- Volume 71, Issue 9, 1990
Volume 71, Issue 9, 1990
- Bacterial
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Terminal redundancy and circular permutation of mycoplasma virus L3 DNA
More LessThis communication reports the physical map of mycoplasma virus L3 (MV-L3) DNA derived from restriction patterns obtained by digestion with seven different restriction endonucleases. The length of the restriction map is 36 200 bp in contrast to the contour length of native MV-L3 DNA molecules which is 39400 bp as determined by electron microscopy. The difference in length of 3200 bp (corresponding to 8·1 % of the native viral DNA contour length) is explained by terminal redundancy. It was possible to clone all fragments from particular restriction patterns into Escherichia coli vector pAT153, an indication of circular permutation within a population of MV-L3 DNA. However clear evidence has been obtained from the molar ratios of fragments and from hybridization experiments. We suppose that viral DNA is packaged from a concatemeric precursor molecule starting at a specific site called pac.
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- Animal
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Antigenic domains on the peplomer protein of avian infectious bronchitis virus: correlation with biological functions
More LessMonoclonal antibodies (MAbs) directed against structural proteins of infectious bronchitis virus (IBV) were produced to analyse the antigenic structure of this virus. Competitive binding of enzyme-labelled and unlabelled MAbs to IBV peplomer protein was analysed in an antibody binding assay to test the relatedness of the epitopes defined by the MAbs. Based on the competition groups, eight epitope clusters were defined (S-A to S-H); six of these clusters (S1-A to S1-F) were located on the S1 subunit and two (S2-G and S2-H) on the S2 subunit of the peplomer protein. Epitope clusters S1-A and S1-B overlapped extensively. The biological activities of the MAbs were determined and correlated to the epitope clusters. Monoclonal antibodies directed against epitope clusters SI-A to S1-E and one MAb directed against cluster S2-G moderately to strongly neutralized IBV at titres higher than 2 log10, whereas the remaining MAbs, directed against SI and S2, neutralized at titres lower than 2 log10. One MAb, directed against cluster S1-D, inhibited the agglutination of chicken erythrocytes.
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Ducks: a new experimental host system for studying persistent infection with avian leukaemia retroviruses
More LessLong-term persistence of the avian leukosis virus (ALV), the transformation-defective mutant of Prague strain Rous sarcoma virus subgroup C (td PR-C) was established in heterologous duck hosts after infection in mid-embryogenesis. Transient viraemia was observed for about 4 weeks after hatching and was lost in most of the infected ducks by about 6 months. Loss of viraemia was accompanied by the increasing synthesis of virus-neutralizing antibodies. In spite of strong virus-neutralizing antibodies, virus was detected by the cocultivation assay in duck tissues throughout the observation period up to 5 years. In the viraemic phase of infection, we found integrated proviruses in various tissues, preferentially in stomach muscle tissue and in the thymus. The long-term persistence of virus was frequently accompanied by liver necrosis and neoplastic diseases. Injection of td PR-C virus into early embryos resulted in more pronounced infection accompanied by an increased copy number of viral DNA per cell, high mortality and remarkable atrophy of thymus tissue in infected ducklings.
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Fusion of simian immunodeficiency virus with liposomes and erythrocyte ghost membranes: effects of lipid composition, pH and calcium
More LessSimian immunodeficiency virus from macaques (SIVmac) is closely related in its structure and biological activity to human immunodeficiency virus, and is the best animal model for the acquired immunodeficiency syndrome. We investigated the kinetics of membrane fusion between SIVmac and phospholipid vesicles and the effects of various parameters on this process. Purified SIVmac was labelled with octadecyl rhodamine B chloride, and fusion was continuously monitored as the dilution of the probe in target membranes. These studies show that SIVmac fusion is strongly dependent upon the liposome composition. Fusion with pure cardiolipin (CL) liposomes is significantly faster than with CL/dioleoylphosphatidylcholine (DOPC) (3:7), phosphatidylserine (PS) or disialoganglioside (GDla)/ DOPC (1·5:8·5) vesicles. SIVmac does not fuse appreciably with pure DOPC liposomes. Reduction of pH from 7·5 to 4·5 greatly enhances the rate of SIVmac fusion with CL, CL/DOPC and PS membranes, but does not affect fusion with DOPC or GDla/DOPC membranes. Calcium stimulates viral fusion with CL liposomes, but not with CL/DOPC or DOPC liposomes. SIVmac fuses with human erythrocyte ghost membranes only slowly at reduced pH. Our results indicate that SIVmac can fuse with membranes lacking the known viral receptor, CD4. Although the mechanism of SIVmac fusion with model and biological membranes remains to be determined, the fusion activity of SIVmac shares similarities with other lipid- enveloped viruses such as Sendai and influenza viruses.
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Sulphoevernan, a polyanionic polysaccharide, and the narcissus lectin potently inhibit human immunodeficiency virus infection by binding to viral envelope protein
Sulphoevernan is a sulphated α-1 →3,1 → 4 polyglucan (M r 20000) with a helical structure. This compound effectively inhibits both human immunodeficiency virus type 1 (HIV-1) and type 2 infection of cells in vitro at concentrations around 0·5 µg/ml. Moreover, the compound completely inhibits HIV-1-induced syncytium formation at a concentration of 1 µg/ml. Competition experiments with 35S-labelled sulphoevernan revealed that the mannose-specific lectin from Narcissus pseudonarcissus prevented binding of sulphoevernan to HIV-1, whereas the antibody OKT4A did not reduce the amount of sulphoevernan bound to MT-2 cells. These data indicate that the non-cytotoxic polymer sulphoevernan binds to the virus rather than to the host cell. In vivo studies, using Rauscher leukaemia virus in NMRI mice, revealed that, at a daily dose of 20 mg/kg, the animals were protected against virus-induced increases in spleen weight. From these in vitro and in vivo data we conclude that sulphoevernan has potential in the treatment of acquired immunodeficiency syndrome.
