- Volume 75, Issue 9, 1994

Recognition of a host cell receptor by a virus is the first and perhaps the most crucial step in initiating the disease process. This study was undertaken to identify the cellular receptor(s) for bovine herpesvirus 1 (BHV-1). Previously, we reported the development and characterization of bovine anti-idiotypic antibodies (anti-ids) that induce neutralizing antibodies to BHV-1. These anti-ids inhibit BHV-1 penetration of permissive cells. We have used these anti-ids, which mimic an epitope on the virus glycoprotein IV (gIV), and gradient-purified virus in immunoprecipitation (IP) as well as photoaffinity labelling (PAL) assays. In the IP assays, both bovine anti-ids and BHV-1 virions coupled to Sepharose precipitated a 60K protein from 125I-labelled BHV-1 permissive cell membrane extracts. Normal bovine IgG or an irrelevant virus, transmissible gastroenteritis virus (TGEV), used as negative controls failed to precipitate this protein. Similarly, in the PAL assays, the 60K cell surface protein was identified on cells permissive for BHV-1 infection, but not on non-permissive cells when 125I-labelled ligands, the anti-ids or BHV-1 were used as probes. The iodinated ligands failed to identify the 60K protein if they had been pretreated with the antibody 1. Pretreatment of the iodinated ligands with an isotype-matched control antibody had no effect on the identification of the 60K protein present on cells permissive for BHV-1 infection. The negative controls, i.e. normal bovine IgG and TGEV, failed to identify this 60K protein on permissive or non-permissive cells. These results suggest that the 60K protein is a cellular receptor recognized by BHV-1 during the infection process.

A marker vaccine elicits an antibody response in the host that can be distinguished from the antibody response induced by a wild-type strain. To obtain a bovine herpesvirus 1 (BHV-1) marker vaccine, we constructed a glycoprotein E (gE) deletion mutant. This was obtained by removing the complete gE coding region from the BHV-1 genome. To attenuate the gE deletion mutant further, we also introduced a small deletion in the thymidine kinase (TK) gene. We selected three mutants: the gE deletion mutant, a TK deletion mutant and a gE/TK double deletion mutant, and examined their virulence and immunogenicity in calves.After intranasal inoculation, the TK deletion mutant showed some residual virulence, whereas the gE and gE/TK deletion mutants were avirulent. The calves inoculated with the deletion mutants were protected against disease after challenge exposure and shed significantly less virus than control calves. Deleting the gE gene, therefore, has little effect on the immunogenicity of BHV-1, but is sufficient to reduce the virulence of BHV-1 in calves. These findings led us to conclude that the gE deletion mutant is a good candidate for a modified live BHV-1 marker vaccine.

One-week-old pigs were infected intranasally with the Ka strain of Aujeszky’s disease virus (ADV) or with mutants that were lacking the non-essential envelope glycoproteins gI, gp63 or gill. The invasion and spread of these strains in the olfactory nervous pathway were examined by assessing virus levels and by localizing viral antigens in the olfactory mucosa representing the first neuronal level, in the olfactory bulb representing the second neuronal level and in the lateral olfactory gyrus, the rostral perforated substance and the piriform lobe, all representing the third neuronal level. The Ka parental strain invaded and spread up to the third neuronal level. The extent of invasion and spread of the gIII− mutant were similar to those of the parental strain. The gp63− mutant replicated normally in the olfactory mucosa, but its spread to all the other levels was limited as compared with that of the parental strain. The gI− mutant showed a defect in infection at all neuronal levels. These results indicate that, of the non-essential envelope glycoproteins, gl plays the major role in neural invasion and spread of ADV in its natural host. The pattern of invasion and spread of these mutants in the olfactory pathway of pigs was similar to that previously observed in the trigeminal pathway. The type of nervous pathway therefore appears not to influence the neuropathogenesis of ADV or mutants deleted in non-essential envelope glycoproteins in the pig.

