Synthesis and antiviral evaluation of nucleic acid-based (NAB) libraries.

Combinatorial assembly of nucleotide libraries and their antiviral evaluation against HSV-1 are described.

Growth of infectious salmon anaemia virus in CHSE-214 cells and evidence for phenotypic differences between virus strains.

Infectious salmon anaemia virus (ISAV) is a new orthomyxovirus-like virus. Thirteen isolates of ISAV (11 from Canada, one from Norway and one from Scotland) were studied for their replication in the CHSE-214 cell line compared with that in the SHK-1 cell line. All isolates replicated in SHK-1 cells, producing CPE between 3 and 12 days post-inoculation (p.i.). Six Canadian isolates also replicated in CHSE-214 cells, with production of CPE between 4 and 17 days p.i. Analysis of a one-step growth curve of ISAV in CHSE-214 cells showed that progeny virions remained predominantly cell-associated, accounting for the focalized nature of the CPE in the cell monolayer. One isolate (HKS 36) replicated in CHSE-214 cells, as shown by positive RT-PCR results of blind passages, but was non-cytopathic. All of the isolates were analysed for genetic heterogeneity by RT-PCR and RFLP with EcoRI and XhoI in a fraction of genome segment 2. The Canadian isolates showed a different RFLP profile to those of isolates Glesvaer/2/90 from Norway and 390/98 from Scotland. Structural proteins of four isolates, 'Back Bay 98', RPC/NB-877, RPC/NB-049 and Glesvaer/2/90, were examined further by SDS-PAGE. All viruses showed four major polypeptides, designated here as VP1-VP4, in Coomassie blue-stained gels. In isolates Glesvaer/2/90 and RPC/NB-877, these viral proteins had estimated molecular masses of 74, 53, 46 and 26.5 kDa, respectively. Viral proteins in isolates 'Back Bay 98' and RPC/NB-049 were of similar sizes, except that VP3 was 43 kDa. Taken together, these results show that there are phenotypic differences among strains of ISAV.

Characterization of monoclonal antibodies against bovine herpesvirus 1 gD fusion protein expressed in E. coli.

A total of 20 hybridoma cell lines secreting monoclonal antibodies (MAbs) against E. coli expressed bovine herpesvirus-1 (BHV-1) gD fusion protein were produced following the fusion of Sp2/0 myeloma cells with splenocytes from BALB/c mice immunized previously with immunoaffinity purified BHV-1 gD fusion protein. An indirect fluorescent antibody test (IFAT) using BHV-1 infected MDBK cells was used for the selection of positive hybridomas secreting specific antibody. The monoclonal antibody isotypes were 11 IgM, six IgG2b, one IgG1 and two IgG3. All MAbs reacted positively with the E. coli expressed BHV-1 gD fusion protein, BHV-1 infected MDBK cell lysates and PCR BHV-1 gD transcription-translation polypeptide antigens by an ELISA.

Improved detection of bovine herpesvirus 1 in artificially infected bovine semen by protein amplification.

Infection with bovine herpesvirus 1 (BHV 1) occurs worldwide and causes serious economic losses due to loss of animals, abortions, decreased milk production, and loss of body weight. There is a real need for sensitive diagnostic procedures for detection of the presence of virus in order to achieve effective control of BHV 1-induced diseases. BHV 1 is frequently found in bovine semen and can be widely transmitted through artificial insemination. Thus the detection of BHV 1 in artificial insemination centers and semen banks is of crucial importance in the control of its dissemination to the cattle industry, worldwide. In the present study, a protein amplification assay following polymerase chain reaction (PCR) of the highly conserved BHV 1 glycoprotein D gene was used in order to improve the sensitivity of direct virus detection in bovine semen. This method of BHV 1 detection is at least 200 orders of magnitude more sensitive than traditional PCR and would have direct clinical applications in antigen-based detection tests. In this method, amplification of the BHV 1 gD gene by PCR is followed by a coupled in vitro transcription translation of a small aliquot from the reaction. When the transcription translation was carried out in the presence of [35S]methionine and the products analyzed by SDS PAGE and autoradiography, 0.0014 TCID50 of virus could be detected in raw bovine semen in contrast to 0.28 TCID50 of virus detected using traditional PCR. Given the limitations in the method used for protein detection, this 'in vitro protein amplification' has the potential of attaining superior sensitivity for direct virus detection in clinical samples.

Bovine herpesvirus 1-induced apoptosis: phenotypic characterization of susceptible peripheral blood mononuclear cells.

