- Volume 68, Issue 12, 1987

Sensitivity to lethal infection with murine cytomegalovirus depends on the H-2 and background phenotype. H-2d appears to confer sensitivity in isolated cells, but sensitive BALB/c (H-2d) mice also exhibit non-specific immunosuppression which may indicate an impaired protective immune response. To determine the significance and mechanism of this immunosuppression, genetic factors controlling the activation, lymphocytostatic potential and accessory cell function of peritoneal macrophages were analysed after sub-lethal infection. In BALB/c mice, the number of peritoneal cells declined by 20% on day 3 post-infection and increased threefold over normal levels by day 7 with a progressive increase in macrophage activation and differentiation. Cells collected on day 7 exhibited lymphocytostatic activity which was not influenced by indomethacin and depressed their ability to act as accessory cells in a proliferative assay. Similar changes in the numbers of activated mature macrophages occurred in moderately resistant BALB.K (H-2k) mice showing an association with the background phenotype. In contrast peritoneal cell counts from resistant CBA (H-2k) mice were depressed by 80% on day 4 and the remaining cells enhanced the proliferation of syngeneic lymphocytes. However, later in the infection the percentage of peritonea] cells releasing virus declined rapidly and fewer cells became lymphocytostatic in both H-2k strains.

The properties of temperature-sensitive (ts), insertion or deletion mutants of herpes simplex virus (HSV) were investigated in an in vitro model system for latency. The studies defined virus gene products required for establishment of latency and for reactivation of latent virus. All mutants tested established latency in human foetal lung fibroblasts and could be reactivated by intertypic superinfection with HSV or with human cytomegalovirus. Two mutants of HSV type 1 used in these studies, tsK and in 1411, failed to synthesize active immediate early (IE) polypeptide Vmw 175 and were blocked at a very early stage of the virus replication cycle, showing that, at most, only limited gene expression is necessary for the establishment of latency. Mutant dl1403, which lacks the gene encoding IE polypeptide Vmw 110, established latency as efficiently as wild-type HSV. Latent HSV type 2 was reactivated by superinfection with tsK or in 1411 but not with dl1403, suggesting that polypeptide Vmw 110, which is known to regulate gene expression by trans-activation, is required for reactivation in the in vitro system.

Three variants of herpes simplex virus type 1 strain 17 were isolated. One variant had a deletion of 2·5 × 106 M r in IRL/UL and 0·8 × 106 M r in TRL such that sequences were deleted from both long repeats. The deletion in UL removed the 20K and 22K open reading frames. The second variant had a deletion of 3·5 × 106 M r in IRL/UL which again removed the 20K and 22K open reading frames. The third variant had a similar deletion in UL, but in this case the deleted sequences were replaced by sequences from the left end of UL, such that the long repeat was extended by 3 × 106 M r and the overall genome size by 2 × 106 M r All three variants grew almost normally in vitro. The analysis of 11 isolates with extensive variation in the long repeats outside the ‘a’ sequence is also reported.

We describe the isolation and characterization of conditional lethal amber nonsense mutants of vesicular stomatitis virus (VSV), Indiana serotype. The mutants were isolated from a chemically mutagenized stock of wild-type virus by their ability to grow on genetically engineered cells which express a Xenopus laevis amber suppressor tyrosine tRNA gene (su + cells) but not on the non-suppressor parental cells (su − cells). Five mutations were assigned to complementation group I (the L gene) and one to complementation group V (the G gene) by complementation analysis using temperature-sensitive mutants representing each of the five VSV cistrons. Four of the group I mutants were observed to synthesize a novel polypeptide species in su + cells. Immunoprecipitation and immunoblotting studies using monospecific antisera directed against the N and C termini of the VSV L protein showed that the novel polypeptide species contain N terminal-but not C terminal-specific sequences and can thus be considered to be truncated versions of the L protein. In addition a protein which again contained N terminal- but not C terminal-specific sequences could be identified for the fifth group I mutant. Revertants of four of the group I mutants were isolated on the su − cells. The revertants all synthesized normal L protein but not the putative truncated version.

