- Volume 69, Issue 1, 1988

Partial N-terminal amino acid analyses of five radiolabelled non-structural (ns) proteins specified by Kunjin (KUN) virus provided positive identification of NS3, NS5 and three previously hypothetical ns proteins of flaviviruses, ns2a, ns2b and ns4b. Their correct gene order was obtained from their deduced amino acid sequences. Thus the gene order for KUN virus relative to that proposed for yellow fever (YF) virus was as follows: KUN 5′…GP44·P19·P10·P71·(?)·P21·P98-3′, YF 5′…NS1·ns2a·ns2b·NS3·ns4a·ns4b·NS5-3′. The identity of GP44 as NS1 was assumed from the known nucleotide and deduced amino acid sequences; ns4a was not identified. The cleavage sites in the polyprotein for KUN NS2B, NS3 and NS5 were identical, Lys-Arg↓Gly, similar in form to the sequence Arg-Arg↓Ser defined at the cleavage sites of YF NS3 and NS5. A new consensus cleavage site for NS1, NS2A and NS4B in the form Val-X-Ala↓, where X is any one of several uncharged amino acids, was found at corresponding sites homologous to those of KUN virus in all published flavivirus sequences (a total of 18 sites). NS1 and NS4B, but not NS2A, were preceded by a putative signal sequence.

We have mapped and determined the nucleotide sequence of the cowpox virus (CPV) gene coding for the protein of A-type inclusion bodies (ATI). The ATI gene was mapped to the central part of the largest HindIII restriction fragment of the CPV genome. Vaccinia virus (VV) also contained a similar sequence at the equivalent position in its genome, which even under stringent conditions hybridized with the CPV ATI gene. Nucleotide sequence analysis revealed that the upstream region of the coding frame contained a sequence closely resembling the VV late promoter consensus sequence and that the gene encoded a protein of M r 150000.

We have determined the complete nucleotide sequence of human rhinovirus 1B and made comparisons with other rhinoviruses. Extensive homology was found with serotypes 2 and 89 but the similarity to serotype 14 was considerably less. Rhinovirus-specific characteristics have been noted, in particular the length of the 5′ non-coding region and the pattern of codon usage, and these may be sufficient to define the rhinoviruses as a distinct genus rather than being considered as members of the enteroviruses as has been suggested previously.

Persistent infection (PI) of YAC-1 cells by coxsackievirus B3 (CBV-3) was characterized. CBV-3 PIs were maintained for 7 months or more, although in two other cases cells were cured of virus at 6 and 6.5 months of PI. The titre of infectious virus peaked during the first week of the infection and then gradually decreased. The proportion of cells producing infectious centres increased to 100% by 48 h after infection, remained at that level up to the seventh day, and then rapidly decreased. Susceptibility to PI by CBV-3 varied widely among 40 clones from uninfected YAC-1 cells as judged by the yield of infectious virus at 6 weeks post-infection. None of the clones was completely lysed by the virus. Clones were not obtained from cells infected for 2 or 7 days. Of six clones obtained from cells infected for 14 days and 24 clones from cells infected for 6 weeks, none was producing virus and all were resistant to reinfection by CBV-3. Six of the clones were serially subcultured and all remained resistant for as long as they were maintained (5 months). During the course of the PI, viral variants which produced smaller plaques and required a longer incubation period for the development of visible plaques replaced the original viral population. Thus the PI involved a carrier culture with a large proportion of resistant cells. The resistant state did not require the continued presence of virus.

