- Volume 70, Issue 1, 1989

The recently established virus family Flaviviridae contains at least 68 recognized members. Sixty-six of these viruses were tested by cross-neutralization in cell cultures. Flaviviruses were separated into eight complexes [tick-borne encephalitis (12 viruses), Rio Bravo (six), Japanese encephalitis (10), Tyuleniy (three), Ntaya (five), Uganda S (four), dengue (four) and Modoc (five)] containing 49 viruses; 17 other viruses were not sufficiently related to warrant inclusion in any of these complexes.

Foot-and-mouth disease virus (FMDV) A22 Iraq 24/64 adapted to grow in BHK monolayer cells induced antibodies which neutralized many isolates belonging to the A serotype. Plaque-purified virus isolated from this stock also induced broadly reactive antibodies, showing that this property is not due to the combined response to a mixture of variants in the original stock virus. However, viruses obtained by passage in suspension BHK cells of either the monolayer cell-adapted virus or a virus cloned from this stock resulted in the selection of virus which induced antibodies with highly specific neutralizing activity. In addition to their antigenic properties the monolayer and suspension cell-adapted viruses could be distinguished by plaque morphology, tendency to aggregate and ability to attach to BHK cells. Monoclonal antibodies (MAbs) induced with the plaque-purified monolayer-adapted virus had neutralizing activity almost as broad as polyclonal serum, showing that this property can be represented by a single epitope on the virus. These neutralizing MAbs recognize a trypsin-sensitive epitope on the virus. Surprisingly, sequence analysis of the structural protein-coding regions of the genomic RNAs of monolayer and suspension cell-adapted viruses showed no amino acid differences in VP1, the protein known to contain the major neutralization epitope in FMDV and to be the only protein susceptible to cleavage by trypsin in the virus particle. Although three coding differences were found in the capsid protein these were all located in VP2.

Epitopes of strain A22 Iraq 24/64 of foot-and-mouth disease virus have been mapped with monoclonal antibodies (MAbs). Three methods were used : (i) an indirect ELISA using an overlapping set of peptides, (ii) production of neutralization escape variants against each MAb and (iii) sequencing of neutralization escape variants. The study has shown that the virus has at least three overlapping linear neutralizing epitopes within a major antigenic site on VP1. The presence of a second, conformational site was demonstrated but its position on the virus particle was not located. Synthetic peptides with sequences representing the major site elicit antibodies which have similar broad cross-neutralizing activity to polyclonal serum or neutralizing MAbs produced with the virus against a range of field isolates.

Polyclonal and monoclonal antibodies have been raised against a fusion protein containing β-galactosidase and part of the major capsid protein L1 of the human papillomavirus (HPV) type 16. The polyclonal antibodies cross-reacted with the L1 protein of several HPV types including HPV-1, -2, -6 and -11 when reacted with virus-infected tissue sections, and with HPV-6 and -18 L1 fusion proteins on Western blotting. Monoclonal antibodies against the L1 fusion protein of HPV-16 reacted only with HPV-16 L1 fusion proteins on Western blots and with HPV-16-containing biopsy sections as assessed by in situ DNA–DNA hybridization. These antibodies did not detect HPV-6 L1 protein after Western blotting or in HPV-6-infected tissue sections, although one did react with an HPV-18 fusion protein after Western blotting. The monoclonal antibodies were able to detect HPV-16 antigens in routine formaldehyde-fixed, wax-embedded sections of cervical intraepithelial neoplasia sections. HPV-16 L1 proteins were seen in one-third of biopsies that were positive using the polyclonal cross-reacting antisera. Polyclonal antibodies to fusion proteins containing part of the minor capsid protein L2 of HPV-6 or -16 appeared to be more type-specific as no cross-reactivity was seen when these antibodies were reacted with HPV-1- and -2-infected tissue sections.

