- Volume 70, Issue 4, 1989

The complete nucleotide sequence of the fusion protein (F) mRNA of the virulent SBL-1 strain of mumps virus has been determined by sequencing cDNA clones and mRNA and confirmed by partially sequencing the genomic RNA. The mRNA was 1721 nucleotides long excluding the poly(A) sequence and had one long open reading frame which encoded a protein of 538 amino acids with a calculated M r of 58 791. The predicted amino acid sequence had a proteolytic cleavage/activation site, Arg Arg His Lys Arg, cleavage at which yields proteins F2 and F1. The uncleaved protein contained three highly hydrophobic regions: (i) the amino-terminal signal peptide, (ii) the amino-terminal region of F1 and (iii) the carboxy-terminal membrane anchorage domain. There were seven potential N-glycosylation sites, two in F2 and five in F 1. Comparison of the virulent strain F protein sequence with that of an avirulent strain of mumps virus showed a difference of 14 amino acids. Among paramyxoviruses, mumps virus fusion protein shows the highest degree of homology with the fusion proteins of simian virus 5 and Newcastle disease virus.

The influence of the route of infection, regime of inoculation and virus preparation on murine IgG subclass responses to the haemagglutinin of influenza A virus were examined. Virus preparations inoculated by the intraperitoneal, intravenous, intramuscular and intranasal routes were found to induce different IgG subclass profiles after a primary and secondary dose. IgG2a was found to be a major contributor to the responses elicited by all virus preparations irrespective of the route of inoculation. The magnitude of the response varied with the number of doses of virus and the route of inoculation; the intravenous and intramuscular routes produced the largest responses after two doses of virus. Evidence for the local production of antibody is presented and the influence of antigen presentation on the induction of different subclasses is discussed.

Spleen cells primed by Prospect Hill (PH) or Puumala (Pu) virus could cross-react with Hantaan virus (HV) 76-118 strain-infected target cells after in vitro stimulation with HV-infected cells, although anti-PH or anti-Pu immune serum showed no cross-reactive neutralizing (NT) activity to HV without complement. These results and our previous findings with cross-reactive cytotoxic T lymphocytes (CTLs) suggest that some epitopes recognized by CTLs might be common among the hantavirus genus, while the epitopes related to NT activity were mainly specific to each virus of this genus. Next, to evaluate the cross-reactive immunities demonstrated by in vitro study, we investigated the effect of transferring T lymphocytes and sera from BALB/c mice immunized with PH or Pu virus into nude mice before HV inoculation. Transferring T lymphocytes primed by PH or Pu virus reduced HV titres in lungs and spleens of nude mice, corresponding with the results of the in vitro CTL assays. Transferring anti-Pu immune serum also decreased HV titres in nude mice, which seemed to reflect complement-dependent NT activity. Moreover ICR mice previously immunized with PH or Pu virus showed resistance to challenge with a lethal dose of the HV KHF strain, indicating that cross-reactive immunity induced by PH or Pu virus could protect ICR mice against pathogenic HV infection.

Certain scrapie strains cause obesity in several strains of mice. The potential association between obesity and altered glucose tolerance was assessed by monitoring body weight and glucose tolerance throughout the incubation period in scrapie strain–mouse strain combinations that do and do not produce obesity. Virtually all obese mice showed reduced glucose tolerance as shown by significantly higher blood glucose levels 2 h after a glucose overload. Mice injected with a scrapie strain that did not cause obesity showed normal tolerance. The scrapie infectivity titre of the pancreas of obese mice clinically affected with scrapie was very low. Adrenalectomy prevented both the increase in weight and aberrant glucose tolerance but had no other effect on the course of the disease. Following increasing dilution of the inoculum, the increase in body weight and the development of aberrant glucose tolerance reached an end-point that was similar to that of scrapie infectivity. The system described provides an inducible model of obesity with altered glucose tolerance.