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Inhibition of human immunodeficiency virus replication in cell culture by endogenously synthesized antisense RNA
More LessAntisense RNA, which has a sequence complementary to mRNA, may provide the basis for antiviral therapies of high selectivity. We have explored the inhibitory effect of six antisense RNAs upon the replication of human immunodeficiency virus (HIV) in cell culture. We chose regions of the HIV genome to test whether sequences required for splicing or for translation initiation were more susceptible to antisense RNA interference. Our results suggest that inhibitory antisense RNAs contain sequences complementary to the AUG initiation codon of the tat gene and have a comparatively low tendency to form intramolecular base pairs which would interfere with intermolecular duplex formation. Inhibition can be substantial (over 70%) but is transient. Transience does not result from mutation of the input virus. Inhibition was not a consequence of the induction of interferon by antisense RNA-mRNA duplex formation. Our results suggest that at least part of the inhibitory effect is at the posttranscriptional level.
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The immunodominance of epitopes within the transmembrane protein (gp41) of human immunodeficiency virus type 1 may be determined by the host’s previous exposure to similar epitopes on unrelated antigens
Six major epitopes have been recognized within the transmembrane gp41 molecule of human immunodeficiency virus type 1 (HIV-1). The immunodominant epitope is also recognized by antibodies in sera from laboratory personnel and is similar to a linear sequence of amino acids in the genome protein of two rhinovirus serotypes. The hypothesis is presented that immunodominance is produced by multiple priming of the host, following repeated infections with viruses unrelated to HIV-1, which share similar epitopes.
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Proteolytic cleavage of microtubule-associated proteins by retroviral proteinases
More LessAspartic proteinases from human immunodeficiency virus type 1 (HIV-1) and avian myeloblastosis virus (AMV) were found to interfere with microtubule assembly. Preincubation of the proteinases with purified brain microtubule proteins (tubulin and microtubule-associated proteins) at low ionic strength (pH 6·8), completely inhibited microtubule assembly. Analysis of microtubule proteins after incubation with proteinase showed no effect on tubulin but extensive cleavage of the microtubule-associated proteins 1 and 2 was observed. The digestion by the two proteinases differed. In the presence of HIV-1 proteinase, a fragment with an M r of approximately 300000 appeared, as well as at least three other new fragments, with M r values of 188000, 124000 and 73000. In the presence of AMV proteinase, the microtubule-associated proteins were extensively digested to many small fragments. The extending microtubule-associated proteins normally seen by electron microscopy on the microtubule surface disappeared after treatment with AMV proteinase. Our results show that retroviral proteinases are not restricted to cleavage of viral polyproteins in vitro. It is suggested that proteolysis of microtubular proteins by viral proteinases is an important step in viral pathogenicity and that it may be part of a mechanism causing degenerative effects in infected cells.
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Rapid detection and further characterization of infection with hepatitis B virus variants containing a stop codon in the distal pre-C region
More LessRecently, hepatitis B virus (HBV) replication in the absence of HBe antigenaemia has been attributed to HBV variants with a TAG stop codon in the distal pre-C region associated with one or two point mutations. We describe here a rapid detection method for the diagnosis of such HBeAg-negative HBV variants using selective oligonucleotide hybridization. The entire pre-C region was amplified by the polymerase chain reaction and hybridized under stringent conditions with non-mutated (M0), one (M1) and two (M2) point-mutated oligonucleotide probes. Of the 15 HBeAg-positive (group I) and 20 HBeAg-negative (group II) serum samples studied, 14 samples in group I and one sample in group II hybridized with M0 only and 18 samples in group II hybridized with M1 or M2, or both.The remaining two samples (from groups I and II, respectively) failed to hybridize with any of the three probes. DNA sequencing confirmed mixed distal pre-C sequences in samples hybridizing with more than one probe and also revealed novel mutations in the distal pre-C region of the two samples which failed to hybridize with any of the probes. The latter sample had a +2 frameshift and hence represented a new type of HBeAg-negative HBV variant. This method may therefore prove useful in the diagnosis of infections by HBeAg-negative HBV variants resulting from common mutations in the pre-C region, as well as for the identification of less common variants with novel mutations in the same region.
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Isolation and purification of a non-A, non-B hepatitis-associated microtubular aggregates protein
Blood-borne type non-A, non-B (NANB) hepatitis- associated microtubular aggregates protein was isolated and partially sequenced. The microtubular aggregates were isolated from the hepatocytes of NANB- infected chimpanzees and were found to have a buoyant density in sucrose solution of 1·21 to 1·23 g/ml. A single protein, recognized by our anti-microtubular aggregates monoclonal antibodies, was found to have an Mr of 44000 (p44). This p44 protein was not found in uninfected chimpanzees. We determined a partial amino acid sequence for p44, and showed that it has no homology to any known proteins.
