- Volume 69, Issue 9, 1988

In order to investigate further possible structural differences among the two subgroups of respiratory syncytial virus (RSV), we analysed the antigenic characteristics and size of structural proteins of 20 subgroup A and 43 subgroup B strains by their reactions with monoclonal antibodies (MAbs) directed against the proteins of RSV using immunofluorescence, ELISA and radioimmunoprecipitation assays. The latter test also enabled determination of the size of different structural components. The 37 MAbs employed were generated by immunization with both subgroup A and B strains. They represented specificities for distinct epitopes on five different structural proteins. The subgroup A strains proved to be relatively uniform. The fusion (F) protein, nucleoprotein (NP) and matrix (M) proteins of all strains tested had the same M r and all except one strain had a phosphoprotein (P protein) of the same M r. The F and P proteins were lower in M r in B strains compared to A strains, which confirmed previous findings. The M r of the large surface glycoprotein (G protein) of subgroup A strains varied slightly, probably on the basis of differing glycosylation. By contrast, the subgroup B strains exhibited substantial variation in the M r of the G and also the P proteins and in reactivity with MAbs directed against the G and F proteins. Three size classes of the P protein were identified in B strains: 33K to 34K, 32K to 33K, and 31K to 32K. Twenty-seven subgroup B strains failed to react with four anti-G MAbs representing a single epitope, G2; the remaining 16 strains reacted with these MAbs. We designated these two sets of variants of B strains B1, which lacked the epitope, and B2, which had the epitope. The B1 strains also varied in the size of the G and P proteins. In contrast, all B2 strains had large G proteins and all except two strains had relatively large P proteins (33K to 34K). All subgroup B1 and B2 strains exhibited the same sizes of NP, F and M proteins. We conclude that the subgroup B strains of RSV include two variants, B1 and B2, and that the major difference between them resides in the G and P proteins.

Pathogenic properties of influenza A viruses introduced into embryonated chicken eggs via the allantoic cavity, the amniotic cavity or the yolk sac were studied using viruses with cleavable or non-cleavable haemagglutinin (HA), or reassortants derived from the highly pathogenic fowl plague virus (FPV) which has a cleavable HA. The various organs, membranes and fluids were analysed for virus yields, and by immunohistochemistry for production of viral nucleoprotein. Virus replication in primary tissue cultures derived from various embryonic organs was also studied. Some of the reassortants, which were previously found to be non-pathogenic for chickens and were temperature-sensitive (ts) at 40 °C, multiplied at 37 °C to the same extent as the highly pathogenic FPV. The spread of other non-pathogenic reassortants in the embryo was restricted. For example, 81/Ho was able to multiply to a reasonable extent only in the chorioallantoic and the allantoamniotic membranes. After inoculation of the A/PR/8/34 strain containing a non-cleavable HA into the amniotic cavity, virus multipled only in the inner epithelial layer of the amnion, in superficial epidermal cells and in superficial epithelia of the oropharyngeal cavities, the nasal and paranasal sinuses and the oesophagus. Kidneys were free of virus antigen, although the virus multiplied to high titres in primary tissue cultures derived from embryonic kidneys. Thus influenza A viruses can be non-pathogenic for chickens because they are ts and unable to multiply at the body temperature of chickens (41 °C), or because their spread in the animal is impaired per se.

