- Volume 82, Issue 9, 2001
Volume 82, Issue 9, 2001
- Review Article
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- Animal: RNA Viruses
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Genetic diversity and molecular epidemiology of respiratory syncytial virus over four consecutive seasons in South Africa: identification of new subgroup A and B genotypes
More LessThe molecular epidemiology of respiratory syncytial virus (RSV) was studied over four consecutive seasons (1997–2000) in a single tertiary hospital in South Africa: 225 isolates were subgrouped by RT–PCR and the resulting products sequenced. Subgroup A predominated in two seasons, while A and B co-circulated approximately equally in the other seasons. The nucleotide sequences of the C-terminal of the G-protein were compared to sequences representative of previously defined RSV genotypes. South African subgroup A and subgroup B isolates clustered into four and five genotypes respectively. One new subgroup A and three new subgroup B genotypes were identified. Different genotypes co-circulated in every season. Different circulation patterns were identified for group A and B isolates. Subgroup A revealed more variability and displacement of genotypes while subgroup B remained more consistent.
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Differential permissivity to measles virus infection of human and CD46-transgenic murine lymphocytes
Analysis of measles virus (MV) pathogenesis requires the development of an adequate small animal model of MV infection. In this study, permissivity to MV infection was compared in human and transgenic murine T lymphocytes, expressing different levels of the human MV receptor, CD46. Whereas MV binding and entry correlated with CD46 expression, higher levels of MV replication were always observed in human T lymphocytes. This suggests the existence of intracellular factors, acting posterior to virus entry, that could limit MV replication in murine lymphocytes and should be considered when creating new animal models of MV infection.
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A measles virus glycoprotein-derived human CTL epitope is abundantly presented via the proteasomal-dependent MHC class I processing pathway
Peptides derived from measles virus (MV) are presented by MHC class I molecules at widely divergent levels, but it is currently unknown how functional these levels are. Here, for the first time, we studied the natural occurrence and the underlying processing events of a known MV CTL epitope derived from the fusion glycoprotein (MV-F) and restricted via HLA-B*2705. Using MHC–peptide elution of MV-infected cells followed by sensitive mass spectrometry we determined the naturally occurring sequence to be RRYPDAVYL, corresponding to MV-F438–446. Its level of expression was enumerated at approximately 1500 copies per cell, which is considered to be abundant, but lies within the range described for other viral CTL epitopes in human MHC class I molecules. We found that processing of the MV-F438–446 epitope occurs primarily via the classic MHC class I loading pathway, since presentation to CTL depends on both the transporter associated with antigen presentation (TAP) and the proteasome. Even though it is cotranslationally inserted into the ER, a major part of MV-F is located in the cytosol, where it accumulates rapidly in the presence of proteasome inhibitors. We therefore conclude that a substantial cytosolic turnover of MV-F, together with some excellent processing features of MV-F438–446 precursors, such as precise C-terminal excision by proteasomes, efficient TAP transport and strong HLA binding, dictate the abundant functional expression of the MV-F438–446 CTL epitope in HLA-B*2705 at the surface of MV-infected cells.
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Leucine at position 278 of the AIK-C measles virus vaccine strain fusion protein is responsible for reduced syncytium formation
More LessThe live measles virus (MV) vaccine strain AIK-C was attenuated from the wild-type strain Edmonston by plaque purification at 33 °C. Strain AIK-C grew well at 33 °C with a mixture of small-and medium-sized plaques in Vero cells, but did not grow well at 40 °C. To investigate fusion inducibility, expression plasmids for the fusion (F) and haemagglutinin (H) protein regions of MV strains AIK-C (pAIK-F01 and pAIK-H) and Edmonston (pEdm-F and pEdm-H) were constructed. pEdm-F induced extensive cell fusion in B95a and Vero cells under the control of T7 RNA polymerase, whereas a sharp reduction in syncytium formation was observed when pAIK-F01 was used. Six amino acid differences were determined between pAIK-F01 and pEdm-F. Direct sequencing showed that the seed strain AIK-C contained either Leu or Phe at position 278 of the F protein. Experiments using recombinant F protein plasmids demonstrated that those with Leu at position 278 induced poor syncytium formation, while those with Phe at position 278 (Edmonston type) induced extensive cell fusion. Replacement of Phe with Leu at position 278 of pEdm-F reduced fusion-inducing capability. A full-length infectious clone of AIK-C with Leu at position 278 of the F protein was constructed. The rescued virus produced small plaques in Vero cells. However, the same rescued virus with Phe at position 278 produced large plaques. It was concluded that Leu at position 278 of the F protein of the MV vaccine strain AIK-C is responsible for the formation of small plaques.
