Restriction endonuclease analysis of a porcine isolate of bovine herpesvirus type 1.

Deoxyribonucleic acid (DNA) was extracted from bovine herpesvirus type 1 (BHV-1) isolated from a stillborn porcine fetus, from the Cooper reference strain of BHV-1, and from an Ontario bovine respiratory isolate. Each DNA was digested with the restriction endonucleases HindIII, EcoRI, HpaI and BamHI. Except for very minor differences in the patterns produced after digestion with EcoRI and HindIII, the DNA of the porcine isolate reacted in a similar manner to the bovine viruses, and it was concluded that the porcine virus is genetically similar to bovine isolates of BHV-1.     

Antimicrobial resistance in streptococcal species isolated from bovine mammary glands

Streptococcal species isolated from dairy cows with clinical mastitis were obtained from mastitis research workers in Florida, Louisiana, New York, Vermont, Washington, and West Virginia. Seventy-one streptococcal isolates were tested, including 39 strains of Streptococcus agalactiae, 21 strains of S dysgalactiae, and 11 strains of S uberis. The minimal inhibitory concentration of erythromycin, lincomycin, oxytetracycline, penicillin, spectinomycin, streptomycin, and tetracycline was determined for each isolate. Differences were not detected among strains with respect to geographic origin. None of the strains was resistant to penicillin. Lincomycin was the next most effective antimicrobial, with only 2 resistant strains of each streptococcal species. There were no differences among the streptococcal species with respect to resistance to either penicillin or lincomycin. Streptococcus uberis was more likely to be resistant to erythromycin than were S agalactiae and S dysgalactiae (P less than 0.02). Streptococcus agalactiae and S uberis had similar distributions for resistance to oxytetracycline, tetracycline, spectinomycin, and streptomycin. Strains of S dysgalactiae were more likely to have intermediate resistance to oxytetracycline and streptomycin than were strains of S agalactiae and S uberis, which were highly resistant to oxytetracycline and streptomycin (P less than 0.001). Differences were not detected among the streptococcal species with respect to resistance to spectinomycin. Resistance to multiple antimicrobials was observed in all streptococcal species tested. Although S dysgalactiae appeared to have a greater percentage of strains (73%) that were resistant to multiple antimicrobials than did S agalactiae (31%) or S uberis (45%), differences were not statistically significant.     

Differentiation of vaccine and field strains of bovine herpesvirus type 1 by restriction endonuclease analysis

Bovine herpesvirus type 1 (BHV-1) DNA molecules obtained from a limited number of infected cells were cleaved with a variety of restriction endonucleases. By use of selected DNA fragments in Bgl 1, Pst 1, Pvu 1 and Pvu 11 restriction patterns, one reference, two vaccine and three wild-type strains of BHV-1 were distinguished from one another. The simplified DNA fingerprinting method described here should be most useful, not only to control the genetic stability of BHV-1 vaccines during production, but also to differentiate the vaccine strains from other isolates in clinical cases.     

A bovine herpesvirus isolated from sheep.

A viral agent was isolated from the trachea of a lamb that was suffering from a respiratory disorder. The physical and chemical properties of the isolates are characteristic of the herpesvirus group. It contains DNA in its virion, is ether sensitive, acid labile at pH 3.0 and heat labile at 56 degrees C after five minutes. The cytopathology observed provided further evidence of a herpesvirus isolate. The neutralization of the infectivity of the isolate with antiserum to bovine herpesvirus 1 is evidence that it should be considered an isolate of bovine herpesvirus 1. It is concluded that this is a report of a bovine herpesvirus infection in sheep.     

Restriction site mapping of bovine adenovirus type 1

Physical maps of the DNA of bovine adenovirus (BAV) type 1 were established using ApaI, BamHI, BstEII, EcoRI and KpnI enzymes. The size of the viral genome was found to be around 35,000 base pairs. The orientation of the maps was determined by hybridizing Southern blots of BAV-1 DNA with the cloned hexon gene region of BAV-3.     

Hemagglutinating activity associated with bovine herpesvirus type 1

Using C57BL/HPB mouse erythrocytes, hemagglutination has been observed with the Los Angeles and Colorado-1 strains of bovine herpesvirus type 1 and with 12 other Canadian field isolates as well. The specificity of the hemagglutination observed with the viral strains has been confirmed by a hemagglutination-inhibition assay.     

Molecular cloning of DNA from a bovine herpesvirus 1 strain isolated in Hungary

Molecular cloning of the HindIII fragments of bovine herpesvirus 1 (BHV-1) strain HB144, isolated from infectious bovine rhinotracheitis (IBR) in Hungary, and of an infectious pustular vulvovaginitis (IPV) reference strain (K22) is reported. So far 52% of the IBR viral genome and 28% of the IPV viral genome have been cloned. The analysis of differences between the strains is currently in progress.     

