- Volume 76, Issue 9, 1995

The current hepatitis B virus (HBV) vaccines contain the small (S) and middle (M) viral envelope proteins in particulate form but lack the large (L) protein. Although these particles elicit protective immunity to HBV, inclusion of the immunogenic preS1 region of the L protein may enhance their efficacy. To present preS1-derived epitopes on secretable subviral particles we rearranged the HBV envelope ORF by fusing part or all of the preS1 region to either the N or C terminus of the S protein. Fusion of the first 42 residues of preS1 to either site allowed efficient secretion of the modified particles and rendered the linked sequence accessible at the surface of the particle. Conversely, fusion of preS1 sequences to the C terminus of the M protein completely blocked secretion. This block to secretion could be rescued by provision of a heterologous N-terminal signal sequence. All these particles displayed preS1, preS2 and S protein antigenicity. In mice, each construct elicited high titres of preS1-specific antibodies which recognized the authentic L protein. Particles composed of the modified M protein also induced a preS2-specific response. Unexpectedly, however, neither particle elicited S protein-specific antibodies. Nonetheless, the genetic approach employed here represents a strategy to incorporate preS1-derived epitopes both in high density and in highly immunogenic form into their authentic carrier matrix.

The EBNA-LP protein (also known as EBNA-5) of Epstein—Barr virus (EBV) has been reported previously to colocalize in the nuclei of cells with the pRb protein and to bind in vitro to pRb and to the p53 protein, suggesting a role for EBNA-LP in modulation of the function of these proteins. Here we test in transfection assays whether EBNA-LP expression has any functional consequence for repression of E2F-1 activity by pRb or p107 or for activation of transcription by the p53 protein. No significant effect could be found, although the assay systems were sensitive to the established effects of simian virus 40 large T antigen and human papillomavirus type 16 E6 protein. There was very effective repression of GAL4/E2F-1 transactivation by p107, consistent with earlier reports and indicating that p107 can interact with the E2F-1 transactivation domain, even though p107 has been reported to bind specifically to E2F complexes containing E2F-4. The results indicate that, if the associations of EBNA-LP with pRB and p53 are physiologically relevant, they most likely affect other functions of these proteins or modulate their gene regulatory functions in ways that cannot be detected by transfection into cycling transformed cells.

We describe the mutagenesis of the IRS1-US5 region of the human cytomegalovirus genome, demonstrating the potential of the E. coli guanosine phosphoribosyl transferase (gpt) gene as a selectable marker for insertion and deletion mutagenesis of high passage (AD169, Towne) as well as low passage (Toledo) strains of virus. Despite evidence suggesting that the US3 gene product may play a regulatory role, disruption of this gene with a gpt insert had no effect on growth of any of these strains of virus in resting or dividing human fibroblasts, or in human thymus plus liver implants in SCID-hu mice. Transcripts of the gpt gene, under control of the herpes simplex virus thymidine kinase promoter adjacent to the US3 enhancer in the viral genome, accumulated with delayed early (β) kinetics. Mutants with deletions in the IRS1 and US3-US5 regions were isolated by back-selection against gpt with the drug 6-thioguanine by growing virus in human Lesch-Nyhan (hypoxanthine-guanine phosphoribosyl transferase deficient) skin fibroblasts immortalized with human papillomavirus oncogenes. Thus, we demonstrate a dependable method for insertion and deletion mutagenesis that can be applied to any region of the viral genome.

