- Volume 74, Issue 12, 1993
Volume 74, Issue 12, 1993
- Animal
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Co-replication of several isotypes of foot-and-mouth disease virus
More LessGenome segments of the foot-and-mouth disease virus isolates O1Lombardy and O3Venezuela that encode, among other products, capsid protein VP1 were amplified using PCR, and the products were cloned and sequenced. The alignment of up to 11 O3-specific sequences revealed six silent nucleotide changes as well as six changes that cause amino acid substitutions in capsid protein VP1 at positions 45, 83, 141, 145, 170 and 178. The heterogeneity of three O1-specific sequences consisted of seven silent exchanges and amino acid changes at positions 85 and 134 on VP1. Amplification, subcloning and sequencing of cloned O3-specific cDNA was performed to examine the nature of the sequence heterogeneity. As no difference was found among five subcloned sequences, we conclude that the Taq polymerase copied the DNA correctly. The sequence heterogeneity observed with both virus isolates is, therefore, consistent with the quasispecies structure of foot-and-mouth disease virus. Furthermore, amino acid changes at a number of sites have been found to be involved in the formation or modulation of neutralizing epitopes. The novel aspect of this study is the ability to estimate, by cloning of PCR products, the number of virus isotypes, possibly varying in antigenicity, that are able to co-propagate. Seven isotypes of O3Venezuela were identified. Some are of particular interest because they exhibit a change at VP1 codon 145 that causes the replacement of arginine, possibly essential for virus attachment to cells, by isoleucine.
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HPLC is an effective and fast method for analysis of viral proteins: a study of encephalomyocarditis virus mutants differing in pathogenicity
We investigated the use of HPLC in analysis of picornavirus variants by comparing structural polypeptides of three stable mutants of encephalomyocarditis virus (EMCV). The variants are known to differ in their pathogenicity for mice: plaque variant 2 (PV2) is diabetogenic, PV7 is non-diabetogenic and PV21 induces a generalized lethal infection. We first used HPLC to separate the structural proteins at high purity levels. Detailed analysis of these structural proteins by HPLC-peptide mapping revealed differences in all four viral proteins of PV21 as compared with mutants PV2 and PV7. A single amino acid exchange was found in viral protein 1 between PV2 and PV7. Altered peaks were identified by calculating retention times of tryptic peptides using sequence data and a computer program. Since peak alterations could be attributed to the observed amino acid exchanges, the results correlate well with cDNA sequencing data. Thus HPLC proved to be a useful and fast tool for primary or additional characterization of picornavirus variants at the level of whole virus proteins.
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Molecular epidemiology of dengue 3 viruses and genetic relatedness among dengue 3 strains isolated from patients with mild or severe form of dengue fever in French Polynesia
More LessThe nucleotide sequences of a short fragment of the envelope protein gene encoding amino acids 25 to 89 of 27 dengue 3 viruses were determined by direct sequencing of PCR-amplified products, and the viruses were compared regarding their time of isolation and geographic distribution. Four distinct genotypic groups were discerned at 6% divergence between nucleotide sequences. The first group contained is olates from the South Pacific (1988 to 1992), Singapore (1973) and Indonesia (1973 to 1991). The second group comprised viruses from Asia (1956 to 1989) including the reference strain H-87. The third was composed of one isolate from Thailand (1971), and the fourth included the early strains from French Polynesia (1964 to 1969) and from Puerto Rico (1963). Furthermore, the difference between early and recent strains from the South Pacific was as high as 12.3%. This observation suggests that the recent epidemics in the South Pacific were probably the consequence of the spread of a new variant that emerged from New Caledonia. However, relatedness between nucleotide sequence and disease severity, or between strains from epidemics with mild disease (New Caledonia) and strains from epidemics with severe disease (French Polynesia) could not be demonstrated.
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A new serotype of the outer capsid protein VP4 shared by an unusual human rotavirus strain Ro1845 and canine rotaviruses
The VP4 protein of human rotavirus (HRV) strain Ro1845 and canine rotavirus strains K9 and CU-1 exhibited greater than 98% amino acid identity within their group, but showed less identity with VP4 proteins of other HRV and animal rotavirus strains, the simian rotavirus strain RRV VP4 being most similar to them (90% amino acid identity). To exclude the possibility that these three strains were members of the RRV VP4 serotype P3, neutralization studies were performed using antisera to reassortant viruses containing the VP4 gene from each of Ro1845, CU-1 and RRV. The result established close antigenic similarity among the VP4 proteins of Ro1845, K9 and CU-1 and revealed only a marginal degree of similarity between the VP4 proteins of these three strains and that of strain RRV. These sequence and serological data suggest that the VP4 proteins of Ro1845, K9 and CU-1 represent a new P serotype which we propose to assign P13.
