- Volume 73, Issue 6, 1992
Volume 73, Issue 6, 1992
- Animal
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Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes
More LessA new transfection system for influenza virus was developed using the clone 76 cell line, in which the viral RNA polymerase and nucleoprotein (NP) genes can be expressed in response to dexamethasone. Ribonucleoprotein (RNP) complexes were reconstituted by expressing proteins from a chimeric NS-chloramphenicol acetyltransferase (CAT) RNA consisting of the full-length negative-strand RNA of the CAT gene positioned between the 5′- and 3′-terminal sequences of influenza virus RNA segment 8, and purifying NP from an NP gene-expressing Escherichia coli strain. When the reconstituted RNP was transfected into clone 76 cells, CAT was produced only when the synthesis of the three RNA polymerase subunits and NP was induced by treatment with dexamethasone.
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Origin and evolutionary characteristics of antigenic reassortant influenza A (H1N2) viruses isolated from man in China
During the 1988/1989 influenza season, five antigenic reassortant influenza A (H1N2) viruses not previously isolated from man were isolated in Hebei province, People’s Republic of China. All isolates contained haemagglutinins (HAs) and neuraminidases (NAs) which were antigenically similar to those of the recent Russian (H1N1) and Hong Kong influenza A (H3N2) viruses, respectively. The results of antigenic and nucleotide sequence analyses revealed that the genes encoding the polymerase, nucleoprotein, NA, matrix and non-structural proteins of the reassortant A/Hebei/24/89 (H1N2) virus were derived from the H3N2 parent virus, whereas its HA gene was from the H1N1 parent virus. The nucleotide sequences of the HA (encoding the HA1 subunit) and NA genes of the reassortant viruses were also determined. Phylogenetic trees constructed from these data by the neighbour-joining method revealed that the HA gene of the reassortant virus was closely related to those of recent human H1N1 viruses, whereas the NA gene was related to a recent human Hong Kong (H3N2) virus lineage.
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Influenza virus infection elicits class II major histocompatibility complex-restricted T cells specific for an epitope identified in the NS1 non-structural protein
More LessA T cell epitope of the influenza virus NS1 molecule was identified and shown to be a determinant used in class II major histocompatibility complex-restricted T cell responses to infectious virus. An I-Ed-restricted BALB/c mouse T hybridoma clone recognizing influenza virus A/Puerto Rico/8/34 (PR8; subtype H1N1) but not A/Udorn/72 (subtype H3N2) secreted lymphokines in response to purified recombinant NS1 or fusion proteins containing amino acids 1 to 81 or 1 to 42 of NS1. As expected for recognition of a non-virion protein, the clone failed to respond to u.v.-inactivated virus. The antigenic determinant was localized by synthetic peptides to amino acids 13 to 32 of NS1, explaining the lack of recognition of A/Udorn/72 virus which has an alanine to valine substitution at position 23 within the determinant. A single intranasal dose of infectious PR8 virus was found to elicit T cells that responded to peptide NS1 13–32, suggesting that this determinant is a significant target of T cells in normal infections. To stimulate helper T cell responses similar to those achieved with infectious virus, influenza virus vaccines may therefore have to include NS1 in addition to virion components.
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Influenza virus pyrogenicity: central role of structural orientation of virion components and involvement of viral lipid and glycoproteins
More LessUltraviolet light-inactivated, non-infectious influenza virus is pyrogenic; virion components are probably responsible for this pyrogenicity. To try to identify the pyrogenic component, influenza virions were disrupted with either bromelain or sodium deoxycholate (DOC). Treatment of infectious virions with bromelain, under conditions that removed the surface glycoproteins (spikes), destroyed their pyrogenicity. The supernatant, containing non-aggregated and modified glycoproteins, was also non-pyrogenic. Disruption of virions with DOC considerably reduced pyrogenicity; however, some was retained by the sub-viral cores. Viral nucleoprotein and matrix protein, purified from the supernatant, were non-pyrogenic. Aggregated stellate clusters of surface glycoproteins separated on sucrose gradients were pyrogenic in half of numerous tests performed with different batches of material. Treatment of virus with ether resulted in complete loss of pyrogenicity. Liposomes made from extracted viral lipid were non-pyrogenic. In contrast, virosomes made from the viral lipid and the aggregated stellate clusters of surface glycoproteins were pyrogenic. Hence, optimum pyrogenicity depends upon the integrity of the virus particle, but haemagglutinin and/or neuraminidase appear essential, and lipid may be involved.