The nucleotide sequence of a 2384 bp portion within the unique short (US) region of the herpesvirus simiae (simian herpes B virus; SHBV) genome is presented. A partial and a complete open reading frame (ORF) were found within this nucleotide sequence. The partial ORF encodes the C terminus (147 amino acids) of a protein kinase which is highly conserved in the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and simian agent 8 (SA8) US regions. The complete ORF is located 3′ to the partial ORF within the 2384 bp sequence and encodes a 593 amino acid glycoprotein which appears to be closely related to the SA8 glycoprotein G (gG), but shares little amino acid similarity with gG of HSV-1 and -2. However, the complete ORF shares certain features conserved among most alphaherpesvirus gGs, notably three highly conserved cysteine residues and an adjacent N-glycosylation site. Therefore, it was concluded that this complete ORF encodes the SHBV gG. The 358 amino acid C-terminal portion of SHBV gG was expressed in Escherichia coli as a fusion protein and this was detected by immunoblotting with sera from cynomolgus monkeys which were either experimentally or naturally infected with SHBV. The purified fusion protein was inoculated into rabbits to raise an antiserum which recognized a number of apparently SHBV gG-specific protein bands in extracts from SHBV-infected simian cells.

We have used the vaccinia virus-T7 RNA polymerase-based expression system for studies on the activity of proteases from various retroviruses on homologous and heterologous Gag polyproteins in eukaryotic cells. Proteases from human immunodeficiency virus (HIV) types 1 and 2, equine infectious anaemia virus, human T cell leukaemia virus type 1 and human spumavirus were produced and were shown to cleave their cognate Gag substrates produced in trans. Analysis of cross reactivity revealed that lentivirus proteases cleaved only lentivirus Gag proteins and oncovirus proteases acted primarily on oncovirus Gag proteins. The HIV-2 protease cleaved the HIV-1 Gag precursor almost as efficiently as HIV-1 protease. Expression of the 5′ end of the human spumavirus pol gene revealed that it encodes a functional protease that acts specifically on the human spumavirus Gag polyprotein. This assay will allow further investigation on the activity and specificity of retrovirus proteases in eukaryotic cells.

We constructed a series of human immunodeficiency virus 1 (HIV-1)/simian immunodeficiency virus strain mac (SIVmac) chimeric viruses having vpr and/or nef genes of either HIV-1 or SIVmac based on a chimeric virus with LTRs, gag, pol, vif and vpx derived from SIVmac and tat, rev, vpu and env from HIV-1. All of the chimeric viruses replicated in human and macaque peripheral blood mononuclear cells (PBMCs) and in several CD4+ human cell lines, though their growth potentials were slightly different depending on whether vpr and nef were from HIV-1 or SIVmac, or were defective. The presence of nef accelerated replication in all the cells used and the replication of each chimera appeared to reflect that of the parental virus from which nef was derived. The presence of vpr had no clear effect in human and monkey PBMCs, but the replication of each chimera was influenced by the origin of vpr in H9 and A3.01 cells. NM-3rN, which carries HIV-1 vpr and SIVmac nef, was inoculated intravenously into three rhesus monkeys, three cynomolgus monkeys and two pig-tailed monkeys. From 2 to 14 weeks after inoculation, viruses were consistently re-isolated from all the monkeys and virus loads were as high as that of SIVmac reported previously. The results indicate that infection with NM-3rN is more efficient than any of our previous chimeric viruses and suggest that NM-3rN, having HIV-1 Env, will be a useful challenge virus for evaluating AIDS vaccines based on HIV-1 Env in macaque monkeys instead of chimpanzees.

We have investigated the in vivo state of feline immunodeficiency virus (FIV) transcription in peripheral blood mononuclear cells (PBMC) of chronically FIV-infected, asymptomatic cats. FIV was detected in a high percentage of PBMC but not in the plasma of these cats. By quantitative reverse transcription–PCR (RT–PCR) analysis, FIV transcriptional status in the PBMC was characterized by extremely low or undetectable levels of unspliced or singly spliced mRNAs and predominantly multiply spliced mRNAs. Upon stimulation in vitro, however, the larger mRNA species and infectious virus production were rapidly induced in the PBMC. Furthermore, we demonstrated that viral production was induced in association with differential increases in the levels of each multiply spliced mRNA coding for FIV regulatory proteins. From these results, we suggest that replication of FIV is blocked at an early stage of gene expression in vivo, as described in asymptomatic human immunodeficiency virus (HIV) -infected patients, and that FIV infection in cats may be a useful model for clinical latency of HIV infection in man. Moreover, we propose that the replication of FIV in vivo may be controlled by the differential expression of each multiply spliced mRNA.