Antisera were raised in rabbits against fusion proteins consisting of β-galactosidase and partial amino acid sequences of Semliki Forest virus (SFV)-specific non-structural proteins nsP1, nsP2, nsP3 and nsP4. The antisera were specific since each of them precipitated only one labelled protein of a size expected for nsP1, nsP2, nsP3 or nsP4 from lysates of [35S]methionine-labelled SFV-infected BHK-21 cells. The specific antisera also precipitated p220 (with sequences of nsP1, nsP2 and nsP3), p155 (nsP1 and nsP2) and p135 (nsP3 and nsP4) which have been previously shown to be cleavage products of the polyprotein precursor of the non-structural proteins. nsP1, nsP4 and most of nsP3, together with the virus-specific RNA polymerase activity, were in the mitochondrial pellet (P15) fraction of infected BHK-21 cells whereas nsP2 was evenly distributed between P15 and the supernatant fraction (S15). Only antisera directed against nsP3 sequences precipitated a labelled protein from cells incubated with [32P]orthophosphate during SFV infection. Treatment of the immunoprecipitate with calf alkaline intestinal phosphatase reduced the amount of labelled nsP3 considerably. Immunoprecipitated 32P-labelled nsP3, isolated by SDS-PAGE, was subjected to acid hydrolysis. Both phosphoserine and phosphothreonine but not phosphotyrosine could be identified in the hydrolysate. Approximately twice as much [32P]serine as [32P]threonine was detected in nsP3. P15 and S15 fractions were prepared from [35S]methionine- and 32P-labelled SFV-infected cells and the 35S/32P ratio of nsP3 was determined after immunoprecipitation and SDS-PAGE. The nsP3 in S15 was less heavily phosphorylated (about 50%) than P15-associated nsP3. Anti-nsP3 serum revealed large cytoplasmic vesicles in SFV-infected cells in indirect immunofluorescence microscopy.

Cytotoxic T lymphocytes (CTLs) were generated when spleen cells from mice infected with viruses causing haemorrhagic fever with renal syndrome were stimulated in vitro with syngeneic cells infected with viruses. These cytotoxic effector cells, with Lyt2+ L3T4- markers on their surface, demonstrated H-2 restriction. CTLs induced by Hantaan virus (76–118 strain) or Seoul virus (B-1 strain) showed cross-reactivity with infected target cells. Hantaan virus infection induced a higher CTLs response than Seoul virus infection, although the antibody responses to these two viruses and the replication of the two virus strains in athymic nude mice were not significantly different. Viral antigen detected with a monoclonal antibody reacting with nucleocapsid antigen was observed mainly in the cytoplasm of macrophages infected with Hantaan virus, but in the nucleus of cells infected with Seoul virus. The major viral antigens recognized by CTLs are discussed on the basis of these findings.

Persistent infections with St Louis encephalitis (SLE) virus were established in three mosquito cell lines (Aedes albopictus, A. dorsalis and Culex tarsalis) and were maintained for over 2 years. All three persistently infected cell cultures shared two features: (i) no overt cytopathic effect and (ii) a relatively high proportion of cells infected (41 to 85%). The Aedes persistently infected cultures were resistant to superinfection with the homologous virus but not heterologous viruses. Two significant differences were observed between the Aedes and C. tarsalis persistently infected cell cultures: (i) viral titres in the A. albopictus and A. dorsalis cell cultures decreased slowly over time (the decrease was particularly marked in the A. albopictus cell cultures), whereas titres in the C. tarsalis cell cultures remained relatively constant and (ii) the addition of anti-SLE virus antibody led to decreased virus production in the C. tarsalis cell cultures (one of two cultures was cured of infection), whereas antibody had no effect on the persistently infected Aedes cell cultures. These results suggest that there may be significant differences in the regulation of viral replication and the maintenance of flavivirus persistent infections in mosquito cell lines of different origins.

Viral mutants that appeared during long-term persistent infections of mosquito cell cultures (Aedes albopictus, A. dorsalis and Culex tarsalis) with St Louis encephalitis virus were characterized. Evidence was obtained for the presence of temperature-sensitive mutants in the A. dorsalis and C. tarsalis persistently infected cultures, and small plaque mutants were predominant in all cultures except one of two cell cultures of C. tarsalis. Virus from persistently infected A. albopictus cell cultures was growth-restricted in Vero and C. tarsalis cells. One of two persistently infected A. dorsalis cell cultures also produced viral mutants that were growth-restricted in C. tarsalis cells. Further, Western blots of persistently infected A. albopictus cell extracts showed an overproduction of capsid (C) and envelope (E) structural proteins and reduced production of an M r 27K protein (p27) which was immunologically related to the E protein. In contrast, the production of E and C proteins in persistently infected C. tarsalis cultures was consistent with the amount of infectious virus present, whereas p27 was relatively overproduced. These observations suggest that the host cell has an important influence on both the types and relative quantities of viral mutants that accumulate during long-term persistent infections.

