- Volume 69, Issue 7, 1988

The genome structure of equine herpesvirus 1 (EHV-1) subtype 2 was shown by electron microscopic studies and restriction endonuclease site mapping to comprise two covalently linked segments (L, 109 kbp; S, 35 kbp). The S segment contains a unique sequence (US) flanked by a substantial inverted repeat (TRS/IRS). Thus, the genome structure of EHV-1 subtype 2 is similar to that published previously for EHV-1 subtype 1, but the two subtypes differ in the occurrences of EcoRI and BamHI restriction sites. Hybridization studies using cloned EHV-1 DNA showed that the genome of EHV-1 subtype 2 is colinear with the genomes of EHV-1 subtype 1 and herpes simplex virus type 1. DNA sequence data for four EHV-1 subtype 2 genes, including one potentially encoding a glycoprotein, were obtained by sequencing a 4574 bp BamHI fragment containing the junction between US and TRS. The genome structure, hybridization and sequence data confirm that EHV-1 subtype 2 is of the alphaherpesvirus lineage.

A DNA virus with the characteristics of a herpesvirus has been isolated from woodchuck hepatocytes cultured in vitro. We refer to this virus as herpesvirus of marmots (HVM). Electron microscopy of thin sections of HVM-infected cells showed nucleocapsids with a hexagonal outline and a diameter of 80 nm. Enveloped virions were seen in cytoplasmic vacuoles and outside the cell. Negatively stained virus particles purified from cell supernatants were enveloped with the characteristic appearance of herpesviruses. The DNA was double-stranded with a molecular size of approximately 140 kb and a G+C content of 73%. The virus replicated with a lytic effect in kidney cells of owl monkeys and African green monkeys, baby hamster kidney cells, feline kidney cells and WCH-17, a cell line derived from a woodchuck hepatoma. An indirect immunofluorescence assay has shown the presence of antibody to HVM in seven out of 37 animals tested. An important reason for studying HVM lies in its possible role in infection or the disease produced by woodchuck hepatitis virus, an animal model for human hepatitis B virus.

Canine herpesvirus (CHV) was compared with four other herpesviruses by several serological techniques. Cross-neutralization was demonstrated between CHV and herpes simplex virus types 1 and 2 and pseudorabies virus. Non-neutralizing cross-reactions were found with these viruses and also with equine abortion virus and bovine mammillitis virus. The data suggest that CHV is immunologically more closely related to herpes simplex virus than to the other viruses used in this study.

One (KM91) of a series of isolates of alphaherpesvirus saimiri (αHVS) produced rapidly fatal encephalitis in rabbits following intradermal infection, whereas the others (KM180, KM322 and KM338) were non-lethal and produced ganglionitis and prolonged latency. Alphaherpesvirus saimiri KM91 initially produced ganglionitis but quickly ascended the spinal cord to the brain causing death 10 days post-infection. Prior infection with any of the three benign isolates or inoculation with β-propiolactone (βPL)-inactivated αHVS KM91 protected rabbits from lethal encephalitis when they were subsequently challenged with a lethal dose of αHVS KM91. Each of 20 rabbits co-inoculated in the same site with a lethal dose of αHVS KM91 and either αHVS KM322 (1.5 × 103 to 1.5 × 105 p.f.u.) or βPL-inactivated αHVS KM322 (1 × 107 p.f.u. equivalents) survived. In contrast only half of those co-inoculated with αHVS KM91 and βPL-inactivated αHVS KM91 (1 × 107 p.f.u. equivalents) survived. Co-inoculation of lethal αHVS KM91 (75 p.f.u.) and benign αHVS KM322 (1.5 × 103 p.f.u.) into opposite flanks resulted in protection from encephalitis in one of four rabbits. Alphaherpesvirus saimiri KM91 was shown to have the capacity to become latent in dorsal root ganglia if the rabbit did not die.