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Cloning, sequencing and expression in Escherichia coli of cDNA for a non-A, non-B hepatitis-associated microtubular aggregates protein
A 1·7 kb cDNA encoding a novel antigen (p44; apparent Mr 44K) associated with non-A, non-B (NANB) hepatitis, was isolated from the hepatic cDNA library of a chimpanzee infected with NANB hepatitis. The library was screened with a monoclonal antibody against this antigen. The cDNA cloned contained an open reading frame encoding a 444 amino acid protein with an Mr calculated to be 50468. The cDNA hybridized to a 1·9 kb mRNA obtained from chimpanzee hepatocytes infected with either the NANB or hepatitis delta viruses. It hybridized weakly to mRNA from hepatitis B virus-infected hepatocytes, and not at all to mRNA from normal chimpanzee hepatocytes. Southern blot analysis revealed that p44 is a host protein in chimpanzees, and that an identical gene exists in the human genome.
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DNA sequence of the gene encoding a major secreted protein of vaccinia virus, strain Lister
More LessInfection of tissue culture cells with vaccinia virus results in the specific secretion of several polypeptides into the medium. Previous studies identified a protein of approximate Mr 35000 (35K) which was secreted in large amounts at both early and late times after infection with the Evans strain. We now show that a related protein is secreted by the Lister strain but not by WR, Wyeth nor Tian Tan. The gene encoding the Lister strain 35K protein was mapped within the inverted terminal repeats of the genome. The DNA sequence of this region showed that the ends of this gene are very similar to previously published sequences flanking a gene of WR which encodes a protein of approximate Mr 7500 (7·5K). Our results suggest that the 7·5K polypeptide of WR may have arisen as a result of a deletion event and is a truncated form of the 35K Lister protein. Site-directed mutagenesis demonstrated that the 35K secreted protein encoded by Lister is not essential for growth in tissue culture.
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Prokaryotic expression of the major capsid protein of human cytomegalovirus and antigenic cross-reactions with herpes simplex virus type 1
More LessThe major capsid protein (MCP) of human cytomegalovirus (HCMV) was expressed in three portions as β-galactosidase fusion proteins, covering about 75 % of the open reading frame (ORF). Fusion protein SH 1 contained nucleotides 101 to 1243 of the ORF, fusion protein FS 1 contained nucleotides 1944 to 3089 and fusion protein SS 1 covered nucleotides 2624 to 3793. The recombinant proteins were tested for their immunoreactivity with human sera. Fusion protein FS 1 was found to represent the immunodominant region. The recombinant proteins were used to generate polyvalent rabbit antisera to investigate cross-reactivities with the major capsid protein (VP5) of herpes simplex virus type 1 (HSV-1). A monospecific antiserum raised against the fusion protein close to the N terminus of the MCP, as well as a monoclonal antibody and a monospecific rabbit antiserum directed against the viral MCP, cross-reacted with the VP5 as shown by immunoblotting and immunofluorescence. In order to detect common epitopes of the major capsid proteins of HCMV and HSV-1, the recombinant proteins were conjugated to CNBr-activated Sepharose and taken for purification of MCP antibodies from HCMV and HSV-1 seropositive individuals. Using this affinity chromatography method, cross-reactivity could be observed with HCMV- and HSV-positive human antisera in immunoblot experiments.
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Comparative studies of the proteins of equine herpesviruses 4 and 1 and asinine herpesvirus 3: antibody response of the natural hosts
More LessProteins of purified virions of equine herpesvirus 4 (EHV-4; equine rhinopneumonitis), EHV-1 (equine abortion virus) and asinine herpesvirus 3 (AHV-3) were compared by metabolic labelling with [35S]methionine or [14C]glucosamine during growth of low passage virus in natural host cells (horse or donkey) and high passage virus in an appropriate cell line and analysis by SDS-PAGE. Approximately 25 different proteins (Mr 300K to 21·5K) were clearly resolved for each virus. The three viruses had similar profiles although significant differences were found. The proteins of the cell line-grown viruses were similar to their precursor viruses grown in natural host cells although some small differences, probably related to differences in glycosy- lation by the various cell types, were noted. Six or seven high abundance glycoproteins were identified for EHV- 4, EHV-1 and AHV-3. The profile of seven glycoproteins of AHV-3 was more similar to EHV-1 than to EHV-4. Antigenic relationships of the proteins of the three viruses were examined using radioimmunopreci- pitation (RIP) and Western blot analyses and a series of polyclonal sera raised in colostrum-deprived, specific pathogen-free (SPF) foals which were immunized with inactivated EHV-4 (foal 3) or EHV-1 (foal 1), challenged and cross-challenged; a polyclonal donkey serum to AHV-3 was also used. The ontogeny of the antibody response in the SPF foals was studied and the major immunogenic proteins, as determined by RIP, were correlated with previously determined serum neutralizing antibody titres. Antibodies were first detected 14 days after primary immunization and were directed to EHV-4 proteins of M, 113K, 75K and 56K or EHV-1 proteins of 110K, 78K, 60K and 58K. Antibodies to these same three (EHV-4) or four (EHV- 1) proteins, together with antibodies to the major capsid protein and proteins of 67K (EHV-4) and 87K (EHV-1) were detected in response to primary infection (control foal 2) and these sera had high neutralizing antibody titres. The antigens of the three viruses were extensively cross-reactive with immunodominant proteins in the Mr ranges 150K to 11 OK and 62K to 56K. However, cross-absorption of EHV-4 and EHV-1 SPF foal antisera indicated the presence of significant amounts of type-specific antibody.
