- Volume 65, Issue 8, 1984

This study demonstrates that populations of defective interfering Semliki Forest virus (DI SFV) are heterogeneous particularly in respect of their interference properties. Interference was quantified by two assays, one measuring inhibition of the yield of infectious progeny virus, and the other measuring reduction in virus-directed RNA synthesis; for 11 different DI SFV preparations a ratio of the two interference titres was calculated. These ratios varied up to 46-fold indicating that each DI virus preparation contained an interference activity that varied independently of the other. However, sister stocks made from the same parental inoculum had similar properties. The effects of different DI virus preparations on other parameters (virus polypeptide synthesis, yield of DI virus and yield of infectious virus) were investigated using inocula with interference titres standardized by either assay. Co-inoculation of L929 cells with 50 p.f.u. SFV showed that these parameters varied independently of each other and of the DI virus inoculum. There was no correlation with the number of undiluted passages each DI stock had received. Direct evidence of physical heterogeneity was demonstrated by metrizamide density gradient centrifugation. Although infecting virus sedimented as a narrow band, DI SFV was distributed over a broad region of the gradient. Its position on the gradient indicated that DI SFV has a higher nucleic acid: protein ratio than standard virus. DI virus progeny obtained by using fractions of the gradient as inoculum were as heterogeneous as the unfractionated parent, confirming that DI viruses retain heterogeneity on passage.

SDS-PAGE and immunoprecipitation analyses were carried out on the virion and cell-associated proteins of Hantaan virus, the causative agent of haemorrhagic fever with renal syndrome (HFRS). Purified virions have a density of 1.17 g/ml in sucrose, and contain four proteins with molecular weights of 45000 (45K), 56K, 72K and 200K, confirming recent evidence that the virus is a member of the family Bunyaviridae. Detergent treatment of virions indicates that the 45K protein is the virus nucleoprotein. Both the 72K and the 56K proteins were labelled with [3H]glucosamine and were removed from virions by bromelain treatment, indicating that they are envelope glycoproteins. The 200K protein was found only in [35S]methionine-labelled preparations. By analogy to prototype viruses of the family Bunyaviridae, these proteins were designated N, G1, G2, and L respectively. Three virus-specific proteins (N, G1, G2) were detected in virus-infected cells. These proteins were precipitable by human convalescent serum and by serum of a Rattus norvegicus trapped in the United States. No additional virus proteins were detected in infected cells. These results confirm recent morphological and RNA studies that Hantaan virus is a member of the family Bunyaviridae. Our results also support the suggestion that Hantaan virus be placed in a new genus of Bunyaviridae.

Simian virus 5 (SV5) isolates derived after co-cultivation of human bone marrow aspirates of multiple sclerosis (MS) patients were shown by immunoprecipitation, cross-neutralization and haemagglutination inhibition techniques to be similar antigenically but not identical to the prototype strain. Analyses of human sera (MS and control) showed that about 20% contained neutralizing antibodies to SV5 and immunoprecipitated the specific SV5 HN polypeptide. A competition assay using a specific SV5 monoclonal antibody confirmed that a human serum containing such neutralizing activity also blocked a specific SV5 epitope whereas another human serum with demonstrable antibodies to the related human parainfluenza virus type 2 did not block this epitope. These tests therefore suggested that SV5 can infect humans. However, there was no indication, on the basis of these tests, of any aetiological relationship of the SV5 infection to the induction of MS.

