- Volume 69, Issue 11, 1988

In order to overcome serological cross-reactions among orbivirus serogroups, which can hinder the accurate diagnosis of bluetongue virus (BTV) infection of livestock, a blocking ELISA (B-ELISA) incorporating a monoclonal antibody (20E9B7G2) with specificity for the BTV serogroup was developed. Experimental antisera raised to South African BTV serotypes 1 to 19 were tested in the B-ELISA and all blocked the binding of 20E9B7G2 to BTV antigen. The sensitivity and specificity of the assay was evaluated with a range of experimental and field sera and compared to a sensitive indirect ELISA (I-ELISA) for the detection of BTV-specific antibodies. The specificity of the B-ELISA was absolute for antibodies to BTV, showing no cross-reaction with experimental antisera to serotypes of the closely related orbivirus causing epizootic haemorrhagic disease of deer. The sensitivity of the B-ELISA exceeded that of the I-ELISA. In particular, the B-ELISA detected a BTV-specific antibody response much earlier after infection that the I-ELISA, while still exhibiting full sensitivity to BTV antibody titres several months after infection.

A panel of monoclonal antibodies (MAbs) raised against an Indian strain of Japanese encephalitis (JE) virus was used to map topographically the epitopes on the envelope protein. Two separate clusters of epitopes were revealed. On the basis of reactivity in haemagglutination inhibition (HI), neutralization (NT), passive protection and antibody-dependent plaque enhancement (ADPE) assays with the MAbs, five functional domains (A, B, C, D and E) were delineated. The flavivirus cross-reactive domain for HI (A) was distinct. The JE virus-specific domain for HI (B) was in continuum with those domains representing non-HI JE virus-specific MAbs (C) and flavivirus cross-reactive MAbs (D). Domain E, which mapped close to domain D was represented by two MAbs that reacted with both JE virus and uninfected cell nuclei. Four conclusions can be drawn. (i) Two distinct antigenic domains were associated with HI, (ii) HI and NT in vivo and in vitro were dissociated functions, (iii) ADPE activity was solely linked with the A domain and (iv) all MAbs reacting with epitopes in the B domain had HI/NT/protective activity but failed to show ADPE. The B domain might therefore be considered the most suitable for development of synthetic or genetically engineered vaccines.

Examination of the intestinal contents of free-living Oryzomys nigripes rats by PAGE revealed two sharply defined bands that could be stained by ethidium bromide or by silver nitrate with comparable intensities. The molecules forming these bands were susceptible to digestion by pancreatic RNase A but not by RNase T1 or by DNase I. Their lengths were estimated to be about 2.6 and 1.5 kbp, respectively, by comparison with rotavirus SA11 genome segments. They cosedimented in CsCl gradients at a density of 1.39 to 1.40 g/ml, together with uniform particles approximately 35 nm in diameter with indistinct surface structure. It is suggested that these particles represent an as yet undescribed virus with a bisegmented double-stranded RNA genome, for which the name ‘picobirnavirus’ is proposed.

The maximum proportion of skeletal and/or skin defects induced by the Semliki Forest virus (SFV) mutant ts22 in the 17-day-old foetal mouse occurred following infection of the mother at day 10 of pregnancy. The skeletal defects were detected using a combination of Alcian blue staining for cartilage and Alizarin red staining for bone. Using immunogold-silver staining with anti-SFV IgG and in situ hybridization with a cDNA probe to SFV non-structural sequences, we have shown that mesenchymal cells in the dermis and surrounding developing cartilaginous plates were heavily infected in most foetuses at day 17 of pregnancy, following infection of the mother at day 10. Other infected foetal tissues contained less viral antigen and nucleic acid; they included the liver, muscle (including myocardium), lung and kidney. The central nervous system contained only small amounts of viral antigen and nucleic acid. It is proposed that the skeletal and skin defects induced in mouse foetuses by ts22 infection result from the tropism of the virus for mesenchymal cells involved in the development of such tissue.