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Sequence and in vitro expression of the M2 gene of turkey rhinotracheitis pneumovirus
More LessNegative-stranded virion RNA and oligonucleotide primers complementary to fusion (F) protein gene sequences were used to generate cDNA clones, revealing that the gene 5′-proximal to the F protein corresponded to the M2 (22K) gene, as in respiratory syncytial (RS) virus. The transcription start signal, GGGACAAGU, was identical to that of the F and matrix (M) proteins of turkey rhinotracheitis virus (TRTV). There were two sequences with the potential to function as transcription termination/poly(A) signals, located at nucleotides 751 to 762 and 777 to 787; 15 clones derived from mRNA indicated that the first of these sequences formed the major signal. Part of the next downstream (5′) gene was sequenced; unlike mammalian pneumoviruses the TRTV M2 gene did not overlap the beginning of the 5′-proximal gene. Northern blotting indicated that infected Vero cells contained less M2 mRNA than F mRNA and that about half of the M2 mRNA was present as a F-M2 dicistronic mRNA. The M2 gene contained two overlapping open reading frames (ORFs 1 and 2), as with RS virus. ORF 1 comprised 558 nucleotides with the coding potential for a 186 amino acid polypeptide, M r 20959, eight or nine residues shorter than for human RS virus strains. The overall amino acid identity was 40%, the N-terminal one-third of the proteins sharing 62% of residues, the remainder 29%. A hydropathy plot of the TRTV M2 protein had close similarity to that of the M2 of RS virus. The protein was predicted to have a basic character with no N-terminal signal sequence or other major highly hydrophobic sequences. In vitro translation of a transcript comprising both ORFs 1 and 2 produced a single product of apparent M r 23000, corresponding to the M2 product of ORF 1. Site-directed mutagenesis confirmed that this product was derived from ORF 1 and that frameshifting was not involved. The second ORF was expressed only from a transcript which lacked the AUG codons of ORF 1 and, although occupying a similar position to that in the RS virus M2 gene, had virtually no amino acid identity in its 73 residue length and was approximately 25% shorter than the corresponding RS virus ORF 2. The hydropathy plot of the potential products of the second ORFs of TRTV and RS virus showed little resemblance. Taken together these results suggest that ORF 2 is unlikely to be expressed in vivo. Our accumulated data show that TRTV has the partial gene order 3′ M-F-M2 5′, whereas the corresponding RS virus genes are arranged 3′ M-SH-G-F-M2 5′.
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Genetic relatedness of hepatitis A virus strains recovered from different geographical regions
A pairwise comparison of the nucleic acid sequence of 168 bases from 152 wild-type or unique cell culture-adapted strains of hepatitis A virus (HAV) revealed that HAV strains can be differentiated genetically into seven unique genotypes (I to VII). In general, the nucleotide sequence of viruses in different genotypes differs at 15 to 25% of positions within this segment of the genome. Viruses from four of the genotypes (I, II, III and VII) were recovered from cases of hepatitis A in humans, whereas viruses from the other three genotypes (IV, V and VI) were isolated only from simian species developing a hepatitis A-like illness during captivity. Among non-epidemiologically related human HAV strains, 81 were characterized as genotype I, and 19 as genotype III. Within each of these major genotypes, there were two distinct groups (sub-genotypes), which differed in sequence at approximately 7.5% of base positions. Each genotype and sub-genotype has a characteristic amino acid sequence in this region of the polyprotein, with the most divergent genotypes differing at 10 of 56 residues. Strains recovered from some geographical regions belonged to a common (endemic) genotype, whereas strains from other regions belonged to several, probably imported, genotypes. Thus, HAV strains recovered in North America were for the most part closely related at the nucleotide sequence level, whereas in other regions, such as Japan and Western Europe, HAV strains were derived from multiple genotypes or sub-genotypes. These data indicate that patterns of endemic transmission can be differentiated from situations in which infections are imported due to travel.
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Coxsackievirus B4 infection of the mouse pancreas: the role of natural killer cells in the control of virus replication and resistance to infection
More LessThe role of natural killer (NK) cells in the early immune response to a pancreatropic isolate of coxsackievirus B4 (CVB4) was investigated in a murine model of pancreatitis. Endogenous (background) NK cell activity in fresh spleen effector cells from eight mouse strains was compared with virus-augmented NK cell activity 4 days post-infection (p.i.). A significant virus-induced increase (P < 0.003) in NK cell activity was seen in seven of eight infected mouse strains, when virus titres in the pancreas were beginning to fall. Lesions in the exocrine pancreas were least extensive in the three strains with the highest endogenous NK cell activity. In C3H/HeJ mice that had been depleted of NK cells prior to infection with a low virus concentration, resistance to infection of the pancreas was completely abolished; myocarditis was also observed in one of these animals. Thus, NK cells may limit virus replication in the pancreas and play a role in resistance in C3H/HeJ mice. Virus-specific neutralizing antibody was not detected in the serum until 5 to 6 days p.i. in most strains and did not appear to influence pancreatic virus titres. It may be significant that CVB4 infection did not induce the expression of major histocompatibility complex (MHC) class I molecules on target acinar cells. With certain tumour cells, an inverse relationship between MHC class I expression and susceptibility to NK cell-mediated lysis is well documented.