A mouse interferon alpha gene (MuIFN-α10) was isolated from a BALB/c cosmid genomic library. The gene was located on a 1.8 kb HindIII fragment and a 5.1 kb EcoRI fragment. The coding region and parts of the 5′ and 3′ non-coding regions were sequenced. The results showed that the MuIFN-α10 gene encoded a protein of 167 amino acids. Like most other MuIFN-α species it contained a putative N-glycosylation site at amino acid positions 78 to 80. It also possessed cysteine residues at positions 1, 29, 86, 99 and 129. In the signal peptide, in addition to cysteine 21, which is present in all MuIFN-α species sequenced so far, a cysteine was found at position 22. At the amino acid level MuIFN-α10 showed strong homology to MuIFN-α1 (only 15 out of 167 amino acids were different). The MuIFN-α10 gene was transiently expressed in monkey COS cells under the direction of the simian virus 40 early promoter. The protein product secreted by COS cells was equally active on mouse (L929) and hamster (CHO) cells. However, as compared to MuIFN-α1 and MuIFN-α4 the specific activity on mouse cells of the protein was 10- to 100-fold lower. To find out which region of its structure was responsible for this low activity, hybrids of the genes encoding MuIFN-α10 and MuIFN-α1 were constructed using the two common XmnI sites which correspond to positions between amino acids 67 and 68 and 123 and 124, respectively. The data showed that hybrid constructs which were MuIFN-α1-like from amino acid 68 or MuIFN-α10-like from position 124 to the C terminus possessed high antiviral activity. Other hybrid constructs were hardly active at all. This implied that the amino acid 68 to 123 region was mainly responsible for the low antiviral activity of MuIFN-α10. In this part of the molecule MuIFN-α1 and MuIFN-α10 differed in only five amino acids. A serine at position 110 and a valine at 85 were unique to MuIFN-α10 as compared to all known MuIFN-α and human IFN-α subspecies.

A panel of murine monoclonal antibodies (MAbs) against the two major glycoproteins of bovine viral diarrhoea virus (BDV) was produced and assayed by serum neutralization, radioimmunoprecipitation (RIP) and immunoblotting. Based on their viral polypeptide specificity and on their ability to neutralize viral infectivity, the MAbs in the panel were divided into three classes: Class 1 MAbs reacted with the 56K to 58K glycoprotein and neutralized the virus, class 2 MAbs recognized the 56K to 58K glycoprotein but were not neutralizing, and class 3 MAbs reacted with the 48K glycoprotein and did not neutralize the virus. These results identify the 56K to 58K protein as one of the envelope glycoproteins of BDV. Evidence was obtained indicating that it is responsible for the induction of neutralizing antibodies. No large uncleaved precursors of the 56K to 58K protein could be identified unequivocally by RIP of infected cell extracts, suggesting that this polypeptide is proteolytically processed cotranslationally. A subset of MAbs that reacted with BDV isolates of the noncytopathic biotypes yielded similar results, indicating that these findings are applicable to both biotypes of BDV.

We selected murine coronavirus JHM variants specifically changed in defined antigenic sites of the peplomer protein E2. Variants were isolated from the supernatants of monoclonal antibody hybridoma cell cultures which continued to secrete neutralizing antibodies after being infected with JHM. Comparative antigenic analysis and biological tests were performed in order to refine an operational epitope map and to characterize functional domains important for pathogenicity. The reaction patterns (neutralization, inhibition of cell fusion, immunofluorescence and binding in ELISA) between the variant viruses and the panel of monoclonal antibodies were very similar. Four groups of variants were characterized each of which revealed distinct changes affecting one defined antigenic site. These observations indicated that at least four independently mutable antigenic sites were associated with domains involved in cell fusion, neutralization and pathogenicity (E2-Aa, -Ab, -Ba and -Bb). JHM variants with alterations in the E2-Aa, -Ab or -Bb sites were similar to wild-type virus. These variants caused acute hepatitis and encephalomyelitis in mice. In contrast, JHM variants with changes in site E2-Ba had a strong propensity to induce chronic disease accompanied by demyelination persisting for several months.

The production and properties of monoclonal antibodies raised against herpes simplex virus type 1 (HSV-1)-infected cell nuclei are described. Biological and immunochemical assays revealed that these antibodies recognize four different proteins in HSV-1-infected cells. Four antibodies reacted with the major DNA-binding protein (ICP8) and six with the 65K DNA-binding protein. Two antibodies detected the ICP35 family of proteins and one antibody bound to a protein with an apparent mol. wt. of 60K. Immune electron microscopy showed that the major DNA-binding protein had a patchy distribution, whereas the 65K DNA-binding protein was evenly spread in the infected cell nuclei. The 60K protein as well as the polypeptides of the ICP35 family were preferentially found associated with the viral capsid.