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Evidence for different lineages of rinderpest virus reflecting their geographic isolation
Sequence analysis of part of the fusion protein gene from recent isolates of rinderpest virus revealed that distinct lineages of the virus exist which reflect the geographical location of their isolation in Africa and Asia. Current strains circulating in Kenya and Sudan were most similar, both in terms of nucleotide sequence and pathogenic nature, to viruses isolated in Egypt and in Nigeria in 1983/1984 and they were quite distinct from an East African isolate (RBT-1) from the 1960s. Two older isolates of the virus, the Japanese avianized/lapinized vaccine strain dating from the 1930s and the Old Kabete strain dating from 1911, each differed considerably from the other viruses. The sequence data were derived from the region where the precursor protein is cleaved to yield the biologically active F1/F2 heterodimer; all strains analysed had a highly basic connecting peptide which is required for efficient cleavage by endogenous host cell proteases. No correlation was found between amino acid changes at this site and the rinderpest virus pathogenicity unlike the association reported for Newcastle disease virus.
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Comparative analysis of the immunoprotective abilities of glycosylated and deglycosylated parainfluenza virus type 3 surface glycoproteins
More LessThe role of carbohydrate moieties on the immunoprotective ability of parainfluenza virus type 3 (PIV-3) haemagglutinin—neuraminidase (HN) and fusion (F) glycoproteins was tested in hamsters. HN and F proteins were purified from detergent-solubilized virus by lentillectin affinity chromatography and deglycosylated by treatment with endoglycosidase F (endo F). Immunization of hamsters with either 1 or 5 µg of mocktreated (glycosylated) affinity-purified proteins elicited strong haemagglutination inhibition and neutralizing antibody responses 4 weeks after the primary injection. In contrast, titres were significantly lower with endo F-treated (deglycosylated) proteins. However, following the booster doses with at least 5 µg of antigen, glycosylated and deglycosylated proteins induced comparable antibody titres. There was no significant difference in the ability of the glycosylated or deglycosylated proteins to protect either the upper or lower respiratory tracts of immunized hamsters against PIV-3 challenge. These results suggest that the carbohydrate moieties of the HN and F proteins are not necessary for eliciting a protective response in hamsters.
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Analysis of the ovine respiratory syncytial virus (RSV) G glycoprotein gene defines a subgroup of ungulate RSV
More LessRespiratory syncytial virus (RSV) has been isolated from sheep suffering from respiratory tract disease. Since the greatest differences between bovine RSV and human RSV are found on the attachment G protein, we have determined the nucleotide and deduced amino acid sequences of the G gene of ovine RSV. The latter contained 838 nucleotides and had a major open reading frame encoding a protein of 263 residues, and shared 73% nucleotide sequence identity with that of bovine RSV. The deduced amino acid sequence of the ovine RSV G protein showed only 60% amino acid identity with the G protein of bovine RSV. Despite the low level of identity, there were similarities in the predicted hydropathy profiles of the G proteins of ovine and bovine RSV. The intergenic sequences for the SH–G and G–F gene junctions of ovine RSV showed 64 and 57% identity respectively with the corresponding regions of the bovine RSV. Our results indicate that ovine and bovine RSV might be classified as two subgroups of an ungulate RSV.
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Isolation and immunogenic properties of a monomeric form of the HA1 subunit of the influenza virus haemagglutinin from infected cells
More LessA monomeric, truncated form of the HA1 subunit of the haemagglutinin of fowl plague virus can be isolated from chorioallantoic membranes of infected eggs. This type of soluble HA1 seems to be generated by the elimination of the amino-terminal 19 amino acids from the native HA1, including the disulphide linkage to the HA2 subunit. The same type of truncated HA1 could be isolated from a filtrate of the allantoic fluid of infected embryonated eggs. Antibodies prepared against this monomeric soluble form of HA1 did not inhibit haemagglutination or neutralize viral infectivity, but interfered with virus release and would be expected to impair the spread of virus after infection.