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Recombination of genomic terminus of bovine herpesvirus type 1 with cellular DNA
More LessBovine herpesvirus 1 (BHV-1) has a linear DNA genome of about 135 kb which appears as two isomers, resulting from its short unique segment being present in the two possible orientations with respect to the large unique segment. BHV-1 also circularizes its DNA to form replicative molecules. Definition of the target sequences at the genomic termini involved in the recombination events during genomic replication and isomerization, as well as virus maturation, led to the discovery that 10% of the genome molecules have additional DNA sequences attached to the right-hand terminus, as shown by electron microscopy. Three such tails have been cloned molecularly; they differ in length and nucleotide sequence, and hybridization experiments demonstrate the cellular origin of two of the three tails. The evidence presented here is consistent with a proportion of the BHV-1 genomes recombining their DNA with cellular DNA during lytic infection.
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The role of carbohydrate in the antigenic and immunogenic structure of bovine herpesvirus type 1 glycoproteins gI and gIV
More LessThe role of carbohydrate in the antigenic and immunogenic structure of bovine herpesvirus type 1 (BHV-1) glycoproteins gI and gIV was investigated. Deglycosy- lated proteins induced a significantly lower antibody response in rabbits than native glycoproteins suggesting that the immunogenicity of several epitopes on gI and gIV is carbohydrate-dependent. Loss of carbohydrate from gl also resulted in a significantly decreased ability to induce a serum neutralizing antibody response to BHV-1, due to modifications in three distinct carbohydrate-containing continuous epitopes. Similarly, in vitro lysis of BHV-1-infected cells was significantly reduced when antibodies raised against degly- cosylated gl were employed; this was attributed to changes in two of the three carbohydrate-dependent neutralizing epitopes on gI. The oligosaccharides may be directly involved as actual components of these continuous epitopes, rather than in stabilization of the conformation of the protein. In contrast, carbohydrate removal from gIV did not have a significant effect on the capacity to stimulate a neutralizing antibody response. Accordingly, none of the neutralizing epitopes on gIV appeared to be carbohydrate-dependent. Similarly, lysis of virus-infected cells was not significantly reduced when antibodies specific for deglycosy- lated rather than native gIV were used. In contrast to the humoral response, the delayed-type hypersensitivity response was stronger in rabbits immunized with deglycosylated proteins than in those inoculated with native glycoproteins gI or gIV. Consequently, the carbohydrates on gI and gIV may play a dual role in the host’s immune recognition and response by contributing to certain epitopes, but masking others. The implications for the development of a subunit vaccine against BHV-1 are discussed.
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Mapping of antigenic sites on the bovine ephemeral fever virus glycoprotein using monoclonal antibodies
More LessMonoclonal antibodies (MAbs) were produced against the G, M2 and N proteins of bovine ephemeral fever virus (BEFV) and 29 were selected for further study. Thirteen neutralizing MAbs were assigned to one conformation-independent and at least two conformation-dependent antigenic sites on the G protein by a competitive binding ELISA. The panel of MAbs were tested by neutralization and immunofluorescence with three strains of BEFV and three BEFV-related viruses. The results indicated that BEFV strains from different sources were not identical and that the M2 protein was the least variable of the proteins investigated. Passive protection studies in mice showed that the correlation between neutralizing titre and resistance to challenge was 0· 85 (P < 0· 001).
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Synthesis of bluetongue virus-encoded phosphoprotein and formation of inclusion bodies by recombinant baculovirus in insect cells: it binds the single-stranded RNA species
More LessA DNA clone of RNA segment 8 (S8) of bluetongue virus type 10 (BTV-10), an orbivirus member of the Reoviridae family has been expressed to high levels (20 mg/1 × l09 cells) using an Autographa califomica nuclear polyhedrosis virus expression vector (pA- cYMl). The expressed protein is similar to the authentic BTV phosphoprotein NS2, in its size, antigenicity, and also the manner of phosphorylation (e.g. same peptides and residues). Both mammalian and insect cell-derived NS2 proteins are phosphory- lated at serine residues only. Using affinity column chromatography and a gel retardation assay, the expressed protein has been shown to possess ssRNA- binding ability, a property which is shown to be independent of the phosphorylation state of the protein. In immunoelectron micrographic studies, gold-labelled anti-expressed NS2 antibodies have been used to localize the NS2 protein within the viral inclusion bodies (VIBs) in BTV-infected mammalian cells. Large inclusion bodies, morphologically similar to VIBs, have been identified in the recombinant virus- infected Spodoptera frugiperda cells. These structures have been shown to react with gold-labelled anti-BTV- 10 antisera, demonstrating the first direct evidence of the origin of inclusion bodies in orbivirus infection.
