- Volume 72, Issue 2, 1991

Immunoelectron microscopy was employed to examine the temporal expression and localization of two proteins involved in baculovirus polyhedron assembly (polyhedrin and p10) of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) in infected Lymantria dispar cells. In addition, the association of p10 with the polyhedron envelope (PE) protein was studied. The major capsid protein (p39) was also examined to investigate the association of virion structural proteins with polyhedron formation. In infected cells, p39 did not show a concentrated association with any infected-cell structures other than nucleocapsids and appeared to be randomly distributed over the nucleocapsid surface. Likewise, polyhedrin showed no major concentrations outside of developing or mature polyhedra. The p10 antibody cross-reacted with a protein associated with condensed chromosomes in uninfected cells. In infected cells, p10 is a component of the body of fibrillar structures. The PE protein has been shown to accumulate around the periphery of fibrillar structures. Cells infected with a polyhedrin-minus virus expressing the β-galactosidase gene under the control of the polyhedrin promoter were examined to determine whether the lack of polyhedra would influence the localization of major polyhedron-associated viral proteins. High concentrations of PE protein accumulating on the periphery of fibrillar structures appeared to be the major difference from wild-type virus-infected cells. The β-galactosidase protein appeared to be distributed throughout the nucleus and cytoplasm, in contrast with the specific localization of the viral proteins.

The organization of genes in the 2.95 kb EcoRI-SalI fragment (0.7 to 3.0 map units) located within the HindIII F region of the Autographa californica nuclear polyhedrosis virus genome was studied by a combination of DNA sequencing, Northern blot analysis, S1 mapping and primer extension analysis. In addition to the two divergent overlapping transcripts [leftward early (ES1) and rightward late (ES2)] previously reported, a third transcript which is present at 18 h p.i. and which also runs leftward, overlapping ES1 by 1600 nucleotides (nt) at the 3′ end, was mapped to this region. The DNA sequence revealed the presence of three open reading frames (ORFs) of significant length. ORF-1 and ORF-2 correspond to the leftward transcripts, and code for potential polypeptides of 151 and 329 amino acids, respectively. ORF-3 which codes for a potential polypeptide of 167 amino acids is located on the opposite strand in a region for which no transcript mapping data are available. However, the conserved late gene promoter/cap site sequence (ATAAG) is present 23 nt upstream of the start of ORF-3.

Ten mouse-passaged scrapie lines were initiated from five sheep with clinical scrapie. Of the lines, five were initiated and passaged exclusively in mice with the s7s7 genotype and the remaining five lines were initiated in mice with the p7p7 genotype, with two of these lines subsequently being passaged exclusively in p7p7 mice and two being passaged mainly in p7p7 mice. Lines were passaged three or four times and two parameters were compared: incubation period and the induction of a weight increase during the preclinical period. Considerable variation in the incubation periods was found between the different passage lines at similar passage levels, with a range in s7s7 mice of 113 days to greater than 450 days and a range in p7p7 mice of 219 days to greater than 500 days. All of the lines passaged exclusively in s7s7 mice had shorter incubation periods in this mouse genotype than in p7p7 mice, whereas of the five lines initiated in p7p7 mice, two had shorter incubation periods in p7p7 mouse strains. C57BL mice were used as the indicator strain and most of the lines caused an increase in weight during the preclinical phase of disease compared to control mice injected with normal brain homogenates. For both parameters, incubation period and preclinical weight increase, differences were seen in lines that had identical passage histories, suggesting that an informational molecule separate from host genomic material must specify scrapie strain differences.