Human adenovirus type 2 mutant, H2 ts 111, presented a double phenotype: temperature-sensitive (ts) for initiation and elongation of DNA synthesis, and cytocidal (cyt) by its large-plaque formation and the nucleolytic cleavage of both viral and cellular DNAs. Both characters were recessive since they were efficiently complemented by wild-type or other DNA-negative ts mutants. H2 ts 111 DNA-terminal protein complex formed at 33 °C and chased at 39.5 °C showed a decreased affinity for glass fibre filters, concurrently with the loss of protein-linked DNA ends. H2 ts 111 DNA breakdown occurring upon shift-up to 39.5 °C therefore appeared to start in close proximity to the genome extremities. Marker rescue experiments showed that the ts character was abolished by co-infection with plasmid recombinants containing whole or part of the E2A region, encoding for the 72K DNA-binding protein. The N-terminal domain of this 72K protein has been assigned between 0 and 200 amino acids, and is supposed to have a function in late transcription control. Since H2 ts 111 mapped between 0 and 300 residues (63.6 to 65.9 map units), its mutation was most likely located between 200 and 300 amino acids, namely in the C-terminal domain of the protein, which is involved in DNA replication. Recombination between H2 ts 111 and H5 dl 313 mutant revealed that the cyt function was localized in the E1B zone, between 3.8 and 11.3 map units. The nucleolytic and cytocidal effects were complemented by HEK 293 cells, an H5-transformed cell line expressing the left-most 12.5% of the viral genome. H2 ts 111 appeared, therefore, as a double ts-cyt mutant. The gene product rendered temperature-sensitive by the H2 ts 111 mutation was found to act stoichiometrically, and not catalytically, a result compatible with a lesion in the 72K protein. Although inactive at the non-permissive temperature, the early 72K protein was normally synthesized and stable in H2 ts 111-infected cells at 39.5 °C. Assymetric complementation obtained with the DNA-defective H5 ts 36 implied a certain degree of type specificity in the DNA-binding protein function.

Two stable monoclonal hybridoma cell lines secreting type-specific antibodies against the human papillomavirus type 1 (HPV-1) were isolated. The monoclonal antibodies detected HPV-1 antigens in frozen sections of HPV-1-induced warts, using immunofluorescence or immunoperoxidase techniques, and they reacted with HPV-1 particles in an immunodiffusion test. The two monoclonal antibodies recognized the major structural viral polypeptide, with a molecular weight of 54000, and a minor polypeptide, with a molecular weight of 76000, in both the dissociated viral particles and in the wart extracts.

Treatment of scrapie-infected mice with dextran sulphate (DS) 500 resulted in considerably reduced spleen titres over a long period of time. Subsequently, the central nervous system disease was delayed or even prevented during the 350-day period of observation. Both effects increased after multiple injections of the compound. The potency of DS 500 to protect against scrapie was greatest when treatment and infection were carried out simultaneously. Under these conditions the lethality of 500 to 1000 LD50 was reduced to almost zero. Treatment as early as 10 weeks before infection still prolonged the incubation periods. Of several other polyions tested, dextran sulphate 5 and pentosan polysulphate also impaired scrapie pathogenesis.

The expression of immediate-early (IE) genes of herpes simplex virus type 1 (HSV-1, MP strain) in non-permissive rat XC cells was analysed and compared with the expression of IE genes in permissive HEp-2 cells by the following three methods: analysis of virus polypeptide synthesis in infected cells, Northern blot hybridization between poly(A) nuclear or cytoplasmic RNA and in vitro labelled virus DNA or plasmid-cloned fragments corresponding to IE genes, and ability of poly(A) cytoplasmic RNAs to direct synthesis of virus polypeptides in vitro. ICP4 (175K), ICP0 (110K) and ICP27 (62K) were synthesized in XC cells although in smaller amounts than in HEp-2 cells; ICP4 is functional since early and late polypeptides could be observed. Their corresponding mRNAs were present at low levels in nuclei and in cytoplasm and are functional since the polypeptides were synthesized in a rabbit reticulocyte system. ICP22 (68K) was not detectable in infected XC cells; its mRNA was present in nuclei and in cytoplasm, but it is not functional since the corresponding polypeptide was not synthesized in a rabbit reticulocyte system. This suggests some structural differences in the ICP22 mRNA molecules in infected XC and HEp-2 cells and implicates cellular determinants in the control of the expression of HSV-1 IE genes.

An enzyme activity from herpes simplex virus type 1 (HSV-1)-infected baby hamster kidney cells has been identified which generates large networks of pBR322 DNA from a monomeric DNA substrate. Extracts derived from cells infected at the non-permissive temperature, with the early regulatory mutants of HSV-1, tsK and tsB2, did not contain activity, suggesting that the enzyme is virus-induced and may be virus-specific. The enzyme is similar to the DNA topoisomerases in that network formation was dependent upon the presence of Mg2+ and a DNA condensing agent, and ATP was not required. Following digestion with EcoRI, the networks could be resolved to a single, linear, monomeric species of pBR322 DNA.