Herpes simplex virus (HSV) type 2 (strain HG52) has four XbaI sites at map coordinates (m.c.) 0.45, 0.7, 0.91 and 0.94, i.e. two in the unique long and two in the unique short regions of the genome. Previously, we had isolated a genome containing only the 0.45 and 0.94 XbaI sites. Here we report the isolation of a mutant (JH2611) in which all four XbaI sites have been removed using an enrichment selection procedure, without any loss of viability. Removal of each site has been shown to be due to a base change or small undetectable deletion/insertion of less than 100 bp. In HSV-1, the XbaI site at 0.45 m.c. is in an open reading frame coding for a polypeptide of 14K. Both the 0.7 and 0.94 m.c. HSV-2 XbaI sites are in intergenic positions. The 0.91 m.c. XbaI site has been shown to be within the coding sequence of the glycoprotein gG-2. Synthesis of gG-2 by JH2611 and two other mutants, JH2610 (formerly HG52X163X3) and JH2609 (formerly HG52X163X21), in which the 0.91 m.c. site has been deleted was analysed by immunoprecipitation using the gG-2-specific monoclonal antibodies AP1 and LP5 and the anti-peptide serum 14713. In the mutants JH2610 and JH2611 neither gG-2 nor its precursor were detected but the monoclonal antibodies detected two polypeptides migrating above the normal positions of gG-2 and the gG-2 precursor; these were not precipitated by the anti-peptide serum. With the mutant JH2609 neither gG-2 nor its precursors could be detected by either the monoclonal antibodies or the anti-peptide serum. The results strongly suggest that gG-2 is non-essential for the growth of HSV-2 in vitro.

Viruses in the Eubenangee, Wallal and Warrego serogroups of orbiviruses have been isolated primarily in Australia. Several isolates in these three serogroups were examined by gel electrophoresis and blot hybridization of genomic RNA. Conserved and variant genes were identified by the degree of hybridization between cognate genes. The dsRNA profiles of isolates within a serogroup exhibited heterogeneity in polyacrylamide gels, but they were indistinguishable in agarose gels. Isolates within a serogroup generally showed a high level of cross-hybridization in eight segments with cognate segments 2 and 6 exhibiting hybridization signal variants. Although Pata virus has been classified in the Eubenangee serogroup, it was not closely related to other Eubenangee isolates by hybridization. Similarly, Mitchell River virus was not closely related to other members of the Warrego serogroup. The taxonomic status of Pata and Mitchell River viruses should be reviewed, and in the interim they should be placed in the ungrouped set of orbiviruses. Inter-serogroup relatedness was also examined by blot hybridization. Representatives from the Eubenangee, Wallal, Warrego, bluetongue, epizootic haemorrhagic disease and Corriparta serogroups were examined. Several of the serogroups were distantly related, and the low level of relatedness was suggestive of a common ancestry. While many of these serogroups may co-circulate in nature, these data suggest that each serogroup represents a distinct gene pool. The taxonomic significance of these data is discussed.

Viruses which belong to the epizootic haemorrhagic disease (EHD), bluetongue and Eubenangee serogroups of orbiviruses exhibit low level cross-reactions in some serological tests. Although Pata virus cross-reacts at low levels with members of the EHD and bluetongue serogroups, it was assigned originally to the Eubenangee serogroup. RNA-RNA blot hybridization data, however, suggest that Pata virus is not a member of the Eubenangee serogroup. In this study, the genetic relatedness of the EHD serogroup viruses, bluetongue virus type 10 (BTV-10) and Pata virus was assessed by RNA-RNA blot hybridization and by gene reassortment experiments in vitro. The five members of the EHD serogroup examined were highly related by reciprocal RNA-RNA blot hybridization. Genes 1, 3, 4 and 6 to 9 were highly conserved with three unique types of gene 2, four variant types of gene 5 and two variant types of gene 10. Geographical boundaries could not be correlated with sequence relatedness because viruses isolated from the same locality were distant relatives when compared with another virus isolated on a different continent. The significance of the unique and variant genes is discussed. The EHD isolates, BTV-10 and Pata virus exhibited distinct profiles on agarose and polyacrylamide gel electrophoresis, and they are related distantly as shown by weak hybridization signals in blot hybridization. Gene 2 was a unique gene among the EHD isolates, BTV-10 and Pata virus. One BTV-10 gene hybridized more strongly to gene 9 of the EHD viruses than the other BTV-10 genes, and its role in encoding the cross-reactive antigen is discussed. Intra-serogroup gene reassortment in vitro was demonstrated in the six crosses among EHD serogroup members. In contrast, gene reassortment was not observed in inter-serogroup crosses between EHD 1 and BTV-10, BTV-10 and Pata virus, and between EHD 1 and Pata virus. Correlation of blot hybridization and gene reassortment indicated that viruses must share high sequence conservation in the majority of their genes before genetic interaction is likely. The usefulness of blot hybridization as an indicator of the likelihood of gene reassortment is discussed. These hybridization and gene reassortment data indicated that Pata virus is not a member of the bluetongue or EHD serogroups and that it should be assigned to the ungrouped set of orbiviruses. The hybridization and gene reassortment data suggest that members of the EHD serogroup, BTV-10 and Pata virus represent three distinct gene pools, and the role of reassortment in the generation of genetic diversity is discussed. The minor antigens shared among these gene pools probably reflect their common ancestry.

