- Volume 81, Issue 10, 2000

The antibody response to the envelope proteins of hepatitis C virus (HCV) may play an important role in controlling the infection. To allow molecular analyses of protective antibodies, we isolated human monoclonal antibodies to the E2 envelope glycoprotein of HCV from a combinatorial Fab library established from bone marrow of a chronically HCV-infected patient. Anti-E2 reactive clones were selected using recombinant E2 protein. The bone marrow donor carried HCV genotype 2b, and E2 used for selection was of genotype 1a. The antibody clones were expressed as Fab fragments in E. coli, and as Fab fragments and IgG1 in CHO cells. Seven different antibody clones were characterized, and shown to have high affinity for E2, genotype 1a. Three clones also had high affinity for E2 of genotype 1b. They all bind to conformation-dependent epitopes. Five clones compete for the same or overlapping binding sites, while two bind to one or two other epitopes of E2. Four clones corresponding to the different epitopes were tested as purified IgG1 for blocking the CD81–E2 interaction in vitro; all four were positive at 0·3–0·5  μg/ml. Thus, the present results suggest the existence of at least two conserved epitopes in E2 that mediate inhibition of the E2–CD81 interaction, of which one appeared immunodominant in this donor.

A novel flavivirus, GB virus C (GBV-C)/hepatitis G virus (HGV), has been detected in chronic liver disease patients. It is known that the viral RNA can be detected in ∼5% of American blood donors. However, the implications for liver disease and the sites of virus replication remain unknown. Possible sites of virus replication were studied by using cell lines and/or primary cells derived from human lymphoid cells, myeloid cells, hepatocytes and endothelial cells. RNA was detected by virus strand-specific RT–PCR and GBV-C/HGV antigen was detected with a rabbit polyclonal anti-E2 (envelope 2) antibody by Western blot analysis. Negative-strand RNA, representative of replicating virus, was detected in lymphoid and megakaryocytoid cell lines and primary vascular endothelial cells. In addition, an increase in virus titre over time was demonstrated and viral antigen was detected, and virus could be passaged to infect fresh cells. However, viral RNA or antigen could not be detected in any of the hepatocyte lines tested. These results indicate that the replication site of GBV-C/HGV is not primarily in hepatocytes and that detection of replicating virus in hepatic tissue may reflect virus replication in haematopoietic cells and/or vascular endothelial cells present in the liver.

The complete genomic and predicted amino acid sequence of the Japanese encephalitis virus (JEV) FU strain, a human isolate recovered from the first outbreak of Japanese encephalitis in Australian territory, was determined. Comparison of the FU genome with 15 fully sequenced JEV genomes revealed high levels of sequence identity, ranging from 88·7% (GP78) to 89·7% (K94P05) for nucleotides and 96·8% (K94P05) to 98·0% (JaGAr01) for amino acid sequences. A total of 39 unique amino acid differences were found in the FU strain polyprotein. Phylogenetic analyses were performed on all available full-length JEV genomes and a selection of 64 E gene sequences from temporally and geographically diverse JEV strains. For comparison with the E gene phylogeny, phylogenetic analysis using cognate prM gene sequences was also carried out. The FU strain was found to be most closely related to Korean isolate K94P05 in the full-length analysis and to Southeast Asian strains in the E and prM gene analyses. The E gene analysis corresponded well with the prM gene analysis and with previous genotyping studies using the prM gene. The epidemiological implications of this investigation are discussed.

Polypeptide 2C is essential for picornavirus replication. Although many data on multiple functions of this highly conserved protein are available, the mechanism of RNA binding is still obscure. In this work, protein 2C of echovirus-9 strain Barty was expressed as a histidine-tagged protein in E. coli followed by nondenaturing purification to homogeneity. After incubation of 2C protein with different kinds of RNA fragments, binding was shown in gel retardation assays. Competition experiments revealed that 2C targets linear RNA unspecifically; however, single-stranded linear DNA does not react with this protein. In contrast to poliovirus, protein 2C of echovirus-9 only recognizes RNA with a low content of secondary structures. This may be a first hint of a different binding specificity of 2C in echo- and polioviruses.

A clonal population of BHK-21 cells resistant to infection with the low-neurovirulence BeAn strain of Theiler’s murine encephalomyelitis virus (TMEV) was derived after four cycles of infection and characterized. These cells were resistant to both low- and high-neurovirulence TMEV strains due to a block in virus attachment and entry and not in virus replication, since transfection of these cells with TMEV RNA to bypass the entry step(s) induced virus replication and assembly. The resistance to infection was stable for more than a year, suggesting that it is a heritable property arising from a mutation in the susceptible parent BHK-21 population. This cell line is being used to identify a receptor for TMEV.