Restriction endonuclease analysis of various strains of Mycobacterium paratuberculosis isolated from cattle

Deoxyribonucleic acid (DNA) preparations from 3 reference strains of Mycobacterium paratuberculosis and from 23 isolates of M paratuberculosis obtained from cattle in New Zealand were characterized by restriction endonuclease analysis, using the enzymes BstE II, Pvu II, and Bcl I. Patterns of DNA fragments for strain 18 (one of the reference strains) differed markedly from patterns of other strains, indicating genetic differences between strain 18 and the other strains of M paratuberculosis evaluated. The other 2 reference strains (TMC 1613 and Weybridge strain 316) and all but 1 of the isolates from cattle had identical patterns with the 3 enzymes. These 2 reference strains differed from each other in their dependence on exogenous mycobactin, but this was not reflected in their restriction patterns. The single variant isolate from cattle had patterns identical to those of the other isolates, using Pvu II and Bcl I, and had only 1 fragment line difference with BstE II. Although close genetic homogeneity of cattle strains of M paratuberculosis prevented development of a typing system on the basis of restriction endonuclease analysis, the results provided a basis for genomic comparison with other closely related organisms.     

缺失gE基因的牛Ⅰ型疱疹病毒构建及其体外增殖特性比较

为构建gE基因缺失的牛Ⅰ型疱疹病毒(BHV1),本研究在BHV1全基因组感染性细菌人工染色体克隆pBHV1-ZJ的基础上,利用Red E/T重组技术,分别构建了gE基因缺失、gE和gN双基因缺失的pBHV1突变体,通过转染获得了重组病毒rBHV1-△gE,而双基因缺失突变体pBHV1-△gN-△gE则未能观察到细胞病变。病毒噬斑大小和生长曲线试验表明,在感染细胞内和培养基中,rBHV1-△gE滴度与亲本毒株BHV1无明显差异,而rBHV1-△gN显著降低;重组病毒rBHV1-△gE和前期构建的rBHV1-△gN形成的噬斑均显著小于亲本病毒,其大小分别为亲本病毒的18%和64%。rBHV1-△gE和rBHV1-△gN的构建将有助于阐明gE与gN协同作用机制和为研制BHV1新型基因标记疫苗奠定基础。     

Restriction endonuclease analysis of canine herpesviruses isolated in Japan

Eighteen canine herpesvirus (CHV) isolates from Japan and two reference strains were compared by restriction endonuclease analysis technique using total DNA extracts from cells infected with the viruses. In order to select the suitable restriction endonucleases for differentiation of CHV isolates, ten enzymes were used and three of them, HindIII, XbaI, and PvuII, were found to be useful for strain differentiation. With these enzymes, CHV isolates from unrelated individuals were readily differentiated from each other. In contrast, all the isolates derived from the same litter were not distinguishable on the basis of restriction cleavage patterns. However, slight mobility shifts were observed among the isolates from the same litter or the same individual. The results showed that this method provides a powerful tool for epidemiological surveys of CHV infection.     

Protein display by bovine herpesvirus type 1 glycoprotein B

Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1), a major component of the viral envelope, is essential for membrane fusion during entry and cell-to-cell spread. It is cleaved in the trans-Golgi network by the proprotein convertase furin. Integration of the open reading frame (ORF) encoding a mutated gB with a second furin cleavage site and mature boIFN-α as intervening peptide between the amino-terminal (NH₂) and carboxy-terminal (COOH) gB subunits yielded recombinant BHV-1/gB2FuIFN-α which, unexpectedly, express gB with an enlarged NH₂-subunit of 90 kDa. Here we show that boIFN-α-specific antibodies bind to the 90 kDa gB subunit and efficiently neutralize BHV-1/gB2FuIN-α infectivity. We also show that inactivated BHV-1/gB2FuIN-α virions induce an antiviral state in cells incubated with UV-inactivated particles. These results demonstrate that the 90 kDa protein is a NH₂-subunit/boIFN-α fusion protein whose boIFN-α domain is biologically active. To verify that BHV-1 gB is suitable for the display of (glyco)proteins on the surface of virions we constructed BHV-1 recombinants expressing within gB the first 273 amino acids of the NH₂-subunit (HA1) of avian influenza haemagglutinin, either flanked by two furin cleavage sites or with only one cleavage site between a gB/NH₂_HA1 fusion protein and the COOH subunit. The resulting recombinant BHV-1/gB2FuHA1 expressed gB from which 55 kDa HA1 was excised and secreted. In contrast, gB from BHV-1/gB_NH₂HA1 infected cells retained HA1 as fusion protein with the NH₂-subunit. Immunoblotting and neutralization analyses revealed that HA1 is incorporated into the envelope BHV-1/gB/NH₂_HA1 particles and exposed to the exterior of virions. Thus, this novel approach enables display of polypeptides and (glyco)proteins of at least 273 amino acids on viral particles which is of particular interest for development of novel diagnostics and vaccines as well as for, e.g. gene therapy applications especially when biologically active ligands need to be presented.     

Restriction endonuclease analysis of BHV-1 and BHV-5 strains isolated in argentina

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.     

