- Volume 65, Issue 7, 1984

The antigenic relationships between three bovine rotaviruses, UK, CP-1 and PP-1, and a porcine rotavirus were investigated; their pathogenicity for piglets was also assessed. After propagation and cloning in cell culture, the four viruses were used to produce convalescent and hyperimmune antisera in gnotobiotic animals. For the UK and PP-1 viruses, reciprocal virus neutralization tests gave ratios of homologous to heterologous titres in the range 57 to >2300. Taking a 20-fold difference in titre to be the criterion of heterogeneity, these two viruses could be regarded as separate serotypes. The third bovine virus, CP-1, was identical to PP-1 in reciprocal neutralization tests but gave a one-way cross-reaction with UK convalescent sera, i.e. it appeared to be intermediate between the two serotypes. UK and CP-1 differed antigenically from the porcine virus as shown by homologous:heterologous neutralization titre ratios of 30 to 12600. The bovine virus, PP-1, however, had a closer relationship with the porcine virus, the homologous:heterologous ratios ranging from 4 to 110. Oral inoculation of piglets with the bovine viruses revealed differences in their pathogenicity. Isolates UK and CP-1 caused no clinical disease and could not be passaged. However, on the first and second passages in pigs, the bovine virus PP-1 and the porcine virus produced clinical disease, excretion of virus in the faeces and seroconversion. The results suggest that PP-1 may be a natural hybrid between a bovine and a porcine rotavirus.

The oligosaccharides of the structural glycoprotein (VP7) of calf rotavirus were characterized. The precursor of VP7 produced in infected cells in the presence of tunicamycin migrated on SDS-polyacrylamide gels with an apparent molecular weight 6000 less than the glycosylated glycoprotein. Endoglycosidase (Endo) H digestion of the mature virus resulted in a decrease of 5000 in the molecular weight of VP7 in two discrete stages. Analysis of Endo H-treated, 3H-labelled digestion products of VP7 on Bio-Gel P-4 identified an oligosaccharide of molecular weight 1350 as the predominant form. Further treatment of the digest with mannosidase and analysis on Bio-Gel P-2 columns indicated that the oligosaccharide was digested into a free mannose and an oligosaccharide of molecular weight 400 in the ratio of 6:1. This indicates that the oligosaccharides of VP7 consist of four N-linked (Man)7 residues, two of which occupy more exposed and two more cryptic positions in the VP7 molecule.

Mouse hepatitis virus 3 (MHV3) infection in mice varies according to the mouse strain used; they may show resistance, semi-susceptibility (paralysis) or full susceptibility (lethal acute hepatitis). In order to study the mechanism of inborn resistance, viral infection was carried out in primary cultures of embryonic fibroblasts originating from various mouse strains exhibiting different sensitivities to MHV3 infection. Virus-induced cytopathic effects and cell membrane antigens as well as virus replication and interferon synthesis were studied. Persistent infection was induced in four out of six primary embryonic fibroblast cultures and in six of six secondary cultures. Two primary embryonic fibroblast cultures from susceptible strains underwent total lysis. A high yield of virus was obtained, as tested by viral titres and cell membrane antigen detection. No significant difference in virus production or in interferon synthesis was observed in fibroblast cultures of the various mouse strains tested. Cytopathic effects characterized by cell lysis were related, however, to phenotypes in vivo. This effect was lost after a single trypsinization of the culture. In addition, resistance to virus-induced cell lysis was inhibited by actinomycin D treatment. These results indicate that the intrinsic resistance of fibroblasts is effective not against virus replication but against virus-induced cell lysis. Such a cellular control mechanism may be an important factor for resistance in vivo.

A number of polypeptides synthesized specifically in Trichoplusia ni multiple nucleocapsid nuclear polyhedrosis virus (T. ni MNPV)-infected Spodoptera frugiperda cells are phosphorylated both early and late in infection. Certain non-structural proteins and the major basic internal protein are the main phosphoproteins detected in infected cells. The polyhedron protein was not phosphorylated. Many cell proteins continue to be phosphorylated throughout infection. Pulse-chase experiments have shown that some polypeptides are stably phosphorylated whereas other polypeptides (including the major basic protein) have phosphates which cycle on and off. One polypeptide was substantially labelled only after a chase with unlabelled orthophosphate. Fractionation of cells into nucleus and cytoplasm showed that polypeptides located in both the cytoplasm and nucleus were phosphorylated.