Equine arteritis virus (EAV) (Arteriviridae) encodes several structural proteins. Whether any of these also function in viral RNA synthesis is unknown. For the related mouse hepatitis coronavirus (MHV), it has been suggested that the nucleocapsid protein (N) is involved in viral RNA synthesis. As described for MHV, we established that the EAV N protein colocalizes with the viral replication complex, suggesting a role in RNA synthesis. Using an infectious cDNA clone, point mutations and deletions were engineered in the EAV genome to disrupt the expression of each of the structural genes. All structural proteins, including N, were found to be dispensable for genome replication and subgenomic mRNA transcription. We also constructed a mutant in which translation of the intraleader ORF was disrupted. This mutant had a wild-type phenotype, indicating that, at least in cell culture, the product of this ORF does not play a role in the EAV replication cycle.

The major envelope glycoprotein genes (ORF5) of seven Czech isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were amplified and their nucleotide sequences were determined. ORF5 displayed nucleotide and amino acid identities of 87·5–100% and 87·6–100%, respectively, among the isolates. In a phylogenetic tree, all European isolates were grouped in a genotype distinct from that of reference American strains (VR-2332, IAF-Klop). Among the European isolates, two different clades were identified. Two Czech isolates (V-501 and V-503) and Italian strain PRRSV 2156 fell into one clade. The remaining European strains comprised the second clade. Surprisingly, two separately clustered strains (V-501 and V-516) were isolated from the same herd. Additionally, the possible effect of in vitro cultivation on the nucleotide sequence was analysed. Nine point mutations in the ORF5 region resulted from 152 in vitro passages of the V-502 isolate in MARC-145 cells.

Mice (ddY strain, 4 weeks old) were infected intranasally with the H5N1 influenza viruses A/Hong Kong/156/97 (HK156) and A/Hong Kong/483/97 (HK483) isolated from humans. HK156 and HK483 required 200 and 5 p.f.u. of virus, respectively, to give a 50% lethal dose to the mice when the volume of inoculum was set at 10 μl. Both viruses caused encephalitis and severe bronchopneumonia in infected mice. The severity of lung lesions caused by the viruses was essentially similar, whereas HK483 caused more extensive lesions in the brain than did HK156. This was supported by the results of virus titration of organ homogenates, which showed that the virus titres in brains of HK483-infected mice were more than 100-fold higher than those of HK156-infected mice, while those in lungs were almost equivalent. Both viruses were detected in homogenates of the heart, liver, spleen and kidney and blood of the infected mice. Virus antigen was detected by immunohistology in the heart and liver, albeit sporadically, but caused no degenerative change in these organs. The antigen was not detected in the thymus, spleen, pancreas, kidney or gastrointestinal tract. In contrast, virus antigen was found frequently in adipose tissues attached to those organs. The adipose tissues showed severe degenerative change and the virus titres in the tissues were high and comparable to those in lungs. Thus, infection of HK156 and HK483 in our mouse model was pneumo-, neuro- and adipotropic, but not pantropic. Furthermore, HK483 showed higher neurotropism than HK156, which may account for its higher lethality.

Molecular epidemiology studies have made significant contributions to the control of measles virus infection through the identification of source and transmission pathways of the virus. These studies allow observation of changes in measles virus genotypes over time in a particular geographical location, clarification of epidemiological links during measles outbreaks, separation of indigenous strains from newly imported strains and distinction between vaccine- and wild-type virus-associated illness. A total of 35 wild-type measles viruses identified in Victoria, Australia, between 1973 and 1998 were characterized by nucleic acid sequence analysis of the nucleoprotein gene and, in some cases, the haemagglutinin gene. Relatedness between the viruses was studied and genotypes were assigned using a classification scheme recently proposed by the World Health Organization. Five recognized genotypes (C2, D1, D4, D5 and H) and one previously undescribed genotype, which we propose to be D7, were identified. Successive replacement of measles virus genetic lineages occurred in Victoria, with no evidence of temporal overlap, during this 25 year period. This pattern of circulation is likely to represent serial importation of wild-type measles virus strains from overseas foci of measles virus infections.