Recovery from primary cytomegalovirus (CMV) infection is associated with resolution of the productive infection without clearance of the virus genome from affected organs. The presence of latent CMV genome in multiple organs provides the molecular basis for recurrence of CMV within multiple organs, and explains the diversity in the organ manifestations of recrudescent CMV disease during states of immunodeficiency. As a part of a unifying concept of multifocal CMV latency and recurrence, previous work has demonstrated the importance of primary virus replication for the overall load of latent CMV in organs and the risk of recurrence. In the present report, the establishment of CMV latency was studied in a murine model in which the course of primaiy infection in the immunocompromised host after syngeneic bone marrow transplantation was modulated by a CD8+ T cell immunotherapy. The antiviral CD8+ effector cells limited virus replication in all organs and protected the recipients from lethal CMV disease, but after resolution of the productive infection virus DNA remained. Interestingly, the copy number of latent virus DNA in tissue did not quantitatively reflect the preceding virus production in the respective organ. Specifically, in contrast to the case in the lungs and the salivary glands, virus replication in the spleen was suppressed by CD8+ T cells to below the limit of detection; yet, virus DNA was also detected in the spleen during latency and accordingly, virus recurrence in the spleen could be induced. These findings demonstrate that the control of virus replication in a particular organ does not prevent the establishment of latency in that organ.

In order to study the function of human cytomegalovirus (HCMV) immediate early gene 2 (ie2) (UL122) gene products made at late times during infection, cDNA clones were isolated from an expression library made with 74 h post-infection mRNA. Based on screening of the library, 1 % of transcripts in infected cells at this time were ie2 region-specific, and transcripts encoding γIE2338aa, a 40K late gene product, were more abundant than those encoding IE2 579aa, an a gene product made throughout infection. As expected, the cDNA capable of directing the expression of γIE2338aa was derived from a contiguous genomic region within exon 5 of the ie1/ie2 region. The cDNA clones encoding γIE2338aa and IE2579aa were compared for their ability to trans-activate viral and cellular promoters and to repress expression from the ie1/ie2 promoter via the ie2 cis-repression signal. Unexpectedly, γIE2338aa trans-activated a variety of test promoters when cotransfected with the major a gene product, IE1491aa. Promoters derived from the cellular β-actin gene, the simian virus 40 early region and the human immunodeficiency virus were all responsive to γIE2338aa plus IE1491aa, although several β promoters derived from the HCMV genome were unresponsive. Thus, this abundant late product from the ie2 region may play a role in trans-activation in addition to its role as a repressor of α gene expression.

Uracil-DNA glycosylase encoded in many species functions as a DNA repair enzyme that removes uracil residues from DNA. To investigate the potential function of uracil-DNA glycosylase encoded by human herpesvirus 6 (HHV-6), we sequenced a DNA clone (pSTY09), identified an open reading frame of 765 bp and compared the putative amino acid sequence with other uracil-DNA glycosylases, by computer analysis. The amino acid sequence of HHV-6 had similarities to other uracil-DNA glycosylases, with the highest degree of similarity to those of human cytomegalovirus and Epstein-Barr virus. Two strongly conserved regions in uracil-DNA glycosylase of other species also existed in HHV-6. The gene product which was expressed in Escherichia coli demonstrated uracil-DNA glycosylase activity. This is the first report to identify and characterize the uracil-DNA glycosylase gene in HHV-6.

The herpes simplex virus type 1 (HSV-1) polypeptides encoded by genes UL26 and UL26.5 are thought to form a scaffold around which the capsid shell assembles. The UL26 gene specifies a proteinase that cleaves both itself and the UL26.5 gene product. To study the structure and function of the UL26 and UL26.5 gene products, the proteins were expressed in ceils infected with recombinant baculoviruses containing the genes under the control of the polyhedrin promoter. Both polypeptides were made in large amounts, approaching the levels of polyhedrin protein expressed in wild-type baculovirus. The UL26 polypeptide behaved in a similar manner to the protein made in HSV-l-infected cells, cleaving itself rapidly into the capsid proteins VP21 and VP24 and converting the UL26.5 product more slowly into the capsid protein VP22a. The results of immuno-blot analysis using antisera specific for the amino-terminal region of the UL26 polypeptide suggested that both the first and second ATGs in the UL26 open reading frame were recognized as translational start signals but the first ATG was the preferred initiation codon as is the case in HSV-l-infected cells. Electron microscopic examination of thin section preparations of cells infected with both the UL26.5- and UL26- recombinant baculoviruses revealed the presence of large numbers of small spherical particles, often arranged in a semi-crystalline array. These clusters of scaffold-like particles were not present in cells infected with UL26- recombinant baculovirus but were observed occasionally in UL26.5-recombinant baculovirus-infected cells. The results suggest that the proteinase, in the absence of other HSV capsid proteins, stimulates the formation of large numbers of scaffold-like particles present either as semi-crystalline arrays or as dispersed structures.