Bovine herpesvirus 1 (BHV-1), a member of the Alphaherpesvirinae, induces apoptotic cell death in peripheral blood mononuclear cells (PBMC). To investigate the process by which BHV-1 induces apoptosis, we determined the susceptibility of the three main PBMC subpopulations to BHV-1-induced apoptosis. This study shows that BHV-1 can induce apoptosis individually in T lymphocytes, B lymphocytes and monocytes. This conclusion is based on the following findings: (i) BHV-1 substantially reduces the percentages of viable T and B lymphocytes in PBMCs. (ii) Concomitant detection of cell phenotype and apoptosis indeed showed higher percentages of apoptotic T lymphocytes and B lymphocytes in BHV-1-infected PBMCs than in mock-infected cells. (iii) Each individual PBMC subpopulations (B lymphocytes, T lymphocytes and monocytes) undergo apoptosis when incubated with BHV-1. These data also suggest that BHV-1 does not require the recruitment of one or more individual PBMC subpopulations (e.g. cytotoxic cells) to induce apoptosis. Finally, we observed that BL-3 cells which have been characterized as bovine tumoral B lymphocytes also undergo apoptosis when incubated with BHV-1. Therefore, the use of the BL-3 cell line provides a new experimental model to investigate the apoptotic process induced by BHV-1 in vitro.

Evidence for biotype-specific effects of bovine viral diarrhoea virus on biological responses in acutely infected calves.

The relationship between the two biotypes of bovine viral diarrhoea virus (BVDV) and the biological responses they induce was studied in 3- to 6-month-old calves inoculated intranasally with a homologous pair of non-cytopathic and cytopathic strains. Marked differences in virological and serological events occurred following exposure to a specific BVDV strain. The non-cytopathic biotype was frequently recovered from nasal secretions and blood cells during the first 28 days post-inoculation whereas the cytopathic counterpart was detected infrequently in nasopharyngeal swabs only. There was no correlation of the recovery of infectious virus in vivo with the biotype-specific neutralizing humoral immune response. Furthermore, seroconversion did not correlate with resistance to reinfection as judged by the transient viraemia and/or shedding of virus observed in a challenge experiment.

Analysis of the biochemical properties of, and complex formation between, glycoproteins H and L of the gamma2 herpesvirus bovine herpesvirus-4.

Genes encoding glycoprotein gH and gL homologues were localized in the genome of the gamma-herpesvirus bovine herpesvirus-4 (BHV-4). Both genes were sequenced and glutathione S-transferase fusion proteins were produced and used to immunize rabbits against the translation products of the two genes. The anti-gH serum recognized a protein with an apparent molecular mass (MM) of 110 kDa both in infected cells and in virions. This protein was sensitive to endo-beta-N-acetylglucosaminase-H (endoH) and endoglycosidase F-N-glycosidase F (endoF-PNGaseF) digestion. A protein with the same relative mobility was immunoprecipitated from infected cells radiolabelled with [3H]glucosamine which confirmed that this product (gp110), now designated BHV-4 gH, was glycosylated. Western blotting with the anti-gL serum detected in infected cells a product with an apparent MM ranging from 31-35 kDa and diffusely migrating protein species ranging from 45-65 kDa. Tunicamycin, monensin, endoH or endoF-PNGaseF treatments showed that both the 31-35 kDa and the 45-65 kDa proteins were glycosylated, gp31-35 being a precursor of the 45-65 kDa glycoprotein species. In radioimmunoprecipitation assays, the anti-gL serum immunoprecipitated from infected cells two glycosylated proteins with apparent MMs of 31-35 kDa (gp31-35) and 45-55 kDa (gp45-55). However a third glycoprotein, gp110, was also immunoprecipitated together with gp31-35 and gp45-55. gp110 and gp45-55 were subsequently confirmed to be virion glycoproteins corresponding to mature forms of BHV-4 gH and gL respectively. In addition, the present study clearly demonstrated complex formation between BHV-4 gH and gL both in virions and in infected cells.

Bovine herpesvirus 1-induced apoptosis occurs at the G0/G1 phase of the cell cycle.

We have previously shown that bovine herpesvirus 1 (BHV-1), even when inactivated, induces apoptotic cell death in mitogen-stimulated bovine peripheral blood mononuclear cells (PBMCs) (Hanon et al., 1996, J. Virol. 70, 4116-4120). In order to gain insight into this process, we have investigated the cell cycle phase at which BHV-1 induces apoptosis in PBMCs. Our results show that the percentage of cells that progress through the S phase was always lower in BHV-1-infected PBMCs than in control cells. This effect was not due to a defective activation of mitogen-stimulated PBMCs since BHV-1 only slightly affected the percentage of cells expressing BoCD25, a well-known lymphocyte activation marker. Furthermore, mimosine and cyclosporine A, two chemicals that inhibit entry into the S phase of the cell cycle by different pathways, did not affect the ability of BHV-1 to induce apoptosis. BHV-1-induced apoptosis also occurred in unstimulated PBMCs and interestingly, this was associated with the expression of c-myc and BoCD25 proteins both of which are related to cell cycle progression. All together, these data provide evidence demonstrating that BHV-1-induced apoptosis occurs at the G0/G1 phase of the cell cycle.

