- Volume 65, Issue 11, 1984

Bacillus subtilis bacteriophage SP3 was shown to be flagellotropic. Mechanical deflagellation of host strain 168wt prevented phage adsorption but adsorption was normal when deflagellated 186wt was allowed sufficient time to regenerate flagella. Two host strains lacking flagella (130 fla − and 19 fla −) did not adsorb SP3. Two host strains with paralysed flagella (38mot − and 13mot −) adsorbed SP3 inefficiently. Revertants of fla − and mot − strains to the motile phenotype adsorbed SP3 normally. A temperature-sensitive host mutant, ts46, with reduced numbers of flagella at 46 °C was incapable of efficient SP3 adsorption at this temperature. However, transfection of ts46 by SP3 DNA occurred with virtually identical efficiency at either temperature. Adsorption of a non-flagellotropic phage, SP82, to all bacterial strains used was unaffected by absence of flagella. Isolated flagella adsorbed SP3 inefficiently. Spontaneously non-motile strains of 168wt had lost the ability to support growth of SP3 but SP82 grew normally. Revertants of these strains to the motile phenotype regained the ability to support SP3 growth. Strains derived from surviving bacteria in SP3 lysates of 168wt were non-motile.

MDCK cells were infected with six human influenza C virus strains (isolated between 1947 and 1981) and seven pig influenza C virus strains (isolated in 1981 and 1982) and the virus-specific polypeptides were compared by SDS-polyacrylamide gel electrophoresis and one-dimensional peptide mapping. The major structural polypeptides, i.e. glycoprotein (gp88), nucleoprotein (NP), and membrane protein (M), and one non-structural polypeptide were identified in all strains by radiolabelling infected cells with [35S]methionine. No differences in the electrophoretic migration of the M proteins or NS proteins were observed. The two earliest human isolates, C/Taylor/1233/47 and C/Great Lakes/1167/54, had faster migrating NP proteins, and another human strain, C/Georgia/1/69, displayed a faster migrating gp88. Minor differences in the one-dimensional peptide maps produced by partial digestion of the M proteins with V8 protease were observed between the human and pig isolates, while more marked differences were noted in the peptide maps of the glycoproteins of the C/Georgia/1/69, C/Yamagata/10/81 and C/Yamagata/11/81 viruses compared to the other human strains and the pig strains. The overall conclusion is that the proteins of human influenza C viruses isolated over a 35 year period and those of recent pig influenza C virus isolates are highly conserved.

Human bone marrow cells derived from multiple sclerosis (MS) and control patients were screened for a number of virus antigens by the fluorescent antibody technique using monoclonal antibodies. The results showed that antigens of the paramyxovirus, simian virus 5, were present in about 60% of MS and 25% of control bone marrows. About 25% of the MS and 50% of control bone marrows were found to contain nucleoprotein antigen of the human parainfluenza types 1 and 3. These experiments demonstrated that paramyxoviruses can persist in human tissues possibly in a defective or repressed state.

We have characterized the physiological defect in two temperature-sensitive mutants of the WSN strain of influenza virus which possessed a lesion in the haemagglutinin (HA) gene. In mutant virus-infected cells at the non-permissive temperature, the precursor HA polypeptide containing predominantly mannose-rich carbohydrate chains was not converted to the mature, functional HA polypeptide. Immunofluorescence showed that the HA polypeptide did not appear on the cell surface but was confined largely to the Golgi apparatus. It was concluded that the major physiological defect of these mutants was a block in the transport of the HA polypeptide beyond the Golgi apparatus. The block could be reversed, however, by lowering the temperature to 34 °C, resulting in normal processing of the precursor polypeptide and emergence of infectious progeny virus within 30 min. The HA activity of the two mutants, but not wild-type virus, was rapidly inactivated at 51 °C. Most, but not all, revertants derived from these mutants had HA with the heat stability of wild-type virus, suggesting that the temperature sensitivity and the heat lability of HA were two pleiotropic manifestations of a single lesion in the HA gene.