The growth of vesicular stomatitis virus (VSV) can be inhibited by the antiviral compound tricyclo-decane-9-yl-xanthogenate (D609). On analysing the antiviral mechanism we found no effect on the primary transcription of infecting VSV genomes.In contrast, the processes of replication and transcription during late stages of infection were inhibited. Despite the synthesis of all five virus-coded proteins (41 % to 56% of the uninhibited control), as shown by labelling with [35S]methionine, the phosphorylation of the non-structural (NS) protein was reduced in the presence of the xanthate by a factor of at least 17. The pattern of phosphorylation of the bulk of cellular proteins remained unaltered under the same conditions. A relation between a possible loss of biological activity of the NS protein owing to the lack of phosphorylation and the decreased VSV RNA synthesis is suggested.

The middle-size (M) genomic RNA of a New York State, U.S.A. isolate of La Crosse (LAC) virus has been cloned by a random priming procedure and its nucleotide sequence determined by the dideoxy method. The RNA was found to be 4526 nucleotides in length and to have a base composition of 34·2% U, 27·8% A, 20·6% C and 17·4% G. There is a single, long open reading frame in the viral complementary RNA that contains sufficient information to code for a protein of 1441 amino acids. In these respects, as in many others, the LAC virus M RNA and its encoded protein were very similar, if not identical, to those previously reported by other investigators for the closely related snowshoe hare virus. The M RNAs of the two viruses show 79% nucleotide sequence homology and 89% homology in the amino acid sequence of their encoded proteins. Several algorithms for predicting surface residues, as well as the Chou-Fasman rules for predicting secondary structure, were used to compare the LAC virus and snowshoe hare virus M gene proteins. These analyses identified 39 sites on the proteins as those most likely to be linear antigenic determinants that might contribute to the differences between the two viruses.

DNA of human papillomavirus (HPV) type 45, a new HPV type 18-related papillomavirus of the genital tract, was cloned from a recurrent cervical lesion displaying mild to moderate dysplasia with koilocytosis. HPV-45 DNA was identified in paraffin sections of biopsies of both the initial and recurrent lesions of the patient, taken 7 months apart. HPV-45 DNA hybridized efficiently to that of many different HPV types under low and moderate stringency conditions (Tm − 37 °C to Tm − 25 °C) but with only HPV-18 DNA under high stringency conditions (Tm − 17 °C). HPV-45 DNA was distinguished from HPV-18 DNA by (i) differences in restriction enzyme digest patterns, (ii) lack of hybridization at Tm - 17 °C between HPV-18 and some fragments of HPV-45, (iii) a value of 25 % in liquid reassociation kinetics between HPV-18 and HPV-45 and (iv) differences in intensities of hybridization with selected tissue DNAs. The prevalence of HPV-45 infection in the genital tract was low. In tests of over 600 tissue DNAs from female genital tract lesions, HPV-45 sequences were detected in three additional tissues, one each of invasive cervical carcinoma, condyloma, and normal cervical epithelium. HPV-45 is a newly recognized papillomavirus which rarely infects the genital tract and is associated with lesions across a wide histological spectrum.

The LI and L2 capsid proteins encoded by human papillomavirus types 6 and 16 (HPV-6 and HPV-16) have been synthesized in bacteria. Antisera were raised against the HPV-6 LI- and L2-β-galactosidase fusion proteins and against an HPV-16 LI C-terminal peptide which was 14 amino acids long. The HPV-16 LI peptide antibodies have been shown to be highly reactive with the HPV-16 Ll-β-galactosidase fusion protein but not against the equivalent HPV-6 L1 -β-galactosidase fusion protein. The effectiveness of these antibodies was compared with commercially available antibovine papillomavirus type 1 (BPV-1) antibodies and the results demonstrated that the anti-BPV-1 antibodies reacted well against HPV-6 L1 -β-galactosidase but not against HPV-16 Ll-β-galactosidase. In addition, the L2 portion of the HPV-6 L2- β-galactosidase fusion protein appeared particularly immunogenic, since antibodies raised against this fusion protein were predominantly reactive with the L2 moiety. The HPV-16 LI peptide antibodies described here will be preferred reagents for the specific detection of HPV-16 capsid antigens, which may be particularly important in early diagnosis of HPV-16 infection.

Human papillomaviruses (HPVs) 5, 8, 19 and 25 induce macular skin lesions in patients with epidermodysplasia verruciformis. HPVs 5 and 8 are known to prevail in skin carcinomas, which develop in about one-third of these patients. We compared the viral DNAs by heteroduplex analysis and on the basis of partial nucleotide sequences.

