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Hepatitis C virus (HCV) is a major cause of chronic hepatitis and hepatocellular carcinoma worldwide. The purpose of this study was to determine how the HCV structural proteins affect the dynamic structural and functional properties of hepatocytes and measure the extra-hepatic manifestations induced by these viral proteins. A transgenic mouse model was established by expressing core, E1 and E2 proteins downstream of a CMV promoter. HCV RNA was detected using RT-PCR in transgenic mouse model tissues, such as liver, kidney, spleen and heart. Expression of the transgene was analysed by real-time PCR to quantify viral RNA in different tissues at different ages. Immunofluorescence analysis revealed the expression of core, E1 and E2 proteins predominantly in hepatocytes. Lower levels of protein expression were detected in spleen and kidneys. HCV RNA and viral protein expression increased in the liver with age. Histological analysis of liver cells demonstrated steatosis in transgenic mice older than 3 months, which was more progressed with age. Electron microscopy analysis revealed alterations in nuclei, mitochondria and endoplasmic reticulum. HCV structural proteins induce a severe hepatopathy in the transgenic mouse model. These mice became more prone to liver and lymphoid tumour development and hepatocellular carcinoma. In this model, the extra-hepatic effects of HCV, which included swelling of renal tubular cells, were mild. It is likely that the HCV structural proteins mediate some of the histological alterations in hepatocytes by interfering with lipid transport and liver metabolism.

Porcine adenovirus-3 (PAV-3) was developed as an expression vector using homologous recombination in Escherichia coli BJ 5183. As a prerequisite, the complete genome of PAV-3 was first introduced as a PacI restriction fragment into a bacterial plasmid. The plasmid, when PacI restricted and transfected into swine testicular cells, produces an infectious virus. The potential of this procedure was demonstrated by the construction of several PAV-3 recombinants. Part of the E3 region, which is nonessential for virus replication under cell culture conditions, was identified and deleted from the virus genome. The gene for glycoprotein D (gD) of pseudorabies virus (PRV), which elicits PRV-neutralizing antibodies in pigs, was cloned and expressed from the E3 region of PAV-3. A 50 kDa polypeptide was identified in recombinant PAV-3-infected cell lysates by immunoprecipitation assays using gD-specific monoclonal antibodies. In another experiment, a region between the right inverted terminal repeat and the promoter of the E4 region was used to clone and express the chloramphenicol acetyltransferase (CAT) gene under the control of SV40 immediate early promoter. CAT gene expression was observed irrespective of the orientation of the CAT gene. These results indicate that the helper-independent recombinant PAV-3 could be used as an expression vector and has potential as a recombinant vaccine vector in pigs.

One anticipated advantage of DNA immunization is the potential to create multivalent vaccines. We have examined the effects of mixing plasmids into single formulations using plasmids expressing four different membrane bound glycoproteins from bovine herpesvirus-1 (BHV-1), bovine parainfluenzavirus-3 (bPI3) and human influenza virus (HINF). Plasmids were delivered by intradermal injection into the tails of mice and the types of responses generated were clearly affected by the expressed antigen. Plasmids expressing glycoproteins B and D of BHV-1, and the haemagglutinin/ neuraminidase of bPI3, generated responses with a predominance of IgG1, suggestive of a Th2 type of response. In contrast, the plasmid expressing HINF haemagglutinin induced an antibody response biased towards IgG2a, indicating a Th1 type of response. In most instances the mixing of plasmids had only slight effects on the magnitude or bias of the responses to the individual components. However, under certain conditions we found that addition of a second plasmid converted an IgG2a biased response to a response with primarily IgG1 antibody. The reverse situation (i.e. an IgG1 conversion to IgG2a), however, was not found. These findings have important implications for the development of multivalent vaccines.

Although intramuscular (i.m.) injection of DNA encoding glycoprotein D (gD) of bovine herpesvirus-1 (BHV-1) induces immune responses in cattle, this route of delivery is inefficient. Here we assessed three parameters that may enhance the efficacy of a gD DNA vaccine in cattle. First, the immune response generated by i.m. injected plasmid expressing a secreted form of gD (tgD) was determined and found to be very similar in magnitude to the response induced by gD-expressing plasmid. Secondly, gD- and tgD-expressing plasmids were administered by intradermal (i.d.) immunization, which resulted in a superior immune response to the secreted form, but no improvement in the response to the membrane-associated form. However, the form of gD used for immunization did not influence the immunoglobulin subtype, the ratio of antigen-specific IgG1 to IgG2 being approximately 4:1. Finally, the effect of promoter strength was assessed by replacing the Rous sarcoma virus (RSV) promoter, which was used in the original experiments, with the human cytomegalovirus immediate early promoter and first intron A (HCMV/IA). Although upon transfection in vitro the HCMV/IA promoter appeared to be stronger than the RSV promoter, there was only a 2-fold higher antibody response in vivo upon i.d. injection of cattle. Protection against virus challenge was obtained in the calves immunized i.d. with tgD-encoding plasmid, as shown by a significant reduction in weight loss, virus excretion, temperature response and clinical disease. No significant protection was observed in the animals vaccinated i.d. with the gD-expressing plasmid, which correlates with the lower level of immunity pre-challenge.

Treatment of bovine alveolar macrophages (BAM) with bovine leukocyte interferon (BoIFN-alpha 1) resulted in reduced bovine herpesvirus type 1 replication and spread. This was demonstrated by reduced virus yields and number of cells infected. BoIFN-alpha 1 treatment of BAM also induced enhanced Fc receptor expression and/or avidity by the cells, increased their activity in antibody-dependent cellular cytotoxicity, and augmented their extrinsic antiviral activity as measured by a reduction in the development of plaques in a susceptible cell line. These results are discussed in the context of the possible role of interferon in activation of AM during the early phases of a virus infection.

Non-cytopathic strains of bovine viral diarrhoea virus (BVDV) readily establish persistent infections in cells of bovine origin. The involvement of endogenous interferon (IFN) on the maintenance of the infection level, as well as the effect of exogenous IFN and tumour necrosis factor alpha (TNF-alpha), was studied. Although exogenous IFN suppressed the spread and replication of virus, it did not cure the infection, even when continuously present over many cell passages. TNF-alpha alone had no antiviral effect in this system. However, both TNF-alpha and IFN enhanced the cytopathic effect of cytopathic BVDV, and induced a BVDV-like cytopathic effect in cells infected with non-cytopathic BVDV. These data are discussed with regard to possible mechanisms of pathogenesis during fatal BVDV disease in cattle.

Treatment of calves with bovine recombinant alpha 1 interferon prior to challenge with bovine herpesvirus type 1 increased the animals' ability to withstand a subsequent Pasteurella haemolytica challenge. The reduction in viral-bacterial synergy observed following interferon treatment did not appear to be due to a direct effect of the interferon on virus replication in the upper respiratory tract. Thus, even though interferon-treated animals shed slightly less virus from their nasal passages than did untreated animals, this reduction was not statistically significant. Furthermore, there was no difference in the level of intranasal interferon secreted by control or interferon-treated animals. These results suggest that interferon treatment does not affect the production of endogenous interferon. In contrast, a significant difference was observed between the number of days that control animals were sick, the levels of serum fibrinogen and the functional activity of polymorphonuclear neutrophilic granulocytes obtained from infected calves. These results suggest that bovine recombinant alpha 1 interferon may have a greater immunomodulatory effect than a direct antiviral effect in this model. This is further supported by the observation that bovine herpesvirus type 1 is relatively resistant to the direct antiviral effect of bovine recombinant alpha 1 interferon in vitro.