- Volume 71, Issue 2, 1990

The nucleotide sequence of the S peplomer gene of bovine coronavirus (BCV) has been determined. A single open reading frame of 4089 nucleotides encodes a polypeptide of 150K with 20 potential sites for addition of N-linked oligosaccharides. Expression of the cloned BCV S gene by a recombinant of Autographa californica nuclear polyhedrosis virus resulted in production of a 180K glycosylated polypeptide which was transported to the surface of the cell. Comparison of the BCV S gene with the analogous genes of murine hepatitis viruses shows that the BCV S polypeptide contains a unique domain of 138 amino acids not present in murine hepatitis virus strain JHM, but which has a partially homologous counterpart in strain A59. This domain accounts for most of the differences in size of the S gene products of these coronaviruses.

Four antigenic sites of the E2 glycoprotein of transmissible gastroenteritis virus were defined by competitive radioimmunoassays of monoclonal antibodies (MAbs). Here, we describe the localization of these sites by testing the antigenicity of protein fragments and prokaryotic expression products of E2 gene fragments, and by sequencing of MAb-resistant (mar) mutants. Partial proteolysis of purified E2 protein allowed the isolation of a 28K fragment recognized by both site A- and site C-specific MAbs. An antiserum against this fragment bound to a synthetic peptide containing residues 1 to 18 and to an expression product containing residues 1 to 325. The same expression product was recognized by site C-specific MAbs. These data indicate that residues within the sequence 1 to 325 contribute to site C and possibly also to site A. Sequencing of mar mutants that escaped neutralization by site A-specific MAbs indicated that residues 538 and 543 also belong to site A. The binding of site-specific MAbs to expression products led directly to the localization of sites B and D, between residues 1 to 325 and 379 to 529, respectively. The first 37 % of the polypeptide chain of E2 appears to be more immunogenic than the rest of the sequence.

By the use of liposomes containing the purified surface glycoprotein (G) of rabies virus and the haemagglu- tinin-neuraminidase (HN) and fusion (F) glycoproteins of Sendai virus, the target antigen of anti-rabies virus cytotoxic T lymphocyte (CTL) clones isolated in a previous study was identified as the G protein. Recognition of the H-2K determinant of the class I major histocompatibility complex (MHC) was necessary for target lysis by the CTL clones. One of the CTL clones was examined for the ability to protect mice against a lethal rabies virus infection. CTL were transferred into syngeneic mice which had been infected in the hind footpad with the ERA strain of rabies virus. The infection was converted into a lethal infection by cyclophosphamide treatment 1 day after virus infection. Transfer of CTL 2 to 3 days after virus infection protected approximately 50% of mice during the observation period of 4 weeks. Greater protection was obtained in mice receiving both anti-rabies virus antibodies and CTL cells.

An activity found in bovine brain microtubule-associated proteins (MAPs) which stimulated vesicular stomatitis virus (VSV) transcription in vitro co-eluted from a gel filtration column (Sepharose CL-6B) with a minor MAP subset of an apparent M r of 100000 to 250000. The activity did not appear to be closely associated with MAPs 1, MAPs 2, tau or tubulin. Anti-MAPs 1 and anti-MAPs 2 IgG did not reduce or abolish the stimulatory activity. The bovine brain MAPs stimulatory activity was similar to that found in HeLa cell extracts: both were heat-resistant, and both co-purified with the MAPs fraction of cell extracts; also amounts of each which gave maximum stimulation when used separately did not give additional stimulation when combined. Fractions from the Sepharose CL-6B column that contained the greatest amount of stimulatory activity exhibited little or no cAMP-dependent or -independent kinase activity. The MAPs stimulatory activity required the presence of both L and NS polymerase subunits.

The cDNA sequence of the large dsRNA segment (segment A) of the N1 strain of infectious pancreatic necrosis virus (IPNV) has been determined. The nucleotide and deduced amino acid sequences were compared to the sequences of segment A of the Jasper strain of IPNV and to the sequences of segments A and B (5′ and 3′ flanking regions) of the 002–73 strain of infectious bursal disease virus (IBDV). The comparison demonstrated that the precursor protein of the major structural polypeptide, pVP2, is highly conserved at the N and C termini, whereas the amino acid sequence of an internal segment shows greater diversity between the strains. This internal segment probably carries the serotype-specific epitopes of birnaviruses. An alternative open reading frame (ORF) (444 bp) partly overlapping with the large ORF (2916 bp) of segment A was found to be conserved among the IPNV strains and is probably also present in the 002–73 strain of IBDV. This small ORF may encode a novel birnavirus polypeptide with an M r of 17K. SDS-PAGE of radiolabelled purified IPNV particles revealed a band corresponding to the possible novel 17K polypeptide. Short terminal inverted repeats are found in segment A of the N1 and Jasper strains of IPNV and in segment B of the 002–73 strain of IBDV. Segment A of IPNV and segment B of IBDV also contain adjacent inverted repeats at their 5′-terminal flanking regions.

