- Volume 82, Issue 12, 2001

The low-affinity nerve-growth factor receptor p75NTR interacts in vitro with the rabies virus (RV) glycoprotein and serves as a receptor for RV. The Lyssavirus genus comprises seven genotypes (GTs) of rabies and rabies-related viruses. The ability of p75NTR to interact with the glycoprotein of representative lyssaviruses from each GT was investigated. This investigation was based on a specific binding assay between BSR cells infected with a lyssavirus and Spodoptera frugiperda (Sf21) cells expressing p75NTR on the cell surface. A specific interaction was observed with the glycoprotein of GT 1 RV (challenge virus standard or Pasteur virus strains) as well as wild-type RV and the glycoprotein of GT 6 European bat lyssavirus type 2. In contrast, no interaction was detected with the glycoprotein of lyssaviruses of GTs 2–5 and 7. Therefore, p75NTR is only a receptor for some lyssavirus glycoproteins, indicating that the other GTs must use an alternative specific receptor.

Infectious salmon anaemia virus (ISAV), an orthomyxovirus-like virus, is an important fish pathogen in marine aquaculture. Virus neutralization of 24 ISAV isolates in the TO cell line using rabbit antisera to the whole virus and comparative sequence analysis of their haemagglutinin (HA) genes have allowed elaboration on the variation of ISAV isolates. The 24 viruses were neutralized to varying degrees, revealing two major antigenic groups, one American and one European. Sequence analysis of the HA gene also revealed two groups of viruses (genotypes) that correlated with the antigenic groupings. The two HA subtypes had nucleotide sequence identity of only ⩽79·4% and amino acid sequence identity of ⩽84·5% whereas, within each subtype, the sequence identities were 90·7% or higher. This grouping was also evident upon phylogenetic analysis, which revealed two distinct phylogenetic families. Between the two groups, the amino acid sequence was most variable in the C-terminal region and included deletions of 4–16 amino acids in all isolates relative to ISAV isolate RPC/NB-980 280-2. In order to view the relationships among these sequences and the HA sequences of the established orthomyxoviruses, a second phylogenetic tree was constructed which showed the ISAV sequences to be more closely related to sequences from Influenzavirus A and Influenzavirus B than to sequences from Influenzavirus C and Thogotovirus. The extensive deletions in the gene of European ISAV isolates lead us to speculate that the archetypal ISAV was probably of Canadian origin.

A formalin-inactivated respiratory syncytial virus vaccine was used to immunize infants in the mid-1960s; when these children later were naturally infected by the virus they developed markedly accentuated disease, and two died. For the present work, a new batch of vaccine was prepared using the original formula. Administration of either the old or new vaccines resulted in enhanced lesions in immunized cotton rats subsequently challenged with live virus, although administration of the vaccine reduced virus replication by 90%. Animals primed with formalin-inactivated virus and challenged developed markedly accentuated lesions of the same type as in animals undergoing primary or secondary infection. In addition, the animals with the vaccine-enhanced disease developed alveolitis and interstitial pneumonitis, which appear to be specific markers for the vaccine enhancement. The newly prepared vaccine appears suitable as a reference standard for studying the mechanism of vaccine-enhanced disease caused by this virus. Additionally, we reviewed the lesions in the lungs of the two humans who died with the vaccine-enhanced disease in 1967, and found that they were similar to, but more severe than those seen in the cotton rats.

Peroral vaccination for preventing respiratory infectious diseases was investigated in a murine model using a temperature-sensitive (ts) mutant of parainfluenza virus type 1. The ts mutant virus administered perorally in drinking water neither multiplied nor caused lesions in the respiratory tract or the central nervous system. However, ts virus antigen-positive cells appeared in oropharyngeal lymphoid tissues. This type of antigenic stimulation was capable of inducing both humoral and cellular immune responses, together with an augmentation of interferon production and natural killer cell activity, making it possible to protect the mice against challenge infection with a virulent wild-type virus. These results suggest that the oral cavity, a constituent member of the common mucosal immune system, is a candidate organ applicable as a vaccine route against virus respiratory diseases.

A cell-free system for studying Sendai virus RNA synthesis was reconstituted from N protein:RNA templates and transfected cell extracts in which the viral N, P and L proteins were expressed. Both transcription (mRNA synthesis) and replication (genome and antigenome synthesis) took place concurrently in these reactions. Viral RNA polymerases engaged in replication (replicases) were found to elongate their chains at a constant speed along the genome (1·7 nt/s), in a highly processive manner. In contrast, viral RNA polymerases engaged in transcription (transcriptases), although capable of synthesizing RNA at a comparable speed to replicases, were poorly processive. In this system, therefore, transcriptases require special reaction conditions to promote processivity that are not required by replicases. In addition, during replication, incomplete nascent genome chains were shown to be assembled with N protein, providing direct evidence that the synthesis and assembly of genomes are concurrent events. The strong processivity of replicases, independent of the reaction conditions, may thus be due to the coupling of genome synthesis and assembly. A model is proposed to explain how pausing of viral polymerase on the template is restricted when assembly and synthesis of the nascent chain are coupled.