Effect of genistein on replication of bovine herpesvirus type 1

OBJECTIVE: To study the antiviral activity of genistein, a soya isoflavone, on in vitro replication of bovine herpesvirus type 1 (BHV-1). SAMPLE POPULATION: Madin-Darby bovine kidney (MDBK) cells. PROCEDURE: Effects of genistein on the magnitude and kinetics of inhibition of BHV-1 phosphorylation of glycoprotein E (gE) and in vitro replication of BHV-1 in MDBK cells were evaluated. Antiviral activity of genistein was compared with 2 compounds, estradiol-17beta (EST) and tamoxifen (TAM), that have estrogenic and antiestrogenic activity, respectively. High-performance liquid chromatography (HPLC) was used to determine the concentration of genistein in medium from infected and uninfected MDBK cultures. RESULTS: Genistein reduced BHV-1, but not gE-deleted BHV-1 (BHV-1gEdelta3.1), replication by 90% at 18 hours after inoculation. This inhibition was not sustained through 24 hours after inoculation. The genistein concentration in media from MDBK cells was decreased by 40% during BHV-1 infection, compared with 16% for uninfected cells, at 24 hours after inoculation. Genistein inhibited gE phosphorylation and BHV-1 replication in a dose-dependent manner. Dosing with 25 microM genistein at 0 and 12 hours after inoculation of BHV-1 was optimal for decreasing BHV-1 replication. Estradiol-17beta EST and TAM did not affect BHV-1 replication. CONCLUSIONS AND CLINICAL RELEVANCE: The decrease in genistein concentration was a viral infection-dependent event. Genistein is an inhibitor of BHV-1 replication because of its ability to inhibit tyrosine kinase activity. A possible application may be for the control of BHV-1 infection in cattle by feeding soya products rich in genistein prior to or during periods of stress.     

Phagocytic and bactericidal properties of bovine macrophages from non-lactating mammary glands

Macrophages were isolated from the mammary glands of non-lactating (dry) cows and their ability to phagocytose and kill staphylococci in vitro assessed. Normal bovine serum enhanced the uptake of staphylococci and was required for optimal killing in the bactericidal test. Dry gland secretion interfered with uptake. Secretions taken progressively into the dry period became more inhibitory. The phagocytic ability of macrophages was significantly less than that of neutrophils present in the same gland preparation when tested in the presence of dry gland secretion. A marked variation in the antibacterial activity of macrophages from different cows was noted.     

Serological survey of bovine herpesvirus type 1 infection in China

To understand the nationwide seroprevalence of bovine herpesvirus type 1 (BoHV-1) infection of cows in China, 1344 sera of dairy cows from 29 provinces and 765 sera from 6 herds in Hubei province were collected with stratified random sampling. Another 483 sera from imported cows were included. The serum antibody was tested by BoHV-1 gG ELISA. The results demonstrated that the overall nationwide seroprevalence was 35.8% (481/1344), while the prevalence for individual province ranged from 12.1% to 77.8%. Although each province had positive samples, the prevalence was clustered in areas based on the cow population size. In Hubei Province, the overall seroprevalence was 22.2% (170/765) while the prevalence for individual farms varied greatly from 0.0% to 41.5%. The sera from imported cows had a moderate prevalence of 21.7% (105/483).     

Abortifacient property of bovine herpesvirus type 1 isolates that represent three subtypes determined by restriction endonuclease analysis of viral DNA.

Bovine herpesvirus type 1 (BHV-1) isolates are classified into 3 subtypes by use of restriction endonuclease analysis. Isolates from aborted fetuses have been either subtype 1 or 2a, whereas subtype 2b viruses have not been associated with abortion. We assessed the abortifacient property of isolates representing each of the 3 BHV-1 subtypes by IV inoculation of heifers with the virus 25 to 27 weeks after breeding. Three heifers were given Cooper (subtype 1) isolate, 3 heifers were given FI (subtype 2a) isolate, and 5 heifers were given K22 (subtype 2b) isolate. All heifers developed fever and viremia 2 to 5 days after inoculation. Heifers given Cooper or FI isolate aborted between 17 and 85 days after inoculation. The 5 heifers given K22 isolate delivered full-term calves. Placenta was obtained from 4 of the 5 heifers, and K22 virus was isolated from each placenta. Four calves had BHV-1 neutralizing antibody in precolostral serum, with titer ranging from 1:4 to 1:512.     

DNA restriction enzyme analysis of a bovine herpesvirus 1 strain isolated from encephalitis in Hungary

The DNA of a bovine herpesvirus 1 (BHV-1) strain isolated from calf encephalitis in Hungary was analysed with restriction enzymes. The cleavage pattern of the encephalitis strain Na/67 differed from those of all the other Hungarian BHV-1 isolates investigated so far. The EcoRI and HindIII cleavage patterns of virus strain Na/67 were found to be similar to the patterns of two other encephalitis strains (N569 and A663 from Australia and Argentina, respectively) characterized earlier. Strain Na/67 is the first isolate in Europe which showed the restriction enzyme pattern of BHV-1.3 previously supposed to be characteristic of encephalitis strains.     

Restriction endonuclease analysis of BHV-1 and BHV-5 strains isolated in Argentina.

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.