Alfred Metzler

European isolates of bovine herpesvirus 1: A comparison of restriction endonuclease sites, polypeptides, and reactivity with monoclonal antibodies

SummaryEleven European isolates of bovine herpesvirus 1 (BHV-1), together with two reference virus strains were compared by restriction endonuclease digestion, by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and by their reactivity with a panel of monoclonal antibodies (McAbs). Based on the cleavage pattern of viral DNA with the restriction endonuclease Hind III the strains could be assigned to one of two established major virus types. Analysis by SDS-PAGE of viral polypeptides revealed that four protein species either displayed virus type or subtype specific minor variation of migration characteristics. Of 43 McAbs tested all reacted with all type 1 strains, whereas five antibodies failed to recognize some of the type 2 viruses. The existence of type specific variations among virus specified proteins was further evidenced by the recovery of one McAb recognizing type 1 viruses only. The data show that BHV-1 isolates can be assigned to established virus types according to the SDS-PAGE profile of viral proteins or the selective reactivity with type specific McAbs.

Bovine herpesvirus 1: Molecular and antigenic characteristics of variant viruses isolated from calves with neurological disease

SummaryThis report presents data showing that several virus isolates recovered in Argentina, mainly from calves with non-purulent meningo-encephalitis, represent a hitherto unrecognized antigenic variant of BHV-1. The following experimental approaches have been adopted to demonstrate both the unique features among and the relatedness with BHV-1 of these isolates: i) crossed serum neutralization test with rabbit immune sera, ii) analysis by SDS-polyacrylamide gel electrophoresis of radio-labeled virus induced polypeptides and glycoproteins, iii) discriminating reactivity of a panel of monoclonal antibodies which recognize known virus types, and iv) restriction endonuclease analysis of viral DNA. Another strain of BHV-1, which exhibits a specific neuropathogenic potential [Hallet al., Austral. Vet. J.42, 229–237 (1966)] shares all major features with the viral strains originating from Argentina.Our results imply that antigenic variants of BHV-1 exist and that they can be accurately and easily identified and differentiated by the available methods.

Innate Immune Responses of Calves during Transient Infection with a Noncytopathic Strain of Bovine Viral Diarrhea Virus

ABSTRACT In this study, six immunocompetent calves were experimentally infected with a noncytopathic strain of bovine viral diarrhea virus (BVDV), and the effects of the viral infection on parameters of the innate immune response of the host were analyzed. Clinical and virological data were compared with the temporal activation of the alpha/beta interferon-regulated Mx gene in white blood cells (WBC) and skin as well as the upregulation of the acute-phase serum proteins haptoglobin (Hp) and serum amyloid A (SAA). The viral strain used did provoke transient health impairment, namely, fever and leukopenia that were associated with viremia, viral shedding with nasal secretions, and antiviral seroconversion. Complete recovery was observed within 3 weeks. Elevated levels of SAA and Hp were apparent from days 4 to 13 and 8 to 11, respectively. In WBC, the levels of Mx mRNA and Mx protein were elevated from days 2 to 15. In the context of this study with BVDV, the level of Mx protein expression in WBC provided the most telling diagnostic window to monitor the hosts ongoing innate immune response.

Reactivity of monoclonal antibodies to proteins of a neurotropic bovine herpesvirus 1 (BHV-1) strain and to proteins of representative BHV-1 strains

Summary15 monoclonal antibodies (McAbs) were induced with the neuropathogenic strain N 569 of bovine herpesvirus 1 type 3 (BHV-1.3). Nine of them could be shown by radioimmunoprecipitation assay to react with viral glycoproteins and two of these McAbs were able to neutralize strain N 569. The reactivity of these 15 monoclonals was compared with 11 monoclonal antibodies induced with a BHV-1.1 strain. The available monoclonal antibodies made it possible to characterize BHV-1.3 and to classify BHV-1 into three types, namely BHV-1.1, BHV-1.2 and BHV-1.3. This confirmed the results based upon restriction endonuclease analysis and viral protein patterns obtained earlier. The main antigenic differences of representative virus strains were found on two glycoproteins designated 3 and 12. Caprine herpesvirus 1, included in this study because of its serological relationship to BHV-1, differed fundamentally from BHV-1 on the grounds of McAb reactivity.

Isolation of sabin-like polioviruses from wastewater in a country using inactivated polio vaccine.

ABSTRACT From 2001 to 2004, Switzerland switched from routine vaccination with oral polio vaccine (OPV) to inactivated polio vaccine (IPV), using both vaccines in the intervening period. Since IPV is less effective at inducing mucosal immunity than OPV, this change might allow imported poliovirus to circulate undetected more easily in an increasingly IPV-immunized population. Environmental monitoring is a recognized tool for identifying polioviruses in a community. To look for evidence of poliovirus circulation following cessation of OPV use, two sewage treatment plants located in the Zurich area were sampled from 2004 to 2006. Following virus isolation using either RD or L20B cells, enteroviruses and polioviruses were identified by reverse transcription-PCR. A total of 20 out of 174 wastewater samples were positive for 62 Sabin-like isolates. One isolate from each poliovirus-positive sample was analyzed in more detail. Sequencing the complete viral protein 1 (VP1) capsid coding region, as well as intratypic differentiation (ITD), identified 3 Sabin type 1, 13 Sabin type 2, and 4 Sabin type 3 strains. One serotype 1 strain showed a discordant result in the ITD. Three-quarters of the strains showed mutations within the 5′ untranslated region and VP1, known to be associated with reversion to virulence. Moreover, three strains showed heterotypic recombination (S2/S1 and S3/S2/S3). The low number of synonymous mutations and the partial temperature sensitivity are not consistent with extended circulation of these Sabin virus strains. Nevertheless, the continuous introduction of polioviruses into the community emphasizes the necessity for uninterrupted child vaccination to maintain high herd immunity.