Reassortant formation following coinfection has been suggested as a mechanism of evolution of rotaviruses. This study was designed to examine the selection of reassortants following coinfection of cultured cells with pairs of subgroup 2 human rotaviruses. The three pairs studied (Wa × P, CJN × 31, 62 × 69) were chosen to maximize the number of RNA segments that could be electrophoretically distinguished. After coinfection and multiple passages, reproducible selection of reassortants was observed with each pair. Although more segments were selected from the virus of a pair that grew to higher titre, certain segments were selected independently of the relative growth properties or multiplicities of infection of the coinfecting viruses; selection of other segments was dependent on both. In determining the time and cause of selection it was found that no selection of genomic RNA segments was detectable prior to or during viral particle assembly in coinfected cells. However, selection was evident within the infectious progeny population after a single cycle of replication. Therefore, selection of specific reassortants following coinfection was apparently due to differences in the infectivities of progeny viruses and not in their assembly. This implies that these infectivities were a function of the parental origin of specific genomic segments.

Thermosensitive mutants were isolated from nitrous acid-treated Kilham rat virus (KRV). At a restrictive temperature (39.5 °C), the mutants tested did not produce appreciable amounts of infectious particles, haemagglutinin or progeny single-stranded DNA. Virus antigen accumulation as detected by immunoperoxidase was reduced. Complementation tests revealed two distinct groups. The three members of complementation group 1 synthesized normal amounts of replicative forms but were restricted in single-stranded DNA production and capsid protein expression, exhibiting phenotypes compatible with cap mutations. The sole representative of group 2, KRV ts6, failed to accumulate replicative forms, displaying a rep- phenotype. These mutants provide new tools to test the role of viral products in the biology of autonomous parvoviruses.

Cotransfection of Rachiplusia ou nuclear polyhedrosis virus (RoMNPV) and BamHI or SmaI fragments from the genome of Autographa californica nuclear polyhedrosis virus (AcMNPV) yielded a high frequency of virus recombinants in Galleria mellonella larvae. When larvae were cotransfected either with the entire genome of AcMNPV and RoMNPV restriction fragments or with a mixture of the two viruses, only AcMNPV was produced. In the virus recombinants, the prevalence of certain regions from the genome of the donor virus (AcMNPV) varied considerably, e.g. from 0% in the 35 to 41 map unit region area to a high level in the 20 to 23 map unit region. The AcMNPV polyhedrin gene was shown to be inherited preferentially.

