- Volume 75, Issue 7, 1994
Volume 75, Issue 7, 1994
- Animal
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Superior expression of juvenile hormone esterase and β-galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters
More LessThe expression characteristics of the p10, polyhedrin and basic protein promoters of Autographa califomica nuclear polyhedrosis virus were compared using two reporter enzymes, juvenile hormone esterase (JHE) and β-galactosidase. In these systems, JHE is exported from the cell and β-galactosidase is localized to the cytosol. Expression of JHE from the basic, p10 and polyhedrin promoters was first detected in the medium at 13,19 and 27 h post-infection respectively. The basic protein promoter yielded the highest expression of the three promoters tested for both enzymes, as determined by protein and enzyme activity assays. In addition, yields of β-galactosidase and JHE under control of the p10 promoter are higher relative to expression under control of the polyhedrin promoter. These data highlight the importance of investigation of viral promoters other than the polyhedrin promoter for high yield protein expression in vitro, and for insecticidal use of recombinant baculoviruses requiring high levels of expression. The results support revision of the current concept that very late viral promoters are always optimal for high yield recombinant protein expression.
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Insect iridescent virus type 6 encodes a polypeptide related to the largest subunit of eukaryotic RNA polymerase II
Cytoplasmic DNA viruses encode a DNA-dependent RNA polymerase (DdRP) that is essential for transcription of viral genes. The amino acid sequences of known large subunits of DdRPs contain highly conserved regions. Oligonucleotide primers, deduced from two conserved domains [RQP(T/S)LH and NADFDG- DE] were used in PCR experiments for the detection of the corresponding gene of the genome of insect iridescent virus type 6, also known as Chilo iridescent virus (CIV). A specific DNA product of about 150 bp could be amplified and was used as a hybridization probe against the CIV gene library to identify the corresponding gene. The gene encoding the DdRP was identified within the EcoRI fragments M (7099 bp) and L (7400 bp) of CIV DNA, between map units 0·310 and 0·347 (7990 bp). The DNA nucleotide sequence (3153 bp) of the gene encoding the largest subunit of DdRP (RPO1) was determined. Northern blot hybridization revealed the presence of a 3·4 kb RNA transcript in CIV-infected cells that hybridized to the CIV DdRP gene. This predicted viral protein consists of 1051 amino acid residues (120K) and showed considerably higher similarity to the largest subunit of eukaryotic RNA polymerase II than to the homologous proteins of vaccinia virus and African swine fever virus. Phylogenetic analysis suggested that the putative RPO1 of CIV could have evolved from RNA polymerase II after the divergence of the three types of eukaryotic RNA polymerases. The putative RPO1 of CIV lacked the C-terminal domain that is conserved in eukaryotic, eubacterial and other viral RNA polymerases and in this respect was analogous to the RNA polymerases of Archaea. It is hypothesized that the equivalent of the C-terminal domain may reside in another subunit of CIV DdRP encoded by an unidentified viral gene.
Nucleotide sequence data reported in this paper are deposited in the GenBank data library under accession no. M81388.
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Analysis of C-terminally truncated dengue 2 and dengue 3 virus envelope glycoproteins: processing in insect cells and immunogenic properties in mice
More LessWe constructed two recombinant Autographa califomica nuclear polyhedrosis baculoviruses. Spodoptera frugi-perda (Sf9) cells containing these constructs produce carboxy-terminally truncated envelope E proteins representing dengue (DEN) virus serotypes 2 and 3. The two recombinant proteins contained their homologous signal sequences at the N terminus and were truncated by 71 and 74 amino acids at the C terminus, respectively. This allowed the translocation of the recombinant proteins to the endoplasmic reticulum followed by glycosylation processing and secretion into the extracellular medium. An additional unglycosylated form which was not secreted was detected inside the infected Sf9 cells. Sera from Swiss mice immunized with the infected Sf9 cell lysates gave a DEN cross-reactive response in ELISA and substantial amounts of neutralizing antibodies to the homologous virus. Similar antibody titres were obtained when the two recombinant proteins were inoculated concomitantly. BALB/c mice were vaccinated with three doses of the recombinant E proteins, taken as monovalent or bivalent immunogens, and challenged with mouse-adapted DEN-2 virus. DEN-2 E protein induced a good protection (90 %) against lethal encephalitis and recombinant DEN-3 E protein gave a substantial cross-protection (54%). Eighty-two percent of the mice immunized with a mixture of both recombinant E proteins survived the DEN-2 virus challenge.