DNA from a cosmid-cloned gene library of human cytomegalovirus (HCMV) strain AD169 was found to share sequence homologies with cellular DNA of various origins. Viral DNA fragments subcloned in plasmid vectors enabled localization of the homologous sequences to five regions of the HCMV genome. Four of these regions were found in the long unique (U l ) segment of the viral genome (EcoRI fragments R, I and b, and HindIII fragment S); one region with virus-cell homology was located in the terminal inverted repeats (EcoRI fragments O and H). All hybridization reactions were carried out under stringent reannealing conditions (18 °C to 21.5 °C below average Tm of HCMV DNA). Fragments from these five HCMV DNA regions did not cross-hybridize with each other. The sequence homologies were seen in DNA from normal human placental tissue, liver autopsy material, intestinal tissue, white blood cells, and colon carcinoma cells, and in a series of haematopoietic cell lines. Sequence homologies were also detected in DNA from owl monkeys and Chinese hamsters, but not in mouse DNA. They were not related to human Alu repeats or cloned human c-myc sequences.

Interferon produced by mouse L-929 cells by incubation with poly(rI).poly(rC) is known to be composed of a mixture of MuIFN-α and MuIFN-β. The α component was separated from the β species by affinity chromatography over a monoclonal anti-MuIFN-β agarose column and partially purified by gel filtration. MuIFN-α, prepared by this method was separated into at least five subspecies by chromatofocusing. The approximate pI values of these components are ⩾ 7.5, 6.5, 6.2, 5.9 and 5.6, respectively. Component 3 (pI 6.2) was the most prominent subspecies present in our MuIFN-α preparations, representing 40 to 50% of the total antiviral activity. Component 1 (pI ⩾ 7.5) which accounted for about 5% of the antiviral activity on mouse cells, differed in some properties from the other interferon subspecies. It showed a relatively high antiviral activity on heterologous cells and it was eluted from a Sephadex column after the other α subspecies. Furthermore, it showed a diminished binding to heparin as compared to the other MuIFN-α subspecies, indicating a lower affinity for polynucleotides.

Transfection of Buffalo rat liver cells with closed circular hepatitis B virus DNA resulted in the synthesis of both hepatitis B e and surface antigens. A 14000 mol. wt. peptide bearing hepatitis B e antigenic determinants was isolated from cell culture fluids. Native hepatitis B e antigen was present in multimeric forms in the cell culture fluids and was associated with protein phosphokinase activity. The multimeric forms of hepatitis B e antigen may serve both structural and enzymic functions for the hepatitis B virion with its small genome.

Simultaneous infection of MDCK cells with influenza viruses A/WSN and B/Kanagawa resulted in mutual interference with virus protein synthesis and in significant suppression of A/WSN growth. When infection by one virus preceded the other by 1 or 2 h, growth of the superinfecting virus was selectively inhibited at the level of transcription. Interference by the pre-infecting virus was strongly dependent on the expression of the viral genome but not on haemagglutinin activity. When the replication of both virus types was restricted to primary transcription by cycloheximide, the only translation products following removal of the drug were those of the preinfecting virus. This result was not affected by blocking secondary transcription by actinomycin D. These findings suggest that intertypic interference occurs at the level of primary transcription. This concept was supported further by the observation that a ts mutant of A/WSN (ts-65) with a defect in primary transcription interfered only with superinfection by B/K anagawa at the permissive temperature.

The unique periodic nature of equine infectious anaemia (EIA) is believed to result from the ability of the infecting virus, EIAV, to undergo relatively rapid antigenic variations which circumvent host immune responses resulting in distinct virus populations in sequential clinical episodes in the persistently infected horse. This model was examined by oligonucleotide mapping comparisons of the RNA genomes of selected isolates of EIAV. Variations in oligonucleotide maps could be reproducibly demonstrated (i) after adaptation of the laboratory strain of EIAV to replication in a pony, (ii) after serial passage of virus between two ponies, and (iii) after a prolonged persistent infection in a single pony. In the latter case, the two isolates examined were recovered from different clinical episodes and were shown to be antigenic variants. In contrast, no variations in RNA structure could be detected in oligonucleotide maps of virus isolated after prolonged passage in tissue culture. Thus, these results support our concept that EIAV is a highly mutable virus, which may give rise to antigenic variants in the presence of immune pressures. The degree of variation observed between oligonucleotide maps is similar to that observed previously between variants of visna virus. These similarities between EIAV and visna virus suggest that genomic point mutations producing antigenic variants may be a more important mechanism of retrovirus persistence than was previously recognized.