Short regions at the 3′ and 5′ ends of the genome of Rinderpest virus (RPV) contain signals that regulate transcription of the viral genome, known as the genome promoter and the (complement to the) antigenome promoter, respectively. An RPV minigenome construct carrying the CAT coding sequence was used as a reporter to investigate residues in the 3′-terminal region of the genome important for these functions. Single-base scanning mutagenesis showed that modifications to nucleotides 1, 3, 4, 10 and 19 of the RPV leader had an extremely inhibitory effect on transcription and/or encapsidation of the minigenome, with CAT expression reduced to 0–10% of control values. Changes in any of the other first 22 nucleotides reduced the efficiency of the minigenome to 20–80% of the wild-type control, with the exception of nucleotides 16, 17 and 20, where mutations did not affect CAT expression significantly. Mutagenesis in blocks identified critical residues in positions 23–26, but changes to leader residues 27–48 had no major effect on CAT expression. A region of about 16 nucleotides (49–65) located around the start of the nucleocapsid gene, including the intergenic triplet CTT, was identified as essential for minigenome function. Mutations further into the nucleocapsid gene (nt 66–89) had a moderate effect (CAT activity 20–60% of control), while at least one critical residue was found in positions 93–96. The importance of four highly conserved G residues at positions 79, 85, 91 and 97 was also investigated. G79 was found to be optimal, though not critical, while a purine was required at 85 and 91. Although G97 is conserved in morbilliviruses, all bases were equally effective at this position.

Wild-type strains of measles virus (MV) isolated in B95a cells use the signalling lymphocyte activation molecule (SLAM; also known as CD150) as a cellular receptor, whereas the Edmonston strain and its derivative vaccine strains can use both SLAM and the ubiquitously expressed CD46 as receptors. Among the major target cells for MV, lymphocytes and dendritic cells are known to express SLAM after activation, but monocytes have been reported to be SLAM-negative. In this study, SLAM expression on monocytes was examined under different conditions. When freshly isolated from the peripheral blood, monocytes did not express SLAM on the cell surface. However, monocytes became SLAM-positive after incubation with phytohaemagglutinin, bacterial lipopolysaccharide or MV. Anti-SLAM monoclonal antibodies efficiently blocked infection of activated monocytes with a wild-type strain of MV. These results indicate that SLAM is readily induced and acts as a monocyte receptor for MV.

The genetic consequences of passing three different strains of La Crosse (LAC) virus orally and transovarially in Aedes triseriatus mosquitoes were examined. Two of the LAC strains (WT LAC and LAC ORI) had been passaged numerous times in cell culture; the third strain (SM1-78) had been passaged only once in suckling mice. Genetic changes were monitored in three regions of the LAC genome after oral infection and dissemination in the mosquito, and transovarial transmission (TOT) of the virus to progeny. Sequence analyses were used to characterize the genetic changes occurring in regions of G1, G2 and N open reading frames (ORFs) during passage. Only one mutation was detected in the G1 ORF of SM1-78 virus after mosquito passage; however, numerous nucleotide and amino acid substitutions were detected in the G1 ORF of WT LAC and LAC ORI (cell culture-adapted viruses). In contrast to G1, the N and G2 ORF sequences examined were stable. Mutations introduced into viral genomes during replication in parental mosquitoes were expressed in progeny mosquitoes following TOT. Genetic diversity of virus populations from a single mosquito was examined by single-strand conformation polymorphisms analysis of the variable region of glycoprotein G1. LAC virus RNA genotype diversity was greatest in virus that infected and replicated in the midgut, and declined as virus disseminated from the midgut and infected ovaries and salivary glands.

The complete genome sequences are reported here of two field isolates of bovine coronavirus (BCoV), which were isolated from respiratory and intestinal samples of the same animal experiencing fatal pneumonia during a bovine shipping fever epizootic. Both genomes contained 31028 nucleotides and included 13 open reading frames (ORFs) flanked by 5′- and 3′-untranslated regions (UTRs). ORF1a and ORF1b encode replicative polyproteins pp1a and pp1ab, respectively, that contain all of the putative functional domains documented previously for the closest relative, mouse hepatitis virus. The genomes of the BCoV isolates differed in 107 positions, scattered throughout the genome except the 5′-UTR. Differences in 25 positions were non-synonymous and were located in all proteins except pp1b. Six replicase mutations were identified within or immediately downstream of the predicted largest pp1a-derived protein, p195/p210. Single amino acid changes within p195/p210 as well as within the S glycoprotein might contribute to the different phenotypes of the BCoV isolates.

