- Volume 54, Issue 2, 1981

Results of analysing antigenic variation in the haemagglutinin (HA) molecule of naturally occurring influenza A (H1N1) viruses from 1977 to 1979 with monoclonal antibodies were found to be dependent in some instances on the test system used. In several instances A/USSR/90/77 HA-specific monoclonal antibodies had sharply reduced haemagglutination-inhibition (HI) titres with variant virus although they bound to the variant and A/USSR/90/77 HAs with similar efficiencies as judged by titration in a sensitive and accurate solid-phase immunofluorimetric assay. In another instance, the converse situation was observed: monoclonal antibodies having a reduced efficiency of binding to the HA of a variant virus nevertheless had comparable HI titres with the variant and with A/USSR/90/77. The chemical basis and epidemiological significance of these observations remain to be elucidated. Nevertheless, the finding that the reaction of monoclonal antibodies can, in some cases, be markedly dependent on the test system employed is of significance for the efficient design and correct interpretation of immunochemical studies which employ monoclonal antibodies to investigate the basis for variation in influenza strains.

Twenty-eight mutations, representing mutation in five different polypeptide-coding regions of the foot-and-mouth disease genome, were examined for their effect on the virulence of the virus for suckling mice. Five types of mutation were examined: temperature-sensitive (ts), electrophoretic (e), co-variant temperature-sensitive and electrophoretic (ts/e), guanidine-resistant (gs +) and putative co-variant guanidine-resistant and electrophoretic (gs +/e). All the ts mutations and three out of the 11 non-ts mutations produced some reductions in virulence. In the majority of cases this reduction in virulence was shown to co-vary with the mutation. No correlation was observed between the site of a mutation or its ‘cut-off’ temperature and the extent of the reduction in virulence.

Studies of the growth in vivo of a small selection of ts mutants suggested that for most mutants their reduced virulence was a trivial effect of their slow growth rate. With one exception they all eventually grew to parental virus levels, the resulting virus being temperature-sensitive and the disease indistinguishable from that caused by the parental virus. The one exception was an avirulent ts mutant which only grew to one-thousandth the titre of the parent virus. This mutant did not cause disease and was therefore considered to be the only avirulent mutant. Its mutation was in the coat protein-coding region of the genome, probably the region coding for VP3.

We describe a simple, rapid and reproducible assay for defective-interfering Semliki Forest virus (DI SFV) which is based on the inhibition of synthesis of virus-specified RNAs in SFV-infected cells. Using the assay, we have been able to show that DI virus is generated by a single passage in baby hamster kidney (BHK) cells in an inoculum which contained no detectable DI virus and we have calculated the u.v. target size of the interfering activity.

We have studied the virus-like 30S (VL30) RNA sequences of mice. Previous work has shown that these sequences are coded in the mouse genome, expressed in some normal cells and released as pseudotypic particles from cells producing murine C-type retroviruses. VL30 sequences have some similarities to standard retrovirus RNA, but differences also exist. To further assess the similarities and differences, several aspects of VL30-specific metabolism were investigated. We studied the initiation of VL30-specific DNA synthesis during an endogenous reverse transcriptase reaction. Short initial VL30-specific cDNA transcripts were covalently attached to RNA as measured by equilibrium banding in caesium sulphate density gradients. Therefore, reverse transcription of VL30-specific cDNA is initiated by an RNA primer. The intracellular synthesis of VL30 RNA was investigated by pulse labelling uninfected JLS-V9 cells with 3H-uridine. Hybridization of the pulse-labelled nuclear RNA indicated that the major VL30-specific RNA evident after a 15 min label was the same size as the mature VL30 RNA. Thus, VL30 RNA is apparently not synthesized via a higher mol. wt. precursor. Both of these results demonstrate similarity of VL30 RNA sequences to standard retroviruses. One unique feature of VL30 RNA was detected. JLS-V9 cells contained both the monomeric VL30 RNA and a hydrogen-bonded 38S form which yielded the monomer when denatured. This contrasts with standard murine leukaemia virus which is only found as a monomer within cells.

Human leukocyte interferon (IFN-α) was induced in cultures of human peripheral blood mononuclear leukocytes (PBML) from seropositive healthy donors infected by mumps virus, or by a human lung carcinoma cell line (Pc-10) persistently infected with mumps virus (Pc-10/MpV). Adherent cells (Mϕ), non-T, non-B cells and B cells all produced IFN in response to mumps virus or Pc-10/MpV cells. The non-T, non-B cells co-cultured with Pc-10/MpV cells produced high-titred IFN. However, T cells did not produce any antiviral factors even in the presence of Mϕ. Furthermore, T cells activated with phytohaemagglutinin (PHA-P), concanavalin A or treated with u.v.-irradiated mumps virus, or Pc-10/MpV cells pretreated with mitomycin C did not produce any antiviral factors in response to either mumps virus or Pc-10/MpV cells.

