- Volume 84, Issue 4, 2003

Immunization via the oral route with an attenuated Yersinia enterocolitica strain expressing a fragment of the measles virus nucleocapsid protein (aa 79–161) via its type III protein secretion system induced a T helper type 1 response in immunized C3H mice, which conferred protection against measles virus-induced encephalitis in a time- and dose-dependent manner.

Newcastle disease virus (NDV) was examined for its suitability as a vector for the expression and delivery of foreign genes for vaccination and gene therapy. A reporter gene encoding human secreted alkaline phosphatase (SEAP) was inserted as an additional transcription unit at four different positions in the NDV genome, between the NP and P, M and F, and HN and L genes and behind the L gene. Eight infectious recombinant NDV (rNDV) viruses, four in the non-virulent strain NDFL and four in the virulent derivative NDFLtag, were generated by reverse genetics. SEAP expression levels, replication kinetics and virus yield were examined. Replication kinetics of the rNDV viruses in primary chicken embryo fibroblasts showed that the insertion of an additional gene resulted in a delay in the onset of replication. This effect was most prominent when the gene was inserted between the NP and P genes. With the exception of the strain that carried the SEAP gene behind the L gene, all recombinant strains expressed high levels of SEAP, both in cell culture and in embryonated chicken eggs. In embryonated eggs, the rNDV viruses showed a 2·6- to 5·6-fold (NDFL) or 2·1- to 8·1-fold (NDFLtag) reduction in yield compared with the parent strains. These results show that foreign genes can be inserted at different positions in the NDV genome without severely affecting replication efficiency or virus yield.

The genome of Bunyamwera virus (BUN) consists of three segments of single-stranded RNA of negative polarity. The smallest segment, S, encodes the N protein and a nonstructural protein called NSs. We recently described a mutant virus (BUNdelNSs) that does not express NSs but overexpresses N and grows to lower titres than wild-type (wt) BUN. Here we report a BUNdelNSs variant that expresses lower levels of N protein and grows to higher titres. Sequencing of the 3′ and 5′ termini of the BUNdelNSs S RNA segment and analysis using a minireplicon system show that the N overexpressing phenotype results from a single nucleotide substitution at position 16 in the 3′ terminus. This mutation could also be detected in wtBUN populations, and was isolated by plaquing a ‘wt’ variant carrying the mutation. This variant was found to express increased N and NSs levels, and grew to lower titres than wtBUN.

Three urban rabies outbreaks have been reported in Colombia during the last two decades, one of these is occurring in the Caribbean Region (northern Colombia), while the other two occurred almost simultaneously in Arauca (eastern Colombia) and in the Central Region and ended in 1997. In order to derive phylogenetic relationships between rabies viruses isolated in these three areas, 902 nt cDNA fragments encoding the cytoplasmic domain of protein G and a fragment of protein L were obtained by RT-PCR. These amplicons contained the G–L intergenic region and were sequenced to draw phylogenetic trees. Phylogenetic analysis showed three distinct groups of viruses in the study sample. Colombian genetic variant I viruses were isolated in both Arauca and the Central Region. These viruses are apparently extinct in Colombia. Colombian genetic variant II viruses were isolated in the Caribbean Region and are still being transmitted in that area. The third group of viruses consists of viruses isolated from two insectivorous bats, three domestic dogs and a human. According to sequence analysis, the data here indicate that the isolates in this third group are bat rabies virus variants. This finding is the first that associates bats to rabies in Colombian dogs and humans, showing an unsuspected vector threatening animal and public health.

Infectious hematopoietic necrosis virus (IHNV) is a rhabdoviral pathogen that infects wild and cultured salmonid fish throughout the Pacific Northwest of North America. IHNV causes severe epidemics in young fish and can cause disease or occur asymptomatically in adults. In a broad survey of 323 IHNV field isolates, sequence analysis of a 303 nucleotide variable region within the glycoprotein gene revealed a maximum nucleotide diversity of 8·6 %, indicating low genetic diversity overall for this virus. Phylogenetic analysis revealed three major virus genogroups, designated U, M and L, which varied in topography and geographical range. Intragenogroup genetic diversity measures indicated that the M genogroup had three- to fourfold more diversity than the other genogroups and suggested relatively rapid evolution of the M genogroup and stasis within the U genogroup. We speculate that factors influencing IHNV evolution may have included ocean migration ranges of their salmonid host populations and anthropogenic effects associated with fish culture.