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Complete nucleotide sequences of Nipah virus isolates from Malaysia
More LessWe have completely sequenced the genomes of two Nipah virus (NiV) isolates, one from the throat secretion and the other from the cerebrospinal fluid (CSF) of the sole surviving encephalitic patient with positive CSF virus isolation in Malaysia. The two genomes have 18246 nucleotides each and differ by only 4 nucleotides. The NiV genome is 12 nucleotides longer than the Hendra virus (HeV) genome and both genomes have identical leader and trailer sequence lengths and hexamer-phasing positions for all their genes. Both NiV and HeV are also very closely related with respect to their genomic end sequences, gene start and stop signals, P gene-editing signals and deduced amino acid sequences of nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, glycoprotein and RNA polymerase. The existing evidence demonstrates a clear need for the creation of a new genus within the subfamily Paramyxovirinae to accommodate the close similarities between NiV and HeV and their significant differences from other members of the subfamily.
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Complete nucleotide sequence of avian paramyxovirus type 6 isolated from ducks
More LessThere are nine serotypes of avian paramyxovirus (APMV). Only the genome of APMV type 1 (APMV-1), also called Newcastle disease virus (NDV), has been completely sequenced. In this study, the complete nucleotide sequence of an APMV-6 serotype isolated from ducks is reported. The 16236 nt genome encodes eight proteins, nucleocapsid protein (NP), phosphoprotein (P), V protein, matrix protein (M), fusion protein (F), small hydrophobic (SH) protein, haemagglutinin–neuraminidase (HN) protein and large (L) protein, which are flanked by a 55 nt leader sequence and a 54 nt trailer sequence. Sequence comparison reveals that the protein sequences of APMV-6 are most closely related to those of APMV-1 (NDV) and -2, with sequence identities ranging from 22 to 44%. However, APMV-6 contains a gene that might encode the SH protein, which is absent in APMV-1, but present in the rubulaviruses simian virus type 5 and mumps virus. The presence of an SH gene in APMV-6 might provide a link between the evolution of APMV and rubulaviruses. Phylogenetic analysis demonstrates that APMV-6, -1, -2 (only the F and HN sequences were available for analysis) and -4 (only the HN sequences were available for analysis) all cluster into a single lineage that is distinct from other paramyxoviruses. This result suggests that APMV should constitute a new genus within the subfamily Paramyxovirinae.
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Antigenic variants with amino acid deletions clarify a neutralizing epitope specific for influenza B virus Victoria group strains
More LessTo study the neutralizing epitopes of influenza B virus Victoria group strains, two monoclonal antibodies (MAbs) were used to select antigenic variants of the virus. MAbs 10B8 and 8E6 were found to react with B/Victoria group strains in three tests, peroxidase–antiperoxidase staining, haemagglutination inhibition and neutralization tests; no reactivity with B/Yamagata group strains was observed. Analysis of the deduced amino acid sequences of 10B8-induced variants identified a single amino acid deletion at residue 165 or 170, as well as a single amino acid substitution at residues 164 (Asp→Tyr), 165 (Asn→Ser or Thr) or 203 (Lys→Thr or Asn). A single amino acid substitution at residue 241 (Pro→Ser) was observed in 8E6-induced variants. Three-dimensional analysis showed that the epitopes for both MAbs were situated in close proximity to each other. Since B/Yamagata group strains are characterized by amino acid deletions at residues 164–166, the epitope for MAb 10B8 is strictly specific for B/Victoria group strains.