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Sequence analysis of human T cell lymphotropic virus type I strains from southern India: gene amplification and direct sequencing from whole blood blotted onto filter paper
Human T cell lymphotropic virus type I (HTLV-I) infection in India has been found to be associated with adult T cell leukaemia/lymphoma (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) among life-long residents of southern India. To examine the heterogeneity of HTLV-I strains from southern India and to determine their relationship with the sequence variants of HTLV-I from Melanesia, 1149 nucleotides spanning selected regions of the HTLV-I gag, pol, env and pX genes were amplified and directly sequenced from DNA extracted from whole blood blotted onto filter paper and from peripheral blood mononuclear cells, obtained from one patient with HAM/TSP, two with ATLL and eight asymptomatic carriers from Andhra Pradesh, Kerala and Tamil Nadu. Sequence alignments and comparisons indicated that the 11 HTLV-I strains from southern India were 99.2% to 100% identical among themselves and 98.7% to 100% identical to the Japanese prototype HTLV-I ATK. The majority of base substitutions were transitions and silent. No frameshifts, insertions, deletions or possibly disease-specific base changes were found in the regions sequenced. The observed clustering of the Indian HTLV-I strains with those from Japan, as determined by the maximum parsimony method, suggested a common source of HTLV-I infection with subsequent parallel evolution. Amplification of DNA from blood specimens collected on filter paper may be useful for the study of other blood-borne pathogens.
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Characterization of vaccinia virus gene B12R
More LessWe report the characterization of vaccinia virus gene B12R which is predicted to encode a 33K protein with 36% amino acid identity to the serine/threonine protein kinase encoded by vaccinia virus gene B1R. S1 nuclease protection experiments showed that gene B12R is transcribed early during infection from an initiation site 11 bp upstream of the open reading frame (ORF). The gene encodes a 33K polypeptide that is not required for virus replication in tissue culture nor for virus virulence in a murine intranasal model. Expression of the B12R gene in Escherichia coli produced an abundant 33K polypeptide which lacked protein kinase activity under conditions in which the protein kinases encoded by vaccinia virus gene B1R and African swine fever virus gene j9L are active.
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Analysis of protective immune responses to the glycoprotein H-glycoprotein L complex of herpes simplex virus type 1
More LessA recombinant vaccinia virus expressing both glycoprotein H (gH) and glycoprotein L (gL) of herpes simplex virus type 1 (HSV-1) was used to examine the protective response to gH-gL in immunized mice and to compare these responses with those induced by the highly protective immunogen, glycoprotein D (gD). Weak levels of HSV-1-specific neutralizing antibody were obtained in response to the gH-gL complex, virus clearance from the site of challenge was marginally enhanced compared to that observed following immunization with gH alone, and gH-gL was found to protect mice against acute infection in the ganglia, although not as efficiently as gD.
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Human adenovirus type 5 recombinants expressing simian immunodeficiency virus macaque strain gag antigens
More LessThe p55 gag gene of simian immunodeficiency virus macaque strain (SIVmac) and the core p27 gag component linked to a synthetic AUG codon have been cloned into adenovirus type 5 vectors to generate either viable E3-replacement or defective E1-replacement viruses. The viruses express the expected SIV proteins in both human and, for the non-defective viruses, monkey cells. A considerable proportion of the p55 produced is exported from the infected cell. These viruses should prove useful both in studies of the immune response to SIV and as components of candidate vaccines aimed specifically at provoking cytotoxic T cell responses.
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- Plant
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Susceptibility of transgenic tobacco plants expressing tobacco rattle virus coat protein to nematode-transmitted and mechanically inoculated tobacco rattle virus
More LessTransgenic Samsun NN tobacco plants expressing the coat protein of tobacco rattle virus were exposed to mechanical leaf inoculation with tobacco rattle virus and to viruliferous trichodorid vector nematodes. Whereas plants were resistant to mechanical inoculation the vector nematodes successfully transmitted tobacco rattle virus to the roots as well as to the leaves of these plants. It is suggested that transgenic resistance is overcome either because vector nematodes inject relatively large numbers of virus particles into a cell or because they inject destabilized particles. The results indicate that coat protein-mediated resistance is unlikely to be of value for controlling tobacco rattle virus in field crops.
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Effects of sequence elements in the potato virus X RNA 5′ non-translated αβ-leader on its translation enhancing activity
The 5′ non-translated αβ-leader sequence of potato virus X RNA consists of two regions: the α sequence (41 nucleotides with no G) and the β sequence (42 nucleotides upstream from AUG). The αβ-leader has been shown to enhance strongly the expression of adjacent genes in chimeric mRNAs. This phenomenon has been postulated to be due to the unpaired conformation of the 5′-terminal 30 nucleotides and/or to the presence within the α region of the CCACC pentanucleotide complementary to the 3′-terminal conserved structure of 18S rRNA. Different derivatives of αβ-leader have been constructed for use in determining the contribution of separate elements of the αβ sequence to translational enhancement. It was found that deletion of the α sequence large fragment which was supposed to be unfolded did not reduce the Δαβ-leader enhancement activity. Moreover, translational enhancement was greater for this derivative. Deletion of the β sequence resulted in a considerable increase in activity of the α-leader showing that the β region was dispensable for translation. Disruption or ‘masking’ of CCACC led to inactivation of the αβ-leader as a translational enhancer. Thus, we identified the CCACC pentanucleotide as the primary motif responsible for the translation enhancing ability of αβ-leader.
