- Volume 67, Issue 1, 1986

West Nile virus (WNV)-immune spleen cells produced an inducer of macrophage procoagulant activity (MPCA) on restimulation with WNV in vitro. This response was specific for WNV and depended on the presence of Thy 1+, L3T4+ and also Ia+ cells but not Lyt 2+ cells. It could be induced by culture with large amounts of non-infectious, u.v.-irradiated virus or with high doses of infectious virus. Inoculation of mice with low titres of infectious, but not with u.v.-irradiated, virus primed for this in vitro response. The time of maximum production of procoagulant-inducing factor was also the period of greatest [3H]thymidine uptake and release of interleukin-2 (IL-2)-like activity. In bulk cultures, MPCA-inducing factor was more readily quantified than IL-2. T cells of the helper/delayed-type hypersensitivity class are thus stimulated by WNV and this aspect of cell-mediated immunity can be readily assayed in vitro.

The role of gangliosides in rabies virus infection of chick embryo-related (CER) cells was investigated. Cultured cells were pretreated with neuraminidase to render the cells transiently non-susceptible to viral infection. Incubation of these desialylated cells with gangliosides allowed them to incorporate exogenous gangliosides and they recovered their susceptibility to rabies virus infection. Infection of CER cells was monitored by specific fluorescence 24 h after virus inoculation. The use of individual purified gangliosides or mixtures of two gangliosides to restore cellular susceptibility to viral infection showed that GT1b and GQ1b were the most effective. The disialogangliosides were also active, principally GD1b, whereas GM1, GM3 were poorly active and GD3 inactive. Incubation of rabies virus with gangliosides prior to virus infection reduced the percentage of infected cells. The results indicate that highly sialylated gangliosides are part of the cellular membrane receptor structure for the attachment of infective rabies virus. However, it is possible that other glycoconjugates such as glycoproteins or glycolipids also participate as components of a receptor structure for rabies virus.

Cultured human fibroblasts showed a typical fibrillar organization of microtubules in immunofluorescence, including the vimentin type of intermediate filament as well as actin-containing microfilaments. During infection with herpes simplex virus type 1 (HSV-1), the vimentin organization was maintained whereas actin, myosin and tubulin showed a progressive association with the viral glycoproteins within juxtanuclear structures. These structures could also be revealed with fluorochrome-coupled wheat germ agglutinin. Disruption of the microtubules by demecolcine treatment or their stabilization by taxol treatment did not prevent the aggregation of viral proteins in the cytoplasm. Taxol stabilization of the microtubules allowed the juxtanuclear accumulation of the glycoproteins in HSV-infected cells whereas treatment with demecolcine led to an accumulation of the glycoproteins either in small vesicles in the cytoplasm or in the focal adhesion areas of the cells. Production of infectious intracellular virus particles was reduced in cells treated with demecolcine or with taxol before and during infection. The results of this study indicate that the normal intracellular transport and distribution of the HSV glycoproteins and the formation of infectious virus are dependent on the presence of intact microtubules.

Three groups of monoclonal antibodies which reacted with cells infected by guinea-pig cytomegalovirus (GPCMV) were prepared. The first group of antibodies immunoprecipitated a 50000 mol. wt. (50K) polypeptide of GPCMV-infected cells. This polypeptide was identified as part of the nuclear inclusion by immunofluorescence. This nuclear fluorescence was markedly diminished when the infected cells were incubated in the presence of phosphonoacetic acid. By immunoelectron microscopy the antibodies reacted mainly with filamentous structures (26 to 28 nm in diameter) in nuclear inclusions and occasionally stained nucleocapsids. Neither intracytoplasmic nor extracellular virions reacted with the antibodies. Therefore, the 50K protein with which the monoclonal antibodies reacted was nuclear inclusion-specific and a non-structural protein. The second group of antibodies reacted with a 76K polypeptide of the infected cells which was a matrix protein found in both cytoplasmic inclusions and extracellular dense virions. The third group of antibodies mainly reacted with a virion core protein by immunoelectron microscopy.