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Immunological relationships between phocid and canine distemper virus studied with monoclonal antibodies
More LessThe immunological relationships between distemper viruses, isolated from a seal and mink in Denmark and from a dog in Greenland, were investigated with 39 previously developed monoclonal antibodies (MAbs) directed against four major structural proteins of canine distemper virus (CDV). They were also investigated with 16 newly developed MAbs directed against the fusion (F) and large glycoprotein (named H in analogy with measles virus) of phocid distemper virus (PDV) isolated from a harbour seal (Phoca vitulina). These MAbs were reacted with the three different isolated viruses and with the LEC strain of measles virus, in ELISA and immunofluorescence tests. In addition, immunoprecipitation tests were carried out with some of the cross-reacting antibodies. All 55 MAbs reacted identically with distemper virus isolated from seals or mink. When the MAbs produced against CDV were tested, 37 of 39 antibodies reacted with a virus isolated from a sled dog diseased in an outbreak of distemper in Greenland prior to the epizootic among seals in the North Sea. Of the 39 antibodies, 25 reacted with PDV and distemper virus isolated from mink. Of these antibodies, only three of the nine antibodies directed against the H protein of CDV cross-reacted with PDV and distemper virus from mink. Eleven MAbs, reacting with six epitopes of the H protein of PDV, were produced. All 11 antibodies reacted with distemper virus from mink, two of the antibodies reacted with CDV and none reacted with measles virus. All five antibodies reacting with three different epitopes of the F protein of PDV reacted with distemper virus from mink and CDV. Of these five antibodies three, directed against two epitopes, reacted with measles virus. Of the two envelope proteins, the H protein shows pronounced immunological differences between PDV and CDV. In contrast, immunologically the F protein appears to be well conserved among morbilliviruses. It is concluded that the virus causing the epizootic in seals in the North Sea in 1988 may have infected mink on land, or, alternatively, the virus in the sea may have originated from virus-infected mink.
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Immunological relationships of simian virus 41 (SV41) to other paramyxoviruses and serological evidence of SV41 infection in human populations
More LessAntigenic relationships of simian virus 41 (SV41) to other paramyxoviruses were examined by immunopre-cipitation of isotope-labelled SV41-infected cell lysates with specific antisera. SV41 is closely related to the group comprising human parainfluenza virus 2 (HPIV-2), simian virus 5 (SV5), parainfluenza virus 4 and mumps virus. Slight cross-neutralization was detected between SV41, HPIV-2 and SV5. Anti-SV41 activities were detected in 21 of 1116 human serum specimens, indicating that a proportion of the human population is infected with SV41. The haemagglutinin-neuramini-dase of SV41 was preferentially immunoprecipitated by anti-SV41 positive sera.
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Prediction and identification of a T cell epitope in the fusion protein of measles virus immunodominant in mice and humans
More LessAmino acid residues 288 to 302 of the fusion protein of measles virus were predicted by a variety of methods to represent a putative T cell epitope. This sequence was synthesized and the peptide was injected into mice of six inbred strains to test this possibility. Lymphocytes from peptide-immunized mice from all six H-2 disparate strains were able to mount a proliferative response following in vitro culture with the peptide. In addition, lymphocytes from three strains also proliferated in the presence of live measles virus. The peptide also behaved as a B cell epitope in that immunization with free peptide in adjuvant resulted in anti-peptide antibody production in all mouse strains. However, these antibodies did not react with the virus in either a solid-phase immunoassay or a virus neutralization assay. Peripheral blood lymphocytes from 10 laboratory personnel with a prior history of exposure to measles virus were tested in a proliferation assay with the peptide and with the virus. Lymphocytes from all 10 individuals proliferated in response to culture with the virus and those from eight responded to the peptide. These results give further support to the concept of permissive interaction of antigenic peptides with a wide range of class II major histocompatibility complex molecules both in mice and man and indicate the possibility of designing peptides that could be used as components of a synthetic vaccine for use in man.
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The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli
More LessThe antigenic structures of the envelope protein, E, and the non-structural protein, NS1, of dengue type 1 virus (DENI) have been studied in the form of recombinant fusion proteins expressed in Escherichia coli. Deletion analysis was used to identify two distinct antigenic domains in E that reacted with subsets of antiviral monoclonal antibodies (MAbs). Domain I of E extends from amino acid residues (aa) 76 to 93 of E; domain II extends from aa 293 to 402 and contains an essential disulphide bridge. MAbs also reacted with several determinants clustered near the N terminus of the NS 1 protein (aa 57 to 126). Recombinant fusion proteins containing E. coli trpE sequences and most of the sequences for either E or NS1 were immunogenic in mice. The antibodies elicited by the E fusion protein reacted with a portion of the protein containing domain II, whereas antibodies elicited by the NS1 fusion protein did not react with the antigenic determinants defined by our MAbs.
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Partial nucleotide sequence of South American yellow fever virus strain 1899/81: structural proteins and NS1
More LessWe have partially cloned and sequenced the genome of a Peruvian yellow fever virus isolate (1899/81) and compared the nucleotide and deduced amino acid sequences of this strain with the previously published sequence of the West African yellow fever virus strain Asibi. In the 3594 base region sequenced, which contains the structural genes (C, M, E), all but the 72 3′-terminal nucleotides of the NS1 gene and 108 nucleotides of the 5′ non-coding region, 515 nucleotide substitutions were detected. Nucleotide divergence was lowest in the 5′ non-coding region, 2·8%, compared with an average rate of 14·7% in the coding regions. Over 91 % of the 512 nucleotide changes in the coding region were silent; 44 amino acid substitutions resulted. The capsid protein was the least conserved, whereas the M protein was the most highly conserved (6·7% and 1·3% divergence, respectively). The envelope protein had 18 amino acid changes (3·7% divergence), one of which created an additional site for potential glycosylation of the 1899/81 virus. NS1 protein divergence (3·9%) was similar to that seen in the E protein. Of the 44 amino acid substitutions found, 34 (77%) were conservative. The highest number of nonconservative differences occurred in the envelope glycoprotein. These changes may significantly affect the antigenic and biological functions of the viruses.