In Vitro and In Vivo Detection of Mx Gene Products in Bovine Cells following Stimulation with Alpha/Beta Interferon and Viruses

ABSTRACT This study focused on products of the bovine Mx1 gene as specific markers for acute viral infections. The rationale for this is the fact that viral infections are commonly paralleled by the synthesis, release, and remote action of alpha/beta interferons (IFN-α/β). Released IFN-α/β act through specific receptors present on nucleated cells to transduce signals for the transcription of numerous IFN-regulated genes, such as the ones for double-stranded-RNA-dependent protein kinase, 2′-5′-oligoadenylate synthetase, or the Mx proteins. In this study, cultured MDBK cells and bovine white blood cells (WBC) were treated with recombinant IFN-α or infected with either bovine herpesvirus 1 (BHV-1) or bovine rotavirus (BRV). Treatment of cultured cells with IFN-α was followed within 4 h by a time- and dose-dependent accumulation of intracytoplasmic Mx protein as revealed by immunostaining and Western blot immunoassay. This was preceded by a distinct rise of Mx mRNA in similarly treated cells, as revealed by a newly established quantitative TaqMan PCR technique. The two viruses displayed a cell-dependent in vitro ability to induce Mx proteins, which was limited to bovine WBC with BHV-1 and to MDBK cells with BRV. The established methods were successfully used to show that infection of calves with a noncytopathic strain of bovine viral diarrhea virus, a pestivirus, was followed within 2 days postinfection by strong expression of both Mx mRNA and Mx proteins in WBC.

The malignant catarrhal fever complex

Malignant catarrhal fever (MCF) is defined as a clinicopathological syndrome caused by related herpesviruses and acquired from persistently infected wildebeest and sheep. There is convincing epidemiologic and virologic evidence that Alcelaphine herpesvirus 1 (AHV1) causes the wildebeest-derived disease (WD-MCF). Present knowledge suggests that a herpesvirus related to AHV1 may be associated with some cases of the non-wildebeest-associated disease (NWA-MCF). However, this virus possibly represents a passenger virus not related with the ultimate cause of the disease. Moreover, evidence for the role played by sheep as the reservoir for the agent of NWA-MCF is not convincing and awaits confirmation.

Interactions of bovine and caprine herpesviruses with the natural and the foreign hosts

Bovine herpesvirus 1 (BHV1) and caprine herpesvirus 1 (CapHV1) are useful models to study virus-host interactions, as well as pathogenicity and latency, when comparing the outcome of infection in the natural and the foreign hosts. Molecular seroepidemiological analyses revealed that cross-reacting antibodies were mainly induced by glycoprotein gI (gB analogue), by the major capsid protein and by nonstructural proteins, whereas the most virus-specific antibodies were elicited by glycoproteins gIII and gIV. These glycoproteins, especially gIII (gC analogue), might therefore play an important role in the virus-host-interactions. As a basis for further studies, we re-evaluated observations concerning experimental infections with BHV1 and CapHV1 in the natural and the foreign hosts. All parameters indicated that both viruses were able to infect either host, but that the pathogenicity was restricted to the natural host. Latent virus could be reactivated exclusively from cows infected with BHV1. It was possible neither to reactivate BHV1 from goats, nor to reactivate CapHV1 from either species. The experiments indicated that the outcome of infection in the natural and the foreign host is dependent on host and viral factors, whereby gIII is only one important virus component involved. Further investigations in the host and host cell range of BHV1 and CapHV1 will help to clarify the role of factors responsible for virus-host-interactions.

In vitro growth characteristics of bovine herpesvirus 4 (BHV-4) as revealed by indirect immunofluorescence assay with monoclonal antibodies and polyvalent antisera

SummaryThe bovine herpesvirus 4 (BHV-4) isolate LVR-140 growing in MDBK cell cultures was investigated by indirect immunofluorescence (IF), using bovine antisera and 15 monoclonal antibodies (MAb). The cytopathogenic effect was discernible between 20 and 30 hours post inoculation (p.i.) and developed slowly as has been reported for other BHV-4 strains. According to the protein specificity as revealed by radioimmunoprecipitation and the intracellular localisation of reacting antigens as detected by IF, the MAb could be assigned to one of three groups. The initial antigen expression was first detected at 8 hours p.i. and was limited to a proportion of the infected cells only, despite the high multiplicity of infection used; infectious cell-associated progency virus was first recognized at 12 hours p.i.