The sequence homology between the RNA genomes of Cricket paralysis virus (CrPV) and Drosophila C virus (DCV) strains have been compared using cDNA hybridization analysis. Results show 60 to 85% homology between DCV strains obtained from different geographical locations. There was no detectable sequence homology between DCV and CrPV.

N:NIH(S) II nu/nu (athymic) and +/nu (euthymic) mice were inoculated with coxsackievirus B3 (CBV-3) and examined at various times after infection for virus titres in the heart, myocarditis and serum neutralizing antibodies. Virus was recovered from the hearts of nu/nu mice for up to 94 days post-inoculation, but was not recovered from the hearts of any +/nu mice beyond 14 days. Inflammatory cell infiltration and necrosis were present in the hearts of +/nu mice at all harvest times (7, 14, 21 and 28 days). Inflammation and necrosis did not become evident in nu/nu mice until 14 days post-inoculation, and was then present in mice from each harvest until the end of the experiment (94 days). In athymic mice, myocarditis showed a strong correlation with persistence of CBV-3 in the heart. In N:NIH(S) II mice, the presence (+/nu) or absence (nu/nu) of a thymus had a major influence on the clearance of virus from the heart and on the development of myocarditis.

Swiss mice immunized with formalin-inactivated encephalomyocarditis virus (EMCV) produced antibodies to EMCV and were protected when infected intraperitoneally with EMCV. Administration of a potent sheep anti-mouse interferon-α/β (IFN-α/β) globulin before reinfection did not alter the vaccine-induced protection. Reinfection was associated with a rapid disappearance of the virus at the site of injection and viraemia was not demonstrable. IFN was not detected in the peritoneum or serum. Furthermore, injection of anti-IFN globulin before, during or after vaccination did not modify the humoral response to vaccination or to reinfection. These results indicate that IFN-α/β does not play a significant role in the resistance of mice to reinfection with EMCV.

The ability of mammalian rotaviruses to replicate in BSC-1 and CV-1 cell cultures was facilitated by the presence of 4% chicken serum. Viral plaques tended to be larger and appear more quickly than in cultures without added chicken serum. It is proposed that chicken serum facilitates plaquing of rotavirus due to its lack of trypsin inhibitors.

The structural glycoproteins of calf (BDV 486), human (Wa) and simian (SA11) rotaviruses were compared for sensitivity to endoglycosidase (Endo) H. The calf and human virus glycoproteins were reduced by approximately 5000 molecular weight in two equal stages. The simian rotavirus glycoprotein sustained only one 2500 molecular weight decrease. The effect of the host cell on the viral oligosaccharide composition was examined. Endo H digests of calf rotavirus 82–124 extracted from stool and its progeny propagated and [35S]methionine-labelled in MA104 cells were compared. Endo H had the same effects on the glycoproteins, indicating that the nature of the host cell does not affect the oligosaccharide composition of the virus glycoproteins.

A major virus-specific immediate-early (IE) polypeptide with an apparent mol. wt. of 52000 (52K) was synthesized immediately after the removal of a cycloheximide block applied to owl monkey kidney cell cultures at the time of infection with high multiplicities of herpesvirus saimiri (HVS). The IE 52K polypeptide was phosphorylated and accumulated rapidly and efficiently in the nucleus of infected cells. Monoclonal antibodies to IE 52K and to a major non-structural delayed-early (DE) DNA-binding protein with similar electrophoretic mobility (DE 51K) were used to show that the IE 52K protein and DE 51K proteins are antigenically distinct. The monoclonal antibody to the IE 52K protein of HVS strain 11 reacted in immune precipitation and immunofluorescence tests with polypeptides of similar mol. wt. present as nuclear antigens in cells infected with heterologous strains of HVS and herpesvirus ateles.