A self-modulating mechanism by the hepatitis C virus (HCV) core protein has been suggested to influence the level of HCV replication, but current data on this subject are contradictory. We examined the effect of wild-type and mutated core protein on HCV IRES- and cap-dependent translation. The wild-type core protein was shown to inhibit both IRES- and cap-dependent translation in an in vitro system. This effect was duplicated in a dose-dependent manner with a synthetic peptide representing amino acids 1–20 of the HCV core protein. This peptide was able to bind to the HCV IRES as shown by a mobility shift assay. In contrast, a peptide derived from the hepatitis B virus (HBV) core protein that contained a similar proportion of basic residues was unable to inhibit translation or bind the HCV IRES. A recombinant vaccinia–HCV core virus was used to examine the effect of the HCV core protein on HCV IRES-dependent translation in cells and this was compared with the effects of an HBV core-recombinant vaccinia virus. In CV-1 and HuH7 cells, the HCV core protein inhibited translation directed by the IRES elements of HCV, encephalomyocarditis virus and classical swine fever virus as well as cap-dependent translation, whereas in HepG2 cells, only HCV IRES-dependent translation was affected. Thus, the ability of the HCV core protein to selectively inhibit HCV IRES-dependent translation is cell-specific. N-terminal truncated (aa 1–20) HCV core protein that was expressed from a novel recombinant vaccinia virus in cells abrogated the inhibitory phenotype of the core protein in vivo, consistent with the above in vitro data.

The N-terminal part of VP1 was sequenced for 43 enterovirus isolates that could not initially be neutralized with LBM pools or in-house antisera. Most isolates were found to belong to human enterovirus type A (HEV-A) and HEV-B (18 isolates of each). All HEV-A isolates could be typed by sequencing, with CV (coxsackievirus)-A16 and EV (enterovirus)-71 being dominant (nine and seven isolates, respectively). These types thus seem to have diverged more from their prototypes than the other types. Among the HEV-B isolates, E-18 dominated with five isolates that became typable after filtration. The virus type obtained by molecular typing was verified for 28 of the other patient isolates by neutralization using high-titre monovalent antisera or LBM pools. Twenty-two of the other 30 ‘untypable’ isolates had substitutions in the VP1 protein within or close to the BC loop. Two closely related HEV-B isolates diverged by 19·4 % from E-15, the most similar prototype. Two non-neutralizable HEV-C isolates split off from the CV-A13/CV-A18 branch, from which they diverged by 15·7–18·2 %. Three of the six non-neutralizable isolates, W553-130/99, W543-122/99 and W137-126/99, diverged by >24·2 % from the most similar prototype in the compared region. The complete VP1 was therefore sequenced and found to diverge by >29 % from all prototypes and by >28 % from each other. Strains similar to W553-130/99 that have been identified in the USA are tentatively designated EV-74. The two other isolates fulfil the molecular criterion for being new types. Since strains designated EV-75 and EV-76 have been identified in the USA, we have proposed the tentative designations EV-77 and EV-78 for these two new members of HEV-B.

Ljungan virus (LV) is a suspected human pathogen recently isolated from bank voles in Sweden. This study describes the genetic characterization of a virus, M1146, which was isolated in 1962 from another vole species (Microtus montanus), trapped in Oregon, USA. Based on antigenic properties, M1146 was postulated previously as a putative member of the family Picornaviridae. The near complete genomic sequence verifies that M1146 is a member of the Picornaviridae, most closely related to LVs isolated in Sweden. The strain M1146 possesses typical LV genomic organization, including a cluster of two 2A homologues. There are significant differences throughout the capsid protein region, while the non-structural region of M1146 is closely related to the Swedish LV genomes. Genetic and phylogenetic analyses show that M1146 represents a new genotype within the distinct LV cluster. Isolation of LV from both Swedish and American voles trapped over a period of 30 years suggests a continuous worldwide presence.