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Persistent infection of a glioma cell line generates a Theiler’s virus variant which fails to induce demyelinating disease in SJL/J mice
More LessTheiler’s murine encephalomyelitis virus (TMEV) induces demyelinating disease which is associated with persistent virus infection of the central nervous system. To study the interaction between TMEV and host cells, we infected the G26-20 glioma cell line in vitro, and this resulted in a lytic infection in which most, but not all, cells were killed. Surviving cells divided and formed a viable monolayer in which a small proportion of cells displayed viral cytopathic effects. Levels of virus produced by these cultures over a 6 month period fluctuated between 6 and 8 log10 p.f.u./ml as measured by viral plaque assay. Similarly, the percentage of cells producing both viral antigen and viral RNA, as measured by a simultaneous immunoperoxidase/in situ hybridization technique, varied between 5 and 30%. Although persistently infected cultures were susceptible to challenge by both vesicular stomatitis virus and herpes simplex virus, they were resistant to infection by homologous viruses. Interferon activity was not identified. TMEV isolated from passage 12 produced smaller plaques than wild-type Daniels strain virus (wt-DAV) on L-2 cell monolayers. In contrast to demyelination induced in SJL/J mice after intracerebral inoculation with wt-DAV, mice infected with the small plaque variant virus failed to develop viral persistence or chronic demyelination. However, following immunosuppression by total body irradiation, SJL/J mice infected with the small plaque variant developed viral persistence but no demyelination. Characterization of the biochemical and molecular determinants of the variant will lead to a better understanding of determinants important in viral persistence.
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Echoviruses include genetically distinct serotypes
More LessWe have studied the genetic relationships of echoviruses using nucleotide sequencing and hybridization analysis. The nucleotide sequence of the echovirus 11 (EV11) P2 and P3 regions, which encode the nonstructural proteins, was shown to resemble closely those of coxsackie B viruses (CBV) and coxsackievirus A9 (CAV9). EV11, CBV and CAV9 have a similar organization in the 3′ non-coding region when compared to polioviruses and CAV21. In contrast, the 3′ end of EV22 shares only minimal sequence homology with other sequenced enteroviruses, and the 3′ non-coding region has a unique secondary structure. Thirty-three echovirus reference strains were tested by nucleic acid hybridization using cDNA probes from the genomes of EV6, 11, 18 and 22. It was shown that a great majority of the strains belongs to the same subgroup as serotypes 6, 11 and 18, whereas EV22 and EV23 are genetically not closely related to this major subgroup.
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Detection and differentiation of picornaviruses in clinical samples following genomic amplification
A polymerase chain reaction (PCR) assay was used to detect and differentiate picornaviruses (PVs), using primers homologous to the 5′ non-coding and VP2 regions of the PV genome. The PCR resulted in a 530 bp PCR product for human rhinoviruses (HRVs) and a 650 bp product for polioviruses, coxsackieviruses (CV) or echoviruses. The PCR assay could detect as little as 1 p.f.u. of virus in either cerebrospinal fluid (CSF) or stool, using ethidium bromide-stained gels. Standard strains of poliovirus, CV, echovirus and HRV were detected, with the exception of echovirus type 22. In contrast, heterologous viruses, such as herpes simplex virus, human cytomegalovirus, adenovirus, influenza virus and rotavirus, as well as human and monkey cell DNA, were not amplified. In nasal swabs taken from patients with respiratory infections, the PCR detected 27 of 28 HRV isolation-positive specimens. All specimens from which viruses other than HRVs were isolated were negative by PCR. The PCR definitively identified poliovirus and CVs from the CSF or stool of patients with aseptic meningitis, as well as CV in the pericardial fluid of a patient who had suffered a myocardial infarction. Specimens taken from patients with similar pathologies, and from which heterologous viruses were isolated, were uniformly negative by PCR.
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Inhibitory effect of protein kinase C inhibitor on the replication of influenza type A virus
More LessThe growth of influenza virus A/PR/8/34 in MDCK cells was inhibited by l-(5-isoquinolinesulphonyl)- 2-methylpiperazine dihydrochloride (H7) which is a potent inhibitor of protein kinase C, but not by an effective inhibitor of cyclic nucleotide-dependent protein kinases. Analysing the inhibitory effect of H7 during the replication cycle of influenza virus, we found that the primary transcripts were sufficiently synthesized in infected cells exposed to H7. The primary transcripts synthesized in the presence and absence of H7 were active in directing the synthesis of viral polypeptides both in a cell-free system and in the system containing H7. In the system where infected cells were exposed to H7, the viral positive-sense RNAs were also significantly amplified 6 h after infection. However, the synthesis of viral proteins other than nucleoprotein from viral primary or amplified (secondary) mRNAs was extremely restricted. The synthesis of host cellular proteins in mock-infected cells was significantly retained in the presence of H7. These results suggest that the selective inhibition of influenza virus translation following the transcription of viral mRNA was induced by H7 in infected cells.
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Influenza B virus mRNA synthesis in vivo: efficient transcription of vRNAs 1, 2 and 3
More LessInfluenza B virus genomic RNA (vRNA) segments encoding polymerase proteins were shown to be efficiently transcribed in vivo, unlike those of influenza A virus. The results are discussed in connection with encoding polymerase proteins were shown to be the primary structure of the 3′ends of vRNA segments.