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A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout
More LessViral haemorrhagic septicaemia (VHS) is a fish rhabdo- virus infection of world-wide importance. Control policies have been established but the disease still causes heavy losses in fish farming. The development of a recombinant subunit vaccine was initiated to produce a Safe and effective vaccine to protect fish against VHS. The VHS virus (VHSV) glycoprotein, which induces neutralizing antibodies in rainbow trout, was chosen for expression in insect cells using a baculovirus vector. The M r of the recombinant protein estimated by SDS-PAGE was slightly lower than that of the native viral protein. The recombinant protein displayed different degrees of glycosylation and was recognized in ELISA by neutralizing antibodies. It was transported to the plasma membrane of insect cells where its ability to induce membrane fusion was preserved. The efficacy of the recombinant protein as a vaccine was compared with those of an inactivated and an attenuated vaccine. When injected intraperitoneally into rainbow trout, the baculo- virus-encoded protein was shown (i) to induce the synthesis of VHSV-neutralizing antibodies and (ii) to confer protection against virus challenge. Immunization performed by immersion failed. This is the first report of a recombinant vaccine that protects fish against VHSV.
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Circulating cytotoxic T lymphocyte precursors in maedi-visna virus-infected sheep
More LessMaedi-visna virus (MVV)-specific cytotoxic T lymphocytes (CTL) were detected, after in vitro culture with MVV antigen and recombinant human interleukin-2, in the efferent lymph and peripheral blood of sheep chronically infected with MVV. Cytotoxicity was mediated by CD8+ lymphocytes and was specific for particular strains of MVV. These precursor CTL were detected in the blood between day 23 and day 100 after infection via the skin. In one out of seven persistently infected sheep MVV-specific cytotoxicity was seen in uncultured peripheral blood cells. Again the effector population consisted of CD8+ lymphocytes. The only other viral infections in which CTL have been detected in peripheral blood mononuclear cells prior to secondary stimulation are those caused by the simian and human immunodeficiency viruses.
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Definition of the range and distribution of human immunodeficiency virus macrophage tropism using PCR-based infectivity measurements
More LessThe tropism of human immunodeficiency virus (HIV) for macrophages (mø)is a well recognized phenomenon, but the range and distribution of m𝜙-tropic phenotypes have not been defined by quantitative means. This study uses a PCR-based infectivity assay to derive an index of m𝜙 tropism for several common strains of HIV. The results show that m𝜙 tropism varies over about six orders of magnitude and that the most m𝜙-tropic strains have a higher infectivity for m𝜙 than for peripheral blood lymphocytes. Strains were distributed throughout this range, suggesting that m𝜙 tropism is a continuously variable phenotypic property. Although the degree of tropism was strongly influenced by the mode of isolation and propagation of virus strains, there was no evidence for the existence of distinct m𝜙-tropic or non-m𝜙-tropic phenotypes. Finally, the tropism of two selected strains was found to be determined by an early step in replication, probably virus entry.
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DNA found in human immunodeficiency virus type 1 particles may not be required for infectivity
More LessWe have studied the presence and significance of retroviral genome-derived DNA in the core of human immunodeficiency virus (HIV) particles produced from transfections of HXB2 expression vectors in COS-7 cells and from HIV type 1 IIIB chronically infected H9 cells. Viruses purified by sucrose cushion centrifugation and treated with DNase I contained 1000-fold more viral RNA than DNA. However protease-defective viruses that contained only p160 gag−pol had less than 100 times the amount of DNA in their cores than wild-type viruses suggesting that the p66/p51 form of reverse transcriptase was responsible for DNA transcription. Viruses produced by transfections in the presence of 3′-azido-3′-deoxythymidine (AZT) contained the viral RNA genome but only DNA of premature length because of the chain terminating effects of AZT. However such viruses were as infectious for CD4+ cells as wild-type virus. We conclude that retrovirus-derived DNA in HIV-1 particles is not required for infection and does not play a significant role in this process.
