- Volume 68, Issue 9, 1987

Immune-stimulating complexes (iscoms) have been prepared containing the major S gene products (HBsAg) of the hepatitis B virus genome. Immunization of B ALB/c mice with a single dose of hepatitis B iscoms in saline resulted in a high titre antibody response to HBsAg. In contrast, the original HBsAg preparation required an adjuvant to produce equivalent amounts of antibody. Analysis of sera from mice immunized with hepatitis B iscoms revealed antibodies directed against the major a determinants of HBsAg. High secondary antibody responses were observed in immunized animals previously inoculated with a sub-immunogenic dose of HBsAg indicating that hepatitis B iscoms may represent a suitable immunogen for use in individuals in whom a course of immunization with currently licensed hepatitis B vaccines has failed to produce a significant anti-HBs response.

A cDNA clone of the infectious bronchitis virus (IBV) spike protein gene has been recombined into vaccinia virus. Cells infected with the recombinant virus synthesized IBV spike antigen which was recognized by antibody raised against purified spike protein. Immunofluorescence showed that the IBV spike antigen was transported to the infected cell surface membrane and immunoprecipitation showed the presence of the glycosylated 180K mol. wt. polypeptide precursor of the two spike subunits S1 and S2 that comigrated with this antigen from IBV-infected cells. Vaccinated mice produced antibody that recognized the IBV spike antigen by ELISA and which neutralized IBV infectivity as shown by ciliostasis tests on tracheal organ cultures.

The location of a promoter (PF) in the HindIII F region of the vaccinia virus genome was mapped by introducing deletions into this region of the DNA. Modified promoters were fused to the herpes simplex virus (HSV) thymidine kinase (TK) gene in plasmids facilitating the construction of recombinant vaccinia viruses, and promoter function was monitored by the ability of such plasmids to rescue TK+ vaccinia viruses from cells infected with TK− virus. Deletions from the 3′ end of the promoter region produced mutants for which function was either not inhibited or abolished, allowing the 3′ promoter boundary to be defined to within 13 nucleotides. As indicated by the presence of the PF transcript in early RNA and the kinetics of HSV TK expression in recombinant vaccinia viruses, transcription from PF occurred primarily at early times during infection. The major transcript was initiated at a site within 20 nucleotides of the 3′ end of the promoter and nine bases upstream of the probable translation initiation codon. In one mutant for which a small but reproducible increase in promoter function was detected, the transcription start site was deleted. Nevertheless, transcription still appeared to begin at the equivalent position with respect to the promoter, despite the altered nucleotide sequence. The location of the start site for the PF transcript indicated that the HSV TK gene, inserted at the BamHI site following the promoter, was preceded by an initiation codon which could potentially attenuate expression of the inserted gene. Conversion of this ATG codon to TAG did not significantly improve HSV TK expression.

Two bovine rotaviruses, C3-160 and 17/4, which multiplied in calves without inducing disease, were studied for their ability to protect against disease caused by a virulent rotavirus, CP-1. Rotavirus 17/4 and the virulent virus CP-1 cross-neutralized poorly and, on the basis of 20-fold differences in neutralization titres, belonged to different serotypes. Rotaviruses C3-160 and CP-1 were more closely related: neutralization of the CP-1 virus by C3-160 antisera was within 20-fold of the homologous titre although neutralization of the C3-160 virus by CP-1 antisera was not. Nine gnotobiotic calves infected with either C3-160 or 17/4, had rotavirus antibody in their faeces and/or serum 21 days after oral inoculation as detected by indirect immunofluorescence and IgG, IgM and/or IgA antibodies by ELISA. As expected from the antigenic relationships between the viruses, the sera and faeces from the four calves infected with C3-160 contained moderate levels of neutralizing antibody to the virulent virus CP-1 and the sera and most of the faeces from the five calves infected with 17/4 contained undetectable or low levels. When challenged with CP-1 on day 21, four age-matched controls developed disease whereas all of four calves previously infected with C3-160 and four of five calves previously infected with 17/4 were protected from disease. It was concluded that avirulent rotavirus infection provided protection against disease caused by a virulent rotavirus even when one of the avirulent viruses was poorly related to the virulent virus by neutralization. Mechanisms other than neutralizing antibody appeared to be important in protection.