Two mouse cytotoxic T cell (Tc) clones, D5 and H1 la, with specificity for the respiratory syncytial virus (RSV) fusion protein (F) were derived from BALB/c mice primed intranasally (i.n.) with RSV (A2 strain). These clones possessed essentially the same characteristics, and only clone H1 1a is described here. Tc clone H1 1a lysed target cells infected with a recombinant vaccinia virus (VV) expressing the RSV F gene, and similar target cells infected with RSV strains Long, 8/60, or 18537. In addition, two RSV-specific mouse Tc lines are described, from BALB/c mice primed i.n. with RSV A2 (Tc line MJC-A2), or intraperitoneally with a VV–RSV F gene recombinant (Tc line MJC-F). Tc line MJC-A2 was of unknown antigen specificity, failing to lyse targets infected with recombinant VVs expressing the RSV nucleoprotein (N), large glycoprotein, F, 1 A, 1C, or partial matrix protein (amino acid residues 88 to 257) genes. MJC-A2 Tc were cross-reactive for all strains of RSV tested. In contrast, the F-specific MJC-F Tc showed a marked degree of RSV strain specificity, efficiently lysing targets infected with RSV Long or A2 strains, but showing greatly reduced lysis of targets infected with RSV 8/60 or 18537 strains. An anti-RSV human Tc line, IH.K2, also recognized the fusion protein. IH.K2 Tc efficiently lysed autologous Epstein-Barr virus-transformed B cells (BCL) persistently infected with RSV A2 and BCL infected with a VV-RSV F gene recombinant. IH. K2 function was not exclusively RSV F-specific, however, as these cells also lysed autologous BCL infected with a VV-RSV N gene recombinant. These data show that the RSV fusion protein is a target antigen for anti-RSV Tc following infection of mice and humans, and that the F-specific Tc repertoire in mice can be influenced by the method and route of priming.

Human sera containing respiratory syncytial (RS) virus-specific antibodies enhance RS virus infection of the U937 macrophage cell line. There was an increase in the number of cells expressing virus antigen when U937 cells were infected with RS virus in the presence of human serum compared to cells infected in the absence of human serum. Human sera enhanced virus yield, as measured by the cell-released infectious virus, by an average of 50-fold compared to virus infection in the absence of human serum. The comparison of the enhancing activities of paired acute and convalescent human sera showed that the titre of enhancing antibody increased in parallel with the titre of RS virus-specific antibody measured by complement fixation and virus neutralization. An RS virus-specific neutralizing monoclonal antibody directed to the virus F protein enhanced virus infection of U937 cells. A non-neutralizing monoclonal antibody directed to the virus nucleoprotein did not enhance virus infection. The possible role of enhancing antibodies in vivo is discussed.

Infection of the lymphoblastoid CEM cell line with herpes simplex virus (HSV) type 1 results in a persistent infection with production of infectious virus. Evidence suggests that the persistent infection was not maintained by interferon or non-interferon-soluble antiviral inhibitors. Treatment of persistently infected cells with anti-HSV serum (termed CEMACR cells) or elevated temperature (39 °C) for 14 days (termed CEMTCR cells) resulted in loss of evidence of virus. HSV DNA was not detected in CEMTCR or CEMTCR cells by Southern blot or in situ hybridization. The CEMACR or CEMTCR cells, however, were resistant to reinfection with homologous, parental virus (HSV0), but were susceptible to heterologous virus (vesicular stomatitis virus). Resistance to reinfection with HSV was not absolute; CEMACR or CEMTCR cells were less permissive to virus isolated from persistently infected cultures at times early in the course of infection, but were more permissive for HSV isolated at later times. Virus isolated later during persistent infection also displayed progressively increased virulence for the parental CEM cells. These results suggest that persistent infection of a human T lymphoblastoid cell line, CEM, with HSV-1 is maintained by a genetically determined cell-virus equilibrium, in which the resistance of cells and virulence of virus increase during persistence.

The major enhancer, extending from nucleotides –530 to –120 upstream of the transcription initiation site of immediate early (IE) genes 1 and 2 in human cytomegalovirus (HCMV), contains four groups of repeated sequence motifs that consist of 17, 18, 19 or 21 bp, respectively. One of these elements, the 19 bp repeat, is a symmetrical palindrome that is also part of IE regulatory sequences of other cytomegalovirus-type herpesviruses, but not of unrelated members of the herpesvirus group. Synthetic oligonucleotides representing the 19 bp repeat unit strongly reduced the activity of the IE1/2 enhancer/promoter in cotransfection assays after transient expression. The HCMV enhancer can substitute for the 72 bp repeats of simian virus 40 (SV40). Replication-competent deletion mutants of SV40/HCMV enhancer recombinants were constructed that contained a single palindromic 19 bp repeat with a central cleavage site for AhaII. If deletions were introduced into the single remaining 19 bp repeat most of the mutant viruses were still replication-competent in CV-1 monkey kidney cells. Insertion of two nucleotides into the single AhaII site did not significantly alter transient SV40 T antigen expression. Deletion of four nucleotides or more from the single 19 bp palindrome reduced the stimulation of T antigen synthesis by the HCMV enhancer/SV40 promoter unit down to about 50%. More extended deletions (28 to 80 bp) did not further reduce T antigen expression. All mutants without an intact 19 bp repeat contained the 18 bp and/or the 21 bp sequence motif. DNase I footprinting and gel retardation assays indicated sequence-specific protein binding by the 19 bp palindrome. Altered palindromes, correlating with reduced enhancer activity, lost most of their protein-binding properties. Thus, the 19 bp repeat element is one of several protein-binding sites that contribute to enhancer strength. However, the 19 bp sequence motif can be deleted entirely to leave reduced activity. The HCMV IE1/2 upstream sequence appears to be the perfect model of an enhancer as a complex of multiple binding sites for trans-activating proteins in a modular fashion.