Significant protection against respiratory syncytial virus (RSV) infection was induced in mice vaccinated intramuscularly (i.m.) with DNA encoding the F or G protein of RSV. The amounts of IgG1 of IgG2a antibodies in mice immunized with DNA-G alone were similar. However, the antibody response in mice co-immunized with DNA-G and DNA encoding IL-4 (DNA-IL-4) was strongly biased towards IgG1. In contrast, the antibody response in mice co-immunized with DNA-G and DNA-IL-2, -IL-12 or-IFN-γ was biased towards IgG2a. Mice vaccinated with DNA-F either alone or in combination with DNA encoding cytokines developed a predominant RSV-specific IgG2a response, which was most pronounced in mice co-immunized with DNA-F and DNA-IL-12 or -IFN-γ. Vaccinated mice developed only a slightly enhanced pulmonary inflammatory response following RSV challenge. More significantly, and in contrast to mice scarified with recombinant vaccinia virus expressing the G protein, mice vaccinated i.m. with DNA-G did not develop pulmonary eosinophilia, even when the immune response was biased towards a Th2 response by co-administration of DNA-IL-4.

The majority of neurons in herpes simplex virus (HSV)-infected murine sensory ganglia are transiently induced to express MHC-I antigens at the cell surface, whereas only a minority are themselves productively infected. The aim of the current work was to determine whether MHC-I antigens can be expressed on the surfaces of infected neurons in addition to their uninfected neighbours. To address this aim a recombinant HSV type 1 strain, S-130, was used to deliver a mouse H2Kd gene, under control of the HCMV IE-1 promoter/enhancer, into human neuroblastoma cells in vitro and mouse primary sensory neurons in vivo. S-130 expressed H2Kd antigens on the surfaces of IMR-32 cells, a human neuroblastoma cell line that expresses very low levels of MHC-I constitutively. In K562 cells, which do not express MHC-I constitutively, H2Kd and β2-microglobulin (β2m) were shown to be co-expressed at the cell surface following S-130 infection. This observation was taken as evidence that class I heavy chain (αC) molecules encoded by the expression cassette in the HSV genome were transported to the cell surface as stable complexes with β2m. Significantly, after introduction of S-130 into flank skin, H2Kd antigens were detected on the surfaces of primary sensory neurons in ganglia innervating the inoculation site. Our data show that HSV-infected murine primary sensory neurons and human neuroblastoma cells are capable of expressing cell-surface MHC-I molecules encoded by a transgene. From this, we infer that up-regulation of αC expression is, in principle, sufficient to overcome potential impediments to neuronal cell surface expression of MHC-I complexes.

Both famciclovir (FCV) and valaciclovir (VACV) are potent inhibitors of herpes simplex virus type 1 (HSV-1) in a murine cutaneous infection model. The object of the present study was to determine whether either drug had an effect on the anatomical distribution of infected neurons in the peripheral nervous system and to assess the consequences for infected cells during, immediately following and several months after a 9 day period of continuous treatment. Mice were inoculated via the neck with a recombinant strain of HSV-1 expressing the lacZ reporter gene under the immediate-early gene promoter. Sensory ganglia were sampled daily up to day 11 post-inoculation (p.i.) and infected cells were detected by means of the reporter gene product. Ganglia were also removed at 1·5 and 10 months p.i. and latency was assessed by explant co-cultivation and by using in situ hybridization to detect LAT-expressing neurons. While both drugs reduced the severity of acute infection markedly, neither compound completely prevented the relentless distribution of infection among peripheral nervous tissue. Furthermore, there was a difference between the compounds regarding the expression of the reporter gene during and after termination of treatment and in the number of residual LAT-positive neurons.

The UL34 gene of herpes simplex virus type 2 (HSV-2) is highly conserved in the herpesvirus family. The UL34 gene product was identified In lysates of HSV-2-infected cells as protein species with molecular masses of 31 and 32·5 kDa, the latter being a phosphorylated product. Synthesis of these proteins occurred at late times post-infection and was highly dependent on viral DNA synthesis. Immunofluorescence assays revealed that the UL34 protein was localized in the cytoplasm in a continuous net-like structure, closely resembling the staining pattern of the endoplasmic reticulum (ER), in both HSV-2-infected cells and in cells transiently expressing UL34 protein. Deletion mutant analysis showed that this colocalization required the C terminus of the UL34 protein. The UL34 protein associated with virions but not with A, B or C capsids. We treated virions, HSV-2-infected cells and cells expressing the UL34 protein with a protease in order to examine the topology of the UL34 protein. In addition, we constructed UL34 deletion mutant proteins and examined their intracellular localization. Our data strongly support the hypothesis that the UL34 protein is inserted into the viral envelope as a tail-anchored type II membrane protein and is significant for virus envelopment.