Theiler's murine encephalomyelitis virus (TMEV) infects most mammalian cells, but a TMEV receptor has not been identified. Studies have demonstrated that the UDP-galactose transporter (UGT) is critical for TMEV attachment and entry into mammalian cells ( Hertzler et al., Virology 286, 336–344, 2001 ). It was suggested that UGT might function as a TMEV receptor. We have demonstrated that polyclonal rabbit antibodies to human UGT that cross-react with hamster UGT do not block binding to or infection of mammalian cells by either high- or low-neurovirulence TMEV. In addition, incubation of virus with galactose, or blocking galactose on the cell surface with lectins, does not inhibit TMEV binding or infection. Thus, TMEV needs UGT for its transporter activity and galactose for assembly of its co-receptors (attachment factors) but does not bind directly to galactose. Excluding direct involvement of UGT and galactose in TMEV binding and entry provides further insight into how TMEV interacts with the host cell and should facilitate ongoing studies to identify a TMEV receptor.

Japanese patients with sporadic acute hepatitis E are infected with polyphyletic strains of hepatitis E virus (HEV). Hepatitis E is considered a zoonotic disease. Thus far in Japan, only three strains of swine HEV have been identified and an antibody study for HEV antibodies has not been done on Japanese pigs. To determine the prevalence of swine HEV infection in Japan and the extent of genetic variation among Japanese swine HEV strains, we tested serum samples obtained from 2500 pigs from 2 to 6 months of age at 25 commercial swine farms in Japan for the presence of IgG antibodies to HEV and swine HEV RNA. Anti-HEV antibodies were detected in 1448 pigs (58 %). One-hundred-and-thirteen (15 %) of the 750 3-month-old pigs and 24 (13 %) of the 180 4-month-old pigs were positive for swine HEV RNA. The nucleotide sequence of a 412 bp region within open reading frame 2 of the 137 swine HEV isolates was determined. Sequence analyses revealed that the 137 isolates shared 76·6–100 % nucleotide sequence identities and were classifiable into genotype III (93 %) or IV (7 %) and that the isolates from the same farm were ⩾97·1 % similar to each other. Phylogenetic analysis showed that the Japanese swine and human HEV isolates segregated into four clusters, with the highest nucleotide identity being 94·4–100 % between swine and human isolates in each cluster. These results indicate that swine HEV is widespread in the Japanese swine population and further support the hypothesis that swine serve as reservoirs for HEV infection.

Yellow head virus (YHV) is a major agent of disease in farmed penaeid shrimp. YHV virions purified from infected shrimp contain three major structural proteins of molecular mass 116 kDa (gp116), 64 kDa (gp64) and 20 kDa (p20). Two different staining methods indicated that the gp116 and gp64 proteins are glycosylated. Here we report the complete nucleotide sequence of ORF3, which encodes a polypeptide of 1666 amino acids with a calculated molecular mass of 185 713 Da (pI=6·68). Hydropathy analysis of the deduced ORF3 protein sequence identified six potential transmembrane helices and three ectodomains containing multiple sites for potential N-linked and O-linked glycosylation. N-terminal sequence analysis of mature gp116 and gp64 proteins indicated that each was derived from ORF3 by proteolytic cleavage of the polyprotein between residues Ala228 and Thr229, and Ala1127 and Leu1128, located at the C-terminal side of transmembrane helices 3 and 5, respectively. Comparison with the deduced ORF3 protein sequence of Australian gill-associated virus (GAV) indicated 83 % amino acid identity in gp64 and 71 % identity in gp116, which featured two significant sequence deletions near the N terminus. Database searches revealed no significant homology with other proteins. Recombinant gp64 expressed in E. coli with and without the C-terminal transmembrane region was shown to react with antibody raised against native gp64 purified from virions.