The hepatitis B (HB) virus DNA sequences coding for the pre-core (preC) or C antigens (HBpcAg, HBcAg) have been inserted into the baculovirus plasmid transfer vector, pAcYM1, such that the HB viral sequences are under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells infected with either of the derived recombinant plasmids in the presence of infectious AcNPV DNA yielded recombinant, polyhedrin-negative viruses that expressed high levels of the respective HBpcAg or HBcAg (representing approx. 5 to 10% and approx. 40% of the stained cellular proteins, respectively). The particulate 27 nm HBcAgs have been purified to homogeneity from infected cell extracts by density gradient centrifugation. Dual expression transfer vectors containing the HBcAg gene sequences and the coding sequences of the HB viral S antigen (HBsAg), each gene under the control of its own copy of the polyhedrin promoter, have also been constructed and used to derive recombinant viruses. The recombinant with the HB C and S genes expressed high levels of the HBcAg (approx. 40% of the cellular proteins) and low levels of the HBsAg (approx. 2% of the stained cellular proteins). Dual expression, occluded, recombinant baculoviruses that make HBsAg, as well as the AcNPV polyhedrin protein, have been prepared that are highly infectious for Trichoplusia ni caterpillars, allowing reproducible preparation of the antigen in larvae. Using radioimmunoassays (RIAs) and ELISAs, the recombinant HBcAg (RIA) and HBsAg (ELISA) have been used to identify human antibodies to HB virus with results that compare favourably with the data obtained with non-recombinant antigens.

Two split influenza virus vaccines administered intragastrically induced lower titres of haemagglutinin (HA)-specific antibodies in pulmonary secretions than whole virus vaccine or a third split virus vaccine. IgA antibody was the predominant HA-specific Ig class. HA-specific IgA titres decayed substantially within 2 weeks following booster immunization, but persisted for at least another 3.5 months. In contrast, HA-specific IgG was maintained at low titres throughout the 4 month study period. When the total vaccine antigenic mass was administered as one dose or as equally divided doses spread over several days, pulmonary antibody responses were comparable. Mice immunized intragastrically with whole virus vaccine were completely protected against intranasal challenge with a homologous virulent virus of the H3 subtype. Partial protection was obtained when the vaccine used for immunization was a distantly related, antigenically variant strain of the same subtype, but no protection was obtained with a monovalent vaccine of an influenza A subtype different to the challenge virus. These characteristics of the response to intragastric immunization against influenza are consistent with features of a useful vaccine.

At the pH required to trigger the membrane fusion activity of the influenza virus haemagglutinin (HA) the soluble ectodomain of the molecule, BHA, which is released from virus by bromelain digestion, aggregates into rosettes. Analyses of soluble proteolytic fragments derived from the rosettes indicated that aggregation is mediated by association of the conserved hydrophobic amino-terminal region of BHA2, the smaller glycopolypeptide component of each BHA subunit. Further analyses of the structure of the soluble fragments and of HA in its low pH conformation by electron microscopy, spectroscopy and in crosslinking experiments showed that, although the membrane distal globular domains lose their trimer structure at the pH of fusion, the central fibrous stem of the molecule remains trimeric and assumes a more stable conformation. The increase in length of BHA2 at low pH observed microscopically appears to result from movement of the amino-terminal region to the membrane proximal end of the molecule and in virus incubated at low pH the amino terminus may insert into the virus membrane. The consequences of these possibilities for the mechanism of membrane fusion are discussed.

Fusion of rubella virus-infected cells was induced by their brief treatment at pH below 6.0. Exposure of rubella virus to pH 5 caused an irreversible conformational change of the viral envelope glycoproteins, E1 and E2. The change was manifested in the marked reduction in both infectivity and haemagglutinating activity of the virus, the increased resistance of E1 and decreased resistance of E2 polypeptides to proteolytic digestion with trypsin, and the acquisition of liposome-binding activity of the virus. The above changes are presumed to mimic the events occurring in the acidic environment within endosomes following endocytosis of the virus.