Glutamine depletion strongly inhibits the replication of Rauscher murine leukaemia retrovirus (RLV) in vitro. Pseudomonas 7A glutaminase-asparaginase (PGA), capable of depleting glutamine and asparagine for prolonged periods, was used to determine the therapeutic effectiveness of glutamine depletion in mice infected with RLV or Friend virus. During PGA treatment of viraemic animals, serum reverse transcriptase activity fell to control levels and infected animals did not develop splenomegaly. The therapeutic results obtained with PGA compared favourably with those of azidothymidine given intraperitoneally at 30 mg/kg/day. Western blots performed on splenic tissue from control and treated animals indicated that glutamine depletion prevented readthrough of an amber codon at the gag-pol junction, stopping translation of viral mRNA at that point. Treatment of RLV-infected animals with PGA resulted in nearly a 200% increase in mean survival time even when therapy was initiated late in the course of the disease. To our knowledge, this is the first demonstration that a nutrient required for viral replication can be enzymically depleted in vivo to inhibit viral replication.

Bovine leukosis virus (BLV) is associated with the disease complex enzootic bovine leukosis. The infection may remain clinically silent in the form of an aleukaemic state or emerge as a persistent lymphocytosis and more rarely as lymphosacroma. BLV has been considered classically to be a B lymphotropic virus, based upon the absolute increase in B lymphocytes in persistent lymphocytosis, the B lymphocyte phenotype of a majority of the cells making up lymphosarcomas and the identification of viral antigen expressed in B lymphocytes following in vitro culture of peripheral blood mononuclear leukocytes. This association of BLV with B lymphocytes is well established but the mechanism(s) of disease expression is not defined. To examine further the cellular tropism(s) of BLV, T lymphocyte subpopulations from 10 lymphocytotic cattle were established in vitro. Lymphocyte cultures were characterized by their subpopulation phenotype and DNA was extracted for identification of integrated provirus by Southern blot hybridization. Provirus was identified in T lymphocyte cultures derived from seven of 10 lymphocytotic cattle, with both T helper and T cytotoxic/suppressor subpopulations affected.

Inoculation of simian immunodeficiency virus into cultures of primary rhesus monkey macrophages or CD4-bearing transformed T lymphocytes resulted in persistent infection, with minimal virus replication in the macrophages and extensive replication in the lymphocytes. However, uninfected T cells added to infected macrophages underwent rapid fusion and lysis and were almost completely eliminated without the production of virus particles. Lysis required direct contact between the T cells and the infected macrophages, which enabled binding between CD4 on the former and viral gp120 on the latter to occur. This process was blocked by soluble CD4 and dextran sulphate. Neutralizing antibodies in the serum of an infected macaque prevented cell fusion by preventing infection of the macrophages. However, these antibodies did not prevent fusion when added to previously infected macrophages. Infected macrophages were incorporated into the syncytia of lymphocytes and continued incorporation of new lymphocytes into the syncytia required infected macrophages to be metabolically active. One inference from these studies is that infected macrophages in vivo could help mediate the well known depletion of T4 cells in patients with AIDS.

Rotaviruses transcribe mRNA containing a 7mGpppGmp cap at the 5′ end in vitro. Guanylyltransferase activity associated with the viral particle was detected by SDS-PAGE due to the formation of a nucleotide-enzyme complex when the virus was incubated with [α-32P]GTP. Using purified viral particles it was shown that only the core polypeptide VP3 exhibits the ability to form a complex with the nucleotide. The reaction is specific for GTP or dGTP when Mg2+ is used as a cofactor. The reaction also depends on the incubation temperature and the pH, as described for other guanylyltransferases. The GMP-VP3 complex transfers the GMP to pyrophosphate, synthesizing GTP or GDP, resulting in the formation of a GpppG cap. These properties of the complex allowed the core polypeptide VP3 to be identified as the rotavirus guanylyltransferase.

The flavivirus Skalica was isolated from a bank vole in Czechoslovakia in 1976. It can be serologically distinguished from prototype strains of tick-borne encephalitis (TBE) virus and has a decreased virulence for adult mice. We have further defined the relationship of Skalica virus to other members of the TBE serocomplex (TBE European and Far Eastern subtypes, Langat and louping ill virus) by using a panel of 22 monoclonal antibodies, peptide mapping and RNA sequence analyses. By these criteria Skalica virus proved to be distinct from TBE virus and to be very closely related to Langat virus, differing by only two bases among a total of 416 nucleotides compared. The sequence of 22% of the Langat genome was determined and the encoded amino acid sequences were derived. Comparison of these with the corresponding amino acid sequences of TBE virus revealed a similarity of 85%, as opposed to 93% similarity between the European and Far Eastern subtypes of TBE virus.

