with varying levels of sensitivity. The results indicate that with further development this microarray assay could be a valuable tool for the diagnosis of vesicular and vesicular-like diseases.     

In vitro interferon production by bovine tissues: effects of hydrocortisone.

The effect of hydrocortisone on bovine interferon production in vitro was studied. Infectious bovine rhinotracheitis virus was used as an inducer. Interferon was assayed by the plaque-reduction method in bovine fetal kidney cultures, using vesicular stomatitis virus as challenge virus. Hydrocortisone decreased interferon production in bovine fetal spleen and peripheral blood leukocyte cultures. Hydrocortisone did not decrease interferon production by bovine alveolar macrophages, in 1 experiment. Properties of viral inhibitors were those of interferon.     

Evaluation of age-related effects on the antiviral activity of interferon and induction of 2-5A synthetase in testicular cell cultures derived from swine of various ages

The antiviral activity of recombinant DNA-derived bovine alpha 1(-1) interferon on an established swine testicular cell line and primary testicular cell cultures derived from swine of various ages (2 days, 3 weeks, and 5 weeks) was determined. Bovine interferon induced a dose-dependent increase in 2-5A synthetase in testicular cells, regardless of the source of the cells. Furthermore, interferon inhibited replication of vesicular stomatitis virus to an equivalent extent in all testicular cell cultures. The results indicate that 2-5A synthetase is a reliable marker of interferon activity in swine testicular cell cultures and that the induction of 2-5A synthetase and antiviral effects of recombinant bovine interferon in primary testicular cell cultures are not dependent on the age of the donor animal.     

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to vesicular stomatitis virus in cattle in an enzootic region of Mexico.

An ELISA was compared with the plaque-reduction serum neutralization (PRSN) test, for detection of vesicular stomatitis virus (VSV) antibodies in cattle in a vesicular stomatitis enzootic region of Mexico. A total of 325 bovine serum samples were screened for VSV antibodies. The PRSN test was performed, using Vero cells. The ELISA contained gradient-purified VSV Indiana (Lab strain) and VSV New Jersey (Hazelhurst) as the antigens. Regression analysis and weighted kappa statistic were used to estimate measures of agreement between the 2 assays for detection of VSV antibodies. The ELISA method proved useful for serodiagnosis of vesicular stomatitis. The ELISA and PRSN test results were highly correlated for detection of VSV antibodies.     

水泡性口炎病毒抗原决定簇单克隆抗体研究

用RT-PCR的方法扩增水泡性口炎病毒G蛋白抗原决定簇部分基因片段,构建克隆质粒pMD18-T-G_(660)和表达质粒pET-28a-G_(660),转化宿主菌BL21(DE3),经IPTG诱导后得到高效表达,表达蛋白质的分子量为30 Ku,占总蛋白的20.745%。切胶回收蛋白测定蛋白含量,作为免疫BALB/c小鼠的免疫原,免疫BALB/c小鼠4次,按单克隆抗体制备技术,获得3株稳定分泌抗表达的G蛋白的单克隆抗体L细胞株,抗体类型鉴定属于IgG亚类。     

Interferon production by bovine tracheal organ cultures infected with bovid herpesvirus 1 strains.

Studies have been made of antiviral inhibitors produced by bovine tracheal organ cultures inoculated with strains of bovid herpesvirus 1. The inhibitors, which had properties of interferon, were assayed by a plaque-reduction method in bovine turbinate cell cultures with vesicular stomatitis virus as challenge virus. Each of the four strains of bovid herpesvirus 1 studied induced interferon in bovine tracheal organ cultures.     

Effect of strain and serotype of vesicular stomatitis virus on viral shedding, vesicular lesion development, and contact transmission in pigs

OBJECTIVE: To determine whether pigs can be infected with strains of vesicular stomatitis virus New Jersey (VSV-NJ) and vesicular stomatitis virus Indiana (VSV-I) isolated during recent vesicular stomatitis outbreaks that primarily involved horses in the western United States and determine the potential for these viruses to be transmitted by contact. ANIMALS: 128 pigs. PROCEDURE: Pigs were challenged with VSV-NJ or VSV-I from the 1995 and 1997 outbreaks of vesicular stomatitis in the western United States, respectively, or with VSV-NJ (OS) associated with vesicular stomatitis in feral pigs on Ossabaw Island, Ga. Pigs (3/group) were inoculated with each virus via 3 routes and evaluated for viral shedding, seroconversion, and the development of vesicular lesions. In another experiment, the potential for contact transmission of each virus from experimentally infected to na?ve pigs was evaluated. RESULTS: Infection of pigs was achieved for all 3 viruses as determined by virus isolation and detection of seroconversion. In inoculated pigs, all 3 viruses were isolated from multiple swab samples at concentrations sufficient to infect other pigs. However, compared with results obtained with the 2 VSV-NJ strains, viral titers associated with VSV-I were low and the duration of virus shedding was reduced. Results from the contact transmission trials were consistent with these results; virus transmission was detected most frequently with the VSV-NJ strains. CONCLUSIONS AND CLINICAL RELEVANCE: Pigs can be infected with VSV-NJ and VSV-I. Differences in the extent of viral shedding and potential for contact transmission were apparent between serotypes but not between the VSV-NJ strains investigated.     