Eighteen strains of Getah virus isolated from mosquitoes, swine and horses in Japan (1956 to 1981), and one strain isolated in Malaysia (1955), were analysed by RNase T1-resistant oligonucleotide fingerprinting. All fingerprints showed a poly(A) tract. The fingerprint pattern of the Malaysian strain was quite different from those of the Japanese strains. Although most of the recent Japanese isolates shared many large oligonucleotide spots in common, the patterns were not identical even among the strains obtained in one locality in the same year. These results suggest that the Getah virus genome undergoes mutation rather frequently. However, there is a tendency for the isolates of the same year to show greater similarity. The fingerprint patterns of certain host-dependent temperature-sensitive (ts) mutants differed from that of the parental strain. Also, there were some differences in large oligonucleotide spots between strain JaNAr12380M isolated in suckling mouse brain (SMB) and strain JaNAr12380A isolated in C6/36 cells, despite the fact that both strains were derived from the same wild mosquito homogenate. In addition, many host-dependent ts mutants were present in strain JaNAr12380A, whereas no such mutants were observed in strain JaNAr12380M. It is concluded that there is considerable variation in the strains of Getah virus infecting mosquitoes in the wild, and also that the variants or mutants present in mosquitoes might be subject to selection during viral multiplication in the mammalian host.

Certain defective interfering (DI) Semliki Forest virus (SFV) preparations completely protected the majority of mice inoculated with a normally lethal dose of SFV, and the surviving mice showed no signs of disease during the period of observation. Depending upon which DI SFV preparation was used, the survivors were resistant to challenge with 100 LD50 SFV (DI SFV p13a), or were completely sensitive (DI SFV p4), the latter having evidently failed to establish a protective immunity. In this report we compared the ability of these two DI SFV preparations to inhibit multiplication of infectious virus in mice inoculated with 10 LD50 SFV. The following conclusions emerged: (i) virus multiplication was profoundly inhibited in the majority of mice treated with either of the DI virus preparations although there was significant multiplication in most tissues, including brain. The number of mice showing evidence of reduced infectivity titres (58%) correlated well with the 60% which survived without disease in lethality experiments. (ii) Despite the presence of infectivity, no SFV antigen or histopathological lesions were detected in brain or spinal cord. (iii) The DI virus preparations p4 and p13a altered the distribution of infectivity in the mouse in different ways: during the first 2 days of the infection modulated by DI virus p4, the infectivity titres (in brain, olfactory lobes and spleen) were comparatively high, being >1% of those in mice inoculated with standard virus alone. However, from day 3, titres declined precipitously and there was little infectivity in any of the tissues investigated. On the other hand, mice treated with DI SFV p13a had, over the entire duration of infection, greatly reduced though significant infectivity in brain, olfactory lobes and spleen and very little infectivity in serum. (iv) In a minority of mice (14.5%), DI virus p13a altered the distribution of infectivity between different tissues so that there was significantly decreased virus in just one or two of the four tissues investigated, suggesting that the infection was being subtly modulated by the DI virus. (v) Interference assays failed to detect DI SFV in any tissue samples although the effects of DI virus on infection in the mouse were obvious.

After digestion with trypsin, α-chymotrypsin, or chemical cleavage using CNBr, fragments of the tick-borne encephalitis (TBE) virus glycoprotein were isolated which retained their reactivity with neutralizing monoclonal antibodies defining a denaturation-resistant antigenic domain. Upon immunization of mice, these fragments induced antibodies reactive with the immunizing peptide, the denatured glycoprotein and the native glycoprotein as a constituent of the whole virus. The immune sera revealed the same properties as the monoclonal antibodies that were used to select the fragments for immunization: neutralizing activity; haemagglutination-inhibiting activity; blocking of the binding of antibodies used for selection; enhancement of the binding of other monoclonal antibodies defining a denaturation-sensitive antigenic domain. It was shown that the natural immune response against certain functionally important, denaturation-resistant immunogenic domains on the native protein can be closely mimicked by immunization with defined protein fragments. Antigenic sites present on these fragments may therefore represent essential constituents of a synthetic vaccine. The fine specificities of antibody populations in anti-peptide or anti-protein immune sera were analysed on the basis of single antigenic determinants by blocking assays using radiolabelled monoclonal antibodies that define eight distinct epitopes on the TBE virus glycoprotein. Quantitative differences in the blocking of certain monoclonal antibodies were also observed between human convalescent sera. The establishment of such blocking profiles using a panel of well-characterized monoclonal antibodies may represent a general method for dissecting the specificities of antibody populations present in polyclonal immune sera and could allow investigations on determinant-restricted differences of immune responses and its possible implications for the course of the disease.