The DNA sequences of genomes from G + C-rich and A + T-rich lymphotropic herpesviruses [i.e. gammaherpesviruses; Epstein–Barr virus and herpesvirus saimiri (HVS)] are deficient in CpG dinucleotides and contain an excess of TpG and CpA dinucleotides relative to frequencies predicted from their mononucleotide compositions. In contrast, for sequences from genomes of G + C-rich and A + T-rich neurotropic herpesviruses (i.e. alphaherpesviruses; herpes simplex virus and varicella-zoster virus) and human cytomegalovirus (HCMV; a betaherpesvirus) the mean observed frequencies of these dinucleotides are close to those expected from their mononucleotide compositions. Comparisons between DNA sequences that encode proteins conserved in all these viruses also show that sequences of these lymphotropic viruses are CpG-deficient whereas the homologous genes from the neurotropic viruses and the HCMV are not. Analyses of local variations in dinucleotide frequencies reveal some occurrences of clustered CpG dinucleotides in generally deficient genomes (e.g. upstream of the thymidylate synthase gene of HVS) and locally CpG-deficient regions within some generally non-deficient genomes (e.g. the major immediate early genes of human, simian and murine CMVs). A relative deficiency in CpG and an excess of TpG and CpA dinucleotides is a diagnostic feature of higher eukaryotic DNA sequences that have been subjected to methylation of cytosine residues in CpG doublets with the resulting increase in mutations to give TpG (and thereby its complement, CpA). The available evidence implicates the latent genome as the site of methylation of these herpesviruses. We conclude that in the neurotropic herpesviruses the normal latent precursors to infectious progeny are not methylated whereas there is local methylation of the immediate early locus in the latent genomes of CMVs, and the latent genomes of these lymphotropic herpesviruses are extensively methylated.

Penetration of the KOS strain of herpes simplex virus type 1 (HSV-1) and the MS and 333 strains of herpes simplex virus type 2 (HSV-2) into HEp-2 cells at pH 6·3 was at least 100-fold less efficient than at pH 7.4. Penetration of two low passage clinical isolates was completely blocked at pH 6·3. The syncytium-forming HSV-1 strains GC and MP were less sensitive than KOS to the mild acidic conditions. The inhibition was completely reversed upon neutralization of the medium. Penetration was assayed by plaque production following protection from acid inactivation upon virus entry. Penetration of HSV-1 KOS into Vero and HEL diploid fibroblast cells was similarly inhibited. HSV-1 KOS grown in 2-deoxy-d-glucose and monensin was also extensively inhibited at pH 6·3 but virus grown in 2-deoxy-d-glucose penetrated more slowly than normal virus at pH 7·4. Electron microscopy of HSV-1 KOS infection indicated that fusion and endocytosis occur at both pH 7·4 and 6·3 but that fusion predominates at pH 7·4 and endocytosis predominates at pH 6·3. These results indicate that fusion at the plasma membrane is the major route of productive entry for HSV, that strains of HSV can differ in their pH dependence for penetration and this may determine whether virus infection can occur following endocytic uptake.

A genetically engineered herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) deletion mutant has been constructed and used to investigate the role of this gene in pathogenesis. Inoculation of mice with the HSV TK deletion mutant resulted in the establishment of latent ganglionic infection as demonstrated by superinfection of explanted ganglia with wild-type (wt) virus but not by routine explant culture suggesting that the virus-encoded TK is not essential for the establishment of latent infection but may be necessary for either reactivation or virus replication following reactivation. In addition, Southern blot hybridization has been used to demonstrate in vivo complementation of this mutant by wt virus in both peripheral and central nervous system tissues of mice during acute infection and to show that such complementation can result in the establishment and reactivation of latent TK– infection.

The La antigen is known to associate, at least transiently, with a series of small nuclear and cytoplasmic ribonucleoprotein particles (snRNPs and scRNPs), e.g. U1 and U6 snRNPs. In CV-1 cells a monoclonal antibody (MAb), directed against the La protein (La1B5), immunostained intranuclear speckles. These speckles were found to co-localize with speckles that were stained by MAbs directed against either all U snRNPs or only against U1 snRNPs. Two h after infection of CV-1 cells with herpes simplex virus type 1 (HSV-1) (strain HFEM) the staining of nuclear speckles with the anti-La MAb disappeared and the La protein was found quantitatively in the cytoplasm. In contrast nuclear speckles remained stained with the MAbs against the U snRNPs. Similar results were obtained using HSV-1 strains Lenette or 17 syn+ or temperature-sensitive (ts) mutants defective either in DNA synthesis (tsS) or in the immediate early protein (M r 175 K) (tsK). Later in infection the La protein returned to the nucleus. Six h after infection most of the nuclear La protein was found to localize within patchy regions. These areas seem to be related to heterogeneous nuclear RNA transcription and/or processing sites, but not to DNA replication sites.

Immunoglobulin G Fc receptors (IgG FcRs) induced by human cytomegalovirus (HCMV) were isolated from solubilized HCMV-infected cell extracts by IgG affinity chromatography and analysed by Western blotting using 125I-labelled human IgG and Fc. FcR species of M r 130K, 65K, 50K and 38K were found to mediate the binding of IgG. The 130K and 65K FcRs were also detected on HCMV virions. All four FcRs were reactive with a murine monoclonal antibody to herpes simplex virus glycoprotein E. Anti-HCMV antibodies markedly inhibited the binding of 125I-labelled human IgG Fc to the 130K and 50K species but inhibited to a lesser extent the binding to the 65K FcR.