When honey bee pupae from seemingly healthy Australian colonies were injected with various salt solutions, inapparent infections of black queen-cell virus (BQCV), Kashmir bee virus (KBV), sacbrood virus (SBV) and, occasionally, cricket paralysis virus were activated. The activated viruses replicated to detectable concentrations after pupae were incubated at 35 °C for 3 days. Inapparent infections of SBV, but not the other viruses, were also activated merely by incubating pupae at 35 °C. The replication of activated BQCV, KBV or SBV was specifically suppressed by injecting pupae with rabbit antisera against the particles of each virus. However, a greater proportion of activated SBV infections occurred in groups of pupae in which the replication of activated KBV was suppressed than in groups of pupae in which KBV was not suppressed. In addition, a greater proportion of activated BQCV infections occurred in pupae in which the replication of both KBV and SBV was suppressed than in groups of pupae that were not injected with KBV and SBV antisera. Results from one group of pupae treated in this way indicated that about 40% were infected with KBV, about 43% with SBV and about 15% with BQCV. There was no evidence of interference between the viruses in the establishment of inapparent infections of pupae, but clear evidence of interference during or after activation. These results have implications for the use of pupae for virus propagation.

A transcription/translation system for generating poliovirus proteins in vitro has been used to assess the proteolytic activity of various polypeptides containing the virus-coded 3C region towards the poliovirus precursor protein P1. Plasmids containing a phage T7 promoter followed by either the complete poliovirus P1 sequence or various sequences containing the 3C region were used for this purpose. We showed that all except one of the 3C-containing polypeptides had a very restricted activity towards P1, generating only a small amount of VP1 and no VP0 or VP3. The only polypeptide capable of fully processing P1 into VP0, VP3 and VP1 in vitro was protein 3CD, consisting of the complete 3C and 3D sequences.

A purine analogue, 2-aminopurine, reported to act as an inhibitor of protein kinase, selectively, reversibly and in a dose-dependent manner blocked a very early stage in interferon induction. With chick embryo cells and mouse L cells as hosts, and different viral inducers of interferon, maximal effects of 2-aminopurine were observed during the first 4 h of induction. At 10 mm-2-aminopurine there was a 20-fold reduction in the yield of interferon from both cell types. 2-Aminopurine and actinomycin D both prevented interferon induction with the same time course, indicating a transcriptional block to induction; however, only the action of the former was reversed upon removal of the drug. Addition of 2-aminopurine to an agarose overlay resulted in high efficiency plaque formation by vesicular stomatitis virus New Jersey (Hazelhurst) under conditions where endogenous induction of interferon and its feedback action on aged chick embryo cells normally prevented plaque formation. Two other inducible systems, representing genes involved in interferon action (both its development and activation), and those of heat shock, were not affected by 2-aminopurine. A model is presented implicating the interferon-inducible dsRNA-dependent protein kinase as an interferon induction receptor which, on interaction with dsRNA, generates an amplified signal via phosphorylation that ultimately derepresses the interferon gene(s).

Single serotype vaccination of mature cows with nine different strains of bovine, simian and human rotaviruses induced heterotypic milk and serum neutralizing antibodies against two bovine and four human rotavirus serotypes. Immunization with single-shelled simian rotavirus SA11 increased milk and serum neutralization titres fivefold over those of control cows, without inducing antibodies to outer shell polypeptides of rotavirus. Vaccination with double-shelled SA11 virions also elicited cross-reacting antibodies to the outer shell proteins VP3 and/or VP7 which neutralized rotavirus seven times more efficiently than antisera to single-shelled SA11 virus. A related rotavirus similar to simian rotavirus SA11, but from a different host, might thus be an attractive vaccine for immunization of pregnant cows to confer passive immunity to calves.

The nucleotide sequences of the genes that code for the major inner capsid protein, VP6, of the human rotavirus strain 1076 (subgroup I), porcine rotavirus Gottfried (subgroup II), equine rotavirus strain H-2 (non-I/II) and equine rotavirus strain FI-14 (both subgroups I and II) have been determined. The sixth segment positive-stranded RNA encodes a protein of 397 amino acids in all strains with the exception of strain H-2 in which it encodes a protein of 399 amino acids. Alignment of amino acid sequences of the VP6 protein of strain FI-14 and subgroup II rotaviruses (Wa and Gottfried) indicates a high degree of homology (94%), while homology between strain FI-14 and subgroup I rotaviruses (SA-11, RF and 1076) was somewhat less (90 to 92%). On the other hand a high degree of conservation of amino acid sequence (95 to 97%) was observed between the H-2 strain and subgroup I rotaviruses. Five regions that may contribute to subgroup epitopes were identified. Region A (amino acids 45, 56) and region C (amino acids 114, 120) may contribute to subgroup I epitopes and regions B (amino acids 83, 86, 89, 92), D (amino acids 312 or 314, 317 or 319) and E (amino acids 341 or 343, 350 or 352) may contribute to subgroup II epitopes. When analysed using the Western blot technique monoclonal antibodies specific for VP6 epitopes shared by all rotaviruses were observed to react with both monomeric and trimeric forms of VP6, while monoclonal antibodies specific for a subgroup I or II epitope reacted only with the trimeric form of VP6. This observation and the sequence analyses suggest that subgroup antigenic specificity is determined by conformational epitopes produced by the folding of VP6 or the interaction between VP6 monomers.