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Phylogenetic relationship between human immunodeficiency virus type 1 (HIV-1) long terminal repeat natural variants present in the lymph node and peripheral blood of three HIV-1-infected individuals
More LessPCR has been used to amplify a 540 base pair fragment of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat region encompassing the U5/R/U3 regions from proviral HIV DNA that was present in the lymph nodes and peripheral blood mononuclear cells of three HIV-infected individuals. The resulting PCR products were cloned and the DNA sequence of multiple clones from each reaction was determined to enable the distribution and phylogenetic relationship between the variants in each body site to be assessed. The results of bootstrapped parsimony phylogenetic analyses showed that a distinct polarization was evident between peripheral blood and lymph node variants in the patient who possessed a histologically intact lymph node. In contrast, similar analyses conducted on samples from two patients with extensive lymph node disruption showed similar variants in lymph node and peripheral blood. In the patient with an intact lymph node, lymph node variants were significantly more heterogeneous than those present in blood samples taken either simultaneously or 11 months later. No differences in heterogeneity between lymph node and peripheral blood were observed in patients with disrupted lymph nodes. The significance of these results for our understanding of HIV pathogenesis is discussed.
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Calcium-mediated inhibition of phorbol ester and Tax trans-activation of the human T cell leukaemia virus type 1
More LessHuman Jurkat T cells containing a stably integrated human T cell leukaemia virus type 1 (HTLV-1) long terminal repeat (LTR) reporter gene construct were used to study the role of calcium-dependent cellular activation pathways in LTR trans-activation. Treatment of these cells with the calcium ionophore ionomycin resulted in a reduced basal response of the LTR and reduced responses to 12-O-tetradecanoylphorbol-13-acetate- and Tax-mediated trans-activation. This effect was also observed for virus production in the HTLV-1-producing T cell line MT-2. Experiments designed to determine the events underlying this inhibition, using inhibitors of calcium-related events, revealed that the ionomycin- induced repression of the LTR was alleviated in all cases by cyclosporin A. This compound was also effective in preventing the ionomycin-induced reduction in virus production in MT-2 cells. These results suggest a role for calcium-related events in the down-regulation of HTLV-1 expression.
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Characterization of the temporal accumulation of minute virus of mice replicative intermediates
More LessWe have characterized the temporal appearance and accumulation of minute virus of mice (MVM) replicative forms (RF) in highly synchronized single rounds of infection using a combination of restriction endonuclease analysis and two-dimensional agarose gel electrophoresis. Between 4 and 12 h after release of infected cells into the S-phase, both monomer (mRF) and dimer RF (dRF) increased exponentially at similar rates such that the ratio of mRF relative to dRF remained unchanged. These DNA forms accumulated at a faster rate than MVM RNAs, suggesting that the number of DNA templates available for replication is limiting, not the expression of MVM gene products, and that the majority of DNA templates are likely to be destined for DNA amplification rather than transcription and further gene expression. During this exponential DNA amplification phase, approximately 65 % of mRF were in a fully extended form, whereas most of the remaining mRF were covalently closed in the left end and extended in the right end. Although MVM replication presumably generates right-hand turn-around mRF, only a low level of this form persists (5 to 10 % of total mRF) at all times examined, suggesting that this form must be quickly converted to the extended form. Greater than 90% of dRF, which have right-hand palindromes on both ends of the molecule, were extended on both ends. A significant proportion of dRF and higher concatemers are nicked in the left-hand palindrome, suggesting that resolution of dRF into two mRFs may occur via single-stranded nicks rather than a double-stranded cut. An additional replicative form, previously termed band X, has been identified as an RNA-DNA duplex. This band is formed predominantly intracellularly, before cell lysis but its biological significance remains unclear. Our results provide direct experimental support for many of the predictions of the current models of parvovirus replication and suggest that the kinetic hairpin transfer model should be adjusted to include a strand-transfer or similar mechanism for the resolution of dRF to account adequately for the production of left-end turn-around forms.