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Induction of cervical neoplasia in the mouse by an extract of cells infected by varicella-zoster virus
More LessSince several human herpesviruses, including varicella-zoster virus (VZV), have been demonstrated to transform mammalian cells in vitro, VZV was tested in a mouse model of virus-induced cervical neoplasia to determine whether it is oncogenic in vivo. Herpes simplex viruses types 1 and 2 and cytomegalovirus have been previously shown to induce cervical neoplasia in this mouse model. VZV was propagated in WI-38 cell cultures and inactivated by ultraviolet irradiation. Control material was prepared in an identical manner from uninfected cell cultures. Cotton tampons, saturated with inactivated virus or control material, were inserted into the vaginas of C57BL mice three times a week for 60 weeks. Cervical dysplasia was detected in 40 % and invasive carcinoma in 34 % of virus-exposed mice by histological examination. No lesions were detected in control animals. These observations indicate that VZV, or some product of virus-infected cells, is oncogenic in vivo for the mouse cervix.
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Identification of antigenic sites on pseudorabies virus glycoprotein gp50 implicated in virus penetration of the host cell
M. Eloit, H. Bouzghaia and B. TomaFive monoclonal antibodies specific for glycoprotein gp50 of pseudorabies virus were used to make a topographical map of gp50 and to determine the biological function of the different antigenic domains. Three antigenic domains were identified by competition binding assays and additivity assays (IA, IB, II). Domain IA corresponds to a continuous epitope, whereas domains IB and II consist of one or several discontinuous epitopes, identified by their resistance to heat or reducing treatments. Domains IA and IB correspond to sites highly involved in virus neutralization. Neutralization experiments by monoclonal antibodies recognizing domains IA and IB and performed before or after adsorption of virions to cells showed that these domains have a role in penetration of virus into the cell.
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Increased antibody responses to human papillomavirus type 16 L1 protein expressed by recombinant vaccinia virus lacking serine protease inhibitor genes
The L1 gene of human papillomavirus type 16 (HPV- 16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome. Insertion into the thymidine kinase (TK) gene was achieved by selection of TK− mutants in BUdR on TK− cells. Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7·5K promoter and selection of mycophenolic acid-resistant recombinant viruses. Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics. However, immunization of mice with these recombinant viruses induced different levels of antibody to the LI protein. Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene. The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the LI protein of HPV-16.
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The sequence of the nucleocapsid protein (N) gene of Piry virus: possible domains in the N protein of vesiculoviruses
More LessA number of independent cDNA clones of the Piry virus N gene message were identified and sequenced. From the resulting sequences and previously published data, we derived the sequence of the mRNA for this protein. Sequence similarities of the translated region of Piry virus with that of other viruses suggest that Piry virus is as distantly related to Chandipura virus as it is to the vesicular stomatitis viruses of Indiana and New Jersey serotypes. Based on the relative conservation of the amino acid sequence of the nucleocapsid protein of these vesiculoviruses, the N protein can be subdivided into at least three regions, possibly indicative of functional domains.
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Categorizing some early and late transcripts directed by the Autographa californica nuclear polyhedrosis virus
More LessUsing an SI mapping assay on RNA from Spodoptera frugiperda cells infected by the Autographa californicanuclear polyhedrosis virus in the presence and absence of cycloheximide and aphidicolin, we can distinguish three classes of transcripts. First, there are those whose synthesis is blocked by the DNA synthesis inhibitor aphidicolin and which are therefore late transcripts. These include the late transcript of the 39K gene and a late leftward transcript across the XhoI site in the HindIII-F region. Second, there are those whose synthesis is not blocked by aphidicolin, but whose accumulation is inhibited by the protein synthesis inhibitor cycloheximide and which are therefore presumably delayed early genes. These include the p26 transcript(s), the early 39K transcript and the a2 transcript in the HindIII-K/Q region. Third, there are those whose accumulation is not affected or is enhanced by cycloheximide. These are not necessarily immediate early transcripts, but their response to cycloheximide is clearly different from that of those in the second class. They include the α1, and α3 transcripts in the HindIII-K/Q region and the early leftward transcript across the XhoI site in the HindIII-F region.
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Nucleotide sequence and genomic organization of melon necrotic spot virus
More LessCloned cDNA copies of the genomic RNA of melon necrotic spot virus (MNSV) have been sequenced and the sizes and locations of predicted viral proteins have been deduced. The genome consists of at least 4262 nucleotides and the positive strand contains three to five open reading frames (ORFs) which may be expressed. The 5′ proximal ORF encodes a 29K protein (p29) and terminates with an amber codon which may be read through to produce an 89K protein (p89). Two small centrally located ORFs each encode a 7K protein (p7A and p7B). As p7A is in frame with p7B, readthrough of the amber codon terminating p7A may occur, producing a 14K protein (pi4). The 3′ proximal ORF encodes the 42K coat protein. The genomic organization of MNSV, its probable translation strategy and the amino acid sequences of its putative proteins closely resemble those of known carmoviruses, suggesting that MNSV should be classified as a member of the carmovirus group. Unusual properties of the putative MNSV replicase (p89) suggest that MNSV should be classified in a new virus supergroup with several other viruses sharing these properties.