The rotavirus genome encodes two glycoproteins, one structural (VP7) and one non-structural (NSP4), both of which mature and remain in the endoplasmic reticulum (ER). While three amino acids in the N terminus have been proposed to function as a retention signal for VP7, no information is yet available on how NSP4 remains associated with the ER. In this study, we have investigated the ER retention motif of NSP4 by producing various C-terminal truncations. Deleting the C terminus by 52 amino acids did not change the intracellular distribution of NSP4, but an additional deletion of 38 amino acids diminished the ER retention and resulted in the expression of NSP4 on the cell surface. Brefeldin A treatment prevented NSP4 from reaching the cell surface, suggesting that C-terminal truncated plasma membrane NSP4 is transported through the normal secretory pathway. On the basis of these results, we propose that the region between amino acids 85 and 123 in the cytoplasmic region of NSP4 are involved in ER retention.

The intrinsic recombination rate of human immunodeficiency virus (HIV) exceeds the point mutation rate by a factor of 10. As the majority of infected cells in vivo harbour multiple proviruses, the stage is set for rampant recombination. Therefore, it may be presumed that phylogenic relationships and mutation frequencies will probably be affected by recombination. However, the proportion of homoplasies arising from recombination and mutation is not known. By studying the evolution of the hypervariable regions of the simian immunodeficiency virus envelope gene among four macaques, it is shown that homoplasies arise more from recombination than from point mutation. When recombination is accounted for, the minimum number of substitutions in a sequence set may be reduced by as much as 45 %. In fact, the true number of point mutations in a set of HIV sequences tends to the number of discrete substitutions. Hence, lineages are younger than anticipated previously, although not in proportion to the ratio of the intrinsic recombination/point mutation rate. Recombination also inflates codon polymorphisms.

Human T-cell leukaemia virus type 1 (HTLV-I), the aetiological agent of adult T-cell leukaemia (ATL) and tropical spastic paraparesis (TSP/HAM), transforms human T-cells in vivo and in vitro. The Tax protein of HTLV-I is essential for cellular transformation as well as viral and cellular gene transactivation. The interaction of Tax with cellular proteins is critical for these functions. We previously isolated and characterized a novel Tax-binding protein, TRX (TAX1BP2), by screening a Jurkat T-cell cDNA library. In the present study, we present evidence that the tumour suppressor p53 targets the TRX protein for proteasome degradation. Pulse–chase experiments revealed that p53 enhanced the degradation of TRX protein and reduced the half-life from 2·0 to 0·25 h. p53 mutants R248W and R273H enhance TRX degradation suggesting a transcriptionally independent mechanism. Both HTLV-I Tax and the proteasome-specific inhibitor MG132 inhibited p53-mediated TRX protein degradation. These results suggest that TRX degradation is mediated through activation of the proteasome protein degradation pathway independent of transcriptional function of p53. Our results provide the first experimental evidence that Tax inhibits transciption-dependent and independent functions of p53.

The nucleotide sequence of the Friend murine leukaemia virus variant FIS-2 LTR has high identity with the closely related Friend murine leukaemia virus (F-MuLV) LTR, except for the deletion of one direct repeat, a few point mutations and the generation of a glucocorticoid response element (GRE) in the U3 region. The GRE can mediate gene induction by glucocorticoids, mineral corticoids, progesterone and androgens, and it has been shown that incorporation of a GRE(s) within the LTR can increase the transcriptional activity of retroviral enhancers. We have previously reported an increased early virus replication in male mice compared with female mice when infected with a virus containing the FIS-2 LTR and have proposed that the GRE might contribute to this sex difference. In the present study, we introduced a single point mutation in the GRE and performed comparative studies in NIH 3T3 cells and in young adult male and female NMRI mice. We found that significantly more virus was produced from NIH 3T3 cells infected with wt FIS-2 than from cells infected with the FIS-2 GRE mutant and that this difference was further augmented by glucocorticoids. The glucocorticoid antagonist RU486 inhibited virus production in a dose-dependent manner. The wt FIS-2 disseminated significantly faster than the FIS-2 GRE mutant in both male and female mice. There was no significant difference in the dissemination rate between male and female mice infected with the FIS-2 GRE mutant. Hence, the GRE in the FIS-2 LTR is one determinant of the significant sex difference in susceptibility to FIS-2 infection.