An in vitro transcription assay was used to study transcription from synthetic RNA corresponding to the 3′ terminus of influenza A virus cRNA. Micrococcal nuclease-treated influenza virus ribonucleoprotein was used as a source of active polymerase complex. Mutations at two regions of the 13 nucleotide-long conserved cRNA 3′ terminus were shown to reduce transcription templated by the short added model RNAs. The first region, at positions 1 and 2 from the 3′ terminus, was shown to be affected by the exact nature of the dinucleotide primer used in the in vitro transcription reactions and may not be relevant in vivo. The second region, centred on positions 11 and 12, may be involved in base pairing with conserved nucleotides at the 5′ terminus of the cRNA. Evidence for this comes from the finding that RNA corresponding to 5′ conserved sequences, but mutated to restore the postulated base pairing with the mutated 3′ ends, could partly restore transcription. Binding of the influenza virus polymerase complex to a set of 5′-mutated RNAs was investigated using a photochemical cross-linking assay. Specific binding to two regions of the cRNA 5′ terminus was demonstrated, at positions 1 to 3 and positions 8 to 10. Together, these observations suggest that a panhandle forms from the termini of the cRNA molecule and that this structure may play a role in transcription to produce virion RNA.

A Borna disease virus (BDV)-like agent was isolated from the central nervous system (CNS) of cats with a spontaneous non-suppurative encephalomyelitis (‘staggering disease’). In contrast to the rabbit-adapted BDV strain V, which can be propagated in several primary and permanent cell cultures, the cat virus grew only in embryonic mink brain cells. Infection of adult Wistar rats with feline brain tissue material did not result in clinical disease during a period of 5 months, nor in growth of infectious virus in the brain. However, using the brain suspension of a newborn rat inoculated with feline brain tissue material, it was possible to induce typical Borna disease (BD) in four adult rats. This indicates a possible adaptation of the cat virus during passages in rats. By the use of an RT-PCR technique, BDV-specific RNA could be detected in a majority of brain samples from diseased cats. BDV-specific antigen was demonstrated in feline CNS samples both by immunohistochemistry and ELISA. However, the amount of BDV RNA and BDV antigen was less in the cats as compared to horses with BD, providing further support for the notion that a distinct feline BDV strain exists.

Three murine monoclonal antibodies (MAbs) that neutralize equine arteritis virus (EAV) infectivity were identified and characterized. The antibodies, 93B, 74D(B) and 38F, recognized the major envelope glycoprotein (GL) encoded by open reading frame (ORF) 5 in immunoblots and by immunoprecipitation. All three MAbs were used to compare the Bucyrus isolate of EAV and MAb neutralization-resistant (NR) escape mutants with the vaccine virus and 19 independent field isolates of EAV by virus neutralization. The different abilities of the MAbs to neutralize virus isolates indicated that they recognize non-identical epitopes. Susceptibility to virus neutralization could not be used to distinguish viruses from acutely and persistently infected horses. Comparison of the ORF 5 nucleotide and deduced amino acid sequence from NR and neutralization-sensitive virus isolates revealed amino acid sequence changes at positions 99 and 100 which correlate with the NR phenotype. Additional unique changes in the amino acid sequence of MAb NR viruses at positions 96 and 113 may also contribute to neutralization resistance. The sequence data further showed that the Bucyrus-derived viruses contain one N-glycosylation site, whereas the field isolates DL8 and DL11 possess two sites, both of which are used. Most of the non-conservative amino acid sequence changes were located within the second half of the N-terminal hydrophilic domain. Sequence changes within the first half of the N-terminal ectodomain, the predicted transmembrane domain and the C-terminal hydrophilic domain were mainly silent base substitutions or resulted in conservative amino acid substitutions, suggesting that these regions of the protein are functionally conserved.