The primary vector species for bluetongue virus (BTV) in the United States, Culicoides variipennis, was orally infected with BTV serotype 10, BTV serotype 17, or a mixture of the two viruses. The recovery of virus from the infected flies was low following a period of extrinsic incubation. Electrophoretic analysis of progeny virus from singly infected flies revealed that only the parental electropherotype could be isolated from those flies. In contrast, electrophoretic analysis of virus from mixedly infected flies revealed that eight of the 11 virus-positive flies produced virus progeny with reassortant electropherotypes. The proportion of reassortant progeny varied from 7 to 78% (mean 42%), depending on the individual fly. Analysis of segregation of the parental origin of genome segments in the reassortant progeny virus suggested that, while reassortment of most segments was random, selection for genome segment 8 from the type 17 parent may have occurred. Analysis of segregation in individual mixedly infected flies showed that each fly yielded a relatively unique set of reassortants, but that specific electropherotypes were isolated repeatedly from individual flies. These data indicated that the vector species C. variipennis was a permissive host for high frequency reassortment of genome segments of BTV.

Arboviruses with segmented genomes have the potential to reassort in both their vertebrate hosts and arthropod vectors. Reassortment of Thogoto virus, a tick-borne orthomyxo-like virus, has been demonstrated following dual infection of hamsters by temperature-sensitive mutants. To investigate whether similar events can occur in ticks, Rhipicephalus appendiculatus larvae and nymphs were dually infected by interrupted feeding on viraemic hamsters. Wild-type reassortant virus was isolated from the ticks 12 to 15 days after engorgement. Following moulting, nymphs and adults transmitted reassortant virus to uninfected hamsters. This is the first reported evidence that a tick-borne arbovirus can reassort in vivo in a naturally infected arthropod vector. The relative roles of vector and vertebrate host in generating and perpetuating reassortant viruses in nature are discussed.

The morphology of border disease virus and bovine virus diarrhoea virus in infected bovine embryonic testis cells was examined by electron microscopy. Particles which appeared to be mature virions of both viruses were similar, being roughly circular and approximately 46 nm in diameter with a 20 to 25 nm core. Virus replication took place totally within the cytoplasm in association with structures formed from modified endoplasmic reticulum.

The complete nucleotide sequence of the genes coding for the two membrane glycoproteins E1 and E2 of rubella virus has been determined from cloned cDNA derived from the 40S genomic RNA. A sequence of 2451 nucleotides extending from the poly (A) tract towards the 5′ end is presented. Within one continuous open reading frame E2 is located amino-terminally followed by E1 and a 58 residue long untranslated 3′ region preceding the poly(A) tract. The coding regions of E2 and E1 are unusually G/C rich, 71·4% and 66·4% respectively. At the carboxy-terminal end of the coding region of E1, there is an inverted complementary nucleotide sequence, which could form a 13 base pair hairpin structure. Mature E2 and E1 are both preceded by a stretch of uncharged mainly non-polar amino acids, 21 and 20 residues in length, respectively. These could serve as signal peptides that mediate the membrane translocation of the proteins. At the carboxy termini of both proteins there are stretches of hydrophobic amino acids, 19 and 27 residues in length, which probably represent the transmembrane anchors of the proteins. The size of mature E1 is 481 amino acids (mol. wt. 51502), whereas the exact size of mature E2 could not be established as its carboxy- terminal end could not be located in the sequence. A maximum size of 262 amino acids (mol. wt. 28277) is, however, suggested. Between the E2 and E1 genes, there is a stretch of seven amino acids, five of which are arginines, which may serve as cleavage sites for a trypsin-like protease. E1 contains three and E2 four potential sites for asparagine- linked glycosylation. Both proteins are cysteine-rich (5 %). Comparison of the rubella virus amino acid sequence to those of several alphaviruses indicated no sequence homology.

Goose erythrocyte membranes were isolated and tested for their ability to compete with red cell receptors for vesicular stomatitis virus (VSV) attachment and fusion at acidic pH. Crude membranes, solubilized with Triton X-100, Tween 80 and octyl-β-d- glucopyranoside, showed a dose-dependent inhibitory effect on virus binding and haemolysis. The chemical nature of the active molecules was investigated by enzyme digestion and by separation of purified components. Only the lipid moiety, specifically phospholipid and glycolipid, was found to inhibit VSV attachment; a more detailed analysis of these molecules showed that phosphatidylinositol, phosphatidylserine and GM3 ganglioside were responsible for the inhibitory activity and could therefore represent VSV binding sites on goose erythrocyte membranes. Removal of negatively charged groups from these molecules by enzymic treatment significantly reduced their activity, suggesting that electrostatic interactions play an important role in the binding of VSV to the cell surface. Enzymic digestion of whole erythrocytes confirmed the involvement of membrane lipid molecules in the cell surface receptor for VSV.