In different strains of inbred Syrian golden hamsters (Mesocricetus auratus), the lymphocytic choriomeningitis virus (LCMV) strains WE and Armstrong (ARM) produced systemic infection with infective virus and viral antigens detected predominantly in reticuloendothelial organs. Host and virus strain-dependent fatal wasting disease also occurred. After infection with WE, all MHA and PD4 hamsters died of a progressive wasting disease and infectivity persisted in organs at relatively high titres. LSH and CB strain hamsters resisted lethal disease and totally eliminated infection. LVG and LHC strain hamsters were intermediate in susceptibility to WE; some died of wasting and had persistently infected organs, while others cleared infection and survived. ARM was avirulent causing an inapparent infection in all hamsters. LCMV antibody responses were temporally comparable for all hamsters with either lethal or non-lethal infection. Histologically, lymphoid hyperplasia and low-grade systemic perivascular mononuclear leukocyte infiltration were found in all LCMV-infected hamsters. However, non-necrotic segmental ileal changes, which included vascular congestion, minimal haemorrhage and crypt epithelial growth extension into the intestinal wall, were found in susceptible hamsters when infected with the lethal WE strain.

Twelve Palyam serogroup orbiviruses isolated in Zimbabwe from aborted cattle foetuses, plus one isolated from the visceral organs of a cow and another from vulture faeces, were examined in comparison with known members of the serogroup by complement fixation, indirect immunofluorescence, fluorescent focus reduction neutralization tests and PAGE of the segmented, dsRNA genomes. The viruses were indistinguishable from known members of the serogroup by complement fixation and indirect immunofluorescence, but two novel viruses, for which the names Gweru and Marondera are proposed, and two previously described viruses, Nyabira and Abadina, were identified by neutralization tests. The dsRNA profiles of Abadina serotype isolates differed from that of the Abadina prototype virus, indicating that different electropherotypes may occur within serotypes.

A persistent infection with varicella-zoster virus was established in the Mewo human melanoma cell line. This persistently infected cell line went through periodic crises of virus-induced cell killing and then recovery. Analyses of viral DNA derived from the persistently infected cultures revealed that novel viral nucleic acid rearrangements had been generated. These viral DNA sequences were derived from a specific region of the inverted repeat sequence of the genome flanking the short unique genome segment. The novel DNA was of various lengths, each generated by tandem duplication of an approximately 2760 base pair sub-sequence of the normal viral inverted repeat. These novel sequences were inserted into an otherwise apparently normal genome.

Although all herpesviruses are similar in their temporal regulation of gene expression, the organization of the immediate early (IE) genes varies markedly between the different members of the group. Most of the IE transcripts of human cytomegalovirus originate from a restricted region within the long unique segment of its linear dsDNA genome of 235 kb. One of the predominant transcripts from the IE region is a 5 kb RNA. Northern blot analyses revealed that this class of RNA is continuously present in infected cells. It was detected at high levels in IE and late RNA preparations, and in low amounts in early RNA preparations. It was not confined to the poly(A)+ fraction upon oligo(dT) selection, but also appeared in similar amounts in poly(A)- fractions. Fine mapping of this transcript was done by nuclease protection and primer extension. The RNA appeared to be unspliced, and no signals such as TATA or CCAAT, known to be important elements in eukaryotic RNA polymerase II promoters, were found close to the 5′ end. Sequence analysis revealed multiple stop codons throughout the AT-rich potential coding region. Since no splicing was found to occur, the largest protein deduced from the DNA sequence would be of not more than 12000 M r. However, a computer program designed to detect protein-coding DNA sequences by codon usage did not reveal significant evidence for a protein encoded in this region. Therefore this RNA is likely to represent an unprecedented case of a large non-coding transcript present in cells that are lytically infected by an animal virus.

