Double immunization strategy with a BoHV-4-vectorialized secreted chimeric peptide BVDV-E2/BoHV-1-gD.

A bovine herpesvirus 4 was isolated from the milk cell fraction of a healthy cow and his full genome cloned as a bacterial artificial chromosome. So cloned viral genome was used as a vector platform to deliver in vitro and in vivo an optimized secreted chimeric peptide obtained by the fusion of the bovine viral diarrhoea virus glycoprotein E2 ectodomain with the bovine herpesvirus 1 glycoprotein D ectodomain. Recombinant virus infected cells robustly expressed and secreted the chimeric peptide into the culture medium and inoculated animals with the recombinant virus successfully responded toward antigens, gE2 and gD. Thus, this work has implications for the development of safe and effective polyvalent vaccines.

Exploiting persistent infection for selection of bovine herpesvirus 4 recombinants.

Bovine herpesvirus 4 (BoHV-4) is a gamma-herpesvirus with no clear disease association, and due to its biological characteristics, has been suggested as a gene delivery vector. It was demonstrated previously that recombinant BoHV-4 carrying a neomycin-resistance gene was able to infect a human rhabdomyosarcoma cell line (RD-4), resulting in no detectable cytopathic effect (CPE) and allowing selection of G418-resistant persistently-infected cells containing circular episomal viral DNA [Donofrio, G., Cavirani, S., van Santen, V.L., 2000a. Establishment of a cell line persistently infected with recombinant BoHV-4. J. Gen. Virol. 81, 1807-1814.]. Those cells produce infectious virus and infection is predominantly non-permissive and non-cytopathic. Starting from these results, the ability of RD-4 cells to sustain persistent infection was combined with positive selection activity conferred by the neomycin-expression cassette insert, as an easier way to select recombinants of BoHV-4 following homologous recombination in permissive cells. A tool for selecting BoHV-4 recombinants was developed by drug positive selection.

Susceptibility of bovine mesenchymal stem cells to bovine herpesvirus 4.

Bovine herpesvirus 4 (BoHV-4) is a gamma herpesvirus with no clear disease association. Previous studies have demonstrated that macrophages can harbour persistent BoHV-4. Since mesenchymal stem cells in bone marrow regulate the differentiation and proliferation of adjacent haematopoietic precursors, such as macrophages, the interaction between BoHV-4 and mesenchymal stem cells was investigated. Primary bovine mesenchymal stem cells were highly permissive to support full replication of BoHV-4. This finding could be considered a new important step in studies on the potential pathogenesis related to BoHV-4.

Interaction of a green recombinant bovine herpesvirus 4 with in vitro-produced bovine embryos.

The objective of the present study was to assess whether bovine herpesvirus 4 (BHV-4) is able to infect in vitro-produced bovine embryos. A green recombinant BHV-4 (BHV-4EGFP deltaTK), obtained by insertion of an EGFP gene into the TK locus of BHV-4, was used. The presence of this marker protein made it possible easily to detect infected cells under physiological conditions, without harmful manipulation of the cells or the addition of exogenous substrates, so that the spread of the virus could be followed in real time. Zona pellucida intact (ZP-I) and zona pellucida open (ZP-O) blastocytes were exposed to 10(6) TCID50 viral particles and infection was monitored by fluorescent microscopy for 48 h. Expression of EGFP and degeneration of embryonic cells was observed in three of the 18 ZP-O embryos, but in none of the ZP-I embryos. It was concluded from this preliminary study that BHV-4 has only a low ability to infect in vitro-produced bovine embryos, depending on the absence of ZP, the amount of virus present and the stage of embryonic development. However, embryonic stem cells could be transduced by BHV-4EGFP deltaTK just after differentiation, as shown by expression of EGFP.

Association between Chlamydia psittaci seropositivity and abortion in Italian dairy cows.

Although the seroprevalence of Chlamydia psittaci is widespread in Italian dairy herds, its role in inducing genital disorders has not been elucidated. We therefore set up a case-control study to compare seroprevalence to C. psittaci in an aborted-cow population and in a randomly selected control group in the province of Parma (the Po Valley of northern Italy). The true seroprevalence (45%) in aborted cows was significantly higher than that in the control group (24%) (adjusted odds ratio=2.53).

Detection of bovine herpesvirus 4 (BoHV-4) DNA in the cell fraction of milk of dairy cattle with history of BoHV-4 infection.