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Coxsackievirus B4 infection of the mouse pancreas: acute and persistent infection
More LessThe course of infection of a pancreas-adapted isolate of coxsackievirus B4 was followed over a 10 month period in a murine model. Following intraperitoneal inoculation a typical acute infection was seen in nine of 10 inbred mouse strains. Virus rapidly infected the exocrine pancreas, titres peaking 3 to 4 days post-infection (p.i.). Lesions were almost exclusively confined to pancreatic acinar cells and varied in severity among the inbred strains. Virus shed into the blood-stream was not cell-associated. Evidence of persistent infection was found in nine mouse strains and infective virus was recovered from the pancreas of seven strains for up to 10 months p.i. Approximately 28% of pancreases examined beyond the acute phase showed focal inflammation and 22% showed focal necrosis (cell death). Virus was occasionally recovered from other organs (heart, liver and spleen), but lesions were rarely seen. Virus-specific antigen was localized to small groups of pancreatic acinar cells using an indirect immunogold silver staining technique. These observations suggested that the virus persists in pancreatic tissues because it seems unlikely that virus disseminated from distant sites would cause such localized infection. In three of these strains, the course of infection may have been influenced by superinfection with mouse hepatitis virus.
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Early promoters of genital and cutaneous human papillomaviruses are differentially regulated by the bovine papillomavirus type 1 E2 gene product
The physical state of the human papillomavirus (HPV) genome is usually different in malignant lesions of the skin, in which it is generally found in episomal form, and genital mucosa, in which it is frequently integrated with disruption of the E2 gene. Using chimeric or natural HPV promoters in the presence of the bovine papillomavirus type 1 E2 gene product, we observed transcription activation or repression, depending on the distance of E2-binding motifs from the start site. We found a clear difference in the positions of E2-binding motifs in cutaneous and genital HPVs that may partly explain the selective pressure for genome integration of genital HPV types in malignant lesions.
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Processing of hepatitis B virus surface antigen expressed by recombinant Oka varicella vaccine virus
We have constructed a recombinant Oka varicella vaccine virus expressing hepatitis B virus (HBV) surface antigen (HBsAg). HBsAg was synthesized as 26K and 30K proteins in infected cells and secreted into the culture supernatant as 30K and 35K proteins. Inhibitors and glycosidase treatments, and pulse-chase labelling experiments, revealed the glycosylation process of HBsAg. The latter was synthesized as a non-glycosylated 26K protein and subjected to N-linked glycosylation to form a 30K protein with high mannose glycans. Three species of dimers composed of 26K and 30K subunits were then formed with disulphide bonds. Both subunits of the dimers were further subjected to O-linked glycosylation and conversion from high mannose glycans to complex glycans followed by sialylation. Three species of dimers composed of 30K and 35K subunits were secreted into the culture supernatant as HBsAg particles. HBsAg was synthesized, glycosylated with both N- and O-linked glycans, sialylated, and then secreted into the culture supernatant within 1 h. These modifications of HBsAg by glycans might stabilize its structure and enhance its immunogenicity as a live HBV vaccine.
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Distribution of epitopes within the amino acid sequence of the Epstein—Barr virus major envelope glycoprotein, gp340, recognized by hyperimmune rabbit sera
More LessEpstein—Barr virus (EBV) is a major human pathogen for which the development of an effective vaccine remains an important goal. Rabbits were immunized with one of a set of 10 fusion proteins representing protein fragments from the EBV receptor-binding ligand and candidate subunit vaccine gp340. Sera from recipients of fragments from the amino-terminal half of the polypeptide chain bound gp340 in Western blot assays and ELISA but were not virus-neutralizing. The fine epitope specificity of these sera, and of EBV-neutralizing rabbit sera raised against whole EBV and gp340-containing immune-stimulating complexes, were assessed in a peptide ELISA. All but two of these sera bound peptides located between positions 236 and 327 in the 907 amino acids of the gp340 polypeptide chain. Among these it was possible to identify regions containing candidate virus-neutralizing B cell epitopes. The use of a gp340 fusion protein affinity column to isolate antibodies from EBV-neutralizing rabbit sera specific for this region suggests the presence of both continuous and discontinuous B cell epitopes with potential roles in EBV neutralization.
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Expression of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is required for virus growth and neoplastic transformation
More LessThe amino-terminal domain of the large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) has protein kinase (PK) activity and properties similar to those of growth factor receptor kinases which can be activated to transforming potential. DNA sequences that encode the PK domain cause neoplastic transformation of immortalized cells. The studies described in this report used a spontaneous mutant (ts5-152) temperature-sensitive for the synthesis of ICP10 and the previously described ICP10 expression vectors to study the role of ICP10 expression in HSV-2 growth and neoplastic potential. The titres of the ts5-152 mutant are 1000-fold lower at 39 °C compared to 34 °C after 12 h post-infection. The efficiency of plaquing is 0.003. The growth defect at 39 °C correlates with decreased ICP10 synthesis. Sequence analysis of the PK domain of the ts5-152 ICP10 gene identified a pair of frameshift mutations resulting in a 19 amino acid residue substitution at positions 275 to 293 and a downstream single base pair mutation causing a substitution at position 309. Cloning of the mutant ICP10 gene from ts5-152 into a wild-type HSV-2 isolate resulted in a recombinant (859/152) with growth properties and rates of ICP10 synthesis at 39 °C similar to those of ts5-152. Cells transformed with u.v.-inactivated ts5-152, or the recombinant 859/152, have significantly decreased cloning efficiency in agarose at 39 °C, but only during the first 250 post-transfer population doublings. Anchorage-independent growth was observed in cells transfected with expression vectors pJW17 or pJW32 that express ICP10 or its PK domain, respectively. Cells transfected with the frameshift mutant pJW21 or the ICP10 carboxy-terminal vector pJW31 did not form clones in agarose.