Five thymidine kinase (TK)-deficient mutants (B1 to B5) of bovine herpesvirus type 1 (BHV-1) were isolated by selection for resistance to the nucleoside analogue bromovinyldeoxyuridine. The genetic lesion in mutant B1 was localized in a 2·7 kb SalI-SalI subfragment (fTK2.7) which maps between 0·456 and 0·475 within the HindIII A fragment of the BHV-1 genome. The tk genes from wild-type and the TK– mutants B1 to B5 were cloned and sequenced using eight unique synthetic primers designed from a published sequence. The BHV-1 tk gene sequence for the strain 6660 contained some differences compared with that published previously for strain LA. Alignment of the predicted amino acid sequence of the BHV-1 TK polypeptide with different herpesvirus TKs revealed five strongly conserved regions and also identified putative functional relationships with other enzymes. Several interesting features were apparent in the tk gene sequences from the TK– mutants. The TK mutant B1 was a typical frameshift and chain termination mutant due to the deletion of a single base. The tk gene sequence of mutant B2 revealed the deletion of three bases resulting in the loss of valine at amino acid residue 174 of the TK polypeptide. The tk genes of mutants B3 to B5 contained an identical change of a single base addition resulting in frameshift and premature chain termination. In contrast to wild-type BHV-1, the TK-defective mutants were incapable of adsorbing TK-neutralizing antibodies from serum.

The complete nucleotide sequence of the genome of the enterovirus swine vesicular disease virus (SVDV; H/3 76) isolated from a healthy pig has been determined using molecular cloning and DNA sequencing techniques. The RNA genome was 7400 nucleotides long, excluding the poly(A) tract, and appeared to encode a single polyprotein of 2185 amino acids. The predicted amino acid sequence of the polyprotein showed close homology (around 90%) to that of the previously sequenced coxsackie-viruses B1, B3 and B4, and also showed homology (around 60%) to that of poliovirus. This homology allows us to predict the possible cleavage sites of the polyprotein and to identify other features of structural and functional significance, which seem to be important to the biological integrity of the virus. A detailed analysis of homology between SVDV and coxsackieviruses shows that non-structural proteins are highly conserved whereas the structural proteins are less well conserved. The 5′ and 3′ non-coding regions are also conserved, although there are several divergent nucleotide stretches. These stretches may differentiate SVDV from coxsackieviruses.

A sensitive immunoradiometric assay for murine interferon γ (MulFN-γ) has been developed and used reproducibly to measure low levels of MulFN-γ in lung lavage samples from influenza-infected mice. In control infected mice, IFN-γ peaked on day 6, but transfer of virus-specific cytotoxic T cells or T helper cells, which reduced virus replication in vivo in infected hosts, resulted in an earlier peak on day 4.

Cytomegalovirus (CMV) encodes several glycoproteins reported to be structural homologues of glycoproteins encoded by herpes simplex virus type 1 (HSV-1). To map the antigenic and functional domains on the 907 amino acid CMV glycoprotein B (gB), we cloned and expressed a subfragment of BamHI fragment R of the CMV (Towne) genome into an expression vector and reacted the resulting gene product with a panel of monoclonal antibodies. Our results showed that the DNA fragment encodes related glycoproteins which we previously designated gA and which others have reported to be homologous to HSV-1 gB in CMV (AD169). Analyses of the processing of CMV gB transiently expressed in eukaryotic cells showed that glycosylation occurred independently of viral infection. Ten antibodies with complement-dependent and independent neutralizing activity reacted with a truncated derivative of gB that contained 619 amino-terminal residues but lacked the transmembrane and intracellular regions of the molecule. Twelve additional antibodies reacted with a CHO cell line expressing a 680 amino-terminal derivative of gB. All of the reactive antibodies precipitated the 447 residue carboxy-terminal cleavage product of gB from extracts of CMV-infected cells. These results showed that the neutralizing epitopes map in at least two domains of gB which are located in a discontinuous segment of 219 amino acids between residues 461 and 680 from the amino terminus of the molecule.