Chicken spleen cells latently infected with Marek’s disease virus were cultured with and without conditioned medium (CM) obtained from concanavalin A-stimulated chicken spleen cell cultures. The expression of viral internal antigen(s) (VIA), which is usually associated with cultivation, was prevented or markedly reduced by the CM. This effect required the continued presence of CM, since its removal after 48 h resulted in the subsequent appearance of VIA. Although CM contains both gamma interferon (IFN-γ) and interleukin 2, our studies suggest that the ‘latency-maintaining activity’ (LMF) may not be associated with either of these products of stimulated lymphocytes. However, IFN-γ may also have had some suppressive effect. LMF appears to have an M r greater than 10000 and to be inactivated by heating to 90 °C for 5 min.

We present the nucleotide sequence of the glycoprotein H (gH) gene of herpesvirus saimiri (HVS), a representative of the T lymphotropic herpesviruses of New World monkeys, and compare the predicted amino acid sequence with sequences of homologous proteins from four human herpesviruses. The HVS gH gene is located within a block of genes encoding products conserved in all herpesvirus subgroups as represented by the human herpesviruses herpes simplex virus, varicella-zoster virus, cytomegalovirus and Epstein-Barr virus. In agreement with the biological grouping of HVS as a lymphotropic gammaherpesvirus, its gH amino acid sequence shows greatest similarity to that of the B lymphotropic Epstein-Barr virus, although the nucleotide sequences of their respective gH genes show little similarity given different G+C compositions of 31% and 54%. The similarity observed between the gH amino acid sequences of the two representative gammaherpesviruses is greater than that between the two human alphaherpesviruses varicella-zoster virus and herpes simplex virus. The members of the gH family range in size from 706 to 743 amino acid residues for the beta- and gammaherpesviruses, to 838 to 841 for the alphaherpesviruses, giving non-glycosylated precursors with M r values of 78322 to 93651. The difference in size is due to heterogeneity in the poorly conserved N-terminal regions of the larger alphaherpesviruses compared to the smaller beta- and gammaherpesvirus molecules. Greatest similarity is observed in the C-terminal halves of the proteins including residues surrounding four conserved cysteine residues, a conserved N-linked glycosylation site (within the sequence NGTV) 13 to 18 residues proximal to the membrane-spanning sequences, and a short cytoplasmic domain of seven or eight residues for the beta- and gammaherpesviruses’ and 14 or 15 residues for the alphaherpesviruses’ gH. Thus, the representatives of all subgroups of herpesviruses, including those with a non-human host, encode gH homologues. Together with the observation that gH of these viruses are major targets for virus neutralization by antibody, this suggests that this glycoprotein family is essential among all herpesviruses and represents a major component involved in herpesvirus infectivity.

We have determined the DNA sequence of the long repeat region (RL) in the genome of herpes simplex virus type 1 (HSV-1) strain 17, as 9215 bp of composition 71.6% G + C. In addition, the sequences of parts of the long unique region (UL) adjacent to the terminal (TRL) and internal (IRL) copies of RL were determined (2611 and 3836 bp, respectively). Gene organization in these regions of UL was deduced from the sequences and other available data. It was proposed that the region of UL sequenced, adjacent to TRL, contains three complete genes, none with significant previous characterization, and that the region of UL adjacent to IRL also contains three genes, one encoding the immediate early protein IE63. The RL sequence contains one well characterized gene, for the protein IE110, whose organization we have described previously. Between the downstream end of the IE110 gene and UL there is a 3500 bp segment of RL in which we did not find convincing protein-coding sequences, and which thus remains of obscure functionality. Upstream of the IE110 gene is a region previously proposed by others to contain a gene. However, our sequence data are not compatible with their interpretation. We do consider it possible that the region is protein-coding, but regard gene organization here as still unresolved.