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Antipeptide antisera define neutralizing epitopes on the adenovirus hexon
More LessThe adenovirus (Ad) hexon contains both group- and type-specific antigenic determinants. To identify the latter, peptides were synthesized corresponding to residues 281 to 292 from loop 1 and 441 to 455 from loop 2 of the Ad2 hexon. These sequences display type-specific variation and have been shown by X-ray crystallography to be present on the surface of the virion. Antisera raised against the peptides bound both peptide and the native hexon in ELISA, and blocked virus infectivity, as determined by immunofluorescence or neutralization assays. The loop 1 peptide was shown to inhibit binding of the corresponding antiserum to the native hexon in ELISA and to abolish its neutralizing activity. Neither the loop 1- nor loop 2-specific antiserum neutralized the infectivity of Ad4 or Ad40. Neutralization did not appear to result from aggregation of virus particles and thus their inability to attach to the cell, because virions treated with immune serum were internalized to the same extent as those treated with preimmune serum. Examination of the immune response elicited by Ad2 infection revealed that antibodies directed against the L1 and L2 epitopes were also present in human serum. Thus, the variable regions exposed on the surface of the Ad2 hexon represent type-specific neutralizing antigenic determinants.
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Analysis of polyhedra morphology mutants of Autographa californica nuclear polyhedrosis virus: molecular and ultrastructural features
More LessTwo new mutants of Autographa californica multiple nuclear polyhedrosis virus affected in the morphogenesis of their polyhedra, designated M276 and M934, were investigated. Marker transfer experiments demonstrated that the observed phenotype was due exclusively to alterations in the polyhedrin gene. M276 contained a 229 base insertion near the carboxyl terminus coding region which resulted in synthesis of a truncated protein; M934 had a point mutation substituting phenylalanine for leucine at amino acid 183. Both mutations occurred in highly conserved regions of the protein and prevented the occlusion of virus particles, but did not affect targeting for the intranuclear ring zone. M276 was distinct in that it had prominent cytosolic condensations of polyhedrin, although these were probably due to decreased protein solubility. M934 polyhedrin condensations associated prematurely with calyx material such that it became incorporated into the condensation rather than at the surface. Results confirm that occlusion size and shape are features inherent to the polyhedrin protein, and suggest that polyhedrin conformation may help regulate the occlusion process.
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Dissimilar expression of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus polyhedrin and p10 genes
More LessThe temporal expression of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus polyhedrin and p10 genes in Spodoptera frugiperda cells was studied using virus recombinants in which either gene was replaced by the juvenile hormone esterase (JHE) gene of Heliothis virescens. The JHE served as a highly specific and sensitive reporter for gene expression. Activation of the p10 gene followed a pattern different to that of polyhedrin. The p10 gene was activated a few hours earlier than the polyhedrin gene, but its expression reached a lower maximum level. Northern blot analysis complemented and confirmed the results obtained from the JHE assays. Co-infection of sense recombinants and those containing an anti-sense copy of the JHE gene in place of the polyhedrin or p10 gene resulted in reduced levels of JHE gene expression. These experiments independently supported the hypothesis that the p10 gene promoter is more active at earlier times post-infection than that of the polyhedrin gene. The results also highlight the potential of the antisense strategy as an experimental approach for the study of baculovirus gene regulation and possibly insect metabolism.
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Restriction endonuclease analysis and mapping of the genomes of granulosis viruses isolated from Xestia c-nigrum and five other noctuid species
More LessRestriction endonuclease analysis was performed on the genomic DNA of granulosis viruses isolated from noctuid species of six genera: Xestia c-nigrum, Autographa gamma, Hydraecia amurensis, Celaena leucostigma, Aletia pallens and Pseudaletia separata. All of the isolates gave very similar restriction endonuclease profiles with only minor variations. An isolate obtained from X. c-nigrum was chosen as the reference genotype, and a genomic library was constructed for this isolate using plasmid vectors. The genome was mapped using EcoRI, BamHI and BglII, and Southern hybridization; the size of the genome was estimated to be 179 kbp. Hybridization of labelled clones to fragments of other isolates revealed that genotypic variation among isolates resulted from changes in restriction sites, and from deletion or insertion of DNA. Comparative restriction mapping revealed that all of the isolates were variants of one virus, even though they originated from different host species.
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Nucleotide sequence of the polyhedron envelope protein gene region of the Lymantria dispar nuclear polyhedrosis virus
More LessA 6.4 kb region from the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) genome was sequenced and found to contain open reading frames (ORFs) homologous to the polyhedron envelope (PE) protein coding sequence, and the C-terminal half of ORF 1, which is a gene located upstream of the PE protein gene in other baculoviruses. The proteins predicted from the LdMNPV genes encoding the PE protein, and ORF 1 demonstrated 27 and 34% amino acid sequence identity, respectively, with the corresponding genes in the Autographa californica multicapsid nuclear polyhedrosis virus.