Mumps virus (MuV) is known to be associated with acute arthritis and may also have a role in chronic inflammatory joint disease. The mechanism of induction of joint inflammation is not known but may be associated with direct invasion of joint tissue. To investigate the possibility of persistent intra-articular infection, the interaction of MuV with primary cells from normal human joint tissue was examined. These mixed cultures of synoviral membrane cells and chondrocytes were found to be semi-permissive to the virus; only a small proportion of cells (5 to 20%) were infected and produced low titres of progeny virions. In addition, little viral antigen was detected on the cell surface relative to that found on Vero cells. This restricted infection of synovial membrane cells was related to a severely decreased synthesis of the viral glycoproteins, fusion and haemagglutinin—neuraminidase, and the membrane protein in comparison to the levels found in Vero cells. Persistent infections were readily established and could be maintained for 2 to 3 months. During the first month, the infection remained highly focal and supernatant viral titres were low. Thereafter both the percentage of infected cells and viral titres increased until finally the cultures were killed. No evidence was obtained for the generation of temperature-sensitive mutants or defective interfering particles during long-term infection, but the persistent virus derived from the cultures gave cloudy plaques and induced no fusion in Vero cells until passaged. This study has shown that human synoviral tissue cells have the intrinsic ability to support MuV replication and persistence which may be important in the pathogenesis of mumps arthritis.

The genes encoding the small hydrophobic (SH) proteins of a series of respiratory syncytial (RS) virus strains were amplified using the polymerase chain reaction, cloned and sequenced. Analysis of the SH gene sequences from 12 RS virus strains isolated between 1956 and 1989 confirmed the homogeneity of the two subgroups, A and B, previously defined serologically. Although there is only 76% deduced amino acid sequence identity of SH proteins between subgroups, there was little variation in deduced amino acid sequences within the subgroups; nucleotide homologies within the subgroups ranged between 93% and 99%. Forty-two isolates of RS virus from a single epidemic season (autumn/winter 1989) were also examined to determine their relatedness. For these isolates regions of both the SH and nucleocapsid protein genes of each isolate were amplified and these regions were further analysed by direct nucleotide sequencing or restriction mapping. It was possible to discriminate at least six different lineages (or substrains) of RS virus circulating at the same time and in the same locality.

The rabies virus glycoprotein (G protein) has several important functions and is a major antigenic stimulus of the host immune system following rabies virus infection or vaccination. We developed a model system for studying the role of N-linked glycosylation in the intracellular transport and antigenicity of this molecule. The full-length cDNA of the G protein of the ERA strain of rabies virus was inserted into the eukaryotic shuttle vector pSG5 and then stably transfected into wild-type Chinese hamster ovary (CHO) cells and mutant CHO cell lines defective in glycosylation. Transfected wild-type CHO cells expressed the G protein (detected by immunofluorescence) on the cell surface in a manner similar to rabies virus-infected cells. The transfected wild-type CHO cells were shown by immunoprecipitation to produce a protein of 67K that comigrated with the fully glycosylated G protein isolated from virus-infected cells or purified virions. Treatment of the transfected cell lines with tunicamycin completely blocked surface expression and resulted in the intracellular accumulation of the G protein, suggesting that the presence of N-linked oligosaccharides is important for transport of this glycoprotein to the plasma membrane. The G protein cDNA was also expressed in the lectin-resistant CHO cell lines Lec 1, Lec 2 and Lec 8. In these cells initial N-linked glycosylation does occur, but later steps in processing of the oligosaccharides are blocked. In each case, the G protein was expressed on the surface of lectin-resistant CHO cells in a similar manner to expression on wild-type CHO cells. This suggests that various different N-linked oligosaccharide structures support intracellular transport of this glycoprotein. Thus, stably transfected CHO cell lines will provide a useful model system for further studies of the role of N-linked glycosylation in trafficking and antigenicity of the rabies virus G protein.