An early antigen of human cytomegalovirus (HCMV) was purified from infected cells as a 72K polypeptide by immunoaffinity chromatography using a monoclonal antibody. It was located in both nucleus and cytoplasm, and was non-glycosylated and undetectable on the surface of infected cells. Known seropositive subjects had antibody against the purified protein and it elicited proliferative T cell responses in 10 of 16 subjects. Five of 14 T cell lines established in response to the purified protein were predominantly CD8+ and of these two showed major histocompatibility complex- restricted cytotoxicity against HCMV-infected cells. This provides further evidence that antigens expressed at early times may be targets for the immune response during persistent HCMV infection.

The delayed-type hypersensitivity (DTH) response in mice infected with murine cytomegalovirus (MCMV) was measured by ear swelling following a challenge with heat-treated MCMV. DTH was dose-dependent and could be detected as early as 3 days post-infection with peak responses occurring between days 15 and 21 postinfection. The DTH response was found to be specific for MCMV since it could not be elicited by either herpes simplex virus type 1 or influenza A virus in MCMV-primed mice. The elicited DTH response was greater in mice primed with attenuated compared with virulent MCMV. The DTH response was shown to correlate positively with the genetically determined resistance status of mouse strains to this virus. Previous research has shown that resistance to lethal infection with MCMV is controlled by H-2-linked genes since mice having the k haplotype are more resistant than mice having the b or d haplotype of the H-2 complex. Also, non-H-2-linked genes in CBA, C3H, C57BL/10 and probably other strains confer resistance. Resistant strains (C3H [H-2k], CBA [H-2k]) developed greater DTH responses than those of susceptible strains (BALB/c [H-2d], C57BL/10 [H-2b]) inoculated with the same dose of virus. In addition, the genetically resistant mouse strains BIO. BR [H-2k] and BALB. K [H-2k] gave a significantly greater DTH response than that of the corresponding congenic strains C57BL/10 [H-2b], BALB/c [H-2d] and BALB. B [H-2b] which are genetically susceptible to the virus. Also, the DTH response of C57BL/10 [H-2b] was significantly higher than that of BALB. B [H-2b] which correlates with their relative genetic resistance to MCMV, indicating the importance of non-H-2-linked genes. Furthermore, in addition to the response of greater magnitude, resistant strains (CBA, C3H, B10.BR) produced DTH responses to MCMV by day 3 compared with day 5 post-infection for susceptible BALB/c mice. These findings indicate that the magnitude and the time of appearance of the DTH response correlates positively with the genetically determined resistance status, although the role of DTH responses in controlling MCMV infections remains to be determined.

Herpes simplex virus type 1 (HSV-1) strains F, HF and HFEM were studied with respect to pathogenicity in mice and growth characteristics in vivo and in vitro compared to the neurovirulent HSV-1 strains 17 syn+ and KOS. All three viruses demonstrated reduced virulence in mouse brains and were completely avirulent after footpad inoculation. They were shown to express high levels of thymidine kinase activity. Investigations concerning the virulence phenotype indicated that the defect(s) in strains F, HF and HFEM related to general replication deficiency in mouse cells. It was also shown that although the replication restriction observed for strains F and HF was specific for murine cells, strain HFEM did not replicate well in any cell type tested. Additional studies indicated that the avirulence phenotype which followed peripheral inoculation was related to different genotypes, since strain F complemented HF and HFEM and, as expected, the latter two agents did not complement each other. All three agents were found to complement the non-neuroinvasive strain KOS. Finally, the data also show that two herpesviruses which are highly restricted in murine cells (e.g. strains F and HF) can still interact in the animal and produce a lethal infection.

Induction of the major stress response in chick embryo fibroblasts, which follows infection at 38·5 °C with the herpes simplex virus mutant isK, was investigated. Synthesis of cellular stress proteins occurred only when the mutant form of an immediate early polypeptide, Vmwl75, was overproduced. Infection with mutant inl411, which has an amber (TAG) termination signal inserted between codons 83 and 84 of the gene encoding Vmwl75 and therefore specifies a truncated portion of the polypeptide, failed to stimulate stress protein synthesis. The results suggested that the presence of abnormal forms of Vmwl75 at high concentrations was the signal for induction of the stress response in tsK-infected cells.