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Nucleotide sequence of the p39-capsid gene region of the Lymantria dispar nuclear polyhedrosis virus
More LessA 1.85 kb region, containing an open reading frame (ORF) homologous to the baculovirus p39-capsid gene, was sequenced from the Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) genome. Analysis of the p39-capsid gene demonstrated that it was 39% and 47% identical in amino acid sequence with the homologous genes in the Autographa californica and Orgyia pseudotsugata MNPVs, respectively. Two late promoter elements located upstream of the p39 gene in the LdMNPV genome are conserved with two other baculoviruses, whereas an ORF located downstream is not conserved.
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Genetic engineering of a Lymantria dispar nuclear polyhedrosis virus for expression of foreign genes
More LessA bacterial lacZ gene was inserted into an isolate of the Lymantria dispar nuclear polyhedrosis virus (LdMNPV). The transfer vector was constructed by site-directed mutagenesis of the translation start site of the LdMNPV polyhedrin gene, within the BglII E fragment of the viral genome. A multiple cloning sequence was inserted at this start site and used for the insertion of the lacZ gene into the transfer plasmid. Liposome transfection was used to cotransfect L. dispar tissue culture cells with viral DNA and the transfer plasmid. Recombinant LdMNPV isolates were purified by isolation of plaques producing β-galactosidase but not polyhedra. Restriction enzyme fragment profiles were used to determine the site of the lacZ gene insertion, and DNA sequencing of the 5′ and 3′ ends of the lacZ gene insert and the adjoining polyhedrin promoter and coding regions was performed to identify its precise location. Expression of the lacZ gene was examined by studying virus-induced protein using [35S]methionine pulse-labelling, SDS-PAGE fractionation and autoradiography. Expression of β-galactosidase was examined in tissue culture cells using colorimetric assays. The maximum rate of β-galactosidase production was approximately 50 international units (IU)/106 tissue culture cells/day between 3 and 4 days post-infection (p.i), and the peak total expression was 158 IU/106 cells 5 days p.i. β-Galactosidase activity was first detected 48 h p.i. in haemolymph samples from fourth instar L. dispar larvae injected with 106 p.f.u. of virus. The peak β-galactosidase activity in larval haemolymph samples was 1931 IU/ml of haemolymph at 11 days p.i., just prior to death.
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Hepatitis B virus polymerase gene: expression of the long open reading frame using the baculovirus expression system
More LessA recombinant baculovirus was constructed containing a copy of the hepatitis B virus (HBV) genome which was inserted to produce an in-frame fusion of the precore (pre-C) coding region with the first 11 amino acids of the polyhedrin gene. The recombinant baculovirus expressed the 25K pre-C protein and two novel proteins, of approximately 93K and 72K. Both the 93K and 72K proteins are recognized by an anti-polymerase monoclonal antibody. Northern blot analysis of the mRNA produced during infection of Spodoptera frugiperda cells by the HBV recombinant baculovirus detected only one HBV mRNA species, suggesting that the three HBV-specific proteins expressed are translated from the same mRNA. No larger fusion proteins cross-reacting with either anti-core or polymerase antibodies were detected. These findings suggest that the two proteins encoded within the HBV polymerase gene are not produced via a core-polymerase fusion intermediate but by internal binding of ribosomes. These results are the first clear demonstration of efficient expression of two bona fide unprocessed polymerase proteins in a 1:1 ratio from an unspliced pre-C mRNA-like transcript. With the successful expression of the polymerase gene in insect cells it is now possible to produce large amounts of these proteins, allowing a more detailed structural and functional analysis of these proteins.
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Cloning and sequencing of the structural region and expression of putative core gene of hepatitis C virus from a British case of chronic sporadic hepatitis
More LessWe report the cloning and sequencing of the putative structural region of the hepatitis C virus (HCV) genome (2229 nucleotides) from an isolate derived from a British case of chronic sporadic non-A, non-B hepatitis. The overall sequence shows a higher similarity with one type of HCV, HCV1 (92%), than with HCV2 (80%), is very highly conserved at the 5′ end (99%) preceding the long open reading frame, is well conserved also in the putative core region (90 to 97%), but shows marked variation in the putative envelope region, particularly in the envelope 2/non-structural 1 region (70%). The putative core gene was cloned in pJ3Θ under the early simian virus 40 promoter and expressed in human hepatoma cells. A predominantly cytoplasmic 22K polypeptide was expressed which was antigenically reactive with serum from chronically infected HCV patients.
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Bovine papillomavirus type 1-transformed primary mouse fibroblasts show no correlation between tumorigenicity and viral gene expression, but c-myc gene expression is elevated in tumorigenic cell lines
More LessBovine papillomavirus type 1 (BPV-1)-transformed primary mouse fibroblasts containing episomal or integrated BPV-1 sequences were analysed for virus-specific transcripts and c-myc gene expression. Total BPV-1-specific expression was high in cell lines containing episomal BPV-1 DNA in comparison to lines containing integrated BPV-1 sequences, mainly due to higher expression of the E6/E7 sequences. No correlation was found between the viral transcription and tumorigenicity, although BPV-1 gene expression occurred in all cell lines. High levels of c-myc expression were found in all cell lines exhibiting a tumorigenic phenotype as compared to the non-tumorigenic lines. These data suggest that expression of BPV-1 genes may be essential for transformation but not tumorigenicity, whereas high levels of expression of cellular oncogenes like c-myc may be associated with tumorigenicity.
