- Volume 72, Issue 11, 1991
Volume 72, Issue 11, 1991
- Animal
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Location and nucleotide sequence of the gene encoding the viral enhancing factor of the Trichoplusia ni granulosis virus
More LessThe gene encoding the viral enhancing factor (VEF) of Trichoplusia ni granulosis virus has been cloned from a λgt11 expression library, and the complete nucleotide sequence determined. The VEF gene encodes a protein with a predicted M r of 104K which does not share homology with any previously reported proteins. A possible promoter is located four nucleotides upstream of the initiation codon and represents a consensus baculovirus late promoter (ATAAG). This has been confirmed by the identification of VEF mRNA in Northern blots of infected larvae 6 days but not 3 days post-infection. Using an anti-VEF-TrpE polyclonal antiserum in Western blots of dissolved viral occlusion bodies, related proteins have been identified in both Pseudaletia unipuncta granulosis virus Hawaiian strain (PuGV-H) and Heliothis armigera GV (HaGV), but not in Erinnyis ello GV (EeGV), T. ni singly enveloped nuclear polyhedrosis virus (TnSNPV) or Autographa californica multiply enveloped NPV (AcMNPV). Similar results were obtained with Southern blots of genomic digests. DNA fragments homologous to an internal portion of the VEF gene were found in PuGV-H and HaGV but not in EeGV, TnSNPV or AcMNPV.
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Selection kinetics during serial cell culture passage of mixtures of wild-type Autographa californica nuclear polyhedrosis virus and its recombinant Ac360-β-gal
More LessDetailed analysis of the selection process in serial co-infections of cell cultures by wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) strain E2 (AcNPV/E2) and Ac360-β-gal, a genetically engineered strain, shows that the unaltered strain was clearly dominant even when it initially constituted the minority component in the inoculum. A method of calculating a selection coefficient that quantifies the relative advantage of one strain of virus over the other under specific culture conditions is described. Calculated selection coefficients were relatively homogeneous and almost exclusively favoured the progenitor. Selection pressure was not influenced by the relative proportions of the two strains in the population. Selection coefficients, as determined in the present study, may be useful for evaluating the effect of a genetic alteration on viral fitness under specified conditions. Unexpected high frequencies of mixed phenotype plaques were observed during infectivity titrations of media from early serial passages of co-infected cultures. Statistical evaluation implicates some non-heritable combinational phenomenon. Virus plated from mixed phenotype plaques show high segregation of phenotypes implying that genetic recombination does not contribute in a major way to the high mixed phenotype frequencies. Electron microscopic examination of virion pellets from infected 72 h cell culture media similarly argue against co-envelopment as a major contributory factor to the high frequency of mixed phenotype plaques. The cause remains undetermined.
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Involvement of the influenza A virus PB2 protein in the regulation of viral gene expression
More LessTo determine the function(s) of the PB2 protein of influenza A virus, six temperature-sensitive (ts) mutants of A/Udorn/72 (H3N2) virus, each carrying a ts mutation in the PB2 gene, were analysed for virus RNA and protein synthesis. One of the mutants, ICRC27, exhibited unique phenotypes and was characterized in detail. At the non-permissive temperature, 40 °C, the accumulation of mRNA for each genome segment was reduced severely, leading to delayed and reduced synthesis of viral proteins, complementary and viral RNAs (cRNAs and vRNAs). At the permissive temperature, 34 °C, the mutant virus produced severalfold greater concentrations of both mRNAs and cRNAs of PB2, PB1 and PA segments than wild-type virus. The synthesis of the three polymerase proteins and the induction of RNA polymerase activity were also greatly increased. By contrast, the expression of the haemagglutinin (HA) gene was severely suppressed. The over-production of the polymerase mRNAs was not observed during primary transcription, i.e. in the presence of cycloheximide. The ts+ revertants of ICRC27 did not exhibit the ts defects and also lost most of the non-ts phenotypes at 34 °C. These observations indicate that the PB2 protein participates not only in the synthesis of viral RNAs, but also in the regulation of viral gene expression, i.e. in the down-regulation of the three polymerase genes and the up-regulation of the HA gene during secondary transcription.
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Sequence analysis of the haemagglutinin (HA) of influenza A (H1N1) viruses present in clinical material and comparison with the HA of laboratory-derived virus
We used the polymerase chain reaction to amplify the HA1 coding region of influenza A (H1N1) viruses present in clinical material from recent cases of influenza in the U.K. Previously, we have demonstrated that isolation of human influenza viruses in embryonated hens’ eggs selects variants which have amino acid substitutions in their haemagglutinin (HA) clustering around the receptor-binding site. Such egg-selected variants are often antigenically distinct from each other and from corresponding viruses isolated on mammalian cells. Since in general the virus used for vaccine production is an egg-adapted virus, it is important to determine the extent to which these variants are present in the natural virus which causes disease in man. To achieve this, amplified products from clinical material were cloned and many individual clones sequenced. Our results indicate that the HA of the naturally occurring virus is relatively homogeneous and represented by virus isolated in the laboratory on MDCK cells, whereas the variants isolated in eggs are present only at low levels in clinical material.