We have demonstrated, by PCR and restriction enzyme analysis of the PCR product, the presence of bovine herpesvirus 4 (BoHV-4) DNA in the cell fraction of milk from dairy cattle with a history of BoHV-4 infection. We next evaluated the infectious nature of BoHV-4 DNA in those cells. Cocultivation of a BoHV-4-sensitive cell line with BoHV-4 DNA-positive milk cell samples produced cytopathic effects. The same result was obtained from frozen and thawed milk cell fraction coming from the cell milk fraction PCR-positive cows, ensuring that cells were killed and only infectious virus could be recovered after cocultivation with sensitive cells. This report shows that infectious BoHV-4 can be present in milk cells and that therefore nursing may be one of the transmission routes of BoHV-4.

Molecular typing of a BHV-4 (bovine herpesvirus 4) field isolate.

A new BHV-4 (bovine herpesvirus 4) isolated from a case of bovine interdigital dermatitis was characterized by PCR and restriction enzyme analysis. To determine whether the new isolate (PR/1) belonged to BHV-4, DNA from infected cells was specifically amplified by PCR. We used a set of primers spanning a large 2.571 kb conserved region of the BHV-4 genome, including the 3' end of ORF1 (homologous to the EBV BVRF1 gene), ORF2 (homologous to the EBV BXRF1 gene), ORF3 (TK gene) and ORF4 (gH gene) 5' end, respectively. The identity of the amplified product was confirmed by HindIII restriction enzyme digestion and Southern hybridization. No product was observed from the DNA of other bovine herpesviruses tested. The restriction patterns of the PR/ 1 genome compared to DN 599, MOVAR 33/63 and LVR BHV-4 reference strains showed two kinds of differences, either related or not related to the prDNA (polyrepetitive DNA). Taken together, these data show that PR/ 1 is a new BHV-4. We would consider that the present report provides a scheme of work for diagnosis and typing of BHV-4 isolates.

Transfection of bovine cell culture with bovine herpesvirus 4 DNA obtained by cell nuclear extraction.

Bovine herpes virus 4 (BHV-4) is a gamma-herpesvirus not associated with clearly defined clinical entities in cattle. The BHV-4 genome consists of a linear dsDNA of approximately 145 Kbp which is only partially characterized and sequenced. We set up a rapid and practical method to isolate BHV-4 DNA from infected cell culture. Microfuged infected cells after exposure to high salt concentration and detergent allowed viral DNA to be purified. Electrophoretic analysis of the digested DNA showed a complete digestion, corresponding to a classical EcoRI banding pattern of strains Movar 33/63, LVR and DN 599. Moreover the biological integrity of viral DNA here obtained, was demonstrated by transfection experiments. BHV-4 DNA was capable of forming CPE after transfection into BAE-7372 cells. Transfected cells specifically reacted with a BHV-4 infected cow serum demonstrating the presence of viral particles. The possibility of obtaining infectious viral DNA using this method may facilitate the construction of recombinant viruses. Specifically, through the use of cotransfection experiments with deleted or mutated viral DNA sequences, the infectious clones isolated could provide the basis for an increased understanding of BHV-4 viral gene expression, replication and pathogenesis.

Seroprevalence to bovine immunodeficiency virus and lack of association with leukocyte counts in Italian dairy cattle.

We report herein on the first serological detection of antibodies to bovine immunodeficiency virus (BIV) in Italy. According to criteria of a stratified-random sampling of dairy cattle reared in the Parma area (a province in the Po Valley, Northern Italy), sera from 3166 cows belonging to 272 herds were collected. In addition, sera of 138 bulls from eight artificial-insemination (AI) centres were sampled. Seventy-eight cows (2.5%) from 16 herds (5.8%) and seven bulls (5.1%) from two AI centres were positive for BIV-R29 antibodies in the IFA-test. IFA-positive sera assayed by Western blot had reaction to different viral proteins: 81 out of 85 sera showed antibody to p26 (considered the BIV major internal core protein); four sera reacted to other viral proteins but not to p26. Peripheral blood leukocytes of 60 seropositive and 60 seronegative animals, belonging to eight BIV-infected herds, were enumerated to assess any effect of BIV infection on white-blood cells. No significant differences were detected between the two groups. These data indicate that BIV infection is present in Italian dairy cattle--but the role of BIV in inducing disease remains unclear.