The herpes simplex virus (HSV) gene RL1 encodes the protein ICP34·5, which is a specific neurovirulence factor. Null mutants in RL1 fail to replicate in the central nervous system of mice and are therefore totally non-neurovirulent. Additionally, they fail to replicate in neurons of the peripheral nervous system, although they are capable of establishing and reactivating from a latent infection. As the precise function of ICP34·5 in HSV-neuronal interactions is unknown, we have studied the role of ICP34·5 in vitro by examining in detail the phenotypes of RL1-negative viruses in two defined tissue culture systems. The first was mouse embryo fibroblast 3T6 cells, in which RLl-negative mutants are impaired and the in vivo phenotype is mimicked. This impairment is amplified when the cells are in the stationary state. The second was mouse embryo testicular carcinoma F9 cells which, in the undifferentiated state, provide a reversal of phenotype; wild-type virus fails to grow but RLl-negative virus replicates efficiently. Differentiation results in the ability to support wild-type virus growth. The stage at which the replication cycle is blocked plus the role of cellular factors is addressed in both tissue culture systems. Evidence is provided that cell type and cell state are crucial to ICP34·5-cellular interaction and hence, based on these parameters, ICP34·5 can be defined as a host-range determinant. Identification of cellular proteins that specifically interact with or are homologues of ICP34·5 may lead to the identification of neuron-specific proteins that have a similar role.

Herpes simplex virus type 1 (HSV-1) gingivostomatitis during childhood is known to result in a latent infection of the trigeminal ganglion neurons, which innervate the oral mucosa. During latency the viral genome is maintained in a non-infectious state. However, stimuli such as stress, fever or localized trauma can cause HSV-1 to reactivate in neurons and produce recrudescent disease in the peripheral tissues. Recently, HSV-1 proteins and nucleic acids have been detected in biopsies from human duodenal and gastric ulcers, raising the possibility that HSV-1 latency within the enteric nervous system is involved in this chronic recurrent gastrointestinal disorder. The studies in mice described here were done to determine whether HSV-1 latency could be established in neurons that innervate the murine gut. We found that after either intraperitoneal or oral-oesophageal inoculation of mice, HSV-1 establishes a latent infection in nodose ganglia of the vagus nerve, whose sensory neurons project to the gastrointestinal tract. This animal model of HSV-1 latency in the vagal sensory ganglia will be useful to examine the possible relationship between HSV-1 and recurrent gastrointestinal disease.

This study describes the susceptibility to dengue virus infection of a monocytic cell line at different states of differentiation. Infectious virus titres increased in undifferentiated U937 cells following infection with clinical isolates but only when the cells were infected via their Fc receptors. No c.p.e. was observed and virus was not secreted into supernatant fluid. Once differentiated, cells were susceptible to infection either with virus alone or with virus-antibody complexes. Infection was cytolytic and virus was released into the supernatant fluid. Similar results were obtained with freshly isolated peripheral blood monocytes. Increased blood vessel permeability, which occurs in dengue haemorrhagic fever and dengue shock syndrome patients, has been correlated with secondary heterotypic infections and has been postulated to arise from antibody-enhanced infection of monocytes. The data presented suggest a possible mechanism whereby infected monocytes undergoing diapedesis through blood vessel walls might differentiate sufficiently during the process to release virus and cytokines at localized sites on blood vessels.