Mouse L fibroblasts infected with mouse hepatitis virus, MHV3, and radiolabelled with 35S-methionine, contained, in addition to the three major structural polypeptides, p24, p56 and p180, two additional ones, p22 and p50. Of these total five polypeptides, only three, p22, p56 and p180, were labelled in infected cells during a 2 min 35S-methionine pulse and are, therefore, presumed to be immediate translation products. Pulse-chase and chymotryptic peptide mapping experiments showed apparent precursor-product relationships between p56 and p50 and between p22 and p24. Protein synthesis in infected cells was synchronized at the initiation stage by pre-exposure to hypertonic medium. Using a 0.5 min pulse-10 min chase sequence, to limit incorporation of 35S-methionine to stretches of approx. 100 amino acids adjacent to translational initiation sites, it was found that all three polypeptides, p22, p56 and p180 contained radiolabel. It is thus apparent that translation of the three major structural proteins (or precursors) is initiated independently rather than at a single site as in the cases of other positive-strand RNA viruses such as polio or Semliki Forest virus.

Feline rotavirus was detected by electron microscopy in the faecal samples of a cat, and was propagated in an established cell line of foetal rhesus monkey kidney, MA104, cell cultures. Morphologically, feline rotavirus was indistinguishable from known rotaviruses. Complete particles showed a characteristic ‘spoke-like’ arrangement of inner capsomeres surrounded by an outer layer. Intracytoplasmic inclusion bodies in different sizes and shapes were produced in infected MA104 cells. Reproducible clear-cut plaques were produced by feline rotavirus in MA104 cells under the overlay of carboxymethylcellulose in the presence of trypsin. Feline rotavirus was distinct from human, canine, bovine, porcine and simian rotaviruses by the plaque reduction neutralization test. Feline rotavirus, like canine and simian rotaviruses, was found to be less dependent upon trypsin than human, bovine, porcine, chicken and turkey rotaviruses. A seroepidemiological survey (September 1979 to August 1980) showed that 20 out of 61 (32.8%) randomly sampled hospitalized cats at the Cornell Veterinary Teaching Hospital in Ithaca, New York had antibody titres against feline rotavirus. Oral inoculation of cats with feline rotavirus did not produce any clinical disease, but most cats did mount an immune response to the virus following inoculation.

Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells (SP2) with spleen cells derived from Balb/c mice immunized with purified measles virus. Fifteen independent hybrid cell lines, isolated from two separate fusions, were maintained in culture for up to 5 months without loss of their antibody-secreting activity. Radioimmunoprecipitation and polyacrylamide gel electrophoresis showed that in five of the hybrid lines the antibodies were directed against haemagglutinin, in two against the nucleoprotein, and in one against L protein. The remaining seven hybridomas did not precipitate viral antigens under the experimental conditions employed even though they gave positive immunofluorescence against measles virus-infected cells. Monoclonal haemagglutinin antibodies displayed anti-haemagglutinating activity and neutralized measles virus infectivity but not canine distemper virus (CDV).

During the course of characterizing a series of temperature-sensitive mutants of pseudorabies virus, we found one (designated tsL) that did not produce cytopathic changes in rabbit kidney cells at the non-permissive temperature (41 °C). Although the mutant adsorbed to and penetrated the cells in a normal fashion, virus RNA was not synthesized at 41 °C in the infected cells. However, if the cells were first incubated at the permissive temperature (32 °C), virus RNA synthesis occurred at the non-permissive temperature. This occurred even if, during the incubation period at 32 °C, the expression of viral functions was prevented by treatment with an inhibitor of protein synthesis. The DNA in tsL virions did not appear in the cell nucleus at 41 °C, and full, non-enveloped nucleocapsids could be recovered from the cytoplasm of tsL virus-infected cells. These results show that the nucleocapsids of tsL remain intact at the non-permissive temperature and that tsL is an uncoating mutant.

The nature of the DNA in incomplete particles (IP) synthesized by adenovirus type 2 and the ts4 mutant which accumulates such particles were analysed by agarose gel electrophoresis, restriction endonuclease cleavage and blot hybridization techniques. IP DNA consisted of a heterogeneous population of subgenomic-size DNA (IPSD1) and smaller molecules ranging from about 1000 base pairs to 200 base pairs (IPSD2). IPSD1 from ts4 was more heterogeneous than that from wild-type (wt), but both contained sequences from all parts of the viral genome. IPSD2 contained heterogeneous cellular sequences and viral sequences from the left 4.4% of the genome. An endonuclease activity associated with IP and virions was capable of digesting viral or cellular DNA to IPSD2-like fragments suggesting a possible origin for these molecules.