Lymphoproliferation against foot-and-mouth disease (FMD) virus was examined using peripheral blood mononuclear cells from vaccinated cattle. Ten weeks after revaccination the optimum conditions for proliferation were obtained with 1 μg/ml of purified virus after 5 to 6 days in culture. This contrasted with the response at 20 months post-revaccination, when the response required less antigen and showed a peak response after 3 to 4 days in culture. Proliferation was specific for FMD virus, but was cross-reactive between serotypically distinct strains of the virus. The proliferative response to isolated virus proteins (VP) involved all three major capsid proteins (VP1, −2 and −3), although the proliferation of lymphocytes from heterotypically vaccinated cattle was due to VP3. Furthermore, the response induced by purified virus, chemically fixed virus and subunit virus particles was indistinguishable and thus it is likely that processing was required for the induction of proliferation. Together these data strongly suggest that FMD virus-induced lymphoproliferation is T cell-mediated and that VP3 may contain dominant, cross-reactive sequences.

A panel of nine neutralizing monoclonal antibodies was used to analyse the antigenic properties of 188 plaque- purified type 3 poliovirus strains from 17 faecal specimens, derived from eight people during a 2 month observation period. Most poliovirus specimens consisted of a mixture of antigenically distinct variants and the composition of the mixture was found to change between sequential specimens in many individuals, indicating antigenic evolution. Thirty-five strains representing different antigenic patterns were selected for partial sequencing of genomic RNA. Mutations leading to amino acid substitutions, as well as silent mutations, were seen at and close to the known antigenic sites. The frequency of silent mutations was used to estimate the evolutionary potential of the virus. The largest difference in silent changes between strains isolated from one person was 0 ·8 %, which corresponds to a minimum of about 60 mutations per genome within a period of 3 weeks. The observed incidence of silent mutations between isolates from different persons was usually between 0·8 and 2%. These figures agree with the previously reported overall mutation rates of poliovirus, determined by other methods.

Susceptibility to lethal infection with the KT281/75 strain of the tick-borne nairovirus, Dugbe virus, was similar in an outbred strain and several inbred strains of mice. For the outbred strain, both neural and extraneural routes of virus inoculation resulted in lethal infection, but susceptibility decreased with age and only intracerebral inoculation produced a lethal infection in adults. In newborn mice, subcutaneous (s.c.) inoculation of virus (analogous to a tick-bite) produced a disseminated infection, titres being highest in the upper respiratory tract (URT), spleen and liver at 5 days postinoculation (p.i.), the heart at 7 days p.i. and brain by 8 days p.i. In neonates inoculated intranasally (i.n.), by contrast, virus spread rapidly from the URT to the brain by 2 days p.i., in the absence of a detectable viraemia. Virus was undetectable in the blood of s.c. and i.n. inoculated adults; in the former, virus replication was limited to the site of inoculation, and in the latter virus grew in the respiratory tract and again spread to the brain. Immunosuppression of i.n. inoculated adult mice with cyclophosphamide produced some mortality indicating that host defences are important in protecting the adult, especially as newborn and adult lung tissue were equally able to support the growth of Dugbe virus in culture. The similarity between the pattern of Dugbe virus infection in the mouse and that of other, more pathogenic nairoviruses suggests that, although haemorrhagic disease was not observed, this may be a useful model for studying the genetic basis of nairovirus virulence and for testing vaccines and anti-viral drugs.