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Human papillomavirus (HPV) type 18 E7 protein is a short-lived steroid-inducible phosphoprotein in HPV-transformed cell lines
More LessWe used a capture ELISA to quantify the E7 protein of human papillomavirus type 18 (HPV-18). In HeLa cells, which express low levels of immunoreactive E7 protein (iE7), iE7 had a mean half-life of 13·5 min. In HPV-18 E7 recombinant baculovirus (E7rec BV)-infected Sf21 cells, which express higher levels of E7, the half-life of iE7 was much longer (90 min and > 24 h, with two different E7rec BVs). For two transformed human cervical cell lines expressing HPV-18 E7, exposure of the cells to hydrocortisone resulted in a twofold increase in steady-state levels of the E7 protein: no similar effect was observed with progesterone, oestrogen or testosterone. The half-life of iE7 was unaltered by hydrocortisone or progesterone exposure. An immunoassay which distinguished Ser33-phosphorylated E7 from E7 not phosphorylated at this residue (Ser33dephospho-E7), showed that in HeLa and Sf21 cells the majority of E7 was phosphorylated : the half-life of both species of E7 was similar in HeLa cells, but the half-life of Ser33dephospho-E7 was much shorter (90 min) in Sf21 cells than that of Ser33phospho-E7 (> 24 h). A HeLa- fibroblast fusion cell line with tumorigenic potential (CGL-1) had a similar ratio of dephospho-E7 to total E7 (0·06), as a similar fusion cell line (CGL-4) with no tumorigenic potential (0·03). We conclude that E7 is a labile phosphoprotein, and that the expression and steady-state level of the E7 protein in eukaryotic cells may be influenced by the hormonal environment of the cells.
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Nucleotide sequence of a 55 kbp region from the right end of the genome of a pathogenic African swine fever virus isolate (Malawi LIL20/1)
The nucleotide sequence data in this paper will appear in the EMBL database under the accession no. X71982.
The nucleotide sequence of a 55098 bp region from the right end of the genome of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) has been determined. Translation of the sequence identi fied 67 major open reading frames (ORFs) which are closely spaced and read from both DNA strands. At six positions intergenic tandem repeat arrays are found. Comparison of the predicted amino acid sequences of encoded proteins with protein sequence databases identified a number of homologies. These include three subunits of RNA polymerase, a protein with homology to transcription factor SII (TFSII), a DNA ligase, two subunits of mRNA capping enzyme, a DNA topo- isomerase type II, a dUTPase, a protein kinase, three helicases, a ubiquitin-conjugating enzyme, a protein with homology to the nif S and nif S-like proteins identified in some bacteria and Saccharomyces cerevisiae, a protein with homology to both a myeloid differentiation primary response antigen (MyD116) and to a herpes simplex virus-encoded neurovirulence-associated protein (ICP34.5), a protein with homology to the ASFV-encoded structural protein p22, two proteins with homology to copies of the ASFV-encoded multigene family 360 and one protein with homology to the ASFV- encoded multigene family 110. Four genes encode proteins which have homology to each other and constitute a new multigene family (MGF100). Nine ORFs encode proteins which contain predicted transmembrane domains. The possible functions of these predicted ASFV-encoded proteins are discussed and the evolutionary relationship of ASFV to other viruses are considered. Despite the similarities in genome structure and replication strategy of ASFV with poxviruses, sequence similarity between them is low and the organization of ASFV-encoded genes is not colinear with that of the orthopoxviruses.
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Trans-activation of the mouse cytomegalovirus immediate early gene enhancer by ras oncogenes
The ras gene family encodes 2IK proteins that reside on the inner face of the plasma membrane and bind GTP and GDP with an equally high affinity. Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a viral Harvey-raj (v-Ha-ras) cDNA, together with a plasmid (pCMVCAT) carrying the immediate early (IE) enhancer of the murine cytomegalovirus (MCMV) linked to the chloramphenicol acetyltrans- ferase (CAT) reporter gene strongly stimulated CAT activity. Basal levels of pCMVCAT expression as well as trans-activation by v-ras plasmid were both inhibited by cotransfection of an expression vector containing the dominant inhibitory mutant gene Ha-ras Asn-17. This indicates that the p21 ras protein is responsible for these activities. High pCMVCAT activation was also observed in cell lines carrying stably transfected ras oncogenes, activated by point mutation or amplification. To define the cis-acting DNA elements in the MCMV IE enhancer responsible for this trans-activation by p21 ras protein, we constructed several plasmids containing the CAT gene under control of MCMV IE enhancers that were deleted in different regions. The CAT assays demonstrated that several sequences were responsive to p21 ras protein. These sequences are scattered throughout the IE enhancer, upstream of the transcription start site, and contain responsive elements that are homologous to the binding sites for cellular transcription factors such as NFkB, API, ATF and SP1. Activation of the p2l ras protein may thus be one of the signals that regulate IE genes transcription during MCMV infection.