Trypsin treatment of bovine ova after in vitro exposure to vesicular stomatitis virus

Preimplantation bovine ova were exposed in vitro to vesicular stomatitis virus, Indiana serotype, to document adherence of the virus to the zona pellucida. To determine the efficacy of this treatment, some of the ova were treated with trypsin after exposure to the virus. Vesicular stomatitis virus was isolated from 5 of 10 groups of zona pellucida-intact ova after 12 sequential washes without trypsin treatment. Vesicular stomatitis virus was also isolated from 4 of 11 groups of zona pellucida-intact ova after trypsin treatment.     

Biology of the prolactin family in bovine placenta. II. Bovine prolactin-related proteins: Their expression, structure and proposed roles

The placenta produces various peptides and steroid hormones that regulate placental function and fetal growth. Prolactin‐related proteins are peptides that are produced by the placenta and belong to the growth hormone/prolactin family, and have structural similarity to prolactin and placental lactogen. Although several prolactin‐related protein genes have been detected in bovine placenta, their expression profiles and functions are not clear. The main difficulties in examining their biological function is the similarity between their genes and the lack of information about their proteins. Recently, molecular biology methods have been used to detect some new bovine prolactin‐related proteins, and elucidate their biological functions. This review focuses on the structures, expression profiles and conceivable functions of prolactin‐related proteins in bovine placenta. With respect to their expression profiles, bovine prolactin‐related proteins fall into four groups: (i) those expressed around the implantation period; (ii) those that reach peak expression in the middle of gestation; (iii) those that increase with the progress of gestation, reaching a peak in late gestation; and (iv) those that reach a plateau in early gestation and are maintained at that level throughout gestation. Data indicate that bovine prolactin‐related proteins have different biological roles in different periods of gestation. In situ monitoring suggests that bovine prolactin‐related protein‐I has a role in the attachment of trophoblast cells to endometrium during the early implantation period.     

A prospective study of vesicular stomatitis in cattle in an enzootic region of Mexico

A prospective study of vesicular stomatitis was conducted in two bovine herds in southeastern Mexico. In July 1987, an initial serological screening showed that 64% and 87% of the 654 cattle tested negatively to vesicular stomatitis New Jersey and Indiana antibodies, respectively, using the plaque-reduction serum neutralization test. Most seropositive animals were at least 24 months of age. Based on the initial serological screening, cohorts of seronegative and seropositive cattle were monitered (January–December 1988) for the prevalence of vesicular stomatitis virus (VSV) antibodies using an enzyme-linked immunosorbent assay (ELISA). The serological results, using ELISA, indicated that no VSV activity occured in the two study herds. The seronegative cohort of cattle did not yield a positive seroconversion pattern to either VSV Indiana or New Jersey. The seropositive cohort showed a variable antibody response pattern against the VSV. There were no clinical cases of vesicular stomatitis (VS) in the two herds. The data from the national surveillance program for vesicular diseases suggested that 1988 was a year of low VSV infection incidence in southeastern Mexico.     

Production of interferon by bovine peripheral blood monocytes infected with bovid herpesvirus 2

The production of interferon by bovine peripheral blood leukocytes infected with bovid herpesvirus 2 (BHV-2) was investigated in preparation for studying mechanisms of resistance to BHV-2. It was found that bovine peripheral blood monocytes produced high levels of interferon in response to BHV-2 inoculated at a multiplicity of 1. Virus-induced interferon was not stable at pH 2, was destroyed at 56 degrees C or by incubation with trypsin and was active against both vesicular stomatitis virus and BHV-2. Interferon of high specific activity was produced by incubating monocytes for 5 h with BHV-2 in serum-containing medium, replacing the inoculum with serum-free medium for an additional 16 h, and concentrating the serum-free medium by dialysis against dry polyethylene glycol. Interferon concentrations of 40,000 units per mg of protein were readily attained.     

Bovine respiratory syncytial virus-specific monoclonal antibodies

Five hybridomas were produced which secreted monoclonal antibodies to bovine respiratory syncytial virus (BRSV). Two antibodies (8G12, 15C7) neutralized the virus and inhibited syncytia formation in vitro. These monoclonal antibodies also stained, by indirect fluorescent assay, an external envelope protein of living virus-infected cells, and recognized the 48k subunit of the viral fusion protein by Western blot analysis of bovine respiratory syncytial virus-infected cell lysates. Three other monoclonals (6A12, 14D3, 14E3) stained, by indirect fluorescent assay, acetone-fixed virus-infected cells but not living cells. Three hybridomas (6A12, 8G12, 15C7) secreted monoclonal antibody of isotype IgG1, k; two hybridomas secreted monoclonal antibody of isotype IgG2a, k. This apparently is the first report of monoclonal antibodies specific for BRSV glycoproteins.