Passive administration of neutralizing monoclonal antibody (MAb) to glycoprotein H (gH) of herpes simplex virus type 1 (HSV-1) was found to protect mice from an HSV-1 strain SC16 challenge infection. To investigate further the protective potential of gH, recombinant vaccinia viruses were constructed which expressed the HSV-1 gH open reading frame under the control of the vaccinia virus 7.5K early/late promoter or the 4b late promoter. Immunization with recombinant viruses, however, did not induce the production of neutralizing antisera and the mice were not protected from zosteriform spread or the establishment of latent infection following viral challenge. The gH produced by the recombinant vaccinia viruses differed in electrophoretic mobility and antigenicity from authentic HSV-1 gH. Only one of three neutralizing MAbs specific for conformational epitopes on gH was able to immunoprecipitate gH synthesized in recombinant vaccinia virus-infected cells. In addition cell surface expression of gH was not detected in cells infected with the recombinant vaccinia viruses.

Cicloxolone sodium (CCX) is a broad spectrum anti-viral agent which has a largely non-specific and complex mode of antiviral action. However, the experimental finding that herpes simplex virus type 2 (HSV-2) (strain HG52) is consistently more sensitive to inhibition by CCX than HSV-1 (117 syn+) additionally implies the specific involvement of HSV genes. HSV-1/HSV-2 intertypic recombinants have been utilized to investigate this genetic difference by comparing their CCX ED50 concentrations. No short stretch of HSV-2 DNA was associated with its greater sensitivity to CCX, implying that two or more non-contiguously located HSV genes are involved. Correlating CCX sensitivity with recombinant virus genome structures allowed separate evaluation of the gene regions encoding glycoproteins gB, gC, gD, gE, gG, gH and gI and this suggests that the gene locations encoding gH and gC determine the CCX sensitivity difference. The selective inhibitor of Golgi apparatus glycosylation, monensin, gave results analogous to those obtained with CCX.

The primary structure of glycoprotein B (gB) is conserved strongly among many members of the Herpesviridae, including some that differ vastly in their natural properties. To determine whether the structural similarity between the gBs of herpes simplex virus type 1 (HSV-1) and bovine herpesvirus type 1 (BHV-1) was reflected in functional homology, we constructed pseudodiploid HSV-1 virions which, in addition to their own gene encoding gB, also contained a gene for encoding BHV-1 gB. Two kinds of pseudodiploid viruses were constructed. In one, the coding sequences of the BHV-1 gB gene were linked to the 5′ flanking sequences of the HSV-1 thymidine kinase (TK) gene. In the other, the entire BHV-1 gB gene, including its own flanking sequences, was introduced into the TK gene. In cells infected with the viruses both HSV-1 and BHV-1 gB were made but they could be distinguished immunologically by monoclonal antibodies. Both glycoproteins were inserted into cellular and virion membranes but did not form oligomers with each other. A monoclonal antibody that binds to HSV-1 gB but not BHV-1 gB neutralized the parental HSV-1 and a revertant pseudodiploid virus from which the gene encoding BHV-1 gB had been excised, but was significantly less efficient at neutralizing the pseudodiploid viruses. This suggests that the BHV-1 homologue can complement the HSV-1 gB functions required for infectivity.

A 3698 bp region of the genome of infectious laryngotracheitis virus (ILTV) was sequenced and found to contain the entire glycoprotein gB gene and the C-terminal region of a gene homologous to the ICP18.5 protein gene of herpes simplex virus type 1. The ILTV gB gene encoded a protein with an M r of 100K possessing all the characteristics of a transmembrane glycoprotein. Alignment of the ILTV gB sequence with homologous sequences from six other herpesviruses revealed that 10 cysteine residues on the surface of the molecule were completely conserved and that the positions of several N-linked glycosylation sites were largely conserved. Evolutionary trees based on the gB amino acid sequences from a total of 13 herpesviruses were constructed and the relationships among these herpesviruses were examined.