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The DNA sequence coding for the 5′ untranslated region of herpes simplex virus type 1 ICP22 mRNA mediates a high level of gene expression
The sequence coding for the 5′ untranslated region (UTR) of ICP22 mRNA of herpes simplex virus type 1 has been tested for its ability to regulate gene expression. This sequence was placed in frame with the chloramphenicol acetyltransferase (CAT) coding sequence and under the control of the simian virus 40 early promoter-enhancer. Under these conditions, the sequence coding for the 5 UTR led to an increase of about 13-fold in CAT activity, measured during transient expression. The use of mutants with progressive deletions within the sequence coding for the 5′UTR allowed localization of the sequence responsible for the enhancement of gene expression to the first exon of the ICP22 gene. Precise quantification of hybrid ICP22-CAT mRNA showed that the sequence coding for the 5′UTR induced an increase in the amounts of transcripts, which resulted in a parallel increase in CAT activity. This increase in the level of hybrid ICP22-CAT mRNA is not the result of an increase in mRNA stability, nor is it due to more efficient nucleo-cytoplasmic transport of the transcripts. Moreover, the distribution of hybrid mRNA in the different ribosomal populations indicates that the 5′UTR of ICP22 mRNA does not induce a preferential recruitment of the transcripts by the translational apparatus. Taken together, these results indicate that a cis-acting element located in the sequence coding for the 5′UTR of ICP22 mRNA can mediate a high level of gene expression independently of the viral promoter and of viral trans-acting factors.
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BICP22 of bovine herpesvirus 1 is encoded by a spliced 1·7 kb RNA which exhibits immediate early and late transcription kinetics
More LessKinetic analysis of the two divergent immediate early (IE) transcription units of bovine herpesvirus 1 (BHV-1) revealed an unexpected behaviour. The IE1.7 promoter was not turned off at the end of the IE period but acted as a late promoter, unlike the adjacent IE4.2/2.9 promoter which was active only under IE conditions. The genome region specifying the IE 1.7 gene was sequenced (0·814 to 0·839 map units). The IE1.7 promoter was found to overlap with duplicated sequence elements bearing close similarity to herpesvirus origins of replication, which may explain the biphasic transcription kinetics. Exons 1 and 2 of the spliced IE1.7 transcript were non-coding. Exon 3 was found to contain a single open reading frame encoding a protein of 300 amino acids that was designated BICP22 because of its homology to ICP22 (Vmw68) of herpes simplex virus type 1 and related proteins from other herpesviruses. The protein probably represents IEP-55, the most abundant BHV-1 phosphoprotein observed under IE conditions.
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Identification of novel transcripts complementary to the Marek's disease virus homologue of the ICP4 gene of herpes simplex virus
More LessLibraries of cDNA were generated from polyadenylated RNAs derived from Marek’s disease virus (MDV)- transformed cell lines by directional cloning of oligo- (dT)-primed cDNAs in lambda gt22A. Analysis of the libraries for viral sequences showed that a number of cDNA clones originated from transcripts mapping in the BamHl A region of the MDV genome. Sequencing and line mapping of these cDNAs suggested that the RNA transcripts expressed from this region were either in the sense or antisense direction with respect to the MDV homologue of the ICP4 gene of herpes simplex virus. The longest cDNA clone from antisense transcripts was 2756 bp long and partially overlapped the 5′ end of the coding region of the ICP4 gene. The cDNA clone contained at least four introns, shown by comparison of its sequence with the sequence of the ICP4 gene. The presence of introns was confirmed by PCR analysis. All the introns have the consensus splice donor and acceptor signals at their 5′ and 3′ ends respectively. Northern blot analysis showed that the ICP4 gene homologue of MDV was abundantly transcribed only in lytically infected fibroblasts, whereas transcripts complementary to the ICP4 gene were the major transcripts in MDV- transformed cell lines and lymphomas. The transcripts complementary to ICP4 consist of two major RNA species approximately 15 kb and 1·32 kb long. The results suggest that there might be an inverse relationship between the abundance of ICP4 transcripts and their complementary transcripts in MDV-infected and transformed cells.