Following a survey of hepatitis C virus (HCV) infection recently carried in central Africa (Gabon), we cloned and sequenced PCR products of the 5′ non-coding and capsid-encoding regions of HCV RNA from three randomly selected HCV RNA-positive Gabonese subjects. In the capsid-encoding region, the identity between the three Gabonese isolates was 91 to 98%. The three Gabonese sequences showed a divergence of 11 to 17 % from published HCV genotypes I to IV (1a, 1b, 2a and 2b) isolates and of 6 to 11 % from HCV genotype 4 isolates. Thus the Gabonese isolates, termed HC-G, belong to HCV genotype 4. Based on the sequences of the three isolates, a specific probe (cpsG) was designed to detect the HC-G genotype in 30 randomly selected anti-HCV-positive Gabonese subjects, 14 of whom were HCV RNA-positive. Analysis with cpsG showed that 10 of 14 of the HCV RNA-positive subjects were infected by the HC-G genotype. HC-G is therefore highly prevalent in the HCV RNA-positive Gabonese population. The availability of these Gabonese sequences should facilitate the design of specific serological tests for African HCV isolates.

Tick-borne encephalitis virus (TBEV) encodes a conserved, immunogenic, non-structural protein NS1 that is glycosylated and secreted from infected cells in an oligomeric form. An adenovirus recombinant, RAd51, expressing high levels of TBEV NS1 has previously been demonstrated to protect mice against a lethal challenge with TBEV. We show here that BALB/c mice infected with TBEV experienced a transient viraemia between days 3 to 5 post-inoculation that was detectable prior to the encephalitic phase of infection. Mice vaccinated with RAd51 were protected against both the viraemic and encephalitic infections associated with the TBEV challenge. Protection was demonstrated to be due to NS1 synthesized de novo from RAd51 in the vaccinated mice. Since TBEV NS1 is expressed on the cell surface, antibody-dependent complement-mediated cytolysis (CMC) of infected cells was considered as a possible mechanism of protection. Vaccination with the recombinant adenovirus proved to be effective in a mouse strain carrying a genetic deletion in the complement receptor C5. CMC is therefore not an essential component of the observed protective immune response.

Eight isolates of canine distemper virus (CDV) were obtained from seven dogs suffering from distemper by co-cultivation of their mononuclear cells with a marmoset B lymphoblastoid cell line, B95a. Six of the seven dogs had received one or more vaccinations. All of the isolates readily proliferated in B95a cells, but were not completely neutralized by anti-CDV canine plasma, which had high neutralizing activity against the Onderstepoort laboratory strain of CDV. Furthermore, different reactivities of monoclonal antibodies (MAbs) against CDV were observed between the field isolates and laboratory or vaccine strains of CDV in immunofluorescence studies. Immunoprecipitation analysis using MAbs detected the haemagglutinin protein of each new field isolate as 69K, 75K and 155K forms, and the fusion protein as 64K and 65K forms; the corresponding proteins of the Onderstepoort strain were detected as 75K and 61K proteins respectively. It is apparent from these results that the new field isolates of CDV have very different antigenic properties from the Onderstepoort vaccine strain.

We describe the cloning, nucleotide sequencing and expression in Escherichia coli of the major capsid component (VP60) from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV). The sequence of the 3′ -terminal 2483 nucleotides of the genome was found to be 95·4 % identical to the German RHDV strain, showing ten changes in the deduced VP60 amino acid sequence. The gene coding for this structural polypeptide has been expressed in bacteria as a β-galactosidase fusion protein or using a T7 RNA polymerase-based system. The VP60 fusion protein showed only partial antigenic similarity with native VP60 and did not confer protective immunity. The recombinant VP60 produced in the T7 RNA polymerase-based system was antigenically similar to the viral polypeptide as determined using polyclonal and monoclonal antibodies. When used to immunize rabbits the recombinant VP60 was able to protect the animals against a lethal challenge using purified RHDV.