The pathogenicity of neutralization-resistant mutants of the enteric coronavirus transmissible gastroenteritis virus (TGEV) was examined in the newborn piglet. The parental virus (Purdue-115 strain), as well as several mutants selected using monoclonal antibodies (MAbs) directed to antigenic sites A and B, caused an acute enteritis with 100% mortality. By contrast, most of the site D (MAb 40.1) mutants exhibited a strongly reduced enteropathogenicity, leading to the survival of animals inoculated with up to 1000-fold the 100% lethal dose of parental virus. Such a phenotypical change was correlated with point mutations or a small deletion, all located within the S gene sequence coding for the Pro-145 to Cys-155 segment of the mature polypeptide. These observations suggest that an N-terminal subregion of the S molecule is an essential determinant for pathogenesis in TGEV infection.

The role of flavivirus-cross-reactive T lymphocytes in recovery from and pathogenesis of flavivirus infections is not known. In the present paper, we have defined a flavivirus-cross-reactive epitope recognized by two CD4+ CD8− cytotoxic T lymphocyte (CTL) clones, JK4 and JK43. The T cell clones were established from the peripheral blood T lymphocytes of a dengue-4-immune donor, using a limiting-dilution method with dengue-4 antigen. These two T cell clones were cross-reactive for dengue virus types 1, 2, 3 and 4, yellow fever virus and West Nile virus, and recognized NS3 protein. The smallest synthetic peptide recognized by these T cell clones was an identical 9 amino acid peptide which contains amino acids 146 to 154 (VIGLYGNGV) of dengue-4 NS3. HLA-DR15 was the restriction allele for recognition of this epitope by JK4 and JK43. JK4 and JK43 both used T cell receptor Vα8, but JK4 used Vβ8 and JK43 used Vβ2. This result indicates that this epitope is recognized by two flavivirus-cross-reactive CD4+ T cell clones which originated from different T cells in vivo.

Intravenous infection by Theiler’s murine encephalomyelitis virus strain GD VII causes acute encephalomyelitis and paralysis in infected mice. However, nude mice and cyclophosphamide-treated ddY mice did not show paralysis when they were able to survive until day 20 post-infection (p.i.). Of ddY mice infected with 5 × 107 p.f.u./mouse, 70–80% showed symptoms of paralysis on day 20 p.i. The viral titres in the brain and spinal cord in infected mice were not significantly different between paralytic and non-paralytic mice. In all of the mice infected with the virus, CD4+ lymphocytes and CD8+ lymphocytes had infiltrated the brain on days 10, 12, 14 and 20 p.i. as demonstrated by flow cytometric analysis. In contrast, few T lymphocytes infiltrated the spinal cord in the non-paralytic mice. Administration of an anti-CD4 monoclonal antibody (MAb) or anti-T cell receptor-αβ MAb on day 6 p.i. inhibited paralysis until day 20 p.i., though 20% of the MAb-treated mice and 80% of the control mice showed paralysis. Administration of anti-CD8 MAb was not effective in the suppression of paralysis. The MAb treatment did not significantly augment viral replication in the spinal cord, although the viral titres in the brain of the MAb-treated mice increased significantly. After the transfer of spleen cells from infected C3H mice, the recipient mice infected with a small amount of the virus showed paralysis, though uninfected mice did not. This transfer could be blocked by CD4+ lymphocyte depletion of the donor mice. These results indicate that paralysis caused by acute myelitis in Theiler’s virus strain GD VII infection is induced by CD4+ lymphocytes infiltrating the spinal cord.

Banzi is a mosquito borne flavivirus which belongs to the Uganda S serocomplex. No nucleotide sequence data have previously been reported from any virus of this serocomplex. We have determined the nucleotide sequence of the NS5 gene from Banzi virus and the predicted amino acid sequence was elucidated. Previously identified conserved RNA polymerase, methyltransferase and flavivirus NS5 amino acid motifs were present in the Banzi virus NS5 protein. These data add to the evidence for the functional importance of the regions. The encoded amino acid sequence was compared with the predicted amino acid sequence of other flavivirus NS5 proteins. Analysis of these sequences suggested that Banzi virus is most closely related to the mosquito-borne flaviviruses and, in particular, yellow fever virus. This pattern of similarity is in accordance with the previously suggested serological classification of flaviviruses.