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Efficient in vivo encapsidation of a shuttle vector into pseudo-simian virus 40 virions using a shuttle virus as helper
C. Madzak, A. Margot and A. SarasinWe have designed shuttle vectors containing the late region of simian virus 40 (SV40) DNA (coding for the capsid proteins) which could be encapsidated into pseudo-SV40 virions during passage in monkey cells. We describe here the use of these shuttle viruses as helpers for the encapsidation of another shuttle vector into viral particles. Following cotransfection into monkey cells, the efficiency of encapsidation was similar for the shuttle virus and the other plasmid. The amounts of pseudo-SV40 virions recovered from the two vectors reflected the amounts of their DNAs present in monkey cells. Thus, the presence of the SV40 late region did not confer any significant advantage for encapsidation. The encapsidation of any shuttle vector into pseudo-SV40 virions is therefore possible and efficient, shuttle viruses constituting an interesting alternative to the use of SV40 as helper in this process.
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Analysis of human immunodeficiency virus type 1 LTR-LTR junctions in peripheral blood mononuclear cells of infected individuals
More LessCircularized DNA species containing two long terminal repeat circle junctions were analysed in peripheral blood mononuclear cells of human immunodeficiency virus type 1 (HIV-1)-infected individuals. The circle junction fragments found could be classified into four groups: fragments containing a normal circle junction, fragments with deletions at the circle junction, fragments containing the primer binding site inserted at the circle junction, and fragments containing insertions at the circle junction derived from other regions of the HIV-1 genome.
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Production of feline immunodeficiency virus in feline and non-feline non-lymphoid cell lines by transfection of an infectious molecular clone
An infectious molecular clone of the TM1 strain of feline immunodeficiency virus (FIV) was transfected into each of one feline (CRFK), two simian (COS and Vero) and two human (SW480 and HeLa) non-lymphoid cell lines, and virus production was assayed on feline T lymphoblastoid MYA-1 cells by monitoring reverse transcriptase activity. Infectious virus was produced in CRFK, Vero and HeLa cells, but not in COS and SW480 cells. When the basal promoter activity of the FIV long terminal repeat (LTR) was examined in these cell lines by using a chloramphenicol acetyltransferase assay, the activity correlated with the virus production in each cell line. Furthermore, when the activity of the FIV LTR was compared with those of three primate lentivirus LTRs, the highest activity in all the cell lines examined was produced by the LTR of simian immunodeficiency virus from African green monkey (SIVAGM), suggesting that it has a wide expression range. In COS and SW480 cells, the activity of the FIV LTR was much lower than that of the SIVAGM LTR. These results indicate that, whereas the primary block to FIV infection of certain cells may occur at the cell surface, the FIV LTR may also participate in controlling virus replication, as an intracellular mechanism.
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Cell growth effects of Epstein—Barr virus leader protein
More LessB lymphoblastoid cell lines immortalized with P3HR1/633 Epstein—Barr virus (EBV), which has a deletion in the EBV nuclear antigen leader protein (EBNA-LP) gene, were transfected with a vector expressing wild-type EBNA-LP. The EBNA-LP trans-fectants grew out faster under G418 selection than control cells but expression of EBNA-LP made no significant difference to growth rate or saturation density of the resulting established cell lines. When the cells expressing EBNA-LP were allowed to grow to saturation and then diluted in fresh medium they underwent DNA synthesis more rapidly than control cultures.
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Recombination between a herpes simplex virus type 1 vector deleted for immediate early gene 3 and the infected cell genome
More LessWe have used a vector derived from a herpes simplex virus type 1 (HSV-1) mutant deleted for 3.6 kbp of the essential immediate early gene 3 to transduce the Tn5 neomycin phosphotransferase (neo r) gene into rodent and primate cells in culture. The transgene was flanked by genomic sequences from the human hprt gene. We demonstrate in this study that sequences introduced by infection with the replication-defective HSV vector can be stably inserted into the cell genome by recombination. Both the efficiency of stable transduction, measured by the number of neo r-positive colonies, and the number and length of the transgene sequences inserted into the cell genome were found to be a function of cell type. The transduction efficiency appeared to be influenced, at least in part, by the cytopathic potential of the replication-defective HSV-1 vector, which was more pronounced in primate than in rodent cells.
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Non-permissiveness of synovial membrane cells to human parvovirus B19 in vitro
More LessThe ability of cultured human synovial cells derived from synovial membrane and cartilage to support the replication of human parvovirus B19 was assessed. No viral DNA synthesis nor viral antigens were detected suggesting that B19 virus is not capable of replicating in synovial cells. The significance of this finding in relationship to the pathogenesis of parvovirus arthritis is discussed.