A cellular protein that interacts with the NS5A polypeptide of bovine viral diarrhoea virus (BVDV) was identified in a yeast two-hybrid screen. The NS5A interactor was identified as the α subunit of bovine translation elongation factor 1A (eEF1A). Cell-free binding studies were performed with chimeric NS5A fused to glutathione S-transferase (GST–NS5A) expressed in bacteria. GST–NS5A bound specifically to both in vitro-translated and mammalian cell-expressed eEF1A. Moreover, purified eEF1A bound specifically to GST–NS5A attached to a solid phase. Conservation of this interaction was then analysed using a set of NS5A proteins derived from divergent BVDV strains encompassing known biotypes and genotypes. NS5A from all BVDV strains tested so far interacted with eEF1A. The conserved association of eEF1A with virus molecules involved in genome replication and the postulated role of pestivirus and hepacivirus NS5A in replication indicate that this interaction may play a role in the replication of BVDV.

Epidemic outbreaks of dengue fever (DF) were first recorded in Venezuela in 1978 and were followed by the emergence of dengue haemorrhagic fever (DHF) outbreaks in 1989. To gain a better understanding of the nature of these epidemics, the complete envelope (E) gene sequence of 34 Venezuelan dengue type 2 (DEN-2) viruses, isolated between 1997 and 2000 was determined. Of these isolates, 16 were from patients with DF and 17 were from patients diagnosed with DHF. There were no diagnostic sequence differences between them, suggesting that the E gene alone does not determine disease severity. These sequence data were also used in phylogenetic comparisons with a global sample of DEN-2 viruses, including strains collected previously from Venezuela. This analysis revealed that the ancestors of the Venezuelan viruses were Asian in origin, implying that a DEN-2 virus strain from this region was introduced into Venezuela and the wider Caribbean region during the late 1970s or the early 1980s. The phylogenetic trees further indicate that evolution of DEN-2 virus in Venezuela has occurred in situ, with differentiation into a number of distinct but co-circulating lineages, rather than the repeated introduction of new strains from other localities. By incorporating additional sequence data from the virus capsid, premembrane and membrane genes, evidence is provided that a single Venezuelan strain sequenced previously, designated Mara4, is a recombinant virus, incorporating genome sequence from Venezuelan and Asian parental viruses.

The molecular epidemiology of hepatitis A virus (HAV) was studied by analysing HAV strains recovered from environmental water samples over a 7 year period and strains recovered from patients with acute hepatitis over a 5 year period. A total of 54 samples of raw domestic sewage and 66 samples of river water were collected. HAV particles were concentrated and detected by nested RT–PCR. HAV infection in patients with acute hepatitis was serologically diagnosed in 26 of 74 serum samples, which were also analysed by nested RT–PCR. HAV RNA was detected in 57·4% of sewage samples, 39·2% of Llobregat river water samples, 20% of Ter river water samples and 61·6% of serum samples. The HAV genomes detected were characterized further by directly sequencing a region of the 5′ non-translated region, the VP1/2A junction region and, in some samples, the 2B region. Results showed a 95% prevalence of genotype I, with nearly 50% being either subgenotype IA or subgenotype IB. Various strains were found simultaneously in both environmental and clinical samples. These strains were closely related to those described in distant geographical areas. Genotype IIIA was also found in 5% of sewage samples and in 12·5% of serum samples. Strains belonging to a common endemic genotype were not identified. The abundance of HAV in the environment produces a situation of sanitary risk, especially considering the low prevalence of antibodies in the young population.

To determine an antiapoptotic activity of poliovirus type 1 (PV-1), we examined the effect of PV-1 infection on apoptosis that was induced in HEp-2 cells by the treatment with 1 M sorbitol. The virus did not induce apoptosis in the infected cells and could suppress both the fragmentation of chromosomal DNA and morphological cell and cell nuclei changes in the sorbitol-treated cells, indicating that PV-1 induces an antiapoptotic state. Comparison of the kinetics showed that this ability of the virus appeared in the infected cells at the time of progeny virus formation (maturation step of virus multiplication). Simultaneously with this antiapoptotic activity, PV-1 infection also suppressed non-apoptotic cell death induced by sodium chloride. Electron microscopic observation revealed that the cells killed by the sodium chloride treatment had undergone liquefactive necrosis, indicating that PV-1 can inhibit both apoptosis and necrosis. In addition, PV-1 can grow in the apoptotic cells, although the virus yield was reduced to a quarter of the yield in normal cells.