The major glycoproteins of bovine herpesvirus type 1 showed distinct temporal patterns of expression. The glycoproteins GVP 11 and GVP 6 as well as its cleavage products, GVP 11a and GVP 16, were expressed early in the infectious process, whereas GVP 9 was expressed late. Temperature-sensitive mutants were developed and characterized. Mutants belonging to two complementation groups were unable to synthesize DNA at 40 °C, the non-permissive temperature. In cells infected with these mutants the late glycoprotein GVP 9 was not synthesized at 40 °C, whereasthe synthesis of the early glycoproteins GVP 11 and GVP 6 continued at wild-type levels. These studies suggest that the transition from early to late glycoprotein synthesis is linked to viral DNA synthesis.

A temperature-sensitive (ts) mutant, tsG1, has been isolated from the CVS (Challenge Virus Standard) strain of rabies virus. The ts mutation affects the glycoprotein (G protein); it consists of an amino acid substitution (leucine to phenylalanine) at position 132. tsG1 exhibits a slightly reduced pathogenicity when administered via the intracerebral route and complete avirulence after intramuscular inoculation, associated with a very high protective power for adult mice. The ts mutation does not seem to block the transport of the G protein to the plasma membrane at the non-permissive temperature (39.6 °C). It abolishes the c.p.e. of the virus in cell cultures.

A 6·2 kb BamHI terminal fragment from fowlpox virus has been cloned and the nucleotide sequence was determined. The fragment was cloned by S1 digestion of viral DNA and therefore does not contain the covalently closed terminal loop. The cloned sequences comprise a short (230 bp) unique region at the terminal end, which is adjacent to a 3·87 bp long, AT-rich region consisting of sets of short tandemly repeated units, 32 and 56 bp long. The remainder of the fragment is composed of a 2·18 kb unique region containing three major open reading frames. The amino acid sequence encoded by one of these has some similarity to that of platelet-derived growth factor.

A cDNA clone representing the gene for the E3 glycoprotein, the haemagglutinin, of bovine coronavirus was isolated from a plasmid cDNA library of the viral genome and sequenced. The gene is located immediately 5′ of the E2 glycoprotein gene on the viral genome. Nucleotide sequencing of the E3 gene predicts a polypeptide of 424 amino acids with an M r of 47K. In vitro translation of mRNA transcribed from the cloned E3 gene yielded a polypeptide of M r 45K, similar to that predicted from the nucleotide sequence. In the presence of microsomal membranes, the in vitro product was cotranslationally processed to a 62K polypeptide which comigrated on SDS–polyacrylamide gels with the E3 monomer (gp62) obtained from virus-infected cells. Both the 45K and 62K polypeptides were immunoprecipitated with E3-specific monoclonal antibodies, confirming the identity of the gene as that encoding the E3 glycoprotein. Finally, only monoclonal antibodies to the E3 protein inhibited haemagglutination by the virus thus confirming its identity as the haemagglutinin of bovine coronavirus.

The DNA of a field isolate and of a vaccine strain of canine adenovirus type 2 (CAV-2) were analysed by digestion with several restriction endonucleases. The PstI restriction fragments of the field isolate (CAV-2 Glasgow) and the vaccine strain were cloned into the plasmid pBR322. Physical maps of the two viral genomes were constructed by molecular hybridization of PstI, EcoRI, SmaI, BamHI and KpnI digests of the viral DNA with the cloned PstI fragments. The restriction profile of CAV-2 Glasgow was shown to be virtually identical to those of the two prototype CAV-2 strains, Toronto A26/61 and Manhattan. However, the restriction fragment pattern of the vaccine strain of CAV-2 showed characteristic alterations, in particular additional sequences at or near the genome termini.