Glycoprotein B of bovine herpesvirus 4 is a major component of the virion, unlike that of two other gammaherpesviruses, Epstein-Barr virus and murine gammaherpesvirus 68.

This study reports that in bovine herpesvirus 4, glycoprotein B (gB) is a heterodimer and a major component of the virion, unlike gBs of Epstein-Barr virus (gp110) and murine gammaherpesvirus 68, two other gammaherpesviruses. These are new characteristics with regard to the general features of gB in the Gammaherpesvirinae subfamily.

Structural and functional analysis of bovine herpesvirus 1 minor glycoproteins.

This paper focuses on the structure and functions of bovine herpesvirus 1 minor glycoproteins gH, gE, gG and gp42. It reviews the progress which has been made in their identification and characterization, in the study of their temporal expression and processing in infected cells, and finally in the understanding of their biological activities. In addition, aspects discussed include a comparison with two other alphaherpesviruses, namely herpes simplex virus and pseudorabies virus.

Establishment of a rabbit model for bovine herpesvirus type 5 neurological acute infection.

This study was conducted to evaluate the suitability of the rabbit as a model for bovine, herpesvirus 5 (BHV-5) acute infection. In a preliminary experiment, a total of 24 one-month old New Zealand white rabbits were inoculated with BHV-5 or bovine herpesvirus 1 (BHV-1) by the intraconjunctival, intracerebral or intranasal routes. BHV-5 or BHV-1 inoculated in the conjunctiva induced virus proliferation in the eye mucosae and the nasal cavity of rabbits without meningo-encephalitis. On the other hand, only BHV-5 infection by intranasal or intracerebral routes produced a fatal meningo-encephalitis. The intranasal route was used in a further experiment for the establishment of a rabbit model for BHV-5 infection. A total of 45 rabbits were inoculated intranasally with BHV-5 or BHV-1. The results showed that intranasal inoculation of BHV-5 strain N569 in rabbits was followed by the development of a lethal meningo-encephalitis for 66% of rabbits while all BHV-1 infected rabbits remained healthy throughout this experiment (28 days). Analysis between the mortalities of rabbits infected with BHV-5 and BHV-1 were highly significant (p < 0.001). The presence of BHV-5 in the central nervous system (CNS) was confirmed by virus isolation (essentially the cerebrum, midbrain and pons) and by immunohistochemical staining of BHV-5 antigen (essentially in the neurons of the cerebrum) only in BHV-5 infected rabbits showing clinical signs of meningo-encephalitis. The findings obtained confirmed the suitability of a rabbit model for the establishment of BHV-5 neurological acute infection and also as a valuable tool for the comparative study of BHV-5 and BHV-1 neuropathogenicity.

The distinction of serologically related ruminant alphaherpesviruses by the polymerase chain reaction (PCR) and restriction endonuclease analysis.

The amplification and analysis of a 468bp fragment from the gB gene of the serologically related ruminant alphaherpesviruses bovine herpesvirus-1.1 and 1.2 (BHV-1.1 and BHV-1.2), caprine herpesvirus-1 (CapHV-1), cervine herpesvirus-1 (CerHV-1) and rangiferine herpesvirus-1 (RanHV-1) by PCR and restriction endonuclease analysis is described. As primers, 22bp oligomers selected from the BHV-1 gB gene sequences were used for the amplification of the DNA from the five viruses. The amplification product from each virus was analysed by the restriction endonuclease enzymes BglI, HinfI, SmaI and AvaI. The specific amplification obtained demonstrate the existence of the gB gene sequences for each of the five alphaherpesviruses. However, sequences from some of the fragments were found to be different from those predicted from the gB gene following restriction endonuclease analysis. All five amplification products generated the same number of fragments after digestion with HinfI except for two additional bands evident in CapHV-1. The CerHV-1 and RanHV-1 fragments contained slightly different BglI restriction sites from those of the other three. While BHV-1.1, BHV-1.2, CapHV-1 and CerHV-1 contained SmaI and AvaI restriction sites, the RanHV-1 amplification product lacked both SmaI and AvaI restriction sites.

The replication in vitro of the gammaherpesvirus bovine herpesvirus 4 is restricted by its DNA synthesis dependence on the S phase of the cell cycle.