Thirty-two hybridoma cell lines producing monoclonal antibodies (MAbs) against the three major structural proteins of transmissible gastroenteritis virus (TGEV) have been isolated. Radioimmunoprecipitation of intracellular viral polypeptides showed that 17 hybridomas recognized both the peplomer protein [E2, 220 × 103 mol. wt. (220K)] and a lower mol. wt. species (E′2, 175K), which was characterized as a precursor of E2. Six MAbs selectively immunoprecipitated the E′2 protein. Four hybridomas were directed against the low mol. wt. envelope protein (E1, 29K), and three against the nucleoprotein (N, 47K). All major neutralization-mediating determinants were found to be carried by the peplomers. Several anti-E2 MAbs displayed an intrinsic neutralizing activity close to that of the most potent anti-TGEV polyclonal reagents tested (including ascitic fluid of feline infectious peritonitis virus-infected cats). None of the anti-E′2 MAbs induced significant neutralization, although this protein might be incorporated to some extent into the virions. Immunofluorescence patterns obtained with MAbs directed against either the envelope glycoproteins or the nucleocapsid revealed distinctly different distributions of these antigens within the cells. Comparison of nine TGEV strains using our panel of MAbs confirmed their close antigenic relationship, but revealed the occurrence of distinct antigenic differences.

Comparative antigenic and nucleic acid analyses were carried out on two new atypical rotavirus isolates coming respectively from chickens (D/132) and pigs (E/DC-9). Indirect immunofluorescence showed that each virus carried different group antigens which were also distinct from those of previously described rotavirus groups. By genome profile analysis each virus had a pattern of genomic RNAs clearly distinct from those of the other rotavirus groups. Comparative terminal fingerprinting of corresponding genome segments from the two viruses showed large differences between them, indicating that all of their genomic RNAs had significant differences in sequence both from each other and from the three previously defined rotavirus groups. On the basis of these results, extension of the number of rotavirus groups from three to five is proposed, with isolates D/132 and E/DC-9 being the type members of groups D and E respectively.

The proteins of sheep pox, goat pox, sheep and goat pox and lumpy skin disease (Neethling) viruses were labelled with [35S]methionine. The major structural polypeptides of these viruses co-migrated on polyacrylamide gels, demonstrating the very close biochemical relationship between them. Using the agar gel immunodiffusion (AGID) test with radiolabelled antigen preparations, a major common precipitating antigen was identified. This co-migrated on polyacrylamide gels with one of the major structural polypeptides [mol. wt. 67000 (67K)]. The use of [35S]methionine-labelled antigen preparations considerably improved the sensitivity of the AGID test as a diagnostic test for capripoxvirus antibody detection.

Haemagglutinating (HA) antigens of four strains of virus related to that causing haemorrhagic fever with renal syndrome (HFRS) were prepared from infected tissue culture fluids by ultracentrifugation. The titres of the precipitated antigens were increased considerably by acetone extraction and sonication. Acetone extraction completely inactivated infectious virus in the antigen preparations. The antigens were pH-dependent, with pH optima at 5.8. Good correlations were observed in human and rat sera between the titres obtained by the haemagglutination-inhibition (HI) test and an indirect fluorescent antibody test. Moreover, strong cross-reactions among these strains were demonstrated by the HI test. The HI test has not been used previously with HFRS viruses because of the danger involved in preparing HA antigen, but these results indicate that a safe method is available for serological identification of HFRS.

Fusion between purified [3H]uridine-labelled West Nile virus (WNV) particles and liposomes containing RNase, was assayed by degradation of the viral RNA to trichloroacetic acid-soluble material. Fusion of virus with liposomes containing phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol (at a molar ratio of 1:1:1:1.5) was found to be dependent on pH with maximum fusion occurring at pH 6.7 and below. At pH 6.6 fusion was rapid and was essentially complete within 2 min at 37 °C. At this time, approximately 50% of the viral RNA had been degraded and increasing the concentration of liposomes or time allowed for fusion increased this percentage only slightly. Fusion was dependent on temperature, was almost totally non-leaky and was not dependent on the presence of divalent cations. The lipid composition of liposomes was found to influence both the pH optimum for fusion and the maximum degree of fusion observed. Electron microscopy was used to visualize the fusion reaction between liposomes and virus particles.

An isolate of the non-occluded baculovirus Hz-1, containing a high level of defective particles, was recovered after serial passage in Trichoplusia ni (TN-368) tissue culture cells. DNA from defective virions contained deletions of up to 91 kilobase pairs. Defective particles were shown to interfere with the infection or replication of standard virions. Standard virus appeared to be required for replication of defective interfering (DI) particles. Initiation of both standard and DI viral DNA replication occurred at 4 h post-inoculation. Virus-induced protein synthesis was studied by pulse-labelling with [35S]methionine. During defective virus replication in the presence of low amounts of standard virus, there was a significant reduction in the synthesis of 14 proteins (mol. wt. 109, 93, 92, 90, 85, 69, 57, 50, 40, 27.5, 23, 17.5, 17 and 14, all × 10-3) and an increase in the synthesis of five proteins (mol. wt. 104, 75, 41, 37 and 14.2, all × 10-3) as compared to standard virus infections.