Twelve long-term cell lines were established from peripheral blood mononuclear cells (PBMC) or cerebrospinal fluid cells of patients with human T lymphotropic virus type I (HTLV-I) seropositive tropical spastic paraparesis (TSP) originating from the French West Indies, French Guyana or the Central African Republic. Most of these long-term interleukin-2- dependent cell lines exhibited a pattern characteristic of CD4+-activated T cells with high expression of CD2, CD3 and CD4 antigens, associated with a strong density of TAC and DR molecules. Nevertheless, in five cases CD8 expression was present at a significant level. HTLV-I antigens were never detected in uncultured PBMC, but they were expressed in a few cells after short-term culture and after 4 months the majority of the cells were HTLV-I positive, as demonstrated by indirect immunofluorescence (IF) using polyclonal or monoclonal anti-p19 and anti-p24 antibodies. Low and variable levels of reverse transcriptase activity were detected in supernatant fluids of these cell lines only after 4 months of culture, when at least 50% of the cells exhibited HTLV-I antigens by IF. However, numerous type C HTLV-I-like viral particles were detected, mostly in the extracellular spaces, with rare budding particles. Similar findings were found in three T cell lines derived from West Indian and African patients with adult T-cell leukaemia/lymphoma (ATLL). Differences in high Mt polypeptides were detected by Western blot in cell lysates when comparing TSP- or ATLL-derived T cell lines. Thus a signal of 62K was easily detectable in all the TSP lines, but not in the ATLL lines. In all cell lines bands corresponding to p53, p24 and p19 viral core polypeptides were present, as was the env gene-coded protein p46.

The GM1 strain of feline leukaemia virus (FeLV) was isolated from a naturally occurring case of myeloid leukaemia and induces severe haematopoietic abnormalities, including myeloblastic leukaemia, on inoculation into cats. Molecular clones of FeLV-GMl proviruses were obtained and studied by restriction enzyme mapping, blot hybridization and partial DNA sequence analysis. Two types of clone were isolated; the first was a replication-competent FeLV of subgroup A, resembling other low or minimally pathogenic FeLV-A isolates; the second was replication-defective with extensive deletions and mutations in gag and pol, although it has an intact env gene of subgroup B phenotype. Large segments of the defective proviruses, from the 5′ leader sequence upstream of the gag gene to the 5′ half of the env gene, show structural hallmarks of endogenous FeLV-related proviruses. Infectious FeLV-GMl viruses recovered after transfection were tested for their leukaemogenic potential in newborn cats. Early polyclonal myeloproliferative changes were observed in cats inoculated with FeLV-A/GMl alone, although these were more pronounced in animals receiving the full FeLV-AB/GMl complex reconstituted by cotransfection of the defective virus FeLV-B with its FeLV-A helper. Analysis of viruses in the bone marrow showed that replication of the subgroup B component is delayed and restricted to a proportion of cats. Most of the infected cats developed persistent abnormalities of haematopoiesis and one progressed to disseminated myeloid leukaemia. The defective recombinant FeLV-B/GMl appears to play an indirect but important role in myeloid leukaemogenesis.

Two porcine rotavirus strains (CN86 and CC86) isolated during an epidemiological survey of diarrhoea in swine in Argentina were studied because of several unique characteristics. Both these strains were isolated and cloned from the same faecal sample and the electrophoretic migration of 10 of their 11 genomic dsRNA genomic segments in polyacrylamide gels was identical, but strain CC86 had a supershort electro- pherotype. We analysed biochemical, serological and biological properties of both viruses. In vitro translation of genome segment 11 RNAs showed that both viruses produced a polypeptide with an apparent M r of 26K. No differences in any of the other virus-induced proteins made in infected MA104 cells were found on one- and two-dimensional gels for either strain. In addition, the serotype and the subgroup specificities of both viruses were identical (group A, subgroup I, serotype 5). These results suggest that the rearranged strain was probably generated from the standard one and that the coding capacity of the rearranged segment was conserved. Consistent with this hypothesis, primer extension analysis revealed that the supershort strain had a rearrangement involving partial duplication of genomic segment 11. Biological studies showed differences between these viruses. The rearranged strain (CC86) produced larger plaques in monolayers of MA104 cells and outgrew the standard strain (CN86) when cells were coinfected with both viruses at different relative concentrations and different m.o.i. The possibility that large plaque formation and efficient virus replication can be influenced by the products of genomic segment 11, in addition to segment 4, is discussed.