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Nucleotide sequence and structural analysis of two satellite RNAs associated with chicory yellow mottle virus
More LessThe two satellite RNAs associated with CYMV infections were sequenced. The larger (sCYMV-L1) has only linear molecules 1145 nucleotides long, a poly(A) tail, a long open reading frame (ORF) coding for a protein of M r 39636 resembling in composition those of other large nepovirus satellite RNAs, a 5′ leader sequence of 16 nucleotides and a 3′ non-coding region of 40 nucleotides. In vitro translation of sCYMV-L1 yielded a protein product with a size that corresponded to that predicted from the sequence. The smaller satellite (sCYMV-S1) is 457 nucleotides long, has no ORF of significant length and no in vitro messenger activity. Both linear and circular forms of this satellite RNA were detected in infected tissues. Comparison of the sCYMV-S1 primary structure with the sequences of other small nepoviral satellites reveals large regions of homology. Analysis of the secondary structures derived from the sequences of the plus and minus strands suggests possible consensus sequences for their self-cleavage.
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Cucumber mosaic virus satellite RNA (Y strain): analysis of sequences which affect yellow mosaic symptoms on tobacco
More LessPlants infected with cucumber mosaic virus (CMV) (KIN strain) produce a mild mosaic disease on tobacco whereas infections of CMV with satellite RNA (strain Y) cause a severe yellow mosaic. Analysis of recombinant and mutant forms of satellite RNA identified a site (nucleotides 185/186) in the Y satellite RNA that affects the ability to induce the yellow mosaic in combination with CMV but not with tomato aspermy virus. The location of this site with respect to other mutations in the satellite RNA indicated that polypeptides, which may be encoded by the satellite RNA, have no role in induction of yellow mosaic symptoms. The symptom induction is therefore an effect of the satellite RNA on the host plant with the intervention of the helper virus. In the course of the mutation analysis of satellite RNA we detected several secondary mutations which arose in planta. Two of these were deletions of more than 80 nucleotides. Other forms of mutant satellite RNA were non-functional even though the modifications involved nucleotides completely within the large secondary deletions. These data imply complex intramolecular interactions in the satellite RNA.
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Complete nucleotide sequence of clover yellow mosaic virus RNA
The entire genomic RNA of clover yellow mosaic virus was sequenced from cDNA clones and run-off cDNA transcripts. The genomic RNA is 7015 nucleotides in length [excluding a 3′ poly(A) tail], with six open reading frames (ORFs) greater than 150 nucleotides in length. The first five ORFs encode proteins of M r 19IK, 26K, 12K, 6-5K and 28K, respectively. The sixth ORF lies completely within ORF1 and codes for a protein of M r 14K. The capsid protein coding region (M r 23K) is found within ORF5 which encodes the M r 28K protein. Proteins encoded by ORFs 1 to 3 and ORF5 show strong homology with proteins of other potexviruses, especially papaya mosaic virus.
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Molecular genetic analyses of the soybean mosaic virus NIa proteinase
More LessRecombinant DNA molecules containing cDNA to a soybean mosaic virus (SMV) RNA genome were constructed and partial nucleotide sequences determined for two cDNA inserts, pSMV-34 and pSMV-35. Comparison of the predicted amino acid sequence encoded by the pSMV-34 cDNA insert to other potyvirus protein sequences revealed extensive homology with the region of the genome encoding the NIa proteinase, with conservation of the amino acids proposed to form the catalytic triad of the active site. Cell-free transcription and translation of the cloned cDNA sequence containing the NIa open reading frame and flanking sequences revealed that NIa proteinase sequences, which were expressed as part of a high Mr precursor, were able to undergo proteolytic processing. Alteration of the codon for one of the putative active site residues by site-directed mutagenesis eliminated processing and resulted in the accumulation of a high Mr precursor. Based on predicted amino acid sequences at five putative cleavage sites within the SMV polyprotein, a consensus SMV NIa proteinase cleavage sequence of Glu/Asn- Xaa-Val-Xaa-Xaa-Gln ↓ Gly/Ser was proposed. The SMV NIa proteinase and its putative cleavage sites maintained motifs found in other potyviruses.
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Dependence of groundnut rosette virus on its satellite RNA as well as on groundnut rosette assistor luteovirus for transmission by Aphis craccivora
More LessTransmission of groundnut rosette virus (GRV) by Aphis craccivora is known to depend on the additional presence in the source plants of a luteovirus, groundnut rosette assistor virus (GRAV). Naturally occurring isolates of GRV contain a satellite RNA which is the main cause of rosette symptoms in groundnut, different variants of the satellite being responsible for the green and chlorotic forms of rosette. In extensive glasshouse tests, GRAV-dependent transmission of GRV by A. craccivora occurred only from groundnut plants infected with satellite-containing isolates of GRV. This was true whether the GRV isolates were from groundnut plants from Nigeria or Malawi with either the green or chlorotic forms of rosette and whether they contained homologous or heterologous satellites. Aphid transmission of GRV therefore depends not only on the presence of GRAV but also on that of the GRV satellite RNA. This probably explains why satellite-free isolates of GRV have not been found in nature. This is the first report of satellite RNA mediating aphid transmission of a plant virus.
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Transgenic Nicotiana debneyii expressing viral coat protein are resistant to potato virus S infection
More LessThe coat protein gene from potato virus S (PVS) was introduced into Nicotiana debneyii by leaf disc transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral coat protein were highly resistant to subsequent infection by the ME strain of PVS as indicated by an absence of symptom development and a lack of accumulation of virus in both the inoculated and upper leaves. As in reported experiments with plants expressing potato virus X coat protein, plants expressing PVS coat protein were also protected from inoculation with PVS RNA. These results provide further evidence that coat protein- mediated protection for these two groups of viruses, which share similar genome organizations, may involve inhibition of some early event in infection other than or in addition to virus uncoating.
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