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Glycosylation of measles virus haemagglutinin protein in infected cells
More LessProcessing of the measles virus haemagglutinin (H) protein was analysed by the pulse-chase method, immunoprecipitation with an anti-H monoclonal antibody and SDS-polyacrylamide gel electrophoresis, combined with the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or monensin (inhibitors of intracellular processing of secretory proteins) to cultures and digestion of the protein with endoglycosidase H or neuraminidase. The apparent M r of the H protein was increased from 74K to 78K during the chase period. Addition of either CCCP or monensin to the chase medium inhibited the appearance of the 78K H protein, but not the immunoreactivity of the H protein or dimer formation, suggesting that these two events occur in the rough endoplasmic reticulum. The 74K H protein processed in the presence of CCCP was fully sensitive to endoglycosidase H digestion, whereas the 74K H protein processed in the presence of monensin was partially resistant to endoglycosidase H. In experiments using 3H-labelled sugars, [3H]galactose was incorporated into the 74K H protein in the presence of monensin. Neuraminidase treatment increased the electrophoretic mobility of the 78K H protein to 74K. Only the 78K H protein was detected on the surface of untreated cells, and it was resistant to endoglycosidase H digestion. These data suggest that after galactose addition sialic acid is added to the H protein in the trans-Golgi complex and then the mature 78K H protein is transported to the cell surface.
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Enhancement of human immunodeficiency virus type 1 infection by cationic liposomes: the role of CD4, serum and liposome-cell interactions
More LessWe have reported previously the enhancement of the infectivity of human immunodeficiency virus type 1 (HIV-1) by liposomes composed of the cationic lipid N-[2, 3-(dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA). To determine the mechanism by which this process occurs, we have investigated the role of CD4, serum concentration and liposome-cell interactions in the DOTMA-mediated stimulation of HIV-1 infection of A3.01 cells. Serum alone significantly inhibited the binding and infectivity of HIV-1, but DOTMA-mediated enhancement of infectivity was more pronounced in the presence of serum than in its absence. HIV-1 binding to cells was increased in the presence of DOTMA liposomes, DEAE-dextran and polybrene, all of which also enhanced infectivity to a similar extent at comparable concentrations. Fluorescence dequenching measurements indicated that DOTMA liposomes fused with HIV-1, but not with cell membranes, in the presence of serum. The enhancing effect of DOTMA liposomes on HIV-1 infectivity was CD4-dependent, and appeared to involve virus-liposome fusion and liposome binding to the cell surface. DOTMA liposomes did not mediate infection of the CD4− K562 and Raji cell lines.
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Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human carrier: comparison with reported isolates for conserved and divergent regions
The complete nucleotide sequence of a hepatitis C virus derived from plasma of a human carrier in Japan was determined. The cDNA of the isolate (HC-J6) contained 9481 nucleotides and an additional T stretch of 30 to 108 nucleotides at the 3′ end, and had one large open reading frame coding for a polyprotein of 3033 amino acids. It differed by 31.8 to 32.1% in the nucleotide sequence and by 27.4 to 27.7% in the amino acid sequence from an American isolate and two Japanese isolates previously reported. Among these four isolates, the 5′ non-coding region of 329 to 341 nucleotides was well conserved (>93% identity), whereas the 3′ non-coding region of 39 to 45 nucleotides (T stretches not included) was more variable (>30% identity). An excellent degree of conservation of the 5′ non-coding region would reflect its pivotal role in replication, and primers deduced from this region could be applied for the sensitive and specific detection of viral RNA by polymerase chain reaction. Due to a high degree of similarity in the amino acid sequence of the putative core protein (>90%), antigen probes deduced from it would be suitable for the serological diagnosis of HCV infection. Low sequence similarity in the putative envelope protein (>53% identity), however, would have to be taken into account in considering the immunoprophylaxis of HCV infection.
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In vitro homotypic and heterotypic interference by defective interfering particles of West Nile virus
More LessDefective interfering (DI) particles of the flavivirus West Nile (WN) were generated after as few as two high multiplicity serial passages in Vero and LLC-MK2 cells. Six cell lines (Vero, LLC-MK2, L929, HeLa, BHK-21 and SW13) were used to assay interference by DI particles in a yield reduction assay. Interference was found to vary depending on the cell type used. The highest levels of interference were obtained in LLC-MK2 cells, whereas no detectable effect was observed in BHK-21 and SW13 cells. The ability of DI virus to be propagated varied depending on the cell line used; no detectable propagation of DI virus was observed in SW13 cells. Optimum interference was obtained following co-infection of cells with DI virus and standard virus at a multiplicity of 5. Interference between DI and standard viruses occurred only when they were co-infected or when cells were infected with DI virus 1 h before standard virus. Investigation of heterotypic interference by DI particles of WN virus strains from Sarawak, India and Egypt revealed that interference was dependent on the strain of WN virus or flavivirus used as standard virus. A measure of the similarity between five strains of WN virus and other flaviviruses was made on the basis of interference by DI viruses, and was found to be similar to that based on haemagglutination inhibition tests using a panel of monoclonal antibodies.