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Phylogeny of the Simbu serogroup of the genus Bunyavirus
More LessThe Simbu serogroup of the genus Bunyavirus, family Bunyaviridae contains 25 viruses. Previous serological studies provided important information regarding some but not all of the relationships among Simbu serogroup viruses. This report describes the nucleotide sequence determination of the nucleocapsid (N) gene of the small genomic segment of 14 Simbu serogroup viruses and partial nucleotide sequence determination of the G2 glycoprotein-coding region (encoded by the medium RNA segment) of 19 viruses. The overall phylogeny of the Simbu serogroup inferred from analyses of the N gene was similar to that inferred from analyses of the G2 protein-coding region. Both analyses revealed that the Simbu serogroup viruses have evolved into at least five major phylogenetic lineages. In general, these phylogenetic lineages were consistent with the previous serological data, but provided a more detailed understanding of the relatedness amongst many viruses. In comparison to previous phylogenetic studies on the California and Bunyamwera serogroups of the Bunyavirus genus, the Simbu serogroup displays much larger genetic variation in the N gene (up to 40% amino acid sequence divergence).
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Characterization of hepatitis D virus genotype III among Yucpa Indians in Venezuela
The complete genome sequences of hepatitis D virus (HDV) strains isolated from three Yucpa Amerindians in Venezuela were determined and found to be genotype III. Comparison of these three genotype III sequences demonstrated the presence of a hypervariable region containing numerous substitutions, insertions/deletions and a highly conserved region containing the self-cleavage domains, which have been reported previously for genotypes I and II. Amino acid changes within the first 90 amino acids of the hepatitis D antigen (HDAg) were found in the genotype III sequences, while the remainder of the HDAg-coding sequence was conserved. The secondary structure for the RNA-editing site differed between genotypes I and III. It was concluded that the serious delta hepatitis outbreaks characterized epidemiologically in the Yucpa Amerindians were caused by HDV genotype III isolates that were related to HDV genotype III isolates from other regions of South America.
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Immunogenicity of an E1-deleted recombinant human adenovirus against rabies by different routes of administration
The immunogenic properties of an E1-deleted, human adenovirus type 5 (Ad5) vaccine virus with activity against rabies were examined in mice, foxes and dogs using different routes of administration. NMRI mice received 105·8, 105·3, 104·3, 103·3 and 102·3 TCID50 by peroral or intramuscular (i.m.) administration. Furthermore, six mice received 105·8 TCID50 intracerebrally (i.c.). The construct elicited marked seroconversion in mice after oral administration. Immunoreactivity in mice was even more pronounced i.m. and i.c. After direct oral administration (108·0 TCID50) in foxes, six of eight animals developed rabies virus-neutralizing antibodies (VNA). All foxes immunized by direct injection (107·7 TCID50) in the membrane of the jejunum were shown to seroconvert. Pre-existing immunity against canine adenovirus did not hinder the development of rabies VNA after oral application of the construct (108·0 TCID50). Fox cubs (24–29 days old) born from rabies-immune vixens were shown to develop very high levels of rabies VNA after i.m. administration (108·0 TCID50), indicating that the immunogenicity of the construct could surpass maternally transferred immunity. In dogs, the construct (108·0 TCID50) induced a very strong immune response after i.m. administration. However, no immune response was detectable in dogs after direct oral administration (108·3 TCID50) or after endoscopic deposition in the smaller intestine (108·0 TCID50). Hence, it must be concluded that the construct is not suitable for oral vaccination of dogs against rabies.