The colinear genomes were closely related and showed nucleotide sequence homology in the range of 40% to 90%. Homology values between 70% and 80% were observed throughout roughly 4 kb of the heteroduplex molecules as estimated after ‘calibration’ by partial sequencing. Heteroduplex analysis did not support the hypothesis that the different HPV types arise by recombination and allowed no grouping to correlate with the association with malignant tumours. The upstream regulatory sequences of HPVs 8,19 and 25 appeared highly conserved but differed from those of previously sequenced papillomaviruses in length, in the TATA motif, in the copy numbers and positions of the ACCGN4CGGT palindrome and of a YGCCAA direct repeat, and in two strictly conserved blocks of 33 and 29 nucleotides.

Enhancement of yellow fever virus neurovirulence for mice by specific antibody was studied with the French neurotropic vaccine strain. Experimental conditions for enhancement required mice between 14 and 40 days old and intraperitoneal administration of a selected monoclonal antibody 24 h before or up to 72 h after intracerebral virus challenge. Virus infectivity titrations were similar in brains of antibody-treated and untreated mice. Virus recovered from brains of mice with enhanced viral infections was neither qualitatively nor quantitatively different from standard virus. Humoral immune responses in enhanced infections were normal, macrophages did not become infected and viraemia was not significant. Both hydrocortisone treatment and complement depletion with cobra venom resulted in prolongation of mouse survival times but virulence enhancement persisted. Antithymocyte serum had no effect on enhancement although it reduced the humoral immune response. It is proposed that virulence enhancement is due to the combined effects of virus-specific antibody on infected cells, complement-mediated cytolysis and resultant host anti-cellular activity. There is no analogy between mechanisms effecting increased arbovirus growth in vitro in the presence of specific antibody and increased yellow fever virus neurovirulence in vivo after parenteral administration of antibody.

Monoclonal antibodies (MAbs) specific for the rabies virus nucleoprotein (N protein) and non-structural (NS) protein of the nucleocapsid were introduced into adherent cells (fibroblasts and neuroblastoma) by the scrape-loading technique. After the cells had reattached to the substrate, they were infected with rabies virus. Inhibition of infection was monitored by measuring the intracytoplasmic viral nucleocapsid accumulation with an enzyme immunoassay using anti-N protein rabbit serum and by measuring the release of infectious virus with the plaquing system. Seventeen MAbs defining the three independent antigenic sites on the N protein were able to decrease nucleocapsid accumulation and the release of infectious virus. The MAbs describing the two antigenic sites on NS protein also had an antiviral effect on ERA virus. When an anti-N MAb (0·5 ng per cell) was introduced into CVS-infected cells, virus inhibition was complete if the anti-N MAb was introduced between 0 and 5 h post-infection and ineffective beyond 9 h post-infection. The inhibition was dose- dependent. The MAbs could block virus multiplication either by ‘neutralizing’ newly translated N and NS proteins or by impairing the initial transcription of the genome.

Mouse hybridomas producing antibodies against the structural proteins of strain WV4843, a subgroup B strain of respiratory syncytial (RS) virus, were produced by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of the virus. After immunoprecipitation tests with [35S]methion-ine-labelled extracellular virions, 35 clones found to produce antibodies against the fusion (F) protein, six against the membrane (M) protein, 21 against the nucleocapsid (NP) and eight against the phospho- (P) protein were further characterized. Immunoprecipitation with [3H]glucosamine-labelled intracellular virus polypeptides detected nine hybridoma cell lines producing antibodies against the large glyco- (G) protein of the virus. By competitive binding ELISA tests with monoclonal antibodies against each of the structural components, a minimum of two, 24, four, 15 and three epitopes were detected on the G, F, M, NP and P proteins, respectively. Eleven monoclonal antibodies directed against nine epitopes of the F protein could neutralize the infectivity of the virus. In contrast, none of the nine monoclonal antibodies against G could neutralize the infectivity of the virus. In order to find out more about the antigenic relationship between human and bovine RS virus strains all monoclonal antibodies were reacted with subgroup A RS virus and also with three different strains of bovine RS virus and one strain of caprine RS virus in immunofluorescence, ELISA and immunoprecipitation tests. In addition, 31 previously developed monoclonal antibodies against subgroup A virus were reacted with the bovine and caprine strains. The numbers of monoclonal antibodies of subgroup B specific for the B type of the two human subgroups were 9/9, 3/35, 0/6, 0/21, 0/8, for the G, F, M, NP and P proteins, respectively. No antigenic variations were found between the three bovine strains and the caprine strain. They did not react with the nine monoclonal antibodies against the G protein of subgroup B, nor did they react with nine monoclonal antibodies against subgroup A. Most but not all of the monoclonal antibodies against the other structural proteins of the two human RS virus subgroups reacted with the four strains. All 11 monoclonal antibodies against the F protein of subgroup B that could neutralize the infectivity of subgroup B also reacted with the bovine strains and neutralized their infectivity. It is concluded that although the bovine strains share many epitopes with the two human subgroups, they are antigenically distinct from the human viruses.