Restriction endonuclease patterns of pseudorabies virus (PRV) DNA were examined after each of 11 serial passages of the virus through pigs. Minor variations in the electrophoretic mobility of certain restriction enzyme fragments were observed by the sixth passage. This variability was similar to some of the minor variability observed in field isolates. The variable fragments were mapped to three locations on the PRV genome: the junction between the short unique sequences and the repeat sequences, the terminus of the long unique region, and an internal area of the long unique region.

JC virus was previously isolated from the urine and from diseased brain of a patient with progressive multifocal leukoencephalopathy. The DNAs of these isolates, MAD-7 and MAD-8 respectively, were shown in this study to be mixtures of several different full-length genomes. By differences in their restriction endonuclease cleavage patterns, the genomes were categorized into two types. Type I DNA was defined as that typified by the prototype virus, MAD-1; Type II, that typified by viral DNA molecularly cloned from brain tissue of the MAD-7/8 patient (BrC-8 DNA). MAD-7 DNA was an approximately equal mixture of Type I and Type II; MAD-8 DNA appeared to be homogeneously Type II. These DNAs were molecularly cloned for further studies. Of 17 clones of recombinant MAD-7 (rMAD-7) DNA, nine were Type I and were identical to each other and to MAD-1 DNA. The other eight clones comprised five different species of Type II, none of which was identical to MAD-8 or to BrC-8 DNA. Among 17 recombinant MAD-8 (rMAD-8) clones there were two which contained Type I DNA, which had not been detected in the original viral DNA. The remaining rMAD-8 clones were of Type II. The differences among all these DNAs were primarily in the ‘extra’ PvuII sites and in the insertions and deletions in the hypervariable regulatory region of the genome (0.67 to 0.72 map unit). Possible explanations for multiple genomes in separate isolates from the same patient are discussed. It is concluded that the observed variation originated in vivo and not during isolation in vitro.

To test whether the covalent linkage of simian virus 40 (SV40) A gene DNA sequences might lead to integration of bovine papilloma virus type 1 (BPV-1) DNA sequences, recombinant pBR322 plasmids containing both the transforming region of the BPV-1 genome and the SV40 A gene were used for transformation of rodent cells. Plasmids containing both regions transformed with higher efficiency than plasmids containing either region alone. Various clonal cell lines were established and examined with regard to the physical state and the expression of the viral DNA sequences. BPV-1 RNA sequences were detected and the SV40 T-antigen was expressed. The plasmid sequences persisted in all cases in an unintegrated state. In no case was it possible to find evidence for integration of BPV-1 sequences.

Flow cytophotometry and time-lapse cinematography were used to study viral DNA synthesis and alteration of the cellular DNA content of human cells infected by adenovirus type 2 wild-type and two DNA-negative temperature-sensitive mutants. Cell populations with DNA contents greater than 4n (where n represents the normal haploid DNA content of cells) were found after infection and their origin is discussed with regard to cell cycle changes, viral DNA replication and cell fusion or endomitosis.

Bovine parvovirus readily produced plaques when inoculated into 60% confluent, actively growing bovine embryonic lung cells. Incorporation of DEAE-dextran, MgCl2 and DMSO in the agarose overlay medium was found to improve plaque production, especially with the latter chemical. In contrast, protamine sulphate inhibited plaque development. It was found that plaque titration and plaque inhibition tests could be conveniently carried out in 24-well cell culture plates, using an agarose overlay containing DMSO, DEAE-dextran and foetal calf serum. The procedures were highly sensitive, when compared with other established techniques.

Treatment of lymphocytic choriomeningitis virus with proteolytic enzymes, hyaluronidase, and phospholipase C increased infectious titres. Biochemical analysis of bromelain- and trypsin-treated virus revealed that infectivity was high in spite of the decrease to low or undetectable levels of all viral glycoproteins as well as partial degradation of the nucleoprotein.