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Evidence of Borna disease virus genome detection in French domestic animals and in foxes (Vulpes vulpes)
More LessBorna disease virus (BDV) is an enveloped, non-segmented negative-stranded RNA virus which belongs to the Bornaviridae family. BDV is an aetiological agent of encephalitis in horses, sheep and several other vertebrate species. In order to extend our knowledge about the presence of BDV in France, a study based on BDV RNA detection by RT–nested-PCR was done with 196 animal tissues: 171 brain samples collected from different animal species (75 horses, 59 foxes, 31 cattle, 4 dogs, 1 sheep, 1 roe deer) and 25 horse blood samples. An RNA internal standard molecule was constructed and was co-amplified with the test template. This study reports the first detection of BDV RNA in France in 10 brain samples collected from horses, foxes and cattle, and from 14 horse blood samples. Detection of the BDV genome in the brains of six red foxes is the first evidence of BDV infection in this species.
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A PCR primer system for detecting oncoretroviruses based on conserved DNA sequence motifs of animal retroviruses and its application to human leukaemias and lymphomas
More LessMany C- and D-type retroviruses are known to cause a broad spectrum of malignant diseases in animals. Certain genome regions of these animal retroviruses are highly conserved between different animal species. It should be possible to detect new members of the retrovirus family with consensus PCR primers derived from these conserved sequence motifs. The consensus PCR primers developed in this study are generic enough to detect nearly all known oncogenic mammalian and avian exogenous C- and D-type retroviruses but do not amplify human endogenous retroviral sequences. In contrast to previous investigations, the present study involved highly stringent PCR conditions and truly generic PCR primers. Forty-four samples from patients with various immunophenotyped malignant diseases (acute and chronic T-/B-cell lymphocytic leukaemias, acute myeloid leukaemias, T-/B-cell lymphomas, chronic myeloproliferative disorders) and three cell lines (Hodgkin’s lymphoma, Burkitt’s lymphoma) have thus far been investigated using these PCR primers. The fact that no retroviruses have been found argues against an involvement of known animal oncoretroviruses or related hitherto undetected human retroviruses in the aetiopathogenesis of these diseases. The retrovirus detection system developed here may be used to confirm suspected retroviral involvement in other (malignant or nonmalignant) human diseases as well as to identify new animal retroviruses.
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Induction of simian immunodeficiency virus (SIV)-specific CTL in rhesus macaques by vaccination with modified vaccinia virus Ankara expressing SIV transgenes: influence of pre-existing anti-vector immunity
A major aim in AIDS vaccine development is the definition of strategies to stimulate strong and durable cytotoxic T lymphocyte (CTL) responses. Here we report that simian immunodeficiency virus (SIV)-specific CTL developed in 4/4 macaques following a single intramuscular injection of modified vaccinia virus Ankara (MVA) constructs expressing both structural and regulatory/accessory genes of SIV. In two animals Nef-specific responses persisted, but other responses diminished and new responses were not revealed, following further vaccination. Vaccination of another two macaques, expressing Mamu A*01 MHC class I, with MVA constructs containing nef and gag–pol under the control of the moderate strength natural vaccinia virus early/late promoter P7.5, again induced an early Nef-specific response, whereas responses to Gag remained undetectable. Anti-vector immunity induced by this immunization was shown to prevent the efficient stimulation of CTL directed to the cognate Gag epitope, p11C C-M, following vaccination with another MVA construct expressing SIV Gag–Pol under a strong synthetic vaccinia virus-specific promoter. In contrast, vaccination of a previously unexposed animal resulted in a SIV-specific CTL response widely disseminated in lymphoid tissues including lymph nodes associated with the rectal and genital routes of SIV entry. Thus, despite the highly attenuated nature of MVA, repeated immunization may elicit sufficient anti-vector immunity to limit the effectiveness of later vaccination.