Purified preparations of herpes simplex virus type 1 Angelotti were digested with the exoglycosidases sialidase, β-galactosidase, N-acetyl-β-d-glucosaminidase and α-mannosidase, and with the endoglycosidases Endo-H and Endo-F. It was found that treatment of virions with Endo-F specifically decreased viral infectivity by a factor of 10. This reduction in titre was not associated with any measurable differences in virus adsorption, suggesting a role of N-linked complex type oligosaccharide chains in penetration. In contrast, a reduction in titre observed upon digestion of virions with exoglycosidases could be attributed to a proteolytic contamination in these enzyme preparations. Treatment of virions with Endo-H, demonstrated to be free of proteolytic contamination, did not reduce viral infectivity. Analysis of endoglycosidase-digested virions by monospecific antibodies and immunoblotting revealed a susceptibility of all four major glycoproteins (gC, gB, gE and gD) to Endo-F, but only gB was susceptible to Endo-H treatment. In contrast, of all the exoglycosidases used only sialidase was found to be active towards native viral glycoproteins. Upon analysis of endoglycosidase-digested virions we could not find any evidence for proteolysis, degradation or altered protein composition of viral envelopes. In contrast, vigorous inhibition of glycoprotein glycosylation by tunicamycin led to the formation of physically intact virions almost completely lacking all major glycoproteins. These data show that digestion of intact virions with glycosidases allows an analysis of the functional relevance of carbohydrate residues without any obvious alterations in the virion glycoprotein composition.

Human fibroblast FS-4 cells when infected with herpes simplex virus type 1 (HSV-1) become susceptible to lysis by purified populations of T3- human natural killer (NK) lymphocytes. Blocking of HSV-1 protein synthesis or N-linked glycosylation with pactamycin or tunicamycin, respectively, prevented HSV-1-infected cells from being lysed, suggesting that HSV-1 glycoprotein synthesis is required for recognition by NK cells. However, pactamycin- and tunicamycin-treated cells expressed on their membranes a detectable amount (20 to 40% of the untreated control) of HSV-1 glycoproteins gB, gC and gD, left by the virus during its internalization. Phosphonoformic acid (PFA) blocked HSV-1 DNA replication and inhibited the synthesis and surface expression of newly made gC, gD and gB by 90, 80 and 60% respectively. Despite this reduction, PFA treatment had no effect on NK susceptibility. The target structure recognized seems to be different from those expressed on tumour target cells since there was no competition for the lysis of HSV-1-infected FS-4 by K-562 or HeLa tumour target cells. However, a monoclonal antibody specific for the human transferrin receptor which inhibited NK recognition of tumour cells also blocked NK cytotoxicity of HSV-1-infected cells. In summary our results indicate that although viral glycoprotein synthesis is required, gB, gC and/or gD alone are not the targets for NK recognition of HSV-1-infected cells. In addition, they suggest the involvement of the host cell transferrin receptor in the NK killing process.

The α or immediate early mRNA of herpes simplex virus strain HSV-2(G) had a half-life of about 15 min if made in the absence of viral protein synthesis but was relatively stable if viral protein synthesis occurred, either freely or restricted by the presence of the proline analogue azetidine. In contrast, the α mRNA of other strains of the virus is stable, even in the absence of protein synthesis. Studies with recombinant viruses showed that the region of the viral DNA between 0.58 and 0.65 map units [which includes the gene (vhs, UL41) that controls virion-mediated shutoff of host protein synthesis] is important in determining the survival of α mRNA. In mixed infection experiments HSV-2(G) inhibited α as well as host protein synthesis but the shutoff activity appeared to be short-lived. Within 3 h after infection, as a result of protein synthesis, cells became completely resistant to shutoff by superinfecting virus.

We report on the properties of a family of related herpes simplex virus type 1 polypeptides (designated p40) of M r around 40000. The intracellular localization of these polypeptides has been examined using monoclonal antibodies and their association with viral capsids within the nuclei of infected cells has been demonstrated directly by immunoelectron microscopy. Specific DNA staining and the use of mutants defective for DNA packaging has revealed, in contrast to earlier findings, that p40 is present in empty capsids. Protein p40 is not present as a major component of full capsids or of mature virions indicating that it is transiently associated with capsids and that its removal from capsids is linked with the process of DNA packaging.

