- Volume 62, Issue 2, 1982

Coliphage BA14 was isolated from sewage and shown to be related to phages T7 and T3. It is similar to T3 in that it directs the synthesis of an S-adenosyl-methionine-cleaving enzyme (SAMase) early upon infection. However, it differs from all other known T7-related coliphages by the inability of its RNA polymerase (gene 1 product) to transcribe T7 DNA or T3 DNA. BA14, T7 and T3 also show marked differences in autoradiographic patterns of their gel-electrophoretically separated 35S-labelled intracellular phage proteins, restriction endonuclease HpaI cleavage patterns of their DNAs, and serological specificities of their infectious particles. Other distinctive features became apparent upon simultaneous mixed infection with BA14 and T7 or T3: inability of BA14 to produce genetic recombinants with either T7 or T3; lack of functional complementation between amber mutants of BA14 and T7 or T3; mutual exclusion and depression of the burst size of the mixedly infected cells.

Cytopathological studies have shown that Yaba tumour pox virus (Yaba virus) infection leads to the accumulation of large lipid vacuoles. The rate of accumulation of these vacuoles increased as the infection proceeded. These lipid vacuoles were not seen in control cells or in cells infected with monkeypox virus (MPV) but were seen during Yaba virus infection in the presence of cytosine arabinofuranoside (100 μg/ml). Yaba virus also failed to inhibit host protein synthesis as infection proceeded for prolonged periods. Yaba virus proteins were shown to be substantially different from those of MPV when analysed by two-dimensional electrophoresis. The genome of Yaba virus gave restriction enzyme fragments which differed from those of the MPV genome when cleaved with the enzymes HindIII and XhoI. However, Yaba virus DNA hybridized to the HindIII fragments K, L and M and to the XhoI fragments A, B, C, E and G of MPV DNA.

Radioactively labelled virus particles of intracellular origin were isolated from the cytoplasmic fraction of disrupted NIH/3T3 cells chronically infected with Moloney murine leukaemia virus [NIH/3T3 (MLV)]. Interferon (IFN) treatment for 48 h, which arrested more than 90% of virus release, resulted in a remarkable accumulation of these intracellular virions. However, no major effect of such treatment was apparent on their structural properties. Transmission electron microscopic examination revealed that these intracellular virions were located within cytoplasmic vacuoles. IFN treatment resulted in a considerable increase in the number of virus-containing vacuoles, as well as the total number of vacuolar virions. It seems that IFN inhibits the final release of vacuolar virions from the cells, thus leading to their intracellular accumulation.

Chromosome-mediated transfer of murine leukaemia (MuLV) and murine sarcoma (MuSV) virus genetic information to uninfected recipient cells was investigated. Metaphase chromosomes from AKR MuLV-infected SC-1 mouse cells were incubated with NIH/3T3 cells. After several passages (1 to 3 weeks), infectious virions exhibiting reverse transcriptase activity and the characteristic host range of ecotropic, N-tropic AKR virus appeared in the supernatant fluids of the treated cells. Restriction endonuclease analysis of genomic DNA from transfected cells indicated that AKR proviral DNA was associated with the high molecular weight DNA of the host. These results demonstrate that the AKR MuLV genome can be stably transferred to uninfected recipient cells via isolated metaphase chromosomes. Although AKR virions are not able to infect heterologous cells, chromosome-mediated transfection resulted in the establishment of productive AKR MuLV infection in mink cells. Thus, the use of chromosomes to transfer virus genes can circumvent the natural host restriction barrier. In other experiments, it was shown that normal NIH/3T3 cells were transformed after exposure to metaphase chromosomes isolated from an MuSV-infected, non-producer line. Foci were detected 14 to 21 days after chromosome treatment and were shown to contain true viral transformants since transforming virus was produced after superinfection with MuLV.