A panel of hybridoma antibodies that react with the surface peplomer glycoprotein (E2) of the murine coronavirus JHM were produced to characterize major antigenic domains associated with functions related to virulence. Three groups of hybridoma antibodies were differentiated by immunoprecipitation of lysates from JHM-infected cells. One group precipitated the virion structural proteins gp170 and gp98 together with the intracellular form of E2, gp150. A second group reacted with gp98 and gp150, and a third group precipitated gp150 only. Competition assays with biotinylated hybridoma antibodies allowed the definition of at least six different epitope groups. Only those antibodies which immunoprecipitated both gp170 and gp98 neutralized infectivity, inhibited cell fusion and protected infected rats against acute disease. Another class of antibodies binding to gp170 and gp98 also neutralized JHM virus, but did not inhibit fusion and did not protect against disease. Antibodies that immunoprecipitated gp150 and gp98 revealed only weak neutralization and did not inhibit cell fusion or protect animals. Four epitopes were defined by antibodies that immunoprecipitated gp150, but revealed no biological activity. These data indicate that the site responsible for cell fusion is associated with an epitope group carried by gp170 and gp98. Neutralizing antibodies bind to this and another epitope. Furthermore, protection of JHM-infected rats against acute disease requires both inhibition of cell fusion and neutralization of virus.

Characterization of the viruses produced by the spontaneous T lymphoma cell line SL3 is presented. Using supernatant fluids or direct co-cultivation of cells, the SL3 cell line was found to produce replication-defective viruses in excess of replication-competent viruses. The replication-competent viruses released were predominantly those negative in the XC plaque assay (XC−); XC+ viruses represented a minor population. However, when the SL3-derived viruses were passed in mouse embryo fibroblasts, XC− viruses were rarely recovered, and XC+ viruses were readily isolated. These viruses were all ecotropic and lymphomagenic. Viruses with dual host range and non-oncogenic ecotropic viruses were not isolated from the lymphoma cells. Two replication-defective viruses from SL3 cells were studied. Both could be rescued by non-oncogenic retroviruses and were then lymphomagenic. One defective virus appeared related to XC+ viruses. In these studies, the XC+ and XC− viruses appeared to represent two different interference classes using separate cell receptors. Taken together, these experiments show that the SL3 T lymphoma cells replicate a variety of viruses most of which are lymphomagenic. Virus replication and/or virus integration may be the means of maintaining the malignant phenotype of these T lymphoma cells.

A systematic study has been performed using a series of differentiated rat thyroid epithelial cell lines either uninfected or infected with Kirsten murine sarcoma virus (KiMSV), to determine the levels of the p21 product of the v-ras-Ki oncogene in transformed and normal cell lines. The p21 levels have been assayed by SDS-polyacrylamide gel electrophoresis of immunoprecipitates of 35S-labelled cell extracts and by a GDP binding assay. All cell lines analysed showed a significant increase in the levels of p21 after transformation with KiMSV compared to the p21 levels of uninfected and untransformed differentiated thyroid cells. The results reported here confirm the potential ability of the v-ras-Ki oncogene product to transform epithelial cells. They show, furthermore, that not only is p21 present in some epithelial cells transformed by KiMSV, but also that it is functionally active, as has been shown for fibroblasts transformed by the same virus, and that its functioning is maintained after passaging in vivo of the transformed cells.

Plasma membrane-enriched preparations from scrapie-infected and healthy hamster brains were detergent-extracted, then separated by equilibrium density centrifugation in continuous Nycodenz® gradients. The highest level of infectivity was always associated with the insoluble residue which sedimented through 40% Nycodenz. The degree of aggregation in these insoluble complexes varied depending upon treatment. Centrifugation in gradients containing 2 m- to 8 m-urea resulted in the formation of large insoluble aggregates which seemed to retain a high level of infectivity when measured by the method of incubation interval assay. However, measurement of infectivity in these same samples by endpoint titration of tenfold dilutions resulted in values a thousand times lower. These observations reinforce previous findings that scrapie infectivity exists as a macromolecular complex and, furthermore, they emphasize the necessity for using non-denaturing conditions for purification of the scrapie agent.

The DNA molecule of varicella-zoster virus (VZV) is represented structurally as L-S, where L is a unique sequence and S is a unique sequence flanked by an inverted repeat. S may be present in either orientation in virion DNA molecules, but, to date, L has been found in only one orientation. DNA sequences were determined at the L-S joint and genome termini, which were cloned using methods designed to conserve either the 3′ or 5′ terminal nucleotide. Molecular hybridization experiments and analysis of the sequences showed that: (i) the genome is not terminally redundant; (ii) the unique sequence in L is flanked by an inverted repeat of 88.5 base pairs; (iii) a single unpaired nucleotide is located at each 3′ terminus of the genome, such that fusion of the termini would produce a sequence identical to that at the L-S joint; (iv) approx. 5% of virions contain genomes with L inverted. The implications of these results in possible mechanisms for VZV DNA replication are discussed.