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In situ hybridization and immunolabelling study of the early replication of simian immunodeficiency virus (SIVmacJ5) in vivo
The distribution of virus-infected cells in cynomolgus macaques was determined at 4, 7, 14 and 28 days following intravenous challenge with 1000 TCID50 of the wild-type simian immunodeficiency virus SIVmacJ5 (stock J5C). At each time-point, pairs of macaques were killed humanely and the presence of SIV was determined and quantified in blood, spleen, peripheral and mesenteric lymph nodes, thymus, lung and ileum by virus co-cultivation with C8166 cells, by quantitative DNA PCR or by in situ hybridization (ISH). At day 4 post-infection (p.i.), detection of the virus was sporadic. By day 7 p.i., however, significant SIV loads were detected in the blood and lymphoid tissues by DNA PCR and virus co-cultivation. Large numbers of cells expressing SIV RNA were detected in mesenteric lymph nodes by ISH and significantly fewer (P<0·05) in the spleen. Significant numbers of ISH-positive cells were also observed in sections of ileum. By day 14 p.i., the distribution of SIV was more even in all lymphoid tissues analysed. By day 28, most of the tissues were negative by ISH, but all remained positive by virus isolation and DNA PCR. Immunolabelling of sections of mesenteric lymph node with monoclonal antibodies specific for SIV envelope and Nef largely confirmed the observations from ISH. These results indicate that, even following intravenous challenge, a major site of the initial replication of SIV is gut-associated lymphoid tissue. Vaccines that induce protection at this site may therefore be superior, even against parenteral challenge.
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p53-dependent transcriptional repression of p21waf1 by hepatitis C virus NS3
More LessHepatitis C virus (HCV) NS3 protein is known to affect normal cellular functions, such as cell proliferation and cell death, and to be involved, either directly or indirectly, in HCV hepatocarcinogenesis. In this study, we demonstrated that NS3 protein could specifically repress the promoter activity of p21 in a dose-dependent manner. The effect was not cell type-specific and was synergistic when combined with HCV core protein. Repression of the p21 promoter by NS3 was almost completely lost when p53 binding sites present on the p21 promoter were removed. Furthermore, p53 binding sites were sufficient to confer a strong NS3 responsiveness to an heterologous promoter, suggesting that NS3 represses the transcription of p21 by modulating the activity of p53. Although the NS3 protein domain required for the majority of p21 repression was located on the protease domain, the proteinase activity itself does not seem to be necessary for repression. Both transcription and protein stability of p53 were unaffected by NS3, suggesting that NS3 might repress transcription of p21 by inhibiting the regulatory activity of p53 via protein–protein interaction(s). Finally, the growth rate of NS3-expressing cell lines was at least twice as fast as that of the parent NIH 3T3 cells, indicating that the repression of p21 is actually reflected by the stimulation of cell growth.
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Mutagenesis of hepatitis C virus E1 protein affects its membrane-permeabilizing activity
More LessThe E1 glycoprotein of hepatitis C virus is a transmembrane glycoprotein with a C-terminal anchor domain. When expressed in Escherichia coli, E1 induces a change in membrane permeability that is toxic to the bacterial cell. The C-terminal hydrophobic region (aa 331–383) of E1 is mainly responsible for membrane association and for inducing changes in membrane permeability. These observed changes are similar to those produced in E. coli by influenza virus M2, human immunodeficiency virus gp41 and poliovirus 3AB proteins, whose hydrophobic domains are thought to cause pore formation in biological membranes. To further characterize the activity of E1 at a molecular level, the membrane-permeabilizing ability of a second internal hydrophobic region (aa 262–291) was examined by expressing different deletion mutants of E1 in an E. coli system that is widely used for analysing membrane-active proteins from other animal viruses. Moreover, highly conserved amino acids in the C-terminal hydrophobic region were mutated to identify residues that are critical for inducing changes in membrane permeability. Analysis of cell growth curves of recombinant cultures and membrane-permeability assays revealed that synthesis of this fragment increased the flux of small compounds through the membrane and caused progressive cell lysis, suggesting that this domain has membrane-active properties. Furthermore, analysis of C-terminal mutants indicated that the conserved amino acids Arg339, Trp368 and Lys370 play a critical role in protein function, as both cell lysis and changes in membrane permeability induced by the wild-type clone could be blocked by substitutions in these positions.