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Characterization of a quadruple glycoprotein-deleted pseudorabies virus mutant for use as a biologically safe live virus vaccine
More LessHerpesvirus envelope glycoproteins play important roles in mediating infection initiation and also represent major immunogens. We recently showed that a pseudorabies virus (PrV) mutant lacking the essential glycoprotein gD (gp50), after phenotypic complementation by propagation on genetically engineered PrV gD- expressing cell lines was able to infect primary target cells and spread exclusively by means of direct cell-to- cell transmission. Virions released from non-complementing cells that lacked gD were not infectious because of a defect in penetration and so free infectious virions did not arise after infection of animals by phenotypically complemented gD-negative PrV. This formed the basis for the development of novel non-spreading live herpesvirus vaccines. However, the gD-negative PrV mutant still retained a residual level of virulence, which prevented its use as vaccine, and the need to propagate the gD-negative PrV mutant on trans-complementing cell lines resulted in the appearance of wild-type revertants, rescued by the resident gene in the cell line. To overcome these problems we isolated a PrV mutant designated PrV(376) that, in addition to gD, also lacked the non-essential glycoproteins gG, gl and gE. PrV(376), because of the lack of gD, was also dependent on gD- expressing cells for productive replication. Non-complementing cells infected by phenotypically gD-comple- mented PrV(376) produced non-infectious particles lacking glycoproteins gD and gE as shown by immuno- electron microscopy. Owing to the absence of any homologous sequences between the viral genome and the viral genes resident in the complementing cell line, rescue by homologous recombination was impossible. In cell culture, plaques of PrV(376) were significantly smaller than those of either wild-type, or gD- or gE- deleted mutants, indicating an additive or synergistic effect of the combined deletion on viral cell-to-cell spread capability. Intranasal or intramuscular infection of pigs with phenotypically gD-complemented PrV(376) showed a complete attenuation of viral virulence, with an expected lack of shedding of infectious virus. The PrV(376)-vaccinated pigs exhibited a significant level of protection against challenge infection, measured by survival and weight loss. In summary, PrV(376) represents a novel type of herpesvirus vaccine that combines innocuity, efficacy and biological safety.
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Characterization of immune responses to baculovirus-expressed equine herpesvirus type 1 glycoproteins D and H in a murine model
More LessA murine intranasal infection model for equine herpesvirus type 1 (EHV-1) was used to evaluate immune responses following immunization with insect cells infected by baculoviruses that express EHV-1 glycoproteins. Baculovirus recombinant glycoprotein D (gD) and gH both induced serum antibodies to EHV-1 when measured by ELISA. The gD recombinant also produced a neutralizing antibody response. Protective immunity, determined by accelerated clearance of virus from the target organs in the respiratory tract, was demonstrated in mice immunized with a baculovirus recombinant expressing gD. In addition to the serological response, evidence is presented which shows that cell-mediated responses also play an important role in protection. Both recombinants induced delayed-type hypersensitivity and lymphoproliferation to EHV-1 antigen. The protective effects of T cells were confirmed by adoptive transfer of spleen cells from baculovirus gD- immunized donors to recipients that were challenged with live EHV-1. Depletion of either CD4- or CD8- bearing cells from the gD-immunized donors reduced the ability of the recipients to clear virus from the target organs, although depletion of CD4 cells had a more marked effect.