A serological survey for human T-lymphotropic virus (HTLV)/simian T-lymphotropic virus (STLV) antibodies was performed in 102 wild-caught monkeys and apes from 15 (sub)species originating from Cameroon. Two animals (a Mandrillus sphinx and a Cercocebus agilis) exhibited a complete HTLV-1 seroreactivity pattern while two others lacked either the p24 (a Mandrillus sphinx) or the MTA-1/gp46 bands (a Pan troglodytes). Sequence comparison and phylogenetic analyses, using a 522 bp env gene fragment and the complete LTR, indicated that the two mandrill STLV strains belonged to the HTLV/STLV subtype D clade while the chimpanzee strain clustered in the HTLV/STLV subtype B clade. The Cercocebus agilis STLV strain, the first one found in this species, was closely related to the two HTLV/STLV subtype F strains. Such data indicate that the African biodiversity of STLV-1 in the wild is far from being known and reinforces the hypothesis of interspecies transmission of STLV-1 from monkeys and apes to humans leading to the present day distribution of HTLV-1 in African inhabitants.

Previous findings indicated that HLA-DR is probably one of the most abundant cellular constituents incorporated within the human immunodeficiency virus type 1 (HIV-1) envelope. Given that the life-cycle of HIV-1 has been reported to be modulated by virion-bound host HLA-DR, an improved version of a virus capture technique was developed to assess the degree of HLA-DR incorporation in several clinical isolates of HIV-1 derived from primary human peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDM). Analysis of virus stocks purified from PBMCs and MDM indicated that primary isolates of HIV-1 bearing distinct tropism (i.e. T-, macrophage-, and dual-tropic) all incorporate host cell membrane HLA-DR protein. The amount of incorporated HLA-DR varies among the primary HIV-1 isolates tested. Propagation of some clinical HIV-1 isolates in either autologous PBMCs or MDM resulted in differential incorporation of virion-bound cellular HLA-DR depending on the nature of the virus producer cells. Differences in the degree of HLA-DR incorporation were also noticed when macrophage-tropic isolates of HIV-1 were produced in MDM from different donors. Altogether these data show that the efficiency of HLA-DR incorporation into the envelope of primary isolates of HIV-1 is a multifactorial phenomenon since it is affected by the virus isolate itself, the nature of host cells (i.e. PBMCs or MDM) and the donor source.

Genetic recombination is an important mechanism of retrovirus variation and diversity. Size variation in the surface (SU) glycoprotein, characterized by duplication and insertion, has been observed during in vivo infection with several lentiviruses, including bovine immunodeficiency virus (BIV), equine infectious anaemia virus (EIAV) and human immunodeficiency virus type 1. These duplication/insertion events are thought to occur through a mechanism of template switching/strand transfer during reverse transcription. Studies of RNA recombination in a number of virus systems indicate that cis-acting sequences can modulate the frequency of template switching/strand transfer. The size variable region of EIAV and BIV SU glycoproteins was examined and an AU-rich region and regions of nucleotide sequence identity that may facilitate template switching/strand transfer were identified. An in vitro strand transfer assay using donor and acceptor templates derived from the size variable region in BIV env detected both precise and imprecise strand transfer products, in addition to full-length products. Sequence analysis of clones obtained from imprecise strand transfer products showed that 87·5% had crossover sites within 10 nt of the crossover site observed in vivo. Mutations in the donor template which altered either the AU-rich region or nucleotide sequence identity dramatically decreased the frequency of imprecise strand transfer. Together, these results suggest that cis-acting elements can modulate non-homologous recombination events during reverse transcription and may contribute to the genetic and biological diversity of lentiviruses in vivo.

Full-length DNAs of the Coleman and S7801 strains (pSKY3.0, pSKY5.0) of infectious feline foamy viruses (FFVs) were cloned and sequenced. Parental viruses, designated SKY3.0 and SKY5.0, were secreted following transfection of Crandell feline kidney (CRFK) cells. Production of the rescued parental viruses was enhanced in the presence of trichostatin A. Amino acid sequence similarities between FFV and human foamy virus (HFV) are extremely low for the envelope protein and capsid antigen, as predicted from the two clones. However, a chimeric FFV clone was constructed with the HFV Env substituted for the FFV Env. The chimeric virus (HFFV, SKY4.0) was able to infect and replicate in CRFK cells as well as in peripheral blood mononuclear cells of cats in vivo. Consequently, the chimeric HFFV may be useful for the creation of FV vectors for gene transfer strategies.