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Pathogenesis of avian encephalomyelitis viruses
More LessThe pathogenesis of a field strain, a vaccine strain and the egg-adapted Van Roekel strain of avian encephalomyelitis virus in susceptible chicken embryos and day-old chickens was investigated using enzyme-linked immunosorbent assays for the detection of virus-specific antibody and antigen. The Van Roekel strain was shown to be highly neurotropic whereas the field and vaccine strains were enterotropic. Radioimmunoprecipitation studies using Na125I-labelled purified virus failed to detect any differences in the composition of the structural viral proteins of each strain that could account for these differences. As expected, the field and vaccine strains were more efficient than the Van Roekel strain at inducing antibody following oral administration. Primary cultures of chicken embryo brain cells supported the growth of the Van Roekel strain to a much greater extent than the field and vaccine strains.
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Formation of subviral particles by in vitro translation of subgenomic poliovirus RNAs
More LessRabbit reticulocyte lysates were programmed with either RNA extracted from purified poliovirus or a mixture of mRNAs encoding the capsid precursor, P1, and proteinase 3CD. In both cases, 14S subunits were formed at 30 °C and empty capsids at 37 °C. Both the 14S subunits and empty capsids had the expected polypeptide composition and neutralization epitopes. It is concluded that the proteinase 3CD gene is the only viral genetic information needed for the correct processing of P1 and the formation of 14S subunits, and their assembly into antigenically correct empty capsids.
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Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence
More LessThe 2A region of the foot-and-mouth disease virus (FMDV) polyprotein is only 16 amino acids in length. During synthesis of the FMDV polyprotein a primary proteolytic processing event occurs between the 2A and 2B regions of the polyprotein. The activity responsible for this cleavage is not known but it is thought that either an unidentified virus-encoded proteinase may be responsible, or that 2A acts as a substrate for a host cell proteinase. A series of recombinant FMDV polyproteins has been constructed in which sequences to the N- or C-terminal side of the 2A region have been deleted. Analysis of the processing of these polyproteins shows that a 19 amino acid sequence spanning 2A is sufficient to mediate polyprotein cleavage at a site immediately C-terminal to 2A, whereas deletions extending into the 2A region prevent cleavage.
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Intragenic and intergenic recombination between temperature-sensitive mutants of vaccinia virus
More LessThe frequency of recombination for a complete set of two-factor crosses between vaccinia virus mutations separated by distances of between 54 and 10692 bp was determined. The results show that in intragenic crosses there is a linear relationship between the recombination frequency observed and distances between the mutations of up to 700 bp. However, no length dependence of the recombination frequency in intergenic crosses with a distance between mutations of 328 to greater than 10000 bp is observed. We attribute this lack of dependence to the high rate of viral DNA interchange, which makes some step other than the cross-over event rate-limiting. Furthermore, we believe that the observed difference in recombination frequency between inter- and intragenic recombination is due to complementation between temperature-sensitive mutants at the permissive temperature.
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Frequent detection of bovine polyomavirus in commercial batches of calf serum by using the polymerase chain reaction
More LessTwenty commercial batches of calf serum, obtained from several suppliers, were tested for the presence of bovine polyomavirus (BPyV) DNA and antibodies against the virus. Using polymerase chain reaction (PCR) technology, BPyV DNA was detected in 70% of the batches; no BPyV was detected in any of the negative control samples. The specificity of the amplification reactions was proven by hybridization. PCR results were confirmed by virus isolation experiments performed with five PCR-positive and five PCR-negative serum batches. The results indicate that the use of calf serum to supplement tissue culture media involves a serious risk of contaminating cell cultures with BPyV. No correlation was observed between the presence or absence of anti-BPyV immunoglobulins and the detection of BPyV-specific DNA sequences in the serum batches.
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Mapping of the epitopes of Epstein—Barr virus gp350 using monoclonal antibodies and recombinant proteins expressed in Escherichia coli defines three antigenic determinants
The Epstein—Barr virus (EBV) major surface membrane antigen (MA), gp350/220, induces antibodies that neutralize virus infectivity in vitro. The MA glycoprotein is encoded by nucleotides 1784 to 4504 of the BamHI L fragment of the EBV genome. To define the antigenic epitopes on gp350, sequences encoding portions of the protein were cloned into an Escherichia coli expression system and eight recombinant clones were generated, two overlapping clones representing the C terminus and six overlapping clones representing the N terminus. The epitopes expressed by the recombinant proteins were mapped using 14 anti-MA monoclonal antibodies (MAbs) in a dot blot immunoassay. One of the MAbs reacted with clones that express the C terminus of gp350 and three others reacted with clones expressing the N terminal portion of the protein; the remaining MAbs tested were not reactive with the cloned proteins. The data identify three antigenic determinants on gp350. DNA sequences encoding these epitopes are located between nucleotides 1980 and 2307, 3186 and 3528, and 3528 and 3576 of the BamHI L fragment. In an attempt to elicit neutralizing antibodies, rabbits were immunized with gel-purified recombinant proteins from four of the clones. Neutralization assays indicate that the proteins expressed by these clones do not induce in vitro virus-neutralizing antibodies.