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Determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein NS1 of Murray Valley encephalitis virus
More LessThe 12 cysteine residues in the flavivirus NS1 protein are strictly conserved, suggesting that they form disulfide bonds that are critical for folding the protein into a functional structure. In this study, we examined the intramolecular disulfide bond arrangement of NS1 of Murray Valley encephalitis virus and elucidated three of the six cysteine-pairing arrangements. Disulfide linkages were identified by separating tryptic-digested NS1 by reverse-phase high pressure liquid chromatography and analysing the resulting peptide peaks by protein sequencing, amino acid analysis and/or electrospray mass spectrometry. The pairing arrangements between the six amino-terminal cysteines were identified as follows: Cys4–Cys15, Cys55–Cys143 and Cys179–Cys223. Although the pairing arrangements between the six carboxy-terminal cysteines were not determined, we were able to eliminate several cysteine-pairing combinations. Furthermore, we demonstrated that all three putative N-linked glycosylation sites of NS1 are utilized and that the Asn207 glycosylation site contains a mannose-rich glycan.
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The novel picornavirus Equine rhinitis B virus contains a strong type II internal ribosomal entry site which functions similarly to that of Encephalomyocarditis virus
More LessEquine rhinitis B virus (ERBV) has recently been classified as an Erbovirus, a new genus in the Picornaviridae family. ERBV is distantly related to members of the Cardiovirus and Aphthovirus genera which utilize a type II internal ribosome entry sequence (IRES) to initiate translation. We show that ERBV also possesses the core stem–loop structures (H–L) of a type II IRES. The function of the ERBV IRES was characterized using bicistronic plasmids that were analysed both by transfection into BHK-21 cells and by in vitro transcription and translation in rabbit reticulocyte lysates. In both systems, a region encompassed by nucleotides (nt) 189–920 downstream of the poly(C) tract was required for maximal translation. This sequence includes stem–loops H–L as well as four additional upstream stem–loops. Nt 904 corresponds to the second of three in-frame AUG codons located immediately downstream of the polypyrimidine tract (nucleotides 869–880). Site-directed mutagenesis demonstrated that AUG2 is the major initiation codon despite the appropriate positioning of AUG1 16 nt downstream of the polypyrimidine tract. In direct IRES competition experiments, the ERBV IRES was able to compete strongly for translation factors with the IRES of Encephalomyocarditis virus (EMCV). This was true when the assays were performed in vitro (with the IRESs competing either in cis or trans) and in vivo (with the IRESs competing in cis). A comparative analysis of the strength of several IRESs revealed that the ERBV IRES, like that of the EMCV, is a powerful inducer of translation and may have similar potential for use in mammalian expression systems.
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Distribution of rotavirus-specific memory B cells in gut-associated lymphoid tissue after primary immunization
More LessWe found previously that mice inoculated orally with simian rotavirus strain RRV developed virus-specific memory B cell responses 16 weeks after immunization that were greater than those found 6 weeks after immunization. Memory B cell responses were defined as the quantity of virus-specific IgA detected in small intestinal lamina propria (LP) fragment cultures of immunized mice at various intervals after challenge. Enhanced memory B cell responses correlated with enhanced protection against shedding. In order to understand better the delayed onset of rotavirus-specific memory B cell responses, a method was developed to determine the frequencies of rotavirus-specific memory B cells in gut-associated lymphoid tissues (GALT). We found that protection against rotavirus challenge was determined by the frequency of rotavirus-specific memory B cells in GALT LP.
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