Changes in DNA polymerase activities were studied in pupae of the silkworm Bombyx mori upon infection with nuclear polyhedrosis virus (BmNPV). Two effects were observed in male and female pupae after inoculation of BmNPV: a marked increase in cellular α-polymerase activity and the induction of a new DNA polymerase (BmNPV-polymerase). Activities of both DNA polymerases, α and BmNPV, in the infected pupae increased in parallel, reached a maximum at 72 h post-inoculation and decreased subsequently. The virus infection did not affect the activity of γ-polymerase in the pupae. An increased activity of β-polymerase in the pupae was detected at the late stage of the infection cycle prior to pupal death. The possible roles of the DNA polymerases in BmNPV replication are discussed.

The avian herpesviruses infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as the mammalian herpesvirus pseudorabies virus (PRV) were able to provide complete helper activity for the production of infectious avian adenoassociated virus (AAAV) in chicken cells. The presence of AAAV in the infected chicken cell reduced the multiplication of HVT. ILTV or PRV, however, were not affected if used as helper viruses. Infectious AAAV was determined by an indirect immunofluorescence assay and infectious herpesvirus by plaque assays.

Following intradermal inoculation of bovine papillomavirus type 4 (BPV-4) into a Syrian hamster, a liposarcoma developed at the inoculation site 20 months later. The DNA of this tumour contained multiple copies of the BPV-4 genome which existed in a free unintegrated state. Unintegrated viral DNA and viral DNA isolated from virus particles from bovine alimentary tract papillomas revealed identical cleavage patterns with CpG methylation-resistant and -sensitive restriction enzymes: apparently there was no gross methylation of CpG sites in either case. The entire BPV-4 genome appeared to be represented in the tumour DNA.

Isolates of yellow fever (YF) virus from Africa (Senegambia, Central African Republic, Ivory Coast, Burkina Faso) and from South America (Panama, Ecuador, Trinidad) were examined by oligonucleotide fingerprinting of the 40S genome RNA. Geographically isolated and epidemiologically unrelated viruses were very distinct. On the basis of the T1 oligonucleotide fingerprints of each isolate, four geographical variants (topotypes) of YF virus isolated within the same period of time have been established. The Ivory Coast and Burkina Faso topotypes were similar. In the Central African Republic, two variants could be found exhibiting 70 to 75% homology to one another. In South America, the three analysed strains exhibited only about 70% homology, but could be classified in the same topotype. The oligonucleotide fingerprints of the genome RNA offered a useful tool for the understanding of YF virus variability.

Chick interferon (IFN), produced in primary chick embryo (CE) cells stimulated by u.v.-irradiated Newcastle disease virus, was partially purified by two-step chromatography using both controlled pore glass and Blue Sepharose. The specific activity of the IFN increased about 500-fold by this method and the final recovery from starting material was more than 95%. The partially purified IFN was analysed by SDS-PAGE, and two peaks of IFN activity were observed. The molecular weight represented by the sharp peak was estimated to be 18000 (18K) and a broad peak was found at 20K to 30K. Glycosidase treatment before SDS-PAGE resulted in disappearance of the broad peak and increased the activity of the 18K peak. Anti-CE IFN rabbit serum and a monoclonal antibody against the CE IFN neutralized the antiviral activity of all IFN samples prepared under various conditions.

Circular dichroism studies are reported on the conformation of cucumber mosaic virus particles and of their isolated components. In particular, it was remarkable that the satellite RNA seemed to have less order than genomic RNA and also had a destructuring influence on the organization of the genomic RNA inside the virion.

Serological differentiation indices (SDIs) based on serum titrations in agar-gel double diffusion tests were determined for all ten definitive tombusviruses known at present, namely artichoke mottled crinkle, carnation Italian ringspot, cymbidium ringspot, eggplant mottled crinkle, Moroccan pepper, pelargonium leaf curl, petunia asteroid mosaic, the BS3 and type strains of tomato bushy stunt, and tombus Neckar viruses. More than 2000 titrations using 222 antisera from 36 rabbits were done. The SDIs ranged from 1 to >9, and all intermediate values were found. Simple trigonometry and computer methods were used to classify the results. There was no correlation between the serological relatedness of the particles of these viruses and the homology of their genome nucleic acids previously assessed by hybridization tests. In this respect they resemble the tymoviruses but not the tobamoviruses. The electrophoretic migration of the particles of 12 tombusviruses was studied in 1% agarose containing 20 mm-phosphate buffer pH 7.0. Some serologically indistinguishable tombusvirus strains migrated at different rates.