Fowl plague virus (FPV) ts mutants belonging to six recombination groups and obtained from the Weybridge strain (in the U.S.S.R.) or the Rostock strain (in the U.K.) have been studied in a recombination test. Temperature-sensitive mutants obtained from different FPV strains were revealed which had a ts mutation in gene 1; however, their crossing resulted in ts + recombinants which appeared with a high frequency. This phenomenon was due not to intragenic complementation but to extragenic suppression, when the expression of a ts phenotype of the Rostock strain mutant gene 1 is suppressed by gene 2 products of the Weybridge strain.

The phospholipid metabolism of a measles virus persistently infected cell line, BGM/Hallé, was compared with that of uninfected BGM cells. Synthesis of phospholipid from the isotopic precursor [32P]phosphate was unaffected, but a significant increase in the synthesis of phospholipid from [3H-9,10(n)]palmitic acid was observed in persistently infected cells, reflecting an increase in the palmitic acid content of phospholipid previously described for these cells. The phospholipid composition of measles virus released from persistently infected cells was compared to that of virus from lytically infected BGM cells by radiolabelling to isotopic equilibrium and measuring the incorporation of labelled phosphatides into the virus particle. The incorporation and distribution of [32P]phosphate-labelled phosphatides in virus was similar in lytic and persistent infections. In contrast, the distribution of [3H]palmitic acid among the constituent phosphatides of virus released from the persistent infection was different, partly reflecting the altered saturated fatty acid composition of the phosphatides of the host cell.

The virion proteins and intracellular polypeptides of the murine coronavirus MHV-JHM have been analysed by two-dimensional fingerprinting of their [35S]methionine-containing tryptic peptides. The analysis shows that the virion proteins gp98, gp65, pp60 and p23 are distinct. Virion protein gp25 has the same polypeptide component as p23, and virion protein gp170 has a polypeptide component related to gp98. The six virus polypeptides synthesized in infected cells, 150K, 65K, 60K, 30K, 23K and 14K are also distinct. The 170K and 98K species, which are produced by processing, are related to 150K. The 25K species is a processed form of 23K. The identity of corresponding species in the cell and in the virion has been shown and a model describing the genesis of coronavirus JHM proteins can now be proposed.

Nine virus-specified proteins were identified by SDS-polyacrylamide gel electrophoresis in [35S]methionine-labelled chick embryo cells infected with tick-borne encephalitis (TBE) virus by comparison with mock-infected cells. These proteins were designated P91, p74, p72, P67, GP53(E), P47, p25, P15(C) and P14.5 according to their molecular weights. Peptide mapping of P91, P67, GP53(E) and P47 from TBE virus-infected cells, as well as those of the corresponding proteins from West Nile virus (WNV)-infected cells (previously termed NV5, NV4, V3 and NV3), demonstrated the uniqueness of these proteins. Almost no subtype variability, with respect to the pattern of intracellular proteins, was found when isolates of TBE virus from Austria, Switzerland, Germany, Finland and Czechoslovakia were compared. Peptide mapping of NV5 (P91) and NV4 (P67) from all these isolates using limited proteolysis with α-chymotrypsin and V8 protease revealed completely identical patterns, thus extending our observations that TBE virus seems to represent a very stable member of the flavivirus genus, which was based on the lack of variation found with the structural glycoprotein. On the other hand, the Far Eastern subtype of TBE virus and the closely related louping-ill virus could not only be differentiated from the Western subtype by differences in the peptide maps of their structural glycoprotein but also in those of the non-structural protein NV5, i.e. subtype or subgroup variations are not confined to the virion surface glycoprotein. WNV and Murray Valley encephalitis virus (MVEV) revelaed the expected heterogeneity of virus-specified proteins found in cells infected with different flaviviruses. It is especially interesting that also the largest non-structural protein, NV5, is subject to this heterogeneity, ranging in mol. wt. from 91000 for TBE virus to 98000 for MVEV and that also the peptide maps of NV5, as well as those of NV4, were unrelated. These proteins, therefore, revealed a variability between serologically distinct flaviviruses similar to that observed with the structural glycoprotein.

