Vaginal infections of BALB/c Ann mice with herpes simplex virus type 1 (HSV-1) were studied. Mice were inoculated with virulent strains ANG path and 17 syn+ or low-virulent recombinant strains 27/III and 17-syn3 that differ from parental strains in their glycoprotein B (gB) gene sequences. When low-virulent strains were inoculated separately, no vaginitis/vulvitis was produced despite replication in the vagina. In contrast, after coinfection of mice with the two low-virulent strains, vaginitis/vulvitis was produced and virus could be recovered from the central nervous system (CNS). Two of the CNS isolates produced vaginitis/vulvitis, neuroinvasiveness and death of mice after vaginal infection. Restriction fragment analysis and sequencing were used to assess recombination events in the gB gene sequence of the CNS isolates. After mixed vaginal infection recombination between non-virulent HSV strains occurs, resulting in vaginitis/vulvitis and neuroinvasiveness. No correlation was detected between the syncytial phenotype and local vaginal virulence. Virulence of HSV is not solely dependent on gB function; it seems to be more probable that several genes act in concert to induce virulence and neuroivasiveness.

Previous studies have shown that certain strains of herpes simplex viruses type 1 (HSV-1) are able to induce “fusion from without” (FFWO) which means no transcription or translation of the viral genome happens. The main determinants for FFWO in BHK cells are mutations in the C-terminal part of gB-1. But single mutations in this part of the genome are not sufficient to transfer the FFWO phenotype also to Vero cells. Here, we report that FFWO of HSV strains indeed need additional mutations in the N-terminal part of gD in order to produce the FFWO phenotype in BHK and Vero cells. By marker transfer we are able to show that loss of mutations in the N-terminal part of gD influences the ability to induce FFWO in Vero cells but not in BHK cells. We assume that a mutated gD allows the entrance of a multiple number of virus particles into the cell and enhances therefore the fusion activity of the mutated gB. Mutations in gD alone are not sufficient for fusion activity of HSV.

Glycoprotein C-negative (gC-) mutants of herpes simplex virus type 1 (HSV-1) derived from strains KOS and ANGpath were used to analyse the influence of soluble heparin on the phase of adsorption/attachment of HSV-1 to cells. A dose of 200 micrograms/ml heparin given 20 mins after infection of cells with the gC- positive (gC+) strains KOS and ANGpath at 4 degrees C reduced the adsorption of infective particles to 20-30% of the controls. A weaker heparin effect was observed with gC- mutants. However, also the gC- mutants exhibited a short heparin-sensitive phase. Mutations in amino acids of gB or gD at positions 854 or 25 and 27, respectively, did not alter the attachment capacities of these HSV mutants in the presence of heparin despite their peculiar fusion properties and resistance to soluble gD. We conclude that HSV-1 strains exhibit a heparin-resistant phase of attachment, which is determined by gC. Lack of gC delays the heparin-resistant attachment phase of HSV-1 to cells.

In former studies, we described herpes simplex virus type 1 (HSV-1) strains ANG, ANG path and HSZP as strains with special fusion activities, caused by mutations in the syn 3 locus. In this report, we describe the special penetration properties of these strains: their ability to penetrate at 4 degrees and to overcome the infection block caused by soluble glycoprotein D-1 (gD-1) in the medium. Using intertypic recombinants of HSV-1 strains ANG path and KOS, we showed that these penetration properties are the result of two mutations in amino acids 25 (Leu-Pro) and 27 (Gln-Arg) in the N-terminal part of the mature gD-1 glycoprotein.

In former studies, we described herpes simplex virus type 1 (HSV-1) strains ANG, ANG path and HSZP as strains with special fusion activities, caused by mutations in the syn 3 locus. In this report, we describe the special penetration properties of these strains: their ability to penetrate at 4 degrees and to overcome the infection block caused by soluble glycoprotein D-1 (gD-1) in the medium. Using intertypic recombinants of HSV-1 strains ANG path and KOS, we showed that these penetration properties are the result of two mutations in amino acids 25 (Leu-Pro) and 27 (Gln-Arg) in the N-terminal part of the mature gD-1 glycoprotein.

Syncytial mutations of herpes simplex virus type 1 (HSV-1) strains ANG, ANG path, HFEM, tsB5 and HSZP cause extensive cell fusion and were mapped to the cytoplasmic domain of glycoprotein B (gB), within the syn 3 locus. These strains are so far the only ones which show the phenotype 'fusion from without' (FFWO): 60 min after infection with high m.o.i., cells in a tissue culture are fused without transcription and translation of the viral genome. In this report we detected, using the recombinants 27/III and K-7, that an amino acid exchange from Ala to Val at aa position 854 of gB is the main determinant for FFWO activity of strains ANG, ANG path and recombinant K-7. The transfer of this mutation to wild-type strains KOS and 17 syn+ by co-transfection results in recombinants KOS-854Q, 17-syn3, 17-syn3a and 17-syn3b. As a selection marker we used the cyclosporin A resistance of fusion which was shown to be a unique characteristic of syn 3 locus mutants. The recombinants show the FFWO phenotype in BHK cells but not in Vero cells. FFWO was shown to be cell-type dependent by comparing the number of p.f.u. needed to induce FFWO in various cell types.

Addition of heparin-Na+ as well as related substances of high and intermediate MW (Arteparon and polyanion SP54) 3 h after infection inhibit fusion from within (FFWI) induced by HSV strains with mutations in the syn 3 locus only. The concentration of heparin-Na+ required to inhibit FFWI is 10-fold higher (1 mg/ml) than that needed to inhibit adsorption. Instead of fusion, cell rounding is observed. The effect is readily reversible. A low MW heparin disaccharide is ineffective. Neomycin, at a concentration of 8 mM, inhibits FFWI induced by all HSV-1 but not HSV-2 strains, whereas adsorption is inhibited at 3 mM. We conclude from our observations that cell-cell fusion (FFWI) induced by syn 3 locus mutants of HSV-1 depends on a multistep mechanism. One may be constituted by pre-existing cell-cell connections or microfusions leading to cell rounding, whereas another may be active using newly appearing cell bridges during FFWI; also the three-dimensional structure of the cell membrane may be of importance. Moreover, the molecular mechanisms of FFWI induced by mutations in the syn 3 locus compared to the other 5 syn loci should be different.