mRNA isolated from mumps virus-infected Vero cells was converted into cDNA and cloned into the PstI site of the plasmid pBR322. After screening with 32P-labelled cDNA synthesized from poly(A)+ RNA of uninfected or mumps virus-infected Vero cells, five different groups of virus-specific clones were obtained. The virus specificity of the clones was confirmed by Northern blot analysis, in which the cDNA inserts from the five different groups hybridized to mRNAs of about 2100, 1500, 1450, 2000 and 2200 nucleotides. By the use of oligonucleotides synthesized on the basis of sequences obtained from the five cDNA clones and mRNAs, the sequence of the intergenic and surrounding areas was determined. During genome sequencing, a separate gene was identified between the fusion protein (F) gene and the haemagglutinin-neuraminidase protein (HN) gene. Using oligonucleotides synthesized on the basis of the new gene sequence, cDNA clones with poly(A) were isolated from the cDNA library. The gene order was determined to be 3′ NC-P-M-F-SH-HN-L 5′ (where NC, P, M, SH, and L represent the genes for the nucleocapsid, phosphoprotein or polymerase-associated, matrix or membrane, small hydrophobic and large proteins respectively). There is one nucleotide between the P and M (A), M and F (A), and HN and L genes (G), two between the NC and P (AA) and SH and HN (3′-CG) genes, and seven between the F and SH genes (3′ GAUUUUA) as intergenic sequence. The leader sequence at the 3′ end of the genome has been determined by sequencing the dicistronic leader-NC mRNA using oligonucleotide primers. The sequence from the 3′ terminus to the NC gene start of the mumps virus genome is similar in length (55 nucleotides) to that present in Sendai virus, Newcastle disease virus and parainfluenza virus type 3, and the first five nucleotides are conserved in all negative-stranded RNA virus genomes sequenced to date.

Intergenic and flanking gene regions for the 1C-1B, 1B-N, N-P, M-1A, G-F and F-22K gene junctions of respiratory syncytial virus strain 18537, representing antigenic subgroup B, were determined by dideoxynucleotide sequencing of polycistronic cDNAs and mRNAs. Comparison with their counterparts from the subgroup A strain A2 showed that the intergenic sequences were not conserved within or between the strains. Flanking non-coding gene sequences also were generally not conserved except for the highly conserved gene-start and gene-end transcription signals. The sequence of the overlap between the 22K and L genes was conserved almost exactly between the two subgroups.

A trypsin-resistant mutant of Sendai virus, TR-5, which was obtained by passaging the wild-type (wt) virus in the presence of chymotrypsin followed by plaque purification, also had an enhanced susceptibility to activation by chymotrypsin. A trypsin-sensitive revertant, TSrev-58, derived from TR-5 had a decreased susceptibility to chymotrypsin and was susceptible to trypsin to the same degree as the wt virus. Based on the facts that TR-5 had an amino acid change (Arg→Ile) at residue 116 of the fusion (F) protein, which is the cleavage site for trypsin in the wt virus F protein, and that this change had reverted in TSrev-58, it was concluded that the above amino acid change was responsible for both the enhanced chymotrypsin sensitivity and the trypsin resistance of TR-5. In this paper, the cleavage site of the F protein of TR-5 by chymotrypsin was determined in comparison with those of the wt virus and TSrev-58. The cleavage sites were not different among these viruses, being located between residues 114 (Gln) and 115 (Ser), the two residues at the N-terminal side of the trypsin cleavage site. A possible reason for the increased susceptibility of TR-5 to chymotrypsin is discussed.

Rat cell lines were established in which the bacterial chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus (HIV) long terminal repeat (LTR) was stably integrated. The cell lines showed a repressed phenotype for CAT expression, but could be induced for it by inhibition of protein synthesis, as well as by heat-shock and chemical inducers of the cellular stress response, such as sodium arsenite, 8-hydroxyquinoline and the heavy metals cadmium and copper. A decameric sequence present in the NF-kB binding sites in the HIV LTR (GGGACTTTCC) resembles the cellular heat-shock core sequence and may therefore be involved in the heat-shock response.