Serological analyses of several different cytoplasmic polyhedrosis viruses (CPVs), including two type 1 CPVs from Bombyx mori, type 1 CPV from Dendrolimus spectabilis, type 12 CPV from Autographa gamma, type 2 CPV from Inachis io, type 5 CPV from Orgyia pseudotsugata and type 5 CPV from Heliothis armigera, demonstrated a close correlation between the antigenic properties of the polyhedrin or virus particle structural proteins and the genomic dsRNA electropherotypes. The dsRNAs of these viruses were analysed by electrophoresis in 3% and 10% polyacrylamide gels with a discontinuous Tris–HC1/Tris–glycine buffer system or by 1% agarose gel electrophoresis using a continuous Tris–acetate–EDTA buffer system. Electrophoretic analysis in agarose gels was found to be the most suitable for the classification of CPV isolates into electropherotypes, and the results obtained showed a close correlation with the observed antigenic relationships between different virus isolates. However, electrophoretic analysis in 10% polyacrylamide gels was most sensitive for the detection of intra-type variation and the presence of mixed virus isolates.

The role of the Autographa californica nuclear polyhedrosis virus p10 gene in viral cytopathology and morphogenesis was examined using classes of p10 deletion mutants with and without lacZ (β-galactosidase) gene fusion. Mutant-infected cells did not form the fibrillar cytoplasmic and nuclear structures normally observed late in infection with wild-type (wt) virus, and the cells failed to lyse even at 2 weeks post-infection. Based on wt and mutant cytopathology, we suggest lysis may be facilitated by stepwise exhaustion of the host nuclear membrane, and may require a function resident in the carboxy region of p10; this portion of the molecule is also essential for formation of the p10-rich fibrillar bodies. Additional changes in cytopathology were correlated with the level of p10/LacZ fusion protein expression. The insertional mutant designated Ac229, which encodes 51 N-terminal amino acids of p10 fused to LacZ, caused intranuclear accumulation of granular structures at sites corresponding to the fibrillar bodies of wt viral infections. Occlusion body membranes, which associate with the fibrillar bodies in wt infections, were also formed in mutant virus-infected cells. However, membranes did not associate with occlusion bodies in Ac229 infections, and were aberrantly attached to occlusion bodies in cells infected with mutants having simple p10 deletions (represented by Ac231). Loss of the outer membrane increased sensitivity of the occlusion bodies to disruption by physical stress; a partially attached membrane afforded some protection from disruption.

Functional analyses of the p10 gene promoter from the Autographa californica nuclear polyhedrosis virus (AcNPV) were performed by progressively deleting the 230 nucleotides upstream from the p10 coding sequences towards the ATG codon. Truncated promoter sequences retaining the full 5′ non-coding leader of p10 were inserted in front of the chloramphenicol acetyltransferase (CAT) gene, and promoter activity in transfected AcNPV-infected cells was measured using the transient CAT expression assay. The removal of sequences to a position 101 nucleotides upstream from the p10 ATG did not affect the level of CAT expression. Deletion of a further 13 nucleotides reduced CAT expression by three- to fourfold, but the removal of three more nucleotides, which deleted most of the baculovirus very late gene transcription consensus sequence, almost completely abolished activity. The removal of the TATA motif had no effect on the level of transient expression. We conclude that a sequence of about 101 nucleotides upstream from the ATG codon of p10 is sufficient for high level promoter activity in this transient system.

Piry rhabdovirus is not transmitted from Drosophila melanogaster females to their progeny. However, in mixed infections with sigma, another rhabdovirus, bearing the g+ genetic marker, Piry may occasionally be transmitted to offspring. Thus, an endemic Drosophila virus can act as a helper virus enabling vertical transmission of a virus pathogenic to vertebrates.

Vesicular stomatitis virus (VSV) establishes a non-cytopathic persistent infection in Drosophila melanogaster cells. The synthesis of the viral glycoprotein G was specifically inhibited during a post-transcriptional step, whereas the synthesis and turnover of its mRNA were not modified compared with the other viral mRNAs. Another viral glycoprotein, migrating slightly faster than G protein on an SDS–polyacrylamide gel, was detected in infected Drosophila cells. This protein showed most of the characteristics of the intracellular Gs protein found in infected vertebrate cells. The amounts of G protein integrated into mature virions and of soluble Gs protein secreted into the culture medium were reduced greatly during VSV infection in Drosophila cells.