SUMMARY

Proteins consisting of one, two or four copies of the amino acid sequence 137 to 162, which contains the major immunogenic site of VP1 of foot-and-mouth disease virus, attached to the N-terminus of β-galactosidase have been expressed in Escherichia coli cells. In guinea-pigs the protein containing one copy (P71) of the viral determinant elicited only low levels of neutralizing antibody whereas protective levels were elicited by the proteins containing two (P72) or four (P74) copies of the determinant. Single inoculations of the P72 and P74 proteins containing as little as 2 μg or 0·8 μg of peptide respectively were sufficient to protect all the animals against challenge infection. Moreover, the equivalent of 40 μg of peptide in P74 protected pigs against challenge infection after one inoculation.

Six caprine arthritis-encephalitis virus (CAEV)-free goats kept in strict isolation were inoculated intravenously with a cloned CAEV isolate (virus 020). At 78 weeks post-infection a virus (virus 095) isolated from one of the goats was shown to have the characteristics of CAEV, but was antigenically distinct from virus 020 and two other CAEV isolates by serum neutralization tests. Serum from the goat that had the variant virus neutralized the inoculum virus and the variant virus but serum from other inoculated goats neutralized only the inoculum virus. The variant virus and the inoculum virus were shown to co-exist in the infected goat, but the presence of the antigenic variant did not appear to be associated with an increase in severity of lesions compared with other inoculated goats.

Characterization of a series of mouse monoclonal antibodies (designated αPy C21 to 26) against the region common to the large, middle and small T antigens of polyoma virus has revealed immunological diversities among the N termini of these antigens.

Four of the antibodies (αPy C21 to 24) appeared to recognize all the T antigens present in lytically infected cells, but two of them (αPy C25, 26) failed to immunoprecipitate small T antigen either from 35S- or 32P-labelled cells and recognized only a subset of middle and large T antigens. None of the antibodies recognized the protein kinase activity normally associated with middle T antigen or the 60K mol. wt. antigen-related protein observed in polyoma virus lytically infected or transformed cells. The possible immunodominance of the N termini of the early gene products (as well as VP1) in the natural host of polyoma virus and the observed antigenic heterogeneity are considered.

Primary cultures of human synovial cells shed infectious virus for 14 to 35 days following infection with isolates of Ross River virus which had been passaged in the C6/36 line of Aedes albopictus mosquito cells. No frank cytopathic effect was seen in infected synovial cells and they continued to replicate for the duration of the experiments.

A strain of adenovirus (AV) was isolated from the stools of a child with acute gastroenteritis. This virus (strain 208) was specifically neutralized by antiserum to AV19, a species belonging to subgenus D and previously reported to cause keratoconjunctivitis. However, restriction analysis of strain 208 showed it to be distinct from AV19 and the other 40 human AV serotypes presently known. When calculated from 18 restriction patterns, the proportion of comigrating fragments common to AV19 and strain 208 was only 57%. This percentage was no higher than that obtained by comparison of strain 208 and AV9, a randomly chosen type from subgenus D. Strain 208 therefore appeared to represent a new genome type. The restriction maps of the three viruses for BamHI, ClaI, EcoRI, HindIII, MluI, NdeI and SfiI endonucleases are presented.

We have characterized a set of 15 monoclonal antibodies to pl9gag, one of the internal proteins of avian sarcoma and leukaemia viruses. All the antibodies work in immune precipitations as well as in immunoblotting, though with different efficiencies. We have developed a simple epitope mapping technique, which uses partial chemical cleavages at methionine or tryptophan residues followed by immunoblotting from SDS-polyacrylamide gels, to localize the epitopes of nine of these antibodies. The epitopes fall into at least four classes. The mapping procedure should also be useful for other antigens of known primary structure.