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Bovine respiratory syncytial virus fusion protein gene: sequence analysis of cDNA and expression using a baculovirus vector
More LessThe nucleotide sequence of bovine respiratory syncytial virus (RSV), ATCC strain A51908 fusion (F) glycoprotein gene cDNA was determined. The amino acid sequence deduced was then compared to those of two different isolates of bovine RSV, strains RB 94 and 391–2, and the A and B subtypes of human RSV, strains 18537 and A2. The bovine RSV F protein is highly conserved between the three isolates, A51908 has 97% amino acid identity to RB 94, and 99% identity to 391–2. The F proteins of both the A and B types of human RSV are 81% identical to that of A51908. The cDNA clone was expressed using a baculovirus vector and the expressed recombinant F protein produced in SF9 cells was characterized by Western blot analysis. The recombinant F protein was post-translationally cleaved into the active form and reacted with serum from bovine RSV-infected calves.
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Diversity of the antibody responses produced in ponies and mice against the equine influenza A virus H7 haemagglutinin
More LessA large panel of mouse monoclonal antibodies was produced and tested against field isolates of the equine H7N7 influenza A virus subtype. Only a limited degree of H7 haemagglutinin variation was detected. At least four antigenic sites were identified by selecting variant viruses in eggs. The limited variation in the field did not correlate with the frequency of variant viruses detected in eggs; this frequency was similar to those reported for other influenza viruses. We sought to determine whether the limited amount of variation could be correlated with an epitope-restricted antibody response in vaccinated horses. To this end, limiting dilution cultures were established with peripheral blood leukocytes from vaccinated ponies and the antibodies released into culture supernatants were assayed for binding to variant H7 viruses in ELISA. Three neutralizable antigenic sites mapped by mouse anti-bodies were also recognized by antibodies in pony limiting dilution culture supernatants, indicating that the equine antibody response against the influenza virus H7 haemagglutinin is diverse, and should be effective in selecting variant viruses.
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Changes in specific cleavability of the Sendai virus fusion protein: implications for pathogenicity in mice
More LessSendai virus mutants, KDe-21 and KDe-62, which had undergone multiple cycles of replication in Madin Darby canine kidney (MDCK) cells in the absence of exogenous proteases were isolated. The fusion (F) protein of the mutants regained proteolytic cleavability in MDCK cells and chick embryos, but the F protein remained non-cleavable in other cell lines. Unlike the F protein of wild-type (wt) virus, the mutant F was resistant to trypsin but was sensitive to elastase and, to a lesser extent, to chymotrypsin. Sequence analyses of the F gene and the F protein revealed an amino acid substitution at the cleavage site, Arg(116) to Ile, which conferred trypsin resistance and enhanced cleavability at Ile(116) by elastase and host proteases present in MDCK cells and in chicken embryos. In contrast to the pneumopathogenicity in mice of wt Sendai virus, the KDe mutants were non-pathogenic; cleavage activation of the F protein did not occur in the lungs and thereby infection was terminated after an initial cycle of replication.
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A measles virus isolate from a child with Kawasaki disease: sequence comparison with contemporaneous isolates from ‘classical’ cases
We examined the relationship between a measles virus isolate from a child with Kawasaki disease and two contemporaneous wild-type isolates from children with ‘classical’ measles and the Schwarz vaccine strain. Sequence analysis of 3118 bp from the nucleoprotein, matrix, fusion and haemagglutinin genes of each virus revealed that the isolate from the child with Kawasaki disease was not related to measles vaccine strains and did not contain any of the marked abnormalities previously found in subacute sclerosing panencephalitis isolates, but was more akin to wild-type isolates currently circulating in the U.K. A comparison of our sequences with those obtained from earlier wild-type U.K. isolates suggests significant evolution of measles virus in the U.K. over the last decade.
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The genes encoding the phospho- and matrix proteins of phocine distemper virus
More LessThe nucleotide sequences of the phosphoprotein (P)/V/C and matrix (M) protein genes of phocine distemper virus (PDV) have been determined and the deduced amino acid sequences of the proteins derived from these genes compared with those of the other morbilliviruses. The 1655 nucleotides of the P gene encode a phosphoprotein of 507 amino acid residues (from nucleotide numbers 60 to 1583) which is 75% identical to that of canine distemper virus (CDV). The C proteins of the two viruses are 73% identical. The C protein has the same length, 174 amino acid residues, as C of CDV. The nucleotide sequences of the P genes are 78% identical. The editing site in the P gene is present as a stretch of 18 nucleotides, conserved in all other morbilliviruses. A V-like protein can be accessed by insertion of one G residue at this site. The P and M genes are separated from adjacent genes and each other by the CTT trinucleotide sequence which is totally conserved in morbilliviruses. The M gene is 1447 nucleotides long and encodes a typical paramyxovirus M protein of 335 amino acids (identical in length to those of CDV, measles virus and rinderpest virus). The gene is 78% identical to CDV in the coding region (nucleotides 32 to 1039) but only 34% identical to CDV in the 407 nucleotides of the 3′ untranslated sequence of the M mRNA. The M protein of PDV is 91% identical to the M protein of CDV. The data demonstrate interesting variations in the level of conservation of various genome areas and prove the distinct nature of PDV.