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Replication of the DNA A component of African cassava mosaic virus in a heterologous system
More LessThe capacity for autonomous replication of the DNA A of African cassava mosaic virus (ACMV), a member of the bipartite geminiviruses infecting dicotyledonous plants, has been compared in host and non-host cells. A derivative of the ACMV DNA A was transfected into tobacco and maize protoplasts. Although ACMV is not able to infect maize, replication of the DNA A in maize protoplasts was observed to occur. The efficiency of replication was 10 to 20% of that seen in tobacco protoplasts. In both plant systems, replication was detected after the onset of cell division. ACMV replication in maize cells was compared to that of wheat dwarf virus and found to be 10 to 20% of that observed with the monocotyledon-specific virus. Insertion of 1165 bp of non-viral DNA into the ACMV DNA A prevented replication in maize but not in tobacco.
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Expression of potyvirus proteins in insect cells infected with a recombinant baculovirus
More LessThe N-terminal portion (P1-HC-Pro-P3) of the tobacco vein mottling virus (TVMV) polyprotein was expressed in insect cells and larvae by a recombinant baculovirus. The proteases necessary to process this TVMV polyprotein fragment were active in insect cells, since mature P1, HC-Pro and P3 proteins were detected by specific antisera in Western blots. Antisera to P1, HC-Pro and P3 also recognized polypeptides with apparent M r values predicted for the intermediate processing products of the polyprotein fragment. The results of this study indicate that the autocatalytic processing of TVMV HC-Pro from the polyprotein is supported by insect cells. Helper component activity in extracts of cells infected with recombinant baculovirus was not detected by aphid transmission assay.
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Mutations in zucchini yellow mosaic virus helper component protein associated with loss of aphid transmissibility
More LessZucchini yellow mosaic virus (ZYMV) is a potyvirus transmitted by aphids in a non-persistent manner. Isolates having partially or totally lost their ability to be transmitted by aphids have been isolated and found to be affected in their helper component activities. We have sequenced the helper component coding region of poorly aphid-transmissible (PAT) variants of two strains of ZYMV, E15 and R5A. Mutations have been identified at the nucleotide level leading to two amino acid changes in the E15 PAT variant helper component and to one amino acid change located in the cysteine-rich region (well-conserved among potyviruses) in R5A PAT variant helper component. The mutation in the R5A variant changes the same amino acid as the one identified in potato virus C, a non-transmissible strain of potato virus Y.
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Striking similarities between the nucleotide sequence and genome organization of citrus tatter leaf and apple stem grooving capilloviruses
More LessThe sequence of the 3′-terminal 2956 nucleotides, excluding the poly(A) tail, of the citrus tatter leaf virus (CTLV) genome was determined and compared with that of the apple stem grooving virus (ASGV) genome. The sequence of the 3′-terminal region of CTLV contains two overlapping open reading frames (ORFs) and a 3′-terminal non-coding region of 142 nucleotides. The long, incomplete ORF1 ends at UAG (position 2812) and encodes a protein with at least 938 amino acids (M r > 108 703). This protein contains the GDD motif associated with the RNA polymerase. ORF2, in a different frame within ORF1, starts at AUG (position 1248) and stops at UGA (position 2208) encoding a protein with an M r of 36179 (36K). Partial homologies were found among the 36K protein of CTLV, the 50K protein of apple chlorotic leaf spot closterovirus, the 40K protein of potato virus T and the gene 1 products of caulimoviruses. The arrangement of ORFs in the 3′-terminal region of the CTLV genome is in perfect agreement with that of the ASGV genome. The sequence of the 3′-terminal 2956 nucleotides, excluding the poly(A) tail, of the CTLV genome shows 86.1% identity to that of the ASGV genome. Similarities of amino acid sequences encoded by ORF1 and ORF2 of CTLV with the corresponding regions of ASGV are 86.1% and 97.3%, respectively. These results indicate that CTLV is a capillovirus closely related to ASGV.
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- Corrigendum
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