Antisera were raised against a purified recombinant form of the Epstein—Barr virus (EBV) alkaline deoxyribonuclease (DNase) expressed in Escherichia coli. These sera were shown to be reactive with lymphoblastoid cells expressing EBV antigens (B95-8, P3HR-1 and Raji). Immunostaining studies of cells expressing EBV antigens revealed that the DNase was a component of the restricted early antigen complex of EBV. Western blot analysis of these chemically induced cells revealed that the polypeptide associated with the EBV DNase has an M r of approximately 55000, slightly greater than that of the recombinant form, suggesting that the protein undergoes some form of post-translational modification during virus replication. The DNase enzymic activities observed in B95-8, P3HR-1 and Raji cells following chemical induction were neutralized using the specific antiserum. A detailed examination of protein extracts from the nude mouse-passaged nasopharyngeal carcinoma cell line C-15 failed to detect any antigenic or biochemical evidence for the presence of the DNase. Immunostaining of biopsies of oral ‘hairy’ leukoplakia with the antisera against EBV DNAse revealed high level expression in the more differentiated spinous layers of the epithelium, a pattern of reactivity identical to that observed for other lytic cycle antigens.

An immunodominant Epstein—Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) epitope, represented by peptide 68, has been mapped to the EBV nuclear antigen, EBNA 3. The epitope is recognized by class I-restricted CTLs through HLA-B8 and is functionally active on type A but not type B lymphoblastoid cell lines (LCLs). Herein we show that peptide 68 is not expressed as a functional CTL epitope by type A LCLs infected with an EBV B95-8 isolate. CTLs from cultures stimulated with autologous type A IARC-BL74 or QIMR-WIL LCLs lysed autologous cells stimulated with phytohaemagglutinin (PHA blasts) and coated with exogenous peptide 68. No peptide 68-specific CTLs were generated in cultures stimulated with autologous type A B95-8 or type B AG876 LCLs. However, the B95-8 LCL coated with peptide 68 was effective in the induction of a peptide-specific CTL response. A peptide 68-specific CTL clone failed to lyse the B95-8 LCL, type B AG876 LCL and PHA blasts, although such targets were lysed when coated with peptide 68.

Three structural proteins (4a, 4b and 25K) located within the virion core of vaccinia virus are cleavage products of precursor polypeptides (P4a, P4b and P25K) synthesized late in viral infection. Pulse-chase labelling experiments revealed that cleavage of the core proteins lags considerably behind precursor synthesis and that processing requires continuous protein synthesis. The N-terminal sequences of 4b and 25K, but not 4a, were determined by microsequencing core proteins isolated from purified virions. Comparison of these data with the predicted amino acid sequence of P4b and P25K revealed a conserved Ala-Gly-Ala motif flanking the apparent N termini of both proteins, as well as several additional sequence similarities between the P4b and P25K precursors both upstream and downstream of the putative cleavage site. The Ala-Gly-Ala tripeptide signal was also found in the same region of the amino acid sequences of the homologous proteins of fowlpox virus.

Wild duck populations were investigated over a 4 year period for duck hepatitis B virus (DHBV) infection and liver disease. It appeared that DHBV is endemic in wild migratory mallards from France and the U.S.A., although neither hepatocellular carcinoma nor viral DNA integration could be detected in liver samples examined. The follow up of natural infection indicated that wild mallards developed significantly higher serum titres to DHBV DNA than Pekin ducks. The results of experimental transmission demonstrated that such differences in viraemia were not related to the breed of ducks but to the virus isolate, since the wild mallard-isolated DHBV (DHBVwm) induced significantly higher viraemia in both mallard and Pekin ducklings compared to the domestic Pekin DHBV (DHBVp) isolate. The naturally infected mallard and Pekin ducks had only minor histological lesions of the liver compared with experimentally infected birds. There was no correlation between the intensity of viraemia and the severity of liver lesions, suggesting that as for mammalian hepadnaviruses the hepatic injury in DHBV-infected ducks is probably immunologically mediated.