The virulent strain SFV4 of Semliki Forest virus (SFV), produced from the infectious clone pSP6-SFV4, is lethal after intranasal (i.n.) infection of adult mice and for pregnant mice after intraperitoneal (i.p.) infection. In contrast, the A7 strain of SFV is avirulent when given i.n. to adult mice, but induces fetal death in pregnant mice after i.p. infection. The nucleotide and deduced amino acid sequences of part of the core and all of the envelope region of A7-SFV were determined and compared to those of SFV4. A7 differed from SFV4 at 80 nucleotides (nt) in the coding sequence, 15 of which were associated with amino acid differences and seven of which (two in the E2 protein and five in El) were nonconservative. The 3' non-coding sequence of A7 was longer (415 nt) than that of SFV4 (263 nt) and a divergent sequence of 181 nt was present adjacent to the end of the E1 coding region. The effects on virulence of two mutations in the E2 gene of SFV4, resulting in the non-conservative amino acid substitutions present in A7, were analysed. One mutation (mut 8729 a/c) resulted in only slight attenuation, whereas the other (mut 8902 a/g) resulted in avirulence for pregnant mice. However, mut 8902 a/g was lethal for the majority of developing fetuses after i.p. infection of the mother.

The pathogenicity of the avirulent, demyelinating A7 strain of Semliki Forest virus (SFV) and the virulent SFV4 strain (derived from an infectious clone) for the central nervous system of adult BALB/c mice following intranasal infection was compared. The techniques used included immunocytochemistry using anti-SFV antibody and antibodies to cell markers, in situ hybridization (ISH) using a biotinylated cDNA probe specific for SFV, and immunocytochemistry/ISH double labelling. Whereas SFV4 was lethal at 4 days post-infection, A7-infected mice appeared normal at all times. Neuronal necrosis in the pyriform cortex was present in both infections, but developed sooner and was more severe following infection with SFV4 than with A7. Intact neurons and putative oligodendrocytes contained viral RNA and virus-specific antigen in SFV4 infected mice; viral RNA but not virus-specific antigen was detected in similar cells in A7-infected mice. These results confirm that SFV4 and A7 share similar cell tropisms for the murine central nervous system, but differ in the severity and rate of development of cytolytic damage. Intranasal infection is an efficient monitoring system for studies of the molecular basis of pathogenicity of SFV infection in mice.

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SUMMARYMonospecific polyclonal antisera were raised in guinea-pigs against the calf rotavirus polypeptides VP 1, VP2, VP3* + 4*, VP4.2, VP6, VP7. I, VP7.2 and VP 10. All of the antisera gave a similar pattern of cytoplasmic immunofluorescence in rotavirus-infected cells, but spots of fluorescence of varying intensity with different sera were seen over the nucleus. Immune precipitation, using Staphylococcus aureus to collect immune complexes, showed that VP2 was precipitated by antiserum to VP2 (ct-VP2) and VP6 by ct-VP6, ct-VP7.1 and ct-VP7.2 both precipitated the same range of proteins from infected cells (VP7, VP7.1 and VP7.2) or from virions (VP7.1 and VP7.2). VP 10, either from virions or infected cells, was not precipitated by a-VP 10. The only antiserum which efficiently neutralized infectivity was a-VP7.2. There were low levels of neutralization with a-VP10 (but the results varied from experiment to experiment) and traces with a-VP6, ct-VP7.1 and the other antisera did not neutralize even though a-VP7.1 agglutinated double-shelled particles as seen in immune electron microscopy to a greater extent than ~-VP7.2. Both VP7.1 and VP7.2 were shown to be glycoproteins by tunicamycin treatment of infected cells. Core particles only were agglutinated by a-VP10. All the evidence leads us to conclude that there were major neutralizing antigenic determinants present on VP7.2, a minor component of the outer shell of the virion.