The large envelope (E) protein of flaviviruses is the viral surface protein that contains neutralizing epitopes. We have analysed the E protein of the West Nile (WN) flavivirus for neutralizing epitopes generated from linear segments of the protein; if effective, these might allow the synthesis of peptides suitable for vaccination. For this study we used the E protein and defined fragments thereof as antigens in rabbits. The sera thus obtained, containing antibodies to E protein as shown by Western blot analyses, were tested for neutralizing activity by the plaque reduction neutralization test. If the E protein used as antigen was reduced (the native E protein contains six disulphide bridges) and denatured the resulting antibodies did not consistently demonstrate neutralizing activity. These results show that the E protein of WN virus does not contain a linear segment that is able to induce neutralizing antibodies efficiently. Studies using denatured E protein fragments containing subsets of the intact disulphide bridges showed that the local covalent primary structure of the protein involved in each of the six bridges was also insufficient for inducing the synthesis of neutralizing antibodies. The complete E protein, with all six disulphide bridges intact, purified by preparative SDS–PAGE under the conditions used in our experiments could, however, induce the synthesis of neutralizing antibodies in rabbits. These data indicate that at least one complex epitope which is able to induce neutralizing antibodies is not completely denatured or can be reformed to some extent if the complete E protein has been subjected to SDS–PAGE without prior destruction of the disulphide bridges.

Six monoclonal antibodies (MAbs) to bovine coronavirus (BCV, Quebec isolate) E2 and E3 glycoproteins which were found previously to be neutralizing in vitro were examined for virus-neutralizing activity in vivo. Surgically ligated intestinal loops of newborn colostrum-deprived calves were virus-inoculated, mock-infected or inoculated with a mixture of virus and antibody. Of the six BCV-specific MAbs, four were found to be protective against a virulent field isolate of BCV, as indicated by a reduction in villous atrophy. These MAbs were specific to antigenic domain A and antigenic domains A1 and A2 on the E2 and E3 glycoproteins respectively. MAbs to antigenic domains B and C on the E2 and E3 glycoproteins, respectively, were not protective.

The pattern of human papillomavirus type 16 (HPV-16) integration was studied in 23 invasive carcinomas of the cervix using subgenomic probes. Seventeen tumours contained integrated HPV-16 and in 13 of these there was evidence of disruption within the E1–E2 open reading frames (ORFs). In all cases the upstream regulatory region (URR)–E6–E7 ORFs was maintained intact. Two independently derived tumours were infected with episomal wild-type HPV-16 and an episomal variant of HPV-16 containing a 325 bp deletion within the URR (positions 7598 to 17) and a point mutation at position 20 (A to C). This is the first report of a variant HPV-16 which is likely to be both defective and transmissible. Loss of E2 expression and deletion of a large portion of the URR may be two of the mechanisms leading to altered HPV-16 early gene expression in cervical tumours.

Papillomas, carcinomas in situ and squamous cell carcinomas were induced using ultraviolet irradiation in the hairless mouse strain Mus musculus HRA/Skh. DNA extracted from biopsies was examined using Mastomys natalensis papillomaviral DNA as a hybridization probe at reduced stringency. Sequences homologous to the probe were detected in 16 of 24 papillomas, five of five carcinomas in situ and six of 38 squamous cell carcinomas. A number of tumour DNAs (16/33) also hybridized with mixed DNAs of human papillomavirus types 11, 13, 16 and 18 at reduced stringency. This suggests a role for the hairless mouse as a laboratory model for the study of the involvement of papillomaviruses in malignant transformations.

Genetic variation between clones selected from early passage pools of Wallal virus (Reoviridae, Orbivirus) was investigated. The virus had been isolated in 1970 from a pool of 100 insects (Culicoides dycei), caught in the wild, by laboratory passage in suckling mice. Gel electrophoresis and oligonucleotide fingerprint analysis of clones indicated that multiple reassortant genotypes were present in early passages of the original isolate. The virus, previously described as the prototype strain of Wallal virus, was one of the reassortant clones. A virus recovered during reisolation from the same insect pool was genotypically and serologically distinct from the prototype strain and represents a second Wallal serotype. We have concluded that the clonal variation was due to the presence of two orbivirus serotypes in the original insect pool and that a range of reassortant viruses were generated during early passages of the material in mice.

A cotransfection method has been developed for the generation of recombinant Oryctes baculoviruses. The permissive coleopteran cell-line DSIR-HA-1179 was transfected with a mixture of Oryctes baculovirus DNA (strain PV505) and a transfer vector. The transfer vector, a pUC8-based plasmid, contained the polyhedrin gene from the Autographa californica nuclear polyhedrosis virus, flanked by Oryctes baculovirus DNA from the HindIII fragment N. Oryctes baculovirus does not form plaques with DSIR-HA-1179 cells. Therefore an endpoint dilution method was used to screen for, recover and purify recombinants. There was no phenotypic character that could be used for detecting recombinants, so a dot blot assay was used to screen infected cultures for the presence of recombinants. A recombinant generated by this method contained the entire polyhedrin gene inserted at 98.03 map units from the designated start of the Oryctes baculovirus physical map. No evidence of transcription or translation of the polyhedrin gene was obtained.