Infection with the mouse hepatitis coronavirus (MHV) provides an excellent model for the study of viral diseases of the central nervous system and the gastrointestinal tract. With the ultimate aim of studying mucosal immunity to MHV we have cloned the genes encoding the structural proteins of MHV strain A59 (MHV-A59) into the E3 region of a human adenovirus type 5 vector. Infection of HeLa cells with the resulting recombinant adenoviruses AdMHVS, AdMHVN and AdMHVM revealed the correct expression of the spike (S), nucleocapsid (N) and membrane (M) proteins, respectively. Intraperitoneal inoculation of BALB/c mice with the recombinant viruses elicited serum antibodies which specifically recognized the respective MHV proteins in an immunoprecipitation assay. Only antibodies to the S protein neutralized MHV-A59 in vitro but titres were low. When analysed by ELISA or by immunofluorescence only the antibody response to the N protein was significant; weak responses or no detectable response at all were found for S and M, respectively. Upon intracerebral challenge with a lethal dose of MHV-A59 we found that a significant fraction of animals vaccinated with adenovirus vectors expressing either the S protein or N protein were protected. This protective effect was significantly stronger when the animals were given a booster immunization with the same vector prior to challenge. No protection was induced by AdMHVM. Interestingly, enhanced protection resulted when AdMHVS and AdMHVN were applied in combination as compared to survival after single immunizations. The results indicate that both the N and S proteins generate a protective immune response and suggest that this response is enhanced by combined expression of the two proteins.

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The individual and synergistic antiviral effects of cytokines released by infiltrating immune cells or by cells of the nervous system may play an important role in inhibiting virus spread during infections of the central nervous system (CNS). We examined the antiviral activity against the neurotropic pseudorabies virus (PRV) of interferon-y (IFN-y) and tumour necrosis factor-~ (TNF-ct), and combinations of these cytokines, as compared to that of IFN-fl, in rat nervous tissue cells. PRV replicated efficiently in all neural cell types tested, including neurons, astrocytes and oligodendrocytes. The inhibitory effects were determined by quantifying the inhibition of virus plaque formation, yields of infectious virus at various times after infection and synthesis of viral proteins. At a low m.o.i., IFN-), and IFN-fl inhibited viral plaque formation in all cell types; TNF-~ was effective only in astrocytes but showed synergy with IFN-~. At a higher m.o.i., IFN-fl inhibited yields of infectious virus more effectively than IFN-y, whereas TNF-~ had no effect on virus yields and was only marginally synergistic with the antiviral activity of IFN-~. The yield-reduction assays correlated well with cytokine-induced inhibition of viral protein synthesis. Our results show that both IFN-~ and IFN-fl can induce a state of antiviral resistance in neural cells whereas TNF-~ is effective only in astrocytes at low m.o.i.; they suggest an antiviral role of cytokines in the immune response to virus infections of the CNS.

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