The morphological characteristics of murine cytomegalovirus replication in murine hepatocytes were investigated by electron microscopy and structural evidence of an unusual mode of virus maturation was detected. Nucleocapsids form in the nucleus; those with electron-dense cores bud into the perinuclear cisternae and acquire an outer envelope from the inner membrane of the nuclear envelope. Viral envelopes fuse with the outer membrane of the nuclear envelope releasing nucleocapsids in the paranuclear zone where they aggregate and form cytoplasmic inclusions. These inclusions are invariably surrounded by lamellae and vesicles of the Golgi complex into which nucleocapsids again bud. Virions, now covered with a new membrane envelope, are transported within secondary lysosomes and released by emiocytosis into the extracellular space.

Virions of human cytomegalovirus were shown to contain two discrete membranes. An outer, loose fitting, membrane was sensitive to osmotic shock and could be partially removed by diluting buffered preparations of virions with water. Purified virions were shown, by SDS–polyacrylamide gel electrophoresis of virion membranes labelled by carbohydrate-specific procedures, to contain five glycoproteins with molecular weights of 52, 67, 95, 130 and 250, all × 103. Digestion of virions with endoglycosidases revealed that there were structural differences between the carbohydrate portions of the glycoproteins. All five glycoproteins were recognized by antibodies present in pools of human convalescent sera.

Condylomata acuminata and Buschke-Löwenstein tumours were analysed for the presence of human papillomavirus (HPV) transcripts. HPV DNA and RNA sequences were present in all 13 samples investigated. Ten contained HPV6 and three harboured HPV11. The HPV genomes were found exclusively as extrachromosomal circular molecules. In six biopsy specimens, viral RNA transcripts were not detectable by Northern blot analysis but could be demonstrated in dot blots. From seven HPV6-containing samples it was possible to obtain sufficient amounts of undegraded mRNA. We have found consistently one major species (1.4 kb). Less prominent species of 1.7, 1.85, 2.7 and 3.2 kb, respectively, were also detected. The 3′ ends of the HPV6 mRNAs were located between nucleotides 3917 and 4441 in the putative early region and between nucleotides 7232 and 7696 in the putative late region. The arrangement of the 3′ termini and the adjacent coding areas within the HPV6 genome show that the RNA species are transcribed from one DNA strand.

The DNA of a multiply-enveloped nuclear polyhedrosis virus (MNPV) of the cabbage moth Mamestra brassicae (Mb), isolated from the beet armyworm Spodoptera exigua, was analysed using restriction endonucleases SmaI, BamHI, BglII and PstI. The size of the viral DNA was calculated to be about 152 kilobase pairs. Mapping of restriction sites on the DNA was accomplished by hybridization of 32P-labelled DNA fragments to single and double digests of the MbMNPV genome. Mapping of the putative polyhedrin gene was used to orient the physical map. The relevance of this map to the structural organization of the DNA of MbMNPV isolated from different geographical areas is discussed.

A subcellular system actively replicating Kilham rat virus DNA in vitro was developed. Cellular lysates and isolated nuclei from infected cells showed an amplification of replicative forms in vitro. Solubilized replicative complexes, either partly purified or in the form of a crude extract, were able to synthesize replicative forms and single-stranded DNA. DNA polymerase α played a major role in Kilham rat virus DNA synthesis in vivo and in vitro. Furthermore, a factor present in the cytosol of infected cells increased the polymerizing activity of viral replicative complexes.

Monoclonal antibodies directed against several herpes simplex virus (HSV)-induced DNA-binding proteins were used to investigate protein interactions in HSV-infected cells. Q1 monoclonal antibody, which is specific for the HSV-induced alkaline nuclease, when used in an immunoadsorbant column resulted in the purification of the alkaline nuclease, to which large quantities of the major DNA-binding protein were bound. Conversely, when a monoclonal antibody to the major DNA-binding protein was used in affinity chromatography other polypeptides (including the DNA polymerase and alkaline nuclease) were eluted in addition to the major DNA-binding protein. Similar results were obtained when the experiment was performed using a monoclonal antibody to another HSV-2 DNA-binding protein. These results suggest the possibility that these polypeptides interact as part of the HSV DNA replication complex, and this hypothesis is discussed.