The reaction of foot-and-mouth disease virus (FMDV) with 12S subunit/140S virion cross-reactive (sensitizing) antibody was studied in order to elucidate the requirements for neutralization versus sensitization. The presence of sensitizing antibody in immune serum caused an atypical in vitro neutralization response curve and a non-neutralized fraction. Cell-associated (cytophilic) antibody was not present in the system. Dissociation of the immune complex was not a factor and sensitized virus adsorbed to host cells via the regular virus receptor site(s). This finding led to the conclusion that sensitizing antibody is specific for non-critical sites. Dosing of the neutralization reaction mixtures with fractionated antibody of alternative antigenic specificities had an antagonistic effect on the neutralization response, suggesting steric hindrance. Cell receptor sites were a factor in sensitization since different host systems had different susceptibilities for sensitized antigen. The results suggest that in vitro neutralization of FMDV requires the attachment of multiple antibody molecules as proposed by the multi-hit theory of neutralization. The in vitro measurement of serum neutralizing activity as an indication of the in vivo immune response is discussed.

Monospecific polyclonal antisera were raised in guinea-pigs against the calf rotavirus polypeptides VP1, VP2, VP3* + 4*, VP4.2, VP6, VP7.1, VP7.2 and VP10. All of the antisera gave a similar pattern of cytoplasmic immunofluorescence in rotavirus-infected cells, but spots of fluorescence of varying intensity with different sera were seen over the nucleus. Immune precipitation, using Staphylococcus aureus to collect immune complexes, showed that VP2 was precipitated by antiserum to VP2 (α-VP2) and VP6 by α-VP6. α-VP7.1 and α-VP7.2 both precipitated the same range of proteins from infected cells (VP7, VP7.1 and VP7.2) or from virions (VP7.1 and VP7.2). VP10, either from virions or infected cells, was not precipitated by α-VP10. The only antiserum which efficiently neutralized infectivity was α-VP7.2. There were low levels of neutralization with α-VP10 (but the results varied from experiment to experiment) and traces with α-VP6, α-VP7.1 and the other antisera did not neutralize even though α-VP7.1 agglutinated double-shelled particles as seen in immune electron microscopy to a greater extent than α-VP7.2 Both VP7.1 and VP7.2 were shown to be glycoproteins by tunicamycin treatment of infected cells. Core particles only were agglutinated by α-VP10. All the evidence leads us to conclude that there were major neutralizing antigenic determinants present on VP7.2, a minor component of the outer shell of the virion.

A novel bisegmented double-stranded RNA virus has been isolated from water processed from Thirlmere reservoir. The virus is icosahedral, 58 nm in diam., has a buoyant density of 1.32 g/ml in CsCl, has an S value of 400 and a RNA/protein ratio of 0.087. The two linear segments of RNA have approx. mol. wt. of 2.26 × 106 and 2.09 × 106. The virus contains six polypeptides. The virus was isolated in Drosophila melanogaster cells and fails to replicate in other insect, amphibian, avian, piscine, mammalian and plant cells tested. The virus is biochemically different from infectious pancreatic necrosis virus (IPNV) and Drosophila X virus (DXV). The virus is also serologically unrelated to IPNV (strain Sp) and another invertebrate pathogenic virus, Tellina virus 1. The virus shares common antigens with DXV but is not completely identical.

Interferon production in mouse spleen cells and mouse fibroblasts (L cells) stimulated by three strains of Newcastle disease virus (NDV), Italian, La Sota and Ulster, was investigated. Strain Italian was fully infectious and highly virulent; strain Ulster exhibited very low infectivity and very low virulence; strain La Sota was between these extremes. All of these strains of MDBK cell-grown NDV could induce interferon in mouse spleen cells, and it was concluded that proteolytic cleavage of F0 protein of NDV and, consequently, virus penetration are not necessary for interferon induction in these cells. On the other hand, NDV with uncleaved F0, which was characterized by an apparent lack of haemolytic and cell fusion activity and infectivity for tissue culture cells, had no interferon-inducing ability in L cells. The cleavage of F0 protein was paralleled by an appearance of interferon-inducing activity in L cells.