A cell line (LB59Ly) derived from the leukocytes of a leukaemic cow was established as a monolayer, which spontaneously released large amounts of a retrovirus. This virus was found to be indistinguishable from the bovine leukaemia virus (BLV) produced by the commonly used high-producing heterologous cell line (FLK-BLV). Like the latter, its reverse transcriptase activity was greater in the presence of Mg2+ cations than in the presence of Mn2+ cations; its polyacrylamide gel electrophoresis pattern showed the presence of gp51, p24, p15, p12 and p10, and the antigenicity of the two major proteins completely cross-reacted with those of BLV from FLK-BLV cells. The virus was infectious and induced early and late polykaryocytosis, the specificity of which was demonstrated by use of specific anti-BLV sera.

We have studied the relationship between Friend spleen focus-forming virus (SFFV) and its helper lymphoid leukaemia virus (LLV) by comparing RNase T1 fingerprints of genomic RNAs. Our data indicate that about 70% of the SFFV sequence is a perfect copy of parts of the helper genome. We conclude that our SFFV and LLV isolates have co-evolved very closely and that SFFV-specific sequences are not identical in different Friend virus isolates.

Immobilization of Raji cells on surfaces coated with anti-lymphocyte globulin (ALG) at low cell densities lead to the synthesis of Epstein—Barr virus (EBV) early antigen (EA) in up to 5% of the cells. At higher cell densities the percentage of antigen-positive cells decreased and at confluency no antigen synthesis was observed. Addition of iododeoxyuridine (IdUrd) to low density cultures increased the expression of EA to 20%, whereas in confluent cultures the cells could not be induced to synthesize EA. Treatment of cells in suspension with ALG failed to induce EA synthesis and did not potentiate the effect of IdUrd. Immobilized Raji cells proved to be suitable targets for superinfection with EBV derived from P3HR1 cultures.

Factors influencing the replication of varicella-zoster virus (VZV) in guinea-pig embryo cells were evaluated using both diploid cells (GPEC) and a chemically transformed cell line (GPT). Wild-type and vaccine strains of VZV were successfully isolated and serially propagated in GPEC prepared from early gestation whole embryos (<2 cm in length). Low passage GPEC (≤5 subcultivations) were more susceptible to VZV infection than high passage GPEC (>5 subcultivations), and guinea-pig cells were consistently less permissive than human diploid cells. Cell-free virus was produced from VZV-infected GPEC cultures by sonication and peak yields of 103 p.f.u./ml were obtained. In addition, we report the isolation and propagation of VZV, as well as production of cell-free virus, in GPT. Both GPEC and GPT cells were less susceptible to VZV infection than human cells. However, viral replication was enhanced by incubation of VZV-infected GPT cultures at 32 °C rather than 36 °C.

The hybrid plasmid pTK1 consists of the herpes simplex virus type 1 (HSV-1) BamHI p fragment, which contains the thymidine kinase (TK) gene, inserted into the vector pAT 153. When pTK1 DNA was microinjected into nuclei of Xenopus laevis oocytes, functional HSV-1-specific TK was produced, showing that transcription and translation of the gene occurred. Investigation of pTK1-specific RNA by ‘Southern’ blot hybridization revealed that all regions of the hybrid plasmid were transcribed by RNA polymerase II, but sequences present in TK mRNA were most highly represented in stable transcripts.

In order to understand the mechanism of congenital cytomegalovirus (CMV) infection, we studied the effect of murine cytomegalovirus (MCMV) on murine ectoplacental cone (EPC) cells in vitro. Cytopathic effects (c.p.e.) were seen in many MCMV-infected EPC cell cultures 5 to 7 days after exposure to MCMV. The infected cells showed intranuclear inclusions characteristic of CMV infection when stained with May-Grunwald-Giemsa. Culture fluids collected from MCMV-infected EPC cells after 4 or more days of culture caused c.p.e. when co-cultivated with mouse embryo fibroblasts (MEF). Employment of the anti-complement immunofluorescence (ACIF) test detected MCMV-specific antigens, in situ hybridization localized MCMV DNA and electron microscopy detected the presence of the viral particles in the MCMV-infected EPC cells. Thus, exposure of EPC cells to MCMV in vitro resulted in a productive infection.