The major immediate-early promoter/enhancer (MIEP, −1139 to +52) of human cytomegalovirus (HCMV) is regulated by cell type-specific transcriptional factors, its own MIE proteins (IE2p40, IE1p55, IE1p72 and IE2p86) as well as viral proteins pUL69, pUL82 and pUL84. To investigate the hypothesis that the regulation of HCMV MIEP is modulated by additional viral genes, HCMV (AD169) genomic sublibraries were constructed and in vitro transient co-transfection assays were performed to assess the ability of these sublibraries to modulate MIEP expression. In this study, enhancement of MIEP expression was exhibited by a number of sublibraries, from one of which a genomic clone was selected for augmentation of expression. Subcloning the insert fragment led to the identification of the responsible locus, UL76. To generate a UL76-specific antibody for immunodetection, the UL76 ORF was constructed as a histidine-tagged fusion protein that was produced in prokaryotic cells. A polyclonal antibody raised against the UL76 fusion protein immunoreacts with a protein of 38 kDa (pUL76) in UL76 ORF-transfected cells. Additionally, pUL76 is present in HCMV-infected cells at the immediate-early to late stages of the reproductive cycle. Characterized by its highly basic composition (predicted pI 11·6), a free form of pUL76 tagged with green fluorescent protein was found to localize exclusively to the nucleus. In this report, pUL76 is defined as a novel regulatory protein that modulates both activation and repression of gene expression, depending on the promoter context and the ratio of transfected effector DNA.

Tumour cells of undifferentiated nasopharyngeal carcinoma (NPC) consistently harbour Epstein–Barr virus (EBV) genes. Expression of mRNA transcripts associated with EBV latency has been demonstrated in such cells. However, expression of EBV lytic genes has not been well elucidated, although various lines of evidence have suggested that there is EBV replication in NPC tumour cells. We have studied mRNA expression of representative EBV lytic genes by RT–PCR in nasopharynx biopsies obtained from NPC and control individuals. In both NPC and control biopsies, EBV lytic genes BZLF1, BALF2 and BCLF1 were detected readily. However, BRLF1 was detected in NPC biopsies only. The BRLF1 gene was then cloned and expressed in vitro, and the protein product, Rta, was used as an antigen to detect specific antibodies by immunoprecipitation in plasma samples obtained from NPC patients and healthy controls. IgG antibodies directed against Rta were detected in 44 of 53 NPC plasma samples (83·0%), but only in 1 of 53 control samples (1·9%). Furthermore, the antibody binding regions were found in the C-terminal two-thirds of Rta. This serological result confirms indirectly that BRLF1 is specifically expressed in NPC tumour cells. Rta might play an important role in NPC pathogenesis, considering its multiple functions in EBV replication and cell cycles. Moreover, the detection of IgG antibodies directed against Rta could be developed into a diagnostic parameter for NPC.

Poxviruses encode multiple proteins that enable them to evade host responses. Among these are serine protease inhibitors (serpins). One of the earliest serpins described, cowpox virus crmA, acts to inhibit inflammation and apoptosis. crmA homologous serpins, known as SPI-2, are conserved in rabbitpox, vaccinia and variola viruses. Here, we describe the characterization of ectromelia virus (EV) SPI-2. EV SPI-2 encodes a protein of approximately 38 kDa showing >94% identity with other poxviral homologues. Conservative changes in amino acid sequence were found within the reactive site loop and the serpin backbone. Like crmA, transient expression of SPI-2 protected cells from tumour necrosis factor-mediated apoptosis and inhibited the activity of caspases-1 and -8 but not caspases-3, -6 or granzyme B. Overall, this study demonstrates that EV SPI-2 is functionally similar to crmA, based on in vitro assays.

The genome of frog adenovirus (FrAdV-1) was sequenced and found to be the smallest of all known adenovirus genomes. The sequence obtained was 26163 bp in size and contains a substantial direct repeat near the right terminus, implying that it was derived by recombination from a parental genome of only 25517 bp. The closest relative of FrAdV-1 proved to be turkey adenovirus 3, an avian adenovirus with no previously known near relative. Sequence comparisons showed that the two viruses have equivalent gene complements, including one gene the product of which is related to sialidases. Phylogenetic analyses supported the establishment of a fourth adenovirus genus containing these two viruses, in addition to the established genera Mastadenovirus and Aviadenovirus and the proposed genus Atadenovirus. Sixteen genes were identified as being conserved between these four lineages and were presumably inherited from an ancestral adenovirus.