Because several observations have suggested that replication of the gammaherpesvirus bovine herpesvirus 4 (BHV-4) is influenced by the physiological state of the host cell, a study was carried out to determine the relationship between BHV-4 infection and the cell cycle. The temporal expression of BHV-4 late (L) proteins in unsynchronized cell cultures was first investigated by flow cytometry. Interestingly, L protein expression occurred in a limited number of cells infected with a high multiplicity of infection, and a reciprocal correlation between the percentage of positive cells and the cell density at the time of infection was demonstrated. Moreover, the finding that a BHV-4 early-late protein was expressed in nearly all the cells suggested that a blockage in the viral replication cycle occurred in some infected cells at the stage of viral DNA synthesis or L protein expression. Because this blockage could be the consequence of the dependence of one or both of these events on the cell cycle, they were investigated after infection of synchronized cell cultures. The following findings were made. (i) Cell transition through the S phase quantitatively increased the rate of BHV-4 DNA replication. (ii) BHV-4 DNA synthesis could not be detected in cells arrested in G0. (iii) Synchronization of MDBK cells with Lovastatin before infection increased the percentage of cells expressing L proteins. (iv) In contrast, infection of cells arrested in G0 led to few positive cells. Taken together these results showed that BHV-4 DNA replication and consequently the expression of L proteins are dependent on the S phase of the cell cycle. This dependence could be of importance for several biological properties of BHV-4 infection in vitro and might have implications for the biology of the virus in vivo.

Production and characterization of monoclonal antibodies to cervine herpesvirus-1.

Fourteen hybridoma cell lines secreting monoclonal antibodies (Mab) to cervine herpesvirus-1 (CerHV-1) produced following the fusion of NSO myeloma cells with splenocytes of BALB/c mice previously immunized with gradient purified CerHV-1 were selected using an indirect enzyme-linked immunosorbent assay (ELISA) employing CerHV-1 antigen and tested by the ELISA against four other ruminant alphaherpesviruses from cattle (bovine herpesvirus type 1.1 and 1.2) goat (caprine herpesvirus-2) and reindeer (rangiferine herpesvirus-1). Comparison of all five ruminant alphaherpesviruses with these Mabs confirmed their close antigenic relationships, with two Mabs reacting against all viruses. Ten Mabs which were able to differentiate between the viruses reacted with a 64 kDa polypeptide in a western blot. Four Mabs including two specific only for CerHV-1 with neutralizing activity against the virus used for immunization were directed against a 74 kDa viral protein.

A comparison of serological relationships among five ruminant alphaherpesviruses by ELISA.

Using enzyme-linked immunosorbent assays the cross reactivity of bovine herpesvirus-1.1, bovine herpesvirus-1.2, caprine herpesvirus-2, cervine (red deer) herpesvirus-1 and rangiferine (reindeer) herpesvirus-1 has been examined using rabbit hyperimmune antisera and convalescent cattle and red deer field sera. Significant cross-reactivity among all the five viruses was demonstrated. A detailed analysis showed that: (1) the two bovine herpesviruses are most closely related, (2) the cervine, caprine and rangiferine viruses are more closely related to the bovine viruses than they are to each other, (3) the cervine herpesvirus is more related to the bovine herpesvirus than to the rangiferine or caprine herpesviruses and (4) the rangiferine virus is more related to the cervine virus than to the bovine and caprine viruses. Cattle and red deer sera reacted most strongly with the bovine and cervine viruses respectively.

A quantitative enzyme-linked immunosorbent assay for bovine herpesvirus type 1 (BHV-1) antibody.

A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure antibody to bovine herpesvirus type 1 (BHV-1) in cattle sera. The optical density produced from a single dilution of test serum was compared with a standard curve and the results were read and printed out from a computer interfaced to a multichannel ELISA reader. The printed results were expressed in ELISA units. The ELISA results obtained on 370 cattle sera were compared with those of the serum neutralisation test (SNT). An agreement of 90.5% was obtained when reciprocal SNT titres equal to or greater than 4 and IgG ELISA units equal to or greater than 50 were taken as indicative of a specific reaction. Of the 370 sera, 35 gave discrepant results of which 21 were SNT positive/IgG ELISA negative and 14 were SNT negative/IgG ELISA positive. When the SNT positive sera negative in the IgG ELISA were tested in an IgM ELISA, 19 were found to be positive. Thus, when the IgG and IgM ELISA results were combined the overall agreement between the ELISA and SNT increased to 95.7%. The IgG ELISA had a sensitivity of 82.4% and specificity of 94.4% relative to the SNT, whereas the combined IgG and IgM ELISA results gave a sensitivity and specificity of 98.3% and 94.4% respectively. There was a good positive correlation between the two tests (r = 0.86).