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Comparisons of the genomic sequences of erysimum latent virus and other tymoviruses: a search for the molecular basis of their host specificities
More LessThe nucleotide sequence of the genome of erysimum latent tymovirus (ELV) has been determined. It closely resembles those of the other four sequenced tymoviral genomes in its gene organization and composition, but is the smallest (6034 nucleotides) and most distinct of them. Furthermore the 78 non-coding nucleotides at the 3′ terminus of the ELV genome are unable to form a complete tRNA-like structure like that reported for other tymoviruses. Comparisons of the five tymovirus genomes and their encoded proteins indicate that they have probably evolved from the progenitor tymovirus by independent progressive mutational change without genetic recombination. Comparisons of the sequences of the two non-virion proteins of five tymoviruses, and virion proteins of 17 tymoviruses, revealed no specific similarities between those of ELV and turnip yellow mosaic virus that could explain why their host ranges and symptoms are so similar, yet differ, in this respect, from ononis yellow mosaic, kennedya yellow mosaic and eggplant mosaic tymoviruses.
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The mode of cauliflower mosaic virus propagation in the plant allows rapid amplification of viable mutant strains
More LessWe inoculated the leaves of turnip plants (Brassica campestris spp. rapa cv. Just Right) with two cauliflower mosaic viruses (CaMVs) with different small mutations in a dispensable region of the viral genome, and followed the spread of the virus infection through the plant. Surprisingly, analysis of viral DNA in single primary chlorotic lesions revealed the presence of both mutants. In contrast, the secondary chlorotic lesions and systemically infected leaves contained virus molecules of either one or the other type only. Infection of plants with different ratios of the two reporter viruses showed that this ratio is not conserved during systemic virus spread. Infection with CaMV DNA in the form of heteroduplexes containing a single mismatched base pair, in which each strand carried a distinct diagnostic marker, provided us with evidence that the mismatch was subjected to a repair process in the host plant.
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Rice hoja blanca virus genome characterization and expression in vitro
More LessNo information exists on the organization and mechanisms of expression of the genome of rice hoja blanca virus (RHBV), a member of the tenuivirus group, but here we describe the first steps in its characterization. RHBV contains four ssRNA and three dsRNA species, the sizes of which were estimated by native and denaturing gel electrophoresis. Hybridization analyses using 32P-labelled riboprobes of viral and viral complementary polarities showed that unequal amounts of the two polarities of at least the smallest RNA are present in the virion, and indicated that the dsRNA species contain the same information as the ssRNA species of corresponding size. Total RHBV RNA directs the synthesis of two major proteins of 23K and 21K in vitro. RNA3 directs the synthesis of a 23K protein designated NS3, and RNA4 of a 21K protein designated NS4. The NS4 protein corresponds to the non-structural protein that accumulates in RHBV-infected rice tissue. The nucleocapsid protein is not translated from either total RHBV RNA or any individual RHBV RNA in vitro.
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Relationships among the viroids derived from grapevines
More LessThere have been numerous reports of grapevine viroids, describing physical and biological properties suggestive of similar or identical molecular forms. With consideration of these properties and the application of random-primed and specific cDNA probes, four major groups of grapevine viroids have been defined. Designations which can be used to describe distinct viroids within the four groups include (i) CEVd-g, a grapevine isolate of citrus exocortis viroid, (ii) GVd-c, a grapevine viroid recovered from cucumber, and AGVd, Australian grapevine viroid, (iii) GYSVd-1 and GYSVd-2, two viroids inducing yellow speckle disease and (iv) HSVd-g, a grapevine isolate of hop stunt viroid.
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Conserved terminal nucleotide sequences in the genome of rice black streaked dwarf virus
More LessThe terminal regions of the dsRNA genome segments of rice black streaked dwarf virus (RBSDV) were sequenced. The individual dsRNAs, which were 32P-labelled at their 3′ termini by incubation with [32P]pCp and T4 RNA ligase, were separated by 5% PAGE, and the 10 dsRNA segments were sequenced by two-dimensional electrophoresis. The common 3′-terminal sequences ---GUC 3′ and ---AAAAACUU 3′ were found in the plus and minus strands, respectively. The strictly conserved terminal sequences (5′ AAGUUUUU….GUC 3′) of the genome segments of RBSDV differ from those of the phytoreoviruses and rice ragged stunt virus.
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Complete replication of a satellite RNA in vitro by a purified RNA-dependent RNA polymerase
More LessThe 334 nucleotide R satellite RNA was used as a template for purified RNA-dependent RNA polymerase (RdRp) from cucumber mosaic virus-infected tobacco plants. The products of the reaction were dsRNA and positive-strand RNA of the same size as the R satellite RNA. Similar products were obtained when T7 RNA polymerase positive-strand transcripts of a cDNA clone of the satellite RNA, designed to have the same 5′ and 3′ ends as the satellite RNA, were used as templates. The formation of the positive strands demonstrates complete replication of the satellite RNA. A positive-strand transcript with 65 and 255 additional nucleotides at the 5′ and 3′ ends of the satellite RNA respectively was also utilized as a template by the RdRp, but only dsRNA was formed. However, no products could be detected when the RdRp was programmed with transcripts corresponding to the negative-strand satellite RNA, either with no additional terminal nucleotides or with 24 and 310 additional nucleotides at the 5′ and 3′ ends respectively.
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- Corrigendum
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