The BamHI W YH region of the Epstein-Barr virus (EB V) genome encodes a protein localized to the nucleus of the infected cell, the EBV-determined nuclear antigen EBNA2. We have constructed a series of recombinant vectors that carried the complete EBNA2 gene, or the gene modified so as to contain defined deletions involving presumed exons and regulatory elements of the gene. The recombinant vectors were transfected into COS-1 cells which permit the replication of simian virus 40 origin-containing plasmids to a high copy number, and the transient expression of EBNA2 was analysed. A recombinant plasmid that carried a BglII-NotI subfragment of the BamHI WYH region (nucleotides 44664 to 50628) contained all the information necessary for inducing the expression of a full length EBNA2 polypeptide. Moreover, EBV DNA sequences between nucleotides 45442 and 48337 could be deleted without interfering with the ability of the vectors to induce EBNA2. On the other hand the loss of the left one-third of the BglII-NotI fragment completely abolished the EBNA2- inducing capacity of the vector. A rightward promoter consensus sequence in the BamHI W part of the BglII-NotI fragment was functional in COS-1 cells expressing EBNA2 and essential for EBV-specific RNA synthesis. The results indicated that transcription of the EBNA2 gene was initiated in the BamHI W fragment, that the transcript was spliced and that all of EBNA2 was encoded within the continuous long open reading frame in the BamHI Y and H fragments.

Strain 2 guinea-pigs were inoculated with infectious varicella-zoster virus (VZV) or with immunoaffinity-purified proteins of VZV. Monoclonal antibodies to the VZV gpI (90000/58000 complex) and to a non-glycosylated protein, pl70, were used to prepare the polypeptide antigens. Humoral and cell-mediated immune responses to the infectious virus were compared with those elicited by the gpI and pl70 proteins. Both VZV IgG antibody production and T lymphocyte proliferation to VZV were detected after immunization with infectious VZV and with VZV proteins. The antibody and T lymphocyte responses waned after protein immunization in comparison with the responses induced by infectious VZV but were detected again immediately after reimmunization with gpI or p170.

We have tested a number of herpes simplex virus type 1 (HSV-1) populations for their ability to enter and express virus polypeptides in non-permissive rat XC cells. The viruses tested included 40 intratypic F(MP) recombinants and different batches of virus, belonging to the same strains, that had been produced in two different permissive systems (HEp-2 or Vero cells). Our results indicated that the ability to infect XC cells was determined in part by genetic elements of the virus genome and in part by phenotypic characteristics conferred by the permissive cell that had produced the virus: a virus strain like HSV-1 MP which, when produced in HEp-2 cells, was able to infect XC cells, lost this ability when produced in Vero cells. Working only with viruses produced in HEp-2 cells we showed that the ability to enter XC cells could be transferred from the MP strain to the F strain (which does not normally infect XC cells efficiently) by transfer of the cloned BamHI B (map units 0·745 to 0·81) or BamHI L (map units 0·706 to 0·745) fragments isolated from MP DNA. The implicated locus or loci seemed to segregate, however, from loci controlling gC synthesis and cell fusion, which have been described as mapping in the same region.

In mice, intravenous inoculation of relatively avirulent strains of herpes simplex virus [e.g. P2C6, a mutant of strain CL(101), deficient in thymidine kinase] produced infection in the adrenal gland and mid-spinal cord followed by hind limb paralysis without death. Male mice were less susceptible to paralysis than female mice. Castration of male mice before inoculation increased their susceptibility to that of female animals; treatment with testosterone reversed this change. The differences in susceptibility to paralysis in the various categories of animal were not reflected in differences in growth of virus in the adrenal gland or spinal cord.

Host cell nuclear involvement in an arenavirus infection was examined by immunofluorescence. Both polyclonal antisera and monoclonal antibodies specific for the major nucleocapsid (N) polypeptide revealed virus-specific nuclear inclusions in Pichinde virus-infected Vero cells. Immunoprecipitation of infected cell extracts with the anti-N monoclonal antibodies and subsequent analysis by SDS-PAGE, identified two N-related proteins with mol. wt. of 36000 (p36) and 28000 (p28) in addition to the N polypeptide. Only those monoclonal antibodies which precipitated p28 as well as N and p36 were found to produce nuclear as well as cytoplasmic fluorescence. These findings suggest that either the p28 protein itself or a conformational variant of N was the nuclear antigen detected.