We examined 24 C-type hepatitis specimens from Thailand and detected hepatitis C virus (HCV) RNA in all of them by RT-nested PCR for a portion of the HCV 5′ non-coding (5′ NC) region and a portion of the HCV core region. However, we failed to detect HCV RNA in 11 specimens by RT-nested PCR for a portion in the non-structural protein 5 (NS5) region that has been used commonly for HCV genotyping. We designed a new primer set for a separate portion of the NS5 region. Using this primer set, we succeeded in amplifying this portion in all 24 specimens. Two novel HCV genotypes, tentatively designated HCV-VII and HCV-VIII, were identified by sequencing these amplified regions. Our newly designed primers for RT-nested PCR may be useful for diagnosing infection as well as for genotyping unidentified HCV genomes.

Nine (10%) out of 90 hepatitis C virus (HCV) isolates from hepatitis patients and commercial blood donors in Thailand were not classifiable into any of genotypes I/1a, II/1b, III/2a, IV/2b, V/3a or VI/3b by RT-PCR with type-specific primers deduced from the HCV core gene. These isolates were sequenced over a 1.6 kb stretch of the 5′-terminal sequence and 1.1 kb of the 3′-terminal sequence covering 30% of the entire genome. Based on two-by-two comparison and phylogenetic analyses of the nine Thailand isolates among themselves and with known full or partial sequences of previously reported HCV isolates, the Thailand isolates were classified into five genotypes not reported previously, viz. 6b, 7c, 7d, 9b and 9c. Along with HCV isolates reported already, they make at least nine major genetic groups of HCV which further break down into at least 28 genotypes with sequence similarity in the E1 gene (576 bp) of ⩽80%. As many more HCV isolates of distinct genotypes are expected to be found throughout the world, it will become increasingly difficult to classify them by comparison of any partial sequences of the genome. Complete sequence data will be required for the full characterization and classification of HCV genotypes.

The physical properties of hepatitis C virus (HCV) particles were determined by ultracentrifugation on 20–60% isopycnic sucrose density gradients. We report that (i) two populations of HCV particles were found in the sera of patients with chronic HCV infection [at high density (1.186–1.213 g/ml) and at low density (1.099–1.127 g/ml)], (ii) virus particles with high density values were associated with immunoglobulin, and (iii) virus particles with low density values accumulated base changes within a hypervariable region (HVR) of the E2 envelope domain of the RNA genome. The results indicate that base changes within the HVR of E2 lead to the accumulation of immunoglobulin-free virus particles. Therefore, these findings imply that persistent HCV infection is established as a consequence of sequence variation in the E2 envelope domain.

Intertypic vaccine/vaccine recombinant polioviruses are frequently isolated from vaccine-associated paralytic poliomyelitis cases (VAPP). We identified a vaccine/nonvaccine poliovirus recombinant as the causative agent of a lethal VAPP. Partial RNA sequencing revealed a tripartite recombinant structure of the viral genome. This consisted of a central capsid core of vaccine origin flanked by two units of nonvaccine origin. The first nonvaccine genomic unit spanned the whole 5′ noncoding region, and the second one almost the entire nonstructural protein-coding region and the 3′ non-coding region. Amino acid and nucleotide sequence similarities in the 3′ and 5′ unidentified regions indicated that the viral donor(s) were poliovirus species, suggesting recombination between a vaccine-derived and a wild poliovirus. The nonvaccine donor(s) could not be identified among the investigated wild polioviruses co-circulating in the same geographical area. This is the first report of a natural recombination event occurring in the 5′ genomic extremity of poliovirus. The neurovirulence for transgenic mice and the pathogenicity for humans of the recombinant suggested that the modular genomic organization of this virus might have conferred a selective advantage over its vaccine parent.