The DNA of distinct human papillomaviruses (HPVs) is regularly detected in the majority of human cervical carcinomas. In contrast to benign HPV-induced genital lesions, where the viral genomes are exclusively present as episomes, in cervical carcinomas HPV type 16 (HPV16) DNA was found to be integrated into the host DNA. In order to determine the physical state and expression of HPV DNA sequences at different stages of tumour development, we analysed a series of cervical lesions (mild, moderate and severe dysplasia and carcinoma in situ) that are considered precursors of carcinomas of the cervix. In 66.6% (18 of 27) of the tumours, HPV16 DNA was present. While in mild dysplasias only episomal HPV genomes were found, in all higher grade lesions integration of the viral DNA was detected. There was a close correlation between the episomal state and the expression of the HPV16 genomes:in 15 cases harbouring episomal HPV16 DNA (seven of which also contained integrated genomes) viral transcripts were present. We conclude that integration of HPV genomes takes place very early in cervical cancer development. In addition, the episomal state of the viral DNA depends on viral gene expression. The same conclusion, however, is not applicable in those lesions (three severe dysplasias) containing exclusively integrated HPV16 DNA. Thus, HPV16 DNA can persist in an integrated state without recognizable transcriptional activity. These results point to HPV16 as one potential prerequisite for the first steps in the multistage development of human cervical cancer.

Integrated viral sequences and adjacent cellular sequences from the polyoma virus (Py)-transformed 53-Rat and 82-Rat cell lines which contain two and three partial early regions respectively, each in a single viral insert, have been molecularly cloned. Each of the cloned partial early regions have been subcloned and assessed with regard to their transcription, translation products (T antigens, T Ags) and biological activity including their transforming ability. The 53-Rat 5.3 kb EcoRI fragment is an intact Py EcoRI linear genome (derived from within the tandem duplicated sequences) which transforms rat cells with high efficiency and produces infectious virus when circularized and transfected into mouse cells. The 82-Rat cell line expresses three novel T Ag species of 63K, 40K and 32K in addition to the Py middle and small T Ags. The 63K protein was found to be a truncated form of large T Ag produced as the result of an addition/deletion in early region B sequences unique to large T Ag. The 40K and 32K proteins are hybrid viral-cellular middle and large T Ags respectively, which are expressed from early region A that has been truncated by recombination with rat cellular DNA. Differences in the nuclear and cytoplasmic location of the different 82-Rat early region RNAs are due to RNA stability and/or transport from the nucleus to the cytoplasm most likely as a result of different cellular sequences at their 3′ ends. Finally no common structural feature or sequence specificity was observed at the virus-host DNA joins of the two cell lines.

Eight measles virus strains including four subacute sclerosing panencephalitis (SSPE) isolates were compared on the basis of their growth characteristics in human peripheral blood mononuclear cells and effect on mitogen-stimulated lymphocyte proliferation. The Edmonston strain virus and some other strains of measles virus replicated in phytohaemagglutinin (PHA)-stimulated lymphocytes and released high titres of infectious virus into the culture medium. Inhibition of DNA synthesis was relatively low at the beginning of lymphocyte proliferation and could be detected only at high multiplicity of infection. Three to 4 days after initiation of proliferation a strong inhibition was seen also in cultures infected with low doses of virus, and was apparently related to the production of high titres of new infectious virus. Some virus strains originally isolated from SSPE patients produced only a small amount of infectious virus in lymphocytes at 37 °C but had a very strong inhibitory effect on lymphocyte proliferation leading to early cell death. The inhibitory effect was found over a wide range of virus concentrations, but was strictly dose-dependent and no increase in inhibition with low multiplicities of infection could be seen with longer culture times. The amount of interferon induced by different strains varied from 400 to 6400 international units/ml but the amount of interferon produced in the cultures did not correlate with the inhibitory effect on proliferation or with the amount of released new infectious virus. The Hallé measles strain, originally isolated from an SSPE patient, differed most from the Edmonston strain in its characteristics, and was studied in greater detail. Although only a small amount of infectious virus was produced, the Hallé strain had a very strong inhibiting effect on the proliferation of PHA-stimulated lymphocytes, and extensive syncytium formation was seen leading to cell death within 3 days.

The nucleotide sequences of cDNA clones corresponding to the 3′ OH end of papaya mosaic virus RNA have been determined. The 3′-terminal sequence obtained was 900 nucleotides in length, excluding the poly(A) tail, and contained an open reading frame capable of giving rise to a protein of 214 amino acid residues with an M r of 22930. This protein was identified as the viral capsid protein. The 3′ non-coding region of PMV genome RNA was about 121 nucleotides long [excluding the poly(A) tail] and homologous to the complementary sequence of the non-coding region at the 5′ end of PMV RNA. A long open reading frame was also found in the predicted 5′ end region of the negative strand.