The nucleotide sequence of the genomic segment encoding the major antigen, VP6, of human rotavirus strain RV3 was determined for the virus isolated from faeces. This was compared with the sequence of the cognate gene of tissue culture-adapted candidate vaccine virus that was derived from RV3 and had been passaged 30 times. A single silent nucleotide difference was detected between the two genes and the deduced amino acid sequence for both showed the highest degree of identity (⩾ 97·5%) with the VP6 proteins of rotavirus strains belonging to the same subgroup. The amino acid residues that might affect VP6 subgroup epitope type from RV3 and other viruses of the same or different subgroups were compared. The commonality of particular amino acids among strains of different subgroups suggests that the presence of all subgroup-specific amino acids may be necessary for subgroup determination.

Full-length cDNA of the RNA genome segment coding for the major core protein VP7 of Australian bluetongue virus serotype 15 (BTV-15) has been isolated by reverse transcription-PCR cloning. Comparative analysis indicated that the BTV-15 VP7 sequence had diverged significantly from that of other members of the BTV serogroup. At the amino acid level, BTV-15 VP7 exhibited sequence identities of 80 to 84% with VP7 molecules of other serotypes, significantly lower than the sequence identities of between 93 and 100% observed among other serotypes characterized to date. This was consistent with previous observations that there were significant immunological differences between BTV-15 and other BTV serotypes and that monoclonal antibodies raised against BTV-1 VP7 failed to react with BTV-15 VP7. Recombinant BTV-15 VP7 protein produced from Escherichia coli was largely insoluble, but maintained its immunogenicity. Polyclonal mouse sera raised against the recombinant VP7 protein reacted strongly with VP7 of BTV-15, but weakly with that of BTV-1.

A strain of human immunodeficiency virus type 1 that is strictly tropic for primary human blood cell cultures was constructed in vitro. Mutational studies on the vif vpr, vpu and nef genes of this virus were performed to evaluate their biological functions in natural target cells. For this purpose, replication properties of mutant viruses in peripheral blood mononuclear cells (PBMCs) and macrophages (PBMPs) were determined. Three phenotypes with respect to virus replication were noticed: normal or mildly retarded growth (nef and vpr mutants), impaired growth (vpu mutant), and no growth (vif mutant). These results suggest that the Vif and Vpu proteins are more important than the Nef and Vpr proteins for virus replication in PBMCs and PBMPs.

Human T cell leukaemia virus type I-(HTLV-I) transformed cells are capable of stimulating the proliferation of normal T lymphocytes, or stimulating interleukin 2 expression in Jurkat T lymphoid cells. This effect is mediated by the CD2/lymphocyte function-associated antigen 3 (LFA-3) adhesion/signalling pathway. The current work demonstrates that CD3 is also required for this effect, suggesting that the T cell receptor (TCR)/CD3 complex is mediating this effect. However, this effect does not appear to be due to a superantigen since no change in TCR expression was found after HTLV-I-mediated proliferation, nor was proliferation inhibited by an antibody against the specific TCR expressed on Jurkat cells (TCR Vβ8).

Monoclonal antibodies (MAbs) to equine arteritis virus (EAV) proteins were produced and characterized. The protein specificities of eight MAbs were determined definitively by immunoprecipitation of EAV proteins expressed from vaccinia virus recombinants (WRs). Included were two new WRs produced for this study, expressing the M and the GL proteins, respectively. Three MAbs were determined to be N-specific and five MAbs recognized the GL protein. One GL-specific MAb, 17F5, of the IgA class, efficiently neutralized EAV infectivity. In competitive binding assays (CBAs), the N- specific MAbs defined a single antigenic domain on this protein. Four GL-specific MAbs, including MAb 17F5, demonstrated strong reciprocal competition in binding to the GL protein but differed in their virus-neutralizing ability. Thus the antigenic domain defined by these MAbs is probably composed of overlapping or closely adjacent epitopes. The fifth GL-specific MAb, a nonneutralizing antibody, may define an epitope adjacent to this antigenic domain as reciprocal CBAs demonstrated lower competition.