Both serotypes of herpes simplex virus (HSV), HSV-1 and HSV-2, are aetiological agents of genital herpes, although genital herpes caused by HSV-1 recurs less frequently. The HSV-1 genome contains a number of short, tandemly repeated sequences, and some reiterated sequences can serve as sensitive markers for the differentiation of HSV-1 strains. In the present study, variation in reiterations (assumed to be due to different copy numbers of tandemly repeated sequences) was examined in HSV-1 isolates from genital lesions from the same individual. Six sets (three primary-recurrence sets and three multiple-recurrence sets) of HSV-1 isolates were analysed: the primary-recurrence set consisted of two isolates (one isolated at a primary episode and the other at a recurrent episode) from the same individual; the multiple-recurrence set consisted of plural isolates from different episodes of recurrence in the same individual. Variations in length of the major DNA fragment, containing reiteration I (within the a sequence) and/or reiteration IV (within introns of genes US1 and US12), were detected between isolates of each multiple-recurrence set, but not of the primary-recurrence set. Thus, HSV-1 isolates of multiple-recurrence sets are assumed to have diverged more widely within each set than those of primary-recurrence sets, probably because of more rounds of virus DNA replication. This divergence of reiterations seems to indicate a forward step in the division of HSV-1 from a common ancestor into different lineages.

In early herpes simplex virus (HSV) infection, the virion host shutoff (vhs) protein mediates the degradation of mRNA and subsequent shutoff of host protein synthesis. It is unclear whether vhs acts alone or in concert with virus-induced cellular factors for this activity. This paper examines whether RNase L, a virally induced endoribonuclease, contributes to HSV-induced mRNA decay. Results showed that RNA degradation was comparable in wild-type and RNase L−/− cells, demonstrating that HSV-mediated RNA degradation is independent of RNase L activity. Furthermore, the data show that HSV-1 does not significantly induce RNase L activity in murine embryo fibroblasts.

Identifying cellular genes that promote bovine herpesvirus-1 (BHV-1) productive infection is important, as BHV-1 is a significant bovine pathogen. Previous studies demonstrated that BHV-1 DNA is not very infectious unless cotransfected with a plasmid expressing bICP0, a viral protein that stimulates expression of all classes of viral promoters. Based on these and other studies, we hypothesize that the ability of bICP0 to interact with and modify the function of cellular proteins stimulates virus transcription. If this prediction is correct, cellular proteins that activate virus transcription could, in part, substitute for bICP0 functions. The adenovirus E1A gene and bICP0 encode proteins that are potent activators of viral gene expression, they do not specifically bind DNA and both proteins interact with chromatin-remodelling enzymes. Because of these functional similarities, E1A was tested initially to see if it could stimulate BHV-1 productive infection. E1A consistently stimulates BHV-1 productive infection, but not as efficiently as bICP0. The ability of E1A to bind Rb family members plays a role in stimulating productive infection, suggesting that E2F family members activate productive infection. E2F-4, but not E2F-1, E2F-2 or E2F-5, activates productive infection with similar efficiency as E1A. Next, E2F family members were examined for their ability to activate the BHV-1 immediate-early (IE) transcription unit 1 (IEtu1) promoter, as it regulates IE expression of bICP0 and bICP4. E2F-1 and E2F-2 strongly activate the IEtu1 promoter, but not a BHV-1 IEtu2 promoter or a herpes simplex virus type 1 ICP0 promoter construct. These studies suggest that E2F family members can stimulate BHV-1 productive infection.