We have investigated the molecular basis for differences that we observed in the biological activities of genetically related but distinguishable human papillomavirus type 6 (HPV-6) subtypes. To analyse tissue-specific differences in replication and transcription, and to identify viral gene products important in the benign transformation of epithelial cells, we have modified procedures utilizing guanidinium isothiocyanate and density gradient centrifugation to facilitate the extraction of relatively undegraded DNA and RNA from 21 biopsy specimens of respiratory tract papillomata. Southern transfer analysis was used to characterize the viral genome, and to demonstrate that relative quantities of viral DNA in lesions varied but that the range was similar in lesions induced by HPV-6c, -6d, -6e and -6f. Dot blot analysis of the amount of viral RNA in comparison with the amount of 28S ribosomal RNA indicated that the relative level of viral RNA in each lesion varied considerably and that on average there was approximately twice as much viral RNA in HPV-6c-induced lesions as in HPV-6d-, -6e- or -6f-induced lesions. In dot blot and Northern analyses, hybridization of RNA from HPV-6c-induced lesions with HPV-6c DNA gave a stronger signal than hybridization of the same RNA with an HPV-6e probe, and vice versa. These differences in hybridization intensities with subtype-specific probes indicate that the most abundant RNA species are transcribed from parts of the genome that show sequence divergence between these two subtypes. Northern analysis demonstrated the predominant viral transcript to be about 1200 nucleotides in length in lesions induced by each of the four subtypes.

DNA prepared from a field isolate of African swine fever virus, which causes high mortality and severe disease in domestic pigs, was cloned in bacteriophage lambda and plasmid vectors. Clones containing DNA inserts overlapping with each other and together covering the complete genome, apart from short fragments close to the cross-linked termini of the genome, were obtained. A complete restriction enzyme site map of the genome for three enzymes was deduced.

We report the first complete nucleotide sequence of an adult T cell leukaemia virus/human T cell leukaemia virus type I (ATLV/HTLV-I) isolate from a British patient of Caribbean origin. Sequence comparisons of our proviral clone (HS-35) with other molecular clones are shown. We note the strong sequence conservation between isolates of Caribbean and Japanese origin (2.3% divergence), but demonstrate the higher homologies existing between isolates originating from similar geographical areas (approximately 1% divergence). Implications for the origin, evolution and dissemination of the ATLV/HTLV-I subgroup are discussed. Analysis of defective proviral clones isolated from the same genomic library is also reported, and suggests a pattern of proviral sequence deletions during the biogenesis of defective proviruses.

The reported serological relatedness between the major glycoproteins of human immunodeficiency virus (HIV gp120) and equine infectious anaemia virus (EIAV gp90) was examined using purified antigens in radioimmunoprecipitation (RIP), radioimmunoassay (RIA) and immunoblot assays with reference serum from acquired immunodeficiency syndrome (AIDS) patients, an anti-gp120 goat serum and EIAV-infected horse serum. To assess the contributions of glycoprotein oligosaccharide and peptide components to any observed reactivities, antigens treated with endoglycosidase F to remove carbohydrate were assayed in parallel with the intact glycoprotein. The results of the experiments indicated that the reactivity observed for each antigen was dependent on the immunoassay employed. The RIP and RIA analyses demonstrated that HIV gp120 is equally reactive with the AIDS patient serum, the goat anti-gp120 serum and the EIAV-infected horse serum, whereas the EIAV gp90 reacted only with the horse serum. In immunoblot assays, the HIV gp120 reacted with AIDS patient serum, but not with the EIAV-infected horse serum. Deglycosylation of the HIV gp120 evidently increased its reactivity with the AIDS patient serum, had no significant effect on its reactivity with the goat antiserum, and essentially abolished its reactivity with the EIAV reference serum. Thus, it appears that the serological cross-reactivity observed between HIV gp120 and sera from EIAV-infected horses can be attributed to the oligosaccharide rather than the peptide components of the viral glycoprotein. These studies also emphasize the necessity of employing several assay procedures in assessing lentivirus antigenicity.