In a previous study we showed that cells infected with guinea-pig cytomegalovirus (GPCMV) contain large amounts of a non-structural 50K nuclear protein that is detectable by immunoelectron microscopy using monoclonal antibodies. The present study shows that this 50K protein is a DNA-binding protein, as determined by single-stranded DNA affinity chromatography, and a phosphorylated protein as demonstrated by immunoprecipitation using [32P]orthophosphate-labelled cells. This protein binds both viral and host cellular double-stranded and single-stranded DNA, assayed by a simple method using DNA linked to a nylon membrane. Induction of the 50K protein in GPCMV-infected cells was highly dependent on viral DNA synthesis, which was detected by dot hybridization using a cloned GPCMV DNA probe. Synthesis of the 50K protein was significantly impaired when phosphonoacetic acid was added to the culture medium. Induction of the 50K protein was detected about 6 h before the appearance of the 76K viral matrix protein.

Several new polypeptides were detected in cells infected with CTHV, a cottontail rabbit herpesvirus. All of them (M r 150K, 110K, 93K, 83K, 75K and 35K) accumulated in the nucleus during the infectious cycle, and all except the 150K species bound to DNA-cellulose affinity columns in low-salt buffers. Polyclonal antisera prepared against the 35K DNA-binding protein also recognized the 75K species. Although the 75K protein could be detected earlier in infection than the 35K protein, late in the infectious cycle the latter increased to an abundance approaching that of cellular histones. Treatment of partially purified virions with a non-ionic detergent indicated that the 35K protein, but not the 75K protein, is a component of capsid/tegument structures. The anti-35K/75K serum did not cross-react with herpesvirus sylvilagus virion proteins, which, in an electrophoretic comparison, exhibited both similarities to and differences from the virion proteins of CTHV. Labelling of CTHV-infected cells with [32P]orthophosphate revealed the presence of phosphoproteins electrophoretically comigrating with the 93K, 83K, 75K and 35K proteins.

The gene for the major capsid protein (hexon) of human adenovirus type 41 (Ad41) has been isolated and the complete DNA sequence determined. Comparison of the predicted amino acid sequence with hexons from human Ad2 and Ad5 and bovine adenovirus type 3 reveals regions of high homology at the N and C termini separated by a central region of low homology. Fitting of the Ad41 hexon sequence to the known three-dimensional structure of the Ad2 hexon demonstrates that both hexons have a common architecture. Regions of the hexon which in the trimer constitute the pseudohexagonal base are highly conserved, with the major amino acid changes concentrated in the domains forming the triangular towers which represent the surface of the capsid. Changes in the Ad41 towers therefore permit the virus to present a unique surface to the environment while conservation of residues in the base maintains the integrity of hexon-hexon contacts. A striking difference is the absence in the Ad41 sequence of 32 amino acids which are present in the Ad2 sequence. In Ad2 this region is highly charged and may be responsible for pH-induced conformational changes within the virus capsid. The DNA sequence in the region surrounding the Ad41 hexon gene was also determined and revealed an open reading frame which appeared to code for the homologue of the Ad2-coded endoprotease. Comparison of the predicted amino acid sequences of the Ad41 and Ad2 proteins revealed a high degree of homology suggesting that this protein may have an important role in the infectious cycle of the virus.

The genome of the multiple-embedded nuclear polyhedrosis virus (MNPV) of Lymantria dispar (LdMNPV) was partially characterized by restriction endonuclease analysis and a physical map was constructed using cosmid cloning and Southern cross blot hybridization. Using BamHI, BglII, EcoRI and HindIII, the size of the genome was estimated to be 88.5 × 106 M r or 134.04 kbp. LdMNPV DNA was also analysed using methylation-sensitive restriction enzymes. The resulting restriction profiles suggested that extensive methylation did not occur at the nucleotide sequence recognized by HpaII and MspI. A BamHI restriction map was constructed by comparing overlapping BamHI fragments between cosmid clones containing partial digests of viral DNA. The positions of the BglII, EcoRI and HindIII sites were determined by Southern cross blot hybridizations and aligned to the BamHI restriction map. At least four homologous regions were identified by cross blot hybridizations of BglII-digested LdMNPV DNA and such regions were found to be interspersed along the genome in a fashion similar to that reported for other baculoviruses. Using recombinant plasmids containing the HindIII-V fragment of Autographa californica MNPV to probe Southern blots of LdMNPV DNA, the restriction fragment(s) that contain the polyhedrin gene were identified. Based on these findings the map was oriented with the polyhedrin gene of LdMNPV as the zero point.