Freshly isolated, and established, cultures of bovine peripheral blood mononuclear leukocytes (PBMLs) were exposed to bluetongue virus (BTV) for the purpose of defining potential lymphotropism. PBML cultures were established in the presence of interleukin 2 (IL-2) and mitogen and maintained either as bulk culture or were cloned prior to infectivity studies. All cultures appeared to be of the T cell phenotype based on the following characteristics: binding of T lymphocyte-specific lectins (i.e. peanut agglutinin and Helix pomatia), rosetting of sheep erythrocytes, binding of a putative pan-T monoclonal antibody, and absence of surface immunoglobulin (Ig). T lymphocyte cultures were further characterized by their ability to elicit lectin-dependent cellular cytotoxicity (LDCC). Exposure of established lymphocyte cultures to BTV resulted in productive cytopathic and non-cytopathic infections. Non-cytopathic productive infections were observed in LDCC-negative cultures whereas cytopathic and non-cytopathic infections were observed in LDCC-positive cultures. Exposure of freshly isolated PBMLs to BTV resulted in minimal virus replication; addition of mitogen and IL-2 to such cultures did not augment virus replication. Addition of mitogen and IL- 2 induced negligible blast transformation, whereas PBML viability was minimally affected. These studies establish a tropism of BTV for bovine T lymphocytes with virus replication being limited to those cells undergoing blastogenesis. Establishment of infected lymphocyte cultures, without loss of culture viability, suggest such an interaction may contribute to the long term viraemias associated with BTV infection of cattle.

The interaction between activated bovine T lymphocytes (BTLs) and bovine herpesvirus type 1 (BHV-1) was investigated. BHV-1 infection of BTLs reduced the amplitude of recombinant bovine interleukin 2-induced proliferative responses. This decreased proliferation was caused by a virus-induced lymphocytolysis which was dependent on viable virus and was not inhibited by recombinant bovine interferon-α11. Furthermore, lymphocytolysis was not associated with virus replication or with the synthesis of detectable levels of viral proteins. Electron microscopic examination of virus-infected cells revealed that lymphocytolysis was characterized by early nuclear disintegration resembling apoptosis. These observations suggest that activated T cells, localized at the site of BHV-1 infection, may be susceptible to virus-induced cytolysis.

In 1977 we showed that cells of a human lymphocytic leukaemia-derived T line (Molt-4) have receptors for Epstein-Barr virus (EBV). More recently, EBV- positive human T cell lymphomas have been recognized and human T cell lines containing the EBV genome have been established in vitro. To understand better the interaction of EBV with T cells, we decided to determine first whether human peripheral blood T lymphocytes express receptors for EBV. Using flow cytometry we examined the binding of both lymphocyte-transforming (B95-8) and non-transforming (P3HR-1) strains of EBV to T lymphocyte subpopulations, using a double labelling technique with T cell- specific phycoerythrinated monoclonal antibodies (Leu 2a) and fluoresceinated viral preparation. Our results suggest that, in general, about 50% of the CD8+ (or suppressor/cytotoxic) T cell subpopulation from both EBV-seropositive and -seronegative individuals can bind EBV. EBV receptor expression on these T cells was about 10 and 51 times less than that on Molt-4 and Raji (an EBV receptor-positive B cell line) cells, respectively. The specificity of this binding was demonstrated by the inhibition of attachment of viral preparations preincubated with a monoclonal antibody directed against the viral ligand (gp240/350), and by preincubating these target T cells with unlabelled virus. We were unable to detect EBV-induced antigens in infected T cells, suggesting that, as in Molt-4 cells, virus internalization may not occur in fresh T cells and/or that the virus receptor may not be completely functional. We were also unable to detect C3d (or CR2) receptors on these T cells, or to inhibit virus attachment by treating the targets with an anti-CR2 monoclonal antibody (OKB7), suggesting that the EBV receptor on CD8+ peripheral blood lymphocytes is different from that on B cells.

In previous work, we observed that H-2k-restricted herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTLs) were effectively able to lyse transfected target cells expressing HSV glycoprotein C (gC), but not cells expressing gB, gD or gE. To confirm and extend our observations on the specificity of anti-HSV CTLs, recombinant adenovirus (Ad) vectors able to express HSV-1 gB or gC (AdgB2 or AdgC) were constructed. Syngeneic target cells infected with AdgB2 were efficiently lysed by primary H-2b and H-2d, but not by H-2k-restricted HSV-specific CTL. Limiting dilution studies indicated that 4 to 10% of H- 2b-restricted HSV-specific CTLs recognize gB. H-2k, H-2b and H-2d-restricted anti-HSV-1 CTLs were unable to lyse AdgC-infected syngeneic target cells. To examine the apparent discrepancy between the previous results involving transfected H-2k cells expressing gC and the present results involving AdgC-infected cells, gC-expressing cell lines used in previous experiments were subcloned and retested in CTL assays. DC2 cells which were lysed by HSV-specific CTLs in the previous experiments remained sensitive to anti-HSV CTLs but two other clones derived from the same transfection were not lysed. Further, L cells transfected with the gC or gD gene coupled to the mouse mammary tumour virus promoter and capable of expressing high levels of the glycoproteins following dexamethasone induction were not lysed by H-2k-restricted anti-HSV CTLs. These results suggest that HSV-specific CTLs do not recognize gC, at least when it is expressed using an Ad vector and in most transfected cell lines, whereas a significant proportion of anti-viral CTLs recognize gB presented in some but not all murine haplotypes.