The pH-dependent profiles of haemolysis by influenza viruses AHK/8/68 (HK virus) and APR/8/34 (PR8 virus) were found to possess characteristic differences. Both viruses were highly haemolytic at pH 5.0, but while HK virus-induced haemolysis was undetectable above pH 5.5 to 5.6, PR8 virus-induced haemolysis persisted at higher pH values, becoming undetectable above pH 5.7 to 5.8. In order to determine whether these haemolysis profiles were genetically determined, we studied the pH dependence of haemolysis by eight recombinants of HK and PR8 viruses. The pH profile of each recombinant clearly resembled that of one parent or the other, showing that it is an inherited trait. The pH profile was found to be conferred exclusively by the haemagglutinin (HA) gene.

Polypeptides of ‘trypsin-resistant’ (TR) variants of foot-and-mouth disease virus type O1 (BFS 1860) were analysed by electrofocusing and two-dimensional gel electrophoresis. In contrast to parent O1 virus, trypsin treatment of these variants did not reduce their infectivity and their ability to attach to susceptible cells, although VP1 was cleaved as in the parent virus. In OTR1, one of the cloned isolates, an additional polypeptide (VP a ) with a mol. wt. approx. 31 × 103 (31K), was found which resembled VP1 (28K) in being positively charged and cleaved by trypsinization of the virus into a neutral 18K polypeptide (P18) and a strongly basic fragment (pI > 10) with a mol. wt. of approx. 6K (P6). These findings substantiate the hypothesis that VP a is an elongated VP1. While P18 fragments of both trypsin-treated parent virus and OTR progeny viruses focused at identical (neutral) pH. P6 fragments of trypsinized OTR variants (including OTR1) were even more positively charged than P6 of parent virus. This difference in charge of the P6 polypeptide may be responsible for the retained cell attachment ability of trypsinized OTR viruses. The data are discussed with respect to the known amino acid sequence of VP1 of the closely related O1 Kaufbeuren.

A clone of vesicular stomatitis virus (VSV) isolated after 76 months of persistence exerts specific homologous interference with replication of wild-type or tsG31 (temperature-sensitive) virus in a mixed infection at 37 °C in the absence of defective-interfering particles. Other VSV ts mutants showed little or no specific interfering ability.

Twelve temperature-sensitive (ts) and two morphology mutants of Autographa californica nuclear polyhedrosis virus were generated using the mutagen 5-bromo-2′-deoxyuridine (BrdUrd). The ts mutants grew normally at 25 °C but exhibited abnormal occlusion body formation when grown at 33 °C whereas the morphology mutants produced an abnormal cytopathic effect at 25 °C and 33 °C. None of the mutants was severely restricted for production of non-occluded virus; thus, with the eight ts mutants and the two morphology mutants incubated at 33 °C the drop in titre was 0.5 to 3.0 log10 from that at 25 °C. The mutants fell into three groups based on the amount of polyhedrin synthesized at 25 °C and 33 °C in infected cells. The eight ts mutants were combined with ts mutants constructed in a previous series of experiments and the collection was sorted into complementation groups. The collection comprised ten groups and the new mutants were sorted into seven of these. It is concluded that gene products of these complementation groups have essential roles in occlusion body formation.

The virus particles from four closely related insect nuclear polyhedrosis virus (NPVs) isolated from their homologous Spodoptera spp. hosts were examined using an indirect enzyme-linked immunosorbent assay (ELISA), employing both swine anti-rabbit and protein A conjugate. Results from both systems indicated that S. littoralis NPV is serologically distinct from the other three viruses, S. frugiperda NPV, S. exempta NPV and S. exigua NPV, which are closely related. The relatedness of these viruses is discussed with reference to existing knowledge and the results obtained from both conjugate systems.

Similarities in the trypsin sensitivity of certain polypeptides of adenovirus types 5 and 12 (Ad5 and Ad12) have been used to identify some capsid polypeptides of Ad12. One of the polypeptides suggested to be the IIIa of Ad12 has proved to be the major structural phosphoprotein as in the case of Ad5.