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Expression of HLA class I heavy chains and β 2-microglobulin does not affect human cytomegalovirus infectivity
More LessHuman cytomegalovirus (HCMV) purified from urine or tissue culture supernatant has been reported to contain β2-microglobulin (β2m), which forms the light chain of HLA class I molecules. It has been postulated that HCMV covered with β2m binds to HLA class I α-chains at the cell surface. In the present study we used transfected human and mouse cell lines expressing distinct allelic forms of HLA class I and β2m to determine whether HLA class I molecules could act as cellular receptors for HCMV. The susceptibility of cells to HCMV infection was estimated by calculating the percentage of cells expressing HCMV immediate early antigens. Although the results showed some variation between different transfected cell clones, no correlation was found between expression of HLA class I on the cell membrane and HCMV infection. Preincubation of HLA class I-positive cells with antibodies against HLA class I antigens inhibited HCMV infection after binding and adsorption of HCMV. Trypsin prevented HCMV infection of both class I-positive and class I-negative cells. We conclude that these results do not support the assumption that HLA class I molecules are functional receptors for HCMV.
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Induction of an endothelial cell growth factor by human cytomegalovirus infection of fibroblasts
More LessHuman cytomegalovirus (HCMV) induction of growth factors in fibroblasts was investigated after establishing serum-free culture conditions conducive to viral replication. HCMV infection induced a type II heparin-binding growth factor which stimulated human endothelial cell proliferation.
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Post-translational modification of the tegument proteins (VP13 and VP14) of herpes simplex virus type 1 by glycosylation and phosphorylation
More LessVP13 and VP14, major tegument proteins of herpes simplex virus type 1 (HSV-1) and the products of the UL47 gene, have been shown by partial proteolytic mapping to have closely related protein sequences. These proteins are phosphorylated in virus-infected cells, but not in preparations of purified virus. They also contain O-linked oligosaccharide units which include β-1,4-N-acetyl galactosamine residues, as demonstrated by the binding of Dolichos biflorus lectin. This modification was detected only in purified virus and appears to be restricted to VP13/14 and VP22, another HSV-1 tegument protein.
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Abolition of the RL neurovirulence phenotype of herpes simplex virus type 2 strain HG52 does not require deletion of the DR1 element of the ‘a’ sequence
More LessWe have characterized a spontaneous variant of herpes simplex virus type 2 (HSV-2) strain HG52, 2616, which has 786 bp of both copies of RL deleted, but which retains 782 bp upstream of the 5′ end of immediate early gene 1 and 463 bp downstream of the ‘a’ sequence. Variant 2616 is avirulent following intracerebral inoculation of BALB/c mice, thus showing in vivo characteristics similar to those of variant 2604, described previously (a 1488 bp deletion incorporating the DR1 element of the ‘a’ sequence), but not to those of variant 2701, which has an intermediate neurovirulence phenotype. The deletions in variants 2616 and 2604 remove the conserved HSV-1 and -2 RL open reading frame entirely and extend downstream from it, whereas the deletion in variant 2701 removes only the 5′ part. The data show that deletion of part of the ‘a’ sequence is not required for the production of the RL avirulence phenotype in HSV-2 strain HG52.
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Degenerate primers based on highly conserved regions of amino acid sequence in papillomaviruses can be used in a generalized polymerase chain reaction to detect productive human papillomavirus infections
More LessConserved amino acid sequences within the L1 open reading frame of the human papillomavirus (HPV) genome were used as a basis to design two degenerate primers (GP17 and GP18) and one general probe (GPR22) which direct polymerase chain reaction (PCR) amplification and subsequent detection of a 620 to 660 bp DNA fragment. The conserved nature of the primers and probe was tested experimentally on a panel of 24 cloned HPV DNAs isolated from cutaneous and mucosal lesions, including HPV-2a and -57, which are known to be associated with lesions at both anatomical sites. The sensitivity of this PCR test was at the level of genomic Southern blot analysis, indicating that HPV infections producing high copy numbers can be detected. Positive results were obtained with DNA extracted from clinical samples of genital and cutaneous origin.
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Activation of latent papillomavirus genomes by chronic mechanical irritation
More LessThe skin of animals of a laboratory strain of Mastomys natalensis carrying endogenous, latent papillomavirus genomes was irritated by scratching with glasspaper. Hyperproliferation of the epidermis and amplification of viral DNA followed this treatment, and in approximately 27% of the animals virus-producing papillomas were induced.