During primary ocular infection of mice with herpes simplex virus type 1 (HSV-1) strain McKrae, dendritic corneal ulcers developed and many eyes became permanently damaged. When primary infection had subsided, latent infection was detected in the three parts of the trigeminal ganglion and in the superior cervical ganglion. Such latently infected mice were treated with cyclophosphamide, dexamethasone and u.v. irradiation, or cyclophosphamide and dexamethasone alone. After treatment with immunosuppressive drugs and u.v. irradiation infectious virus was isolated from the ophthalmic part of the trigeminal ganglion, and in eyelids and eyewashings; recurrent herpetic eye disease was seen but only in eyes undamaged by primary infection. After treatment with cyclophosphamide and dexamethasone alone there was a lower incidence of virus isolated from eyewashings and no recurrent disease was seen. There was a good correlation between the pattern and distribution of recurrent lesions and the distribution of cells stained due to the presence of virus antigens.

A cell culture technique with rat dorsal root ganglion neurons cultured in a two-chamber system allowing exposure of neuritic extensions or neuronal cell soma to herpes simplex virus (HSV) infection was exploited for studies of viral neuroinvasiveness. Four groups of HSV strains were investigated. Type 1 strains were isolated from cases of either encephalitis or mucocutaneous infections and type 2 strains were from cases of meningitis or genital infections. The neuroinvasiveness of the type 1 encephalitis strains was significantly greater than the neuroinvasiveness calculated for the type 1 strains of cutaneous origin or for either of the type 2 strains. From our evaluation of the rates of neuritic uptake and axonal transport and the rate of replication in dorsal root ganglion cells and skin fibroblasts, we concluded that the differences observed in neuroinvasiveness probably reflected differences in early virus-nerve cell interactions, i.e. virus attachment and fusion with the neuritic plasma membrane.

Studies with mutant viruses have suggested that the product of gene UL41 of herpes simplex virus type 1 (HSV-1) controls the virion-mediated inhibition of cellular protein synthesis as well as the rate of degradation of viral mRNAs. HSV-1 strain 17+ has a weak host shutoff function, whereas HSV-2 strain G shuts off strongly. A gene of HSV-2(G), judged from its position in the genome to be the probable analogue of gene UL41 of HSV-1, was inserted into the nonessential thymidine kinase gene of HSV-1(17+). The recombinant virus, 17G41, exhibited a strong shutoff function and its immediate early mRNA did not accumulate in the presence of cycloheximide. It resembled HSV-2(G) in these respects and not the parent, confirming the function of the transferred gene. Recombinant virus 17G41 carries the UL41 genes of both strains, 17+ and G, and in this situation the strong shutoff function was dominant. However, after mixed infection with equal multiplicities of 17G41 and HSV- 1(17+) the weak shutoff function was dominant. The recombinant, 17G41, was further modified by insertion of a lacZ expression cassette into the coding region of the original gene UL41 (17+). The resulting virus, 17(41−)G41, also had a strong shutoff activity but grew poorly in tissue culture.

Sera from 75 patients with warts were tested for antibodies against human papillomaviruses (HPVs) in an immunoelectron microscopy test. A group of 40 patients’ sera were tested against a pooled suspension of the HP Vs extracted from their warts and 90% of the sera were found to be antibody-positive. Prior to serological investigation viruses from a second group of 35 patients were typed by dot blot hybridization with HPV-1 and HPV-2 DNA. According to these results the patients were divided into type 1, type 2 and non-1, non-2 virus carriers. Sera of these patients were first tested for antibodies reactive with their homologous viruses and then for antibodies against type 1 virus. More than 80% of the patients possessed antibodies against their homologous viruses, but only a small minority of sera from type 2 and non-1, non-2 virus carriers were reactive with type 1 viruses. These results suggest that the antibody reactive in the immuno- aggregation test is type-specific.