Recombinant DNA clones were constructed in order to study the mechanisms of proteolytic processing and assembly in foot-and-mouth disease virus (FMDV). RNA transcripts from these clones were synthesized using SP6 polymerase and translated in rabbit reticulocyte lysates. Efficient translation occurred in the absence of all 5′ untranslated sequences and processing of the structural proteins occurred in the presence of functional 3C protease which can function in trans. The specificity of 3C protease activity is not limited to Glu-Gly bonds. Translation of correctly processed structural proteins leads to assembly of subviral structures resembling ‘empty’ particles. Further studies on the processing of the FMDV genome show that the primary cleavage (P1–P2) is mediated neither by 3C nor the second FMDV protease L. Preliminary evidence suggests that an initial very rapid cleavage occurs between 2A and 2B with subsequent cleavage of the P1/2A junction probably being carried out by 3C.

Translation of foot-and-mouth disease virus RNA for extended periods in rabbit reticulocyte lysates results in the appearance of a previously undescribed protein. A protein with similar properties can also be detected in BHK cells at late times after virus infection. Specific immunoprecipitation has shown that this protein (Lb′) is closely related to the smaller of the two leader proteins, Lb, although it migrates with an apparently higher M r in SDS-polyacrylamide gels. The conversion of Lb to Lb′ can be mimicked by treatment with carboxypeptidase B. It is suggested that C-terminal trimming of Lb to produce Lb′ results in an increase in negative charge and is responsible for its slower migration in SDS-PAGE.

Foot-and-mouth disease virus challenge experiments in guinea-pigs and immunoassays with a range of peptides equivalent to either or both of the sequences 141 to 158 and 200 to 213 of VP1 showed the most effective structure, in terms of protection, to be one in which both ‘sites’ were present with a minimum of additional amino acids. An 80 residue peptide comprising amino acids 134 to 213 was considerably less effective than 40 or 45 residue peptides. The major site for the induction of protection was deduced to be in the region 141 to 158. Thus, protection with the 40 or 45 residue peptide did not appear to be due to the presence of antibody directed solely to the 200 to 213 sequence. Finally, induction of antibody to the latter site appeared to be dependent on both the size of the peptide and the disposition of ‘sites’ within it.

The sequence of the M gene of the Hu2 strain of measles virus has been determined. In the coding region of the gene, six nucleotide replacements had occurred with respect to the sequence of the M gene of the Edmonston strain. Two of these were silent and only four led to amino acid replacements in the protein sequence even though in SDS-PAGE the mobility of the protein would indicate an M r increase of 2000. No changes were observed in the 5′ untranslated regions of the gene. Two changes were observed in the 3′ untranslated region of the gene. The sequences are compared to those recently published for subacute sclerosing panencephalitis viruses.

We have isolated two plaque size variants of Theiler’s murine encephalomyelitis virus (TMEV) strain DA. One variant, TMEV-C l (C l ), produced large plaques, while the other, TMEV-D s (D s ), produced small plaques in L-2 cells. The D s variant yielded a lower titre in BHK cells and had a significantly slower growth rate when compared to C l and DA. In contrast, D s replicated to a higher titre in the central nervous system (CNS) of infected mice than the large plaque counterpart and DA. Furthermore, the D s (but not C l ) variant was temperature-sensitive, replicating 130- to 500-fold more at 37 °C than at 39 °C. Although D s , C l and DA were able to establish persistent CNS infections in mice, only the D s variant and DA induced demyelinating disease in SJL/J mice. Therefore persistence of TMEV in the CNS is not sufficient to produce demyelinating disease. These two variants of the DA strain of TMEV will be useful for study of the viral genetic elements important in the mechanism of virus-induced demyelination.