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Structural analysis of unstable intermediate and stable forms of recombinant fowlpox virus
More LessThe stability and structure of the products of recombination in a fowlpox virus (FPV) system using the thymidine kinase (TK) gene as the insertion site were examined. A 4.6 kb chimeric DNA fragment from the pUV1 expression vector, containing the bacterial lacZ gene and the vaccinia virus P7.5 promoter, was ligated into the XbaI site of the FPV TK gene. The resulting vector, pFTKlacZb, was transfected into chicken embryo fibroblast cultures infected with FPV at an m.o.i. of 0.1. Recombinants were screened for the expression of β-galactosidase. Five recombinants were isolated and plaque-purified to 80 to 90% for expression of β-glucosidase. Serial cell culture passage of the recombinants led to the gradual reappearance of the non-recombinant parental phenotype. Southern hybridization analysis of EcoRI fragments from all five recombinants indicated that a single cross-over homologous recombination had occurred between either the 5′ or the 3′ end fragments of the TK gene, generating unstable intermediate recombinants incorporating the entire pFTKlacZb vector. Secondary intermolecular or intramolecular recombination of intergenic repetitive sequences within the intermediate recombinants appears to have resulted in frequent regeneration of the parental genotype and an infrequent generation of more stable recombinants. A method was developed to select stable recombinants by passage of the intermediate recombinants in chicken embryo fibroblast cultures treated with 5-bromo-2′-deoxyuridine.
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Prostaglandin A inhibits replication of human immunodeficiency virus during acute infection
More LessAn antiviral effect of prostaglandins (PGs) of the A series on the replication of human immunodeficiency virus (HIV) has been determined. In the T cell line C8166 under single growth cycle conditions, PGA1 reduced the number of infectious progeny 1000-fold in the absence of cytotoxicity. Thus, inhibition of HIV replication by PGA1 represents a true antiviral phenomenon. The number and size of virus-induced syncytia, and the amount of viral antigen were also drastically reduced. The effect was specific for PGAs because PGA2 was also inhibitory, whereas PGB1, PGE1 and PGE2 were inactive. Virus adsorption and penetration do not appear to be targets of antiviral action because PGA1 substantially reduced virus replication, even when added 5 h post-infection. PGA1 did not inhibit viral reverse transcriptase, as determined by in vitro assays, suggesting that its antiviral action is not the consequence of a direct inhibitory effect on this enzyme. PGA1 also inhibited the replication of HIV-1 in CEM × 174 cells, but with less potency. Previously, intravenous infusion of PGA1 into human volunteers has shown no significant deleterious side-effects and thus these observations suggest that PGAs might have potential as antiviral agents in humans.
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Comparison of group B rotavirus genes 9 and 11
More LessGroup B rotaviruses (GBRs) were recognized recently as causative agents of gastroenteritis. Investigations into the relatedness of various heterologous GBR strains have been hindered by the difficulty of growing these viruses in cell culture. Viral RNA extracted from experimentally infected rats was used to prepare cDNA clones. From these, the nucleotide sequences of genes 9 and 11 of the IDIR strain of GBR were determined and compared with the corresponding sequences of the human ADRV strain of GBR. IDIR gene 11 is 643 bp in length with a single open reading frame (ORF) encoding 174 amino acids; IDIR gene 9 is 804 bp in length with a single ORF encoding 246 amino acids. Comparison of the IDIR sequences with those of ADRV showed that nucleotide sequence similarity was 60.6% and 71.9% for genes 9 and 11, respectively. The deduced amino acid sequence similarity was 51.2% for the gene 9 and 66.5% for the gene 11 product. This sequence diversity indicates that GBRs are more distantly related than strains of group A rotavirus.
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cDNA clones of Japanese hepatitis C virus genomes derived from a single patient show sequence heterogeneity
Twelve cDNA clones of Japanese hepatitis C virus (HCV) have been isolated from liver tissue of a single non-A, non-B hepatitis patient. These clones represented the non-structural domains of HCV. The degree of substitution in the nucleotide sequences and deduced amino acid sequences between these clones was 9.5 and 7.7%, respectively. This high level of substitution suggested that repeated infections of different HCVs may have occurred in the patient.
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Recombinant baculoviruses expressing yellow fever virus E and NS1 proteins elicit protective immunity in mice
More LessRecently, we showed that yellow fever virus (YFV) E and NS1 proteins in Spodoptera frugiperda cells infected with a recombinant baculovirus are similar, if not identical to those produced during YFV infection. To study the role of E and NS1 in the induction of protective immunity against fatal YFV challenge, these viral antigens were expressed either alone or in tandem via recombinant baculoviruses Ac-E.NS1, Ac-E1 and Ac-NS1. Swiss mice were immunized with lysates of insect cells infected with the recombinant baculoviruses. Solid protection against lethal YFV encephalitis was achieved after immunization with cell lysates containing the E protein with or without the NS1 protein. Mice inoculated with recombinant protein NS1 alone were not significantly protected but showed an increased survival time. Recombinant E protein expressed alone or in tandem with NS1 elicited a low but significant level of neutralizing antibodies. Although protein NS1 synthesized by recombinant baculovirus expressing E plus NS1 was more immunogenic than that expressed alone, neither strategy induced NS1-specific antibodies with complement-mediated cytolytic activity.
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Monoclonal antibodies differentiate between the haemagglutinating and the receptor-destroying activities of bovine coronavirus
More LessA relatively simple and sensitive method is described which enables the effect of monoclonal antibodies (MAbs) on the receptor-destroying enzyme (RDE) and the haemagglutination (HA) activity of bovine coronavirus (BCV) to be analysed in one assay. A lysate of HRT-18 cells infected with the L9 strain of BCV was found to have a higher RDE:HA ratio than purified virus. At 4 °C the lysate induced an HA pattern which completely disappeared upon raising of the temperature to 37 °C. This L9-infected cell lysate was used to determine the HA inhibition (HAI) titres of MAbs directed against the surface glycoproteins S and HE of BCV. Thereafter, the test plates were incubated at 37 °C to enable the ability of the MAbs to prevent elution of virus from BCV-erythrocyte complexes to be assessed. No inhibition of RDE was detectable with MAbs against glycoprotein S, which had HAI titres ranging from 1:16 to 1:128. On the other hand, MAbs directed against glycoprotein HE had similar HAI titres, but they inhibited elution of 8 HA units of BCV at titres of up to 1:65000.
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Comparison of the 5′ and 3′ untranslated genomic regions of virulent and attenuated foot-and-mouth disease viruses (strains O1 Campos and C3 Resende)
More LessThe complete 5′ and 3′ non-coding regions of two attenuated South American foot-and-mouth disease virus (FMDV) vaccine strains, O1C-O/E and C3R-O/E, and their corresponding virulent parental strains, O1 Campos and C3 Resende, have been cloned from polymerase chain reaction-amplified primary cDNA. Differences observed in the derived nucleotide sequences between attenuated and virulent viruses seem not to affect regulatory signal structures, supporting the theory that genetic variations, primarily in the 3′ halves of the viral genomes, contribute to the attenuation phenotype of the vaccine strains. In addition, this is the first report on the complete sequence of the 5′ untranslated region of a C-type aphthovirus. Approximately 10% of the nucleotides differ from the corresponding known sequences of serotypes A or O.
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Protective effects of monoclonal antibodies against lethal canine distemper virus infection in mice
Monoclonal antibodies (MAbs) against the haemagglutinin (H), fusion protein (F) and nucleoprotein of canine distemper virus (CDV) were examined for their ability to protect mice against lethal CDV infection. One MAb against H and two of six MAbs against F protected mice, the protective effect of the anti-H MAb being stronger than that of the anti-F MAbs. The anti-H MAb showed virus neutralizing activity, but the two anti-F MAbs, which recognized the same epitope, did not. Protection by the anti-F MAbs correlated with cell fusion inhibition, but not with complement-dependent neutralization, complement-dependent cytolysis or antibody-dependent cell-mediated cytotoxicity. These results suggest that neutralization by antibody against H and cell fusion inhibition by antibody against F play important roles in the protective mechanism against CDV infection.
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Acquisition of tomato yellow leaf curl virus by the whitefly Bemisia tabaci
More LessTomato yellow leaf curl virus (TYLCV) genomic DNA can be detected by Southern blot analysis in nucleic acid extracted from a single whitefly. Acquisition of TYLCV by individual whiteflies in relation to the length of the access period, the virus concentration in, and the developmental stage of plant tissues was studied. The frequency of TYLCV detection increased with the length of the access period; DNA was detected in 15% of whiteflies tested after a period of access to infected tissue of 30 min, regardless of whether it had a high or a low virus content (5 ng or 0.05 ng TYLCV DNA/µg plant chromosomal DNA), and in all insects tested after an 8 h period of access to all the plants. Those insects which had access to the youngest leaves of source plants, which have a high virus content, acquired detectable TYLCV DNA within 2 h. Insects which had access to a tissue for the same period acquired variable amounts of TYLCV DNA; insects feeding on plants with a low virus concentration acquired amounts of viral DNA comparable to those acquired by insects feeding on plants containing a 100-fold greater concentration of virus. Viruliferous insects retained TYLCV DNA for at least 13 days when placed on uninfected tomato plants. In these tests, whitefly could not acquire more than 600 million virus genomes (1 ng viral DNA), suggesting the existence of factors controlling the number of virions present in an insect.
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Tubular structures involved in movement of cowpea mosaic virus are also formed in infected cowpea protoplasts
More LessIn cowpea plant cells infected with cowpea mosaic virus, tubular structures containing virus particles are formed in the plasmodesmata between adjacent cells; these structures are supposedly involved in cell-to-cell spread of the virus. Here we show that similar tubular structures are also formed in cowpea protoplasts, from which the cell wall and plasmodesmata are absent. Between 12 and 21 h post-inoculation, tubule formation starts in the periphery of the protoplast at the level of the plasma membrane. Upon assembly, the virus-containing tubule is enveloped by the plasma membrane and extends into the culture medium. This suggests that the tubule has functional polarity and makes it likely that a tubule ‘grows’ into a neighbouring cell in vivo. On average, 75% of infected protoplasts were shown to possess tubular structures extending from their surface. The tubule wall was 3 to 4 nm thick and they were up to 20 µm in length, as shown by fluorescent light microscopy and negative staining electron microscopy. By analogy to infected plant cells, both the viral 58K/48K movement and capsid proteins were located in these tubules, as determined by immunofluorescent staining and immunogold labelling using specific antisera against these proteins. These results demonstrate that the formation of tubules is not necessarily dependent on the presence of plasmodesmata or the cell wall, and that they are composed, at least in part, of virus-encoded components.
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The complete nucleotide sequence of pea seed-borne mosaic virus RNA
More LessThe complete nucleotide sequence of the RNA genome of pea seed-borne mosaic virus (PSbMV) was determined from cloned cDNA and by direct sequencing of viral RNA. The PSbMV genomic sequence was determined to be 9924 nucleotides in length excluding the poly(A) tract. The RNA contained an open reading frame (ORF) of 9618 nucleotides with the potential to encode a polyprotein with a calculated M r of 364000 (364K). The ORF was flanked by a 5′ untranslated leader sequence of 143 nucleotides and a 3′ untranslated region of 163 nucleotides. A comparison of the PSbMV polyprotein with the polyproteins of the potyviruses tobacco etch virus (TEV), tobacco vein mottling virus (TVMV), plum pox virus (PPV) and potato virus Y (PVY) showed that PSbMV had a similar genome organization. The polyproteins had a high level of amino acid identity except in the N-terminal region, which varied in both sequence and length. Putative proteolytic cleavage sites were identified in the polyprotein of PSbMV by comparison with those identified for other potyviruses. The cleavage site between the 6K protein and the 49K proteinase is proposed to occur at the C-terminal side of glutamic acid and not at the C-terminal side of glutamine as in other potyviruses. In addition to the five proteolytic cleavage sites for the 49K proteinase identified previously, a sixth putative cleavage site was identified internally in the 49K proteinase of PSbMV, as well as in the 49K proteinases of TEV, TVMV, PPV, PVY and soybean mosaic virus. Cleavage at this site in the 49K proteinases of TEV, TVMV and PPV would result in an N-terminal protein of 22K to 24K, which is similar in size to the size determined for their VPgs.
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The location of the 5′ end of the potato leafroll luteovirus subgenomic coat protein mRNA
More LessNorthern blot analysis of nucleic acid from potato plant tissues and tobacco protoplasts infected with a Scottish isolate of potato leafroll luteovirus (PLRV) detected the 6 kb genomic RNA and one subgenomic RNA species of about 2.7 kb; RNA extracted from virus particles contained only the genomic species. Blotting with small defined probes suggested that the location of the 5′ end of the subgenomic RNA was between 2380 and 2510 nucleotides from the 3′ end of the PLRV genome (between 3370 and 3500 nucleotides from the 5′ end of PLRV Dutch isolate RNA). When RNA extracted from PLRV-infected or mock-inoculated protoplasts was used as the template for primer extension using primers complementary to the sequence at, or upstream of, the initiation codon of the coat protein gene, a single major infection-specific product was detected. A primer complementary to the sequence between 162 and 179 nucleotides upstream of the coat protein AUG yielded a product of 56 nucleotides. Thus, the subgenomic RNA has a leader sequence of 212 nucleotides, is 2505 nucleotides in length and starts at a position equivalent to 3376 nucleotides from the 5′ end of the PLRV-Dutch genome, 11 nucleotides upstream of the termination codon of the putative polymerase gene. The nucleotide sequence immediately downstream of this position closely resembles that of the 5′ end of the PLRV genomic RNA.
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Infectious in vitro RNA transcripts derived from cloned cDNA of the cucurbit potyvirus, zucchini yellow mosaic virus
More LessA full-length cDNA clone of the RNA genome of the cucurbit potyvirus zucchini yellow mosaic virus (ZYMV) was constructed downstream from a bacteriophage T7 RNA polymerase promoter. A single extra guanosine residue not present in ZYMV RNA was added to the 5′ and 3′ ends. Capped (m7GpppG) ZYMV RNA transcripts were infectious in 10 of 91 Cucurbita pepo test plants; uncapped RNA transcripts were not infectious. The appearance of symptoms in plants inoculated with the infectious transcript was delayed for more than a week compared to plants inoculated with native viral RNA. The progeny virions recovered from infected plants had the same biological properties (aphid non-transmissibility and typical symptoms) as the parental virus. The progeny virions also reacted positively with ZYMV antiserum and ZYMV-specific probes by dot blot hybridization. The authenticity of the progeny virus was verified by identifying a specific molecular marker (C substituted for T in the 3′ non-coding region) using nucleotide sequence analysis.
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Expression of the tobacco mosaic virus movement protein using a baculovirus expression vector
More LessA cDNA clone of the tobacco mosaic virus 30K movement protein (MP) gene was constructed and introduced into an Autographa californica nuclear polyhedrosis baculovirus expression vector. Infection of Spodoptera frugiperda cells with the vector resulted in the synthesis of low levels of MP, which was detected by anti-MP serum as two closely related species of M r approximately 34K and a third species of 32K. The authenticity of the recombinant MP was confirmed by comparison of the protein, on the basis of migration during SDS-PAGE, with authentic MP from several sources. It appeared that the recombinant MP was not modified by N-linked glycosylation, but was phosphorylated. The recombinant MP was produced in both a phosphorylated and an unphosphorylated state and the former species was shown to comigrate with plant-expressed MP during SDS-PAGE.
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Nucleotide sequences of genome segments S8, encoding a capsid protein, and S10, encoding a 36K protein, of rice gall dwarf virus
More LessThe nucleotide sequences of DNAs complementary to the eighth (S8) and the tenth (S10) largest of the 12 genome segments of rice gall dwarf virus (RGDV) were determined. The S8 and S10 segments consist of 1578 and 1198 nucleotides, each with a single open reading frame extending for 1278 nucleotides from nucleotide 21, and 960 nucleotides from nucleotide 22, respectively. S8 encodes a polypeptide of 426 amino acids with an M r of 47419. The amino acid sequences of several peptide fragments of the major outer capsid protein reported as 45K were contained in the predicted polypeptide. This protein, renamed the 47K protein, showed high homology with the outer capsid proteins of rice dwarf virus (RDV) and wound tumour virus (WTV); there was 56, 52 and 48% amino acid sequence identity between RGDV and WTV, RGDV and RDV, and RDV and WTV, respectively. S10 had the coding potential for a polypeptide of 320 amino acids with an M r of 36095 (36K protein), which exhibits 32% and 35% amino acid sequence identity with the predicted translation product of RDV S9 and the P9 capsid protein encoded by WTV S11, respectively. The conserved terminal sequences 5′ GG…GAU 3′ which are present in all genome segments of WTV and RDV so far analysed, and in S9 of RGDV, were also found in RGDV S8 and S10. This conserved sequence together with the segment-specific inverted repeats found in the terminal sequence of RGDV S8 and S10 are thus characteristic structures common to all three phytoreoviruses. The nucleotide sequence of the region surrounding the inverted repeats was more similar between RGDV and WTV than between RGDV and RDV.
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Use of the asymmetric polymerase chain reaction and DNA sequencing to determine genetic variability of bean golden mosaic geminivirus in the Dominican Republic
More LessA combination of the polymerase chain reaction (PCR), asymmetric PCR (A-PCR) and DNA sequencing was used to determine the nucleotide sequence of a hypervariable region of the bipartite genome of bean golden mosaic geminivirus (BGMV). This region, which was part of the intergenic region of the DNA-B component, was amplified using primers designed from the nucleotide sequence of a DNA-B component clone (pDRB1) of an isolate of BGMV from the Dominican Republic (BGMV-DR). pDRB1 is infectious on beans when coinoculated with the DNA-A component of BGMV-DR (pDRA1), and typical bean golden mosaic symptoms are observed on infected plants. Bean leaf tissue infected with BGMV was collected at five separate field locations in the Dominican Republic and the hypervariable region was amplified by PCR, ssDNA was produced using A-PCR, and partial nucleotide sequences were determined. The sequences of the hypervariable region from the field-collected samples ranged from 95% (one sample) to 98% (four samples) identical to the sequence of pDRB1. This contrasts with sequence identities of 86, 75 and 46% between the pDRB1 hypervariable region and the hypervariable regions of BGMV isolates from Guatemala, Puerto Rico and Brazil respectively, and 42% with bean dwarf mosaic geminivirus. These results indicate that Dominican Republic isolates of BGMV are very similar and should be considered isolates of the same virus (BGMV-DR), and that the infectious clones of BGMV-DR are representative of BGMV isolates in the Dominican Republic. The procedures described for DNA extraction from leaf tissue and for production of high quality ssDNA using PCR and A-PCR are rapid and efficient and could be applied to studies of variability and epidemiology of other viruses.
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Analysis of the potential promoter sequences of African cassava mosaic virus by transient expression of the β-glucuronidase gene
More LessDNA fragments from promoter regions of the geminivirus, African cassava mosaic virus, were cloned into pG1, a vector based on pUC18, producing transcriptional fusions with the β-glucuronidase (GUS) gene and nopaline synthase termination sequence. The activity of each promoter construct was assessed by analysing the transient expression of GUS in Nicotiana clevelandii protoplasts. The results demonstrated that constructs containing the common region of DNA A showed much stronger promoter activity in the complementary sense than in the viral sense. These results were supported by the analysis of promoter activity in transgenic N. benthamiana plants. In comparison, in protoplasts a region upstream of the AC2 open reading frame was shown to have moderate promoter activity. Unlike DNA A, the complementary sense DNA B promoter constructs had weak activity; the viral sense DNA B promoter constructs appeared to be regulated by host factors. The implications of these results for the regulation of early and late genes are discussed.
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Detection of the movement protein of red clover necrotic mosaic virus in a cell wall fraction from infected Nicotiana clevelandii plants
More LessThe movement protein of red clover necrotic mosaic virus (RCNMV) was expressed in Escherichia coli as a fusion with a maltose-binding protein using the vector pMAL-cRI and used to produce an antiserum. The RCNMV movement protein was detected in a cell wall fraction obtained from infected Nicotiana clevelandii leaf tissue by immunoblotting using the movement protein antiserum. The movement protein could be detected 6 h after inoculation and reached a maximum after 24 h. In contrast, the virus capsid protein, detected in a soluble fraction by immunoblotting using a capsid antiserum, continued to increase for 72 h after inoculation.
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