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Analysis of the thymidine kinase of a herpes simplex virus type 1 isolate that exhibits resistance to (E)-5-(2-bromovinyl)-2′-deoxyuridine
More LessThe mechanism responsible for the decreased sensitivity of a clinical herpes simplex virus type 1 (HSV-1) isolate, HSV-145, to (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) was examined. Measurements of 50% inhibitory doses of several drugs demonstrated that although HSV-145 was sensitive to phosphonoacetic acid, adenine arabinoside and acyclovir, its sensitivity to BVDU and 5-(2-chloroethyl)-2′-deoxyuridine was significantly less than that normally observed for HSV-1. Analysis of the thymidylate kinase (TMP-K) activity of HSV-145 thymidine kinase (TK) demonstrated a decreased level of TMP-K activity when compared to HSV-1 TK. The TMP-K activity of HSV-145 resembled that observed for HSV-2 and the TK-deficient strain HSV-1 TK−7. When the nucleotide sequence of the HSV-145 TK gene was compared to that of the HSV-1 strains Cl(101) and SC16 a single nucleotide substitution (G changed to A at base position 502) was detected which would result in the substitution of threonine at amino acid position 168 for alanine. The substitution is the same as that for the laboratory- derived BVDU-resistant virus HSV-1 SC16B3. Collectively, these studies highlight the importance of amino acid conservation at position 168 of the HSV-1 TK in conferring efficient TMP-K activity and BVDU sensitivity.
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Herpes simplex virus L particles contain spherical membrane-enclosed inclusion vesicles
More LessThe fine structure of light (L) particles of herpes simplex virus type 1 was examined by cryo-electron microscopy and compared to that of virions. The L particles appeared to be spherical entities with significant variation in size, on average smaller in diameter than virions (140 nm compared to 180 nm). The technique confirmed that L particles are composed of an outer envelope, i.e. a bilaminar membrane with protruding glycoprotein spikes, and a uniformly granular tegument, but lack any nucleocapsid. In addition it revealed the presence of one or occasionally more spherical objects, termed inclusion vesicles (IVs), embedded in the tegument of a large proportion of L particles but not observed in virions, suggesting that presence of IVs is unique to the L particles. The IVs vary in size and appear to be composed of a bilaminar membrane without surface projections and filled with material of relatively low electron density, suggesting that the composition of IVs is distinct from that of the envelope and tegument of the L particles.
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Hepatitis C virus particle detected by immunoelectron microscopic study
To clarify the morphology of hepatitis C virus (HCV), an indirect immunogold electron microscopic study was carried out on two plasma samples with high HCV RNA titres using polyclonal and monoclonal antibodies specific to the putative HCV envelope protein. Spherical virus-like particles, 55 to 65 nm in diameter with spikelike projections, were found in 114 to 116 g/ml fractions after sucrose density gradient centrifugation. These particles were found only in HCV-infected blood donors and had morphological features similar to those of flaviviruses. Moreover, these particles specifically reacted with the polyclonal and monoclonal antibodies to the putative HCV envelope protein. This is the first known report in which the morphology of the HCV particle is clearly shown.
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Entire nucleotide sequence and characterization of a hepatitis C virus of genotype V/3a
The entire nucleotide sequence of a hepatitis C virus (HCV) genome (NZL1) of genotype V/3a was determined from overlapping cDNA clones obtained from a human carrier in New Zealand. It comprised 9425 nucleotides (nt) including a 5-untranslated region of 339 nt, a single large open reading frame encoding a polyprotein of 3021 amino acids, a 3′-untranslated region of 23 nt, and 3 -terminal poly(U) stretches of variable lengths. The NZL1 genome was compared with 15 HCV isolates of other genotypes for which the full- length sequence has been determined. It differed from them by 31·1 to 34·3% in nucleotide sequence identity and by 24·5 to 29·1% in amino acid sequence identity, confirming the distinction of genotype V/3a from the other isolates.
The nucleotide sequence data reported in this paper are deposited in the DDBJ/GenBank/EMBL Data Libraries under the accession no. D17763.
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Sialoglycoproteins that bind influenza A virus and resist viral neuraminidase in different animal sera
Sialoglycoproteins that are resistant to degradation by viral neuraminidase can effectively neutralize influenza A viruses, because they bind irreversibly to the viruses. To detect such proteins in animal sera, we developed an immunochemical assay based on Western blotting techniques. We assessed the binding activity of sialoglycoproteins in sera from nine different animals toward the A/Aichi/2/68 (H3N2) and A/PR