Conditions are defined under which herpes simplex virus (HSV) type 2 produces a characteristic cytopathic effect (c.p.e.) in African green monkey kidney (BS-C-1) cells. The BS-C-1 microplate c.p.e. test was simultaneously evaluated against immunological and biological marker procedures on 359 HSV strains. A BS-C-1 cell suspension was added to serial dilutions of HSV strains in Falcon Micro Test II plates and incubated at 36.5 °C in a 4% CO2 atmosphere. When the infected cultures were examined microscopically, distinctive pseudopod-like extensions were seen in cultures infected with type 2 strains but not seen when the infecting virus was type 1, forming the basis for a differentiating test. The test was 100% accurate in differentiating between HSV-1 and -2 on a one test per specimen basis. The method is simple, rapid and requires no specialized reagents.

Vero cells infected with herpes simplex virus in the presence of actinomycin or cycloheximide, or with u.v.-inactivated virus, suffered a rapid loss of functional mRNA as determined by translation in vitro. It is suggested that a virion-associated factor causes a structural change in the mRNA of the host cell.

The uptake rate of some sugars was demonstrated in human embryo fibroblasts following infection by human cytomegalovirus (HCMV) and a significant increase in glucose uptake was demonstrated. Quantitative and sequential analysis of glucose uptake during the HCMV replication cycle showed that the enhanced uptake began during the first 20 h after infection and occurred even when a high glucose level was constantly present in the medium. The increase in sugar uptake requires an active viral genome, de novo protein synthesis and seems to be dependent only on the early transcription of the viral genome, as it did occur when replication of the viral genome was limited (non-permissive cells or cells infected in the presence of DNA synthesis inhibitors).

Individual cloned HindIII fragments of vaccinia virus DNA were introduced into cells by DNA-mediated gene transfer. The presence of individual fragments in the different transformants was confirmed by Southern blot hybridization analysis. Several of the transformants were found to express viral sequences at various levels. The sizes of the transcripts containing vaccinia virus sequences were highly heterogeneous, with no discrete species of RNA. Positive clones contained vaccinia virus sequences in both the poly(A)+ and poly(A)− RNA fractions, although the prevalence of these sequences was variable in the two fractions. The S1 nuclease map of the 5′ end of the transcripts from transformants containing the HindIII-J fragment revealed a unique 5′ end, similar to RNA from virus-infected cells. In contrast, analysis of the 3′ end of RNAs from these transformants showed a high degree of heterogeneity, which might explain the heterogeneity found in Northern blot patterns. In this report, it is shown for the first time that in cells transformed with vaccinia virus DNA there is proper initiation for, at least, the viral thymidine kinase gene.

Foetal mouse glial cultures were inoculated with murine K papovavirus and subjected to serial subcultivation. Two cell lines were developed. The first of these, KVBCG2A, remained positive for viral infectivity and K virus capsid (V) antigen for over 30 subcultivations. Productive infection was not abolished by serial subcultivation in the presence of antiviral antibody. The second cell line, KVBCG1B, became negative for infectious virus and K virus V antigen, could be cloned from single cells and produced tumours in mice. Sera from tumour-bearing animals produced nuclear fluorescence of KVBCG1B cells and K virus-infected mouse embryo cells but did not react with uninfected mouse embryo cells or with cells infected by polyoma virus. DNA hybridization studies confirmed the presence of K virus DNA in KVBCG1B cells and suggested integration of the viral genome into host chromosomal DNA. K virus produces both persistent infection and cell transformation in glial cultures derived from its natural host.

Seven species of dsRNA were isolated from Nicotiana clevelandii plants infected with tomato bushy stunt virus (TBSV). One of these had a length of 4.8 kilobase pairs (kbp), corresponding to the replicative form dsRNA of TBSV genomic ssRNA; the other six were subgenomic RNA with lengths ranging from 3.3 to 0.44 kbp. Analyses of total and polysomal ssRNA from TBSV-infected tissue by gel transfer hybridization with cDNA probes prepared from genomic TBSV RNA revealed genomic RNA and nine shorter species of ssRNA. Six of the latter were shown to be artefacts probably attributable to the interaction between fragments of TBSV RNA and plant RNA. The remaining three had lengths of 1.8, 1.5 and 1.1 kbp and, since they were found in polysome preparations, may be TBSV subgenomic mRNA species; none of these corresponded exactly in length to any of the subgenomic dsRNA.