Monoclonal antibodies (MAbs) against the major core protein p26 of equine infectious anaemia virus (EIAV) were produced and characterized. Sensitive enzyme-linked immunosorbent assay and Western blot immunoassay were employed to confirm the specificity of these MAbs. Western blot analysis also indicated that MAbs to p26 reacted with another EIAV protein of 55000 apparent M r (designated here as Pr55 gag ) present in density gradient-purified virus preparations. Rabbit antiserum prepared against p26 as well as MAbs to p26 detected Pr55 gag and several other intermediate cleavage products in detergent-soluble lysates of virus-infected cells in Western blot and immunoprecipitation assays. The results suggest that Pr55 gag is the gag polyprotein of EIAV.

The antigenic relationship between a recently isolated porcine respiratory coronavirus (TLM 83) and transmissible gastroenteritis (TGE) virus of swine was studied by neutralization, immunoblotting and radioimmunoassay (RIA) using TGE virus-specific monoclonal antibodies (MAbs) and polyclonal antibodies specific for both viruses. A complete two-way neutralization activity between the two viruses was found. Immunoblotting revealed cross-reactions between TLM 83 and TGE virus antigens at the level of the envelope protein (E1), the nucleoprotein (N) and the peplomer protein (E2). By virus neutralization assays and RIA with TGE virus-specific MAbs, the presence of similar epitopes in the E1 and N proteins and in the neutralization-mediating antigenic site of the E2 protein were demonstrated. E2 protein-specific MAbs, without neutralizing activity and reacting with antigenic sites B, C and D (previously defined), failed to recognize TLM 83. These results indicated a close antigenic relationship and structural similarity between TLM 83 and TGE viruses and also suggested potential ways of differentiating between the two viruses.

Spleen cells from BALB/c (H-2d) mice vaccinated with vgB11, a recombinant vaccinia virus which expresses glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1), lysed EMT6 (H-2d) target cells infected with vgB11 or with HSV-1 but did not lyse uninfected EMT6 cells or infected L-929 (H-2k) target cells. Unlabelled target cell competition of lysis showed that only syngeneic cells infected with vgB11 or HSV-1 inhibited lysis of radiolabelled HSV-1-infected targets. These results demonstrate that vgB11 induces H-2-restricted anti-HSV-1 cytotoxic T lymphocytes and that gB is the target antigen.

The U937 monocytic cell line was used to determine whether antibodies could facilitate infection and replication of the arenaviruses, Pichinde virus (PV) and Lassa fever virus (LFV). When high dilutions of PV-immune serum were added to cultures simultaneously with PV inoculum, virus replication was dramatically (1000-fold) increased. Low dilutions of this antiserum neutralized the virus. LFV also replicated in U937 cells. The presence of LFV-specific immune serum in the growth medium increased the viral titre as much as 10000-fold. Addition of heat-aggregated IgG partially inhibited antibody-mediated enhancement, probably by inhibiting the binding of immune complexes to the monocytic cells.

The genomes of four closely related variants of Artogeia rapae granulosis virus, isolated in England and New Zealand, have been mapped for the restriction endonucleases EcoRI, BamHI, HindIII, KpnI, XhoI, PstI, BglI, and SmaI by secondary digestion of isolated restriction fragments. A total of 69, 70 or 71 fragments were unambiguously mapped for these isolates. A modified system of fragment lettering was adopted so that, for each enzyme, the same letter could be used for colinear fragments from each isolate despite loss or gain of restriction sites. The sizes of the viral DNAs varied slightly, but were all about 110 kbp. The zero point for the maps was taken as the start of the granulin gene which was orientated in the same direction as the polyhedrin gene on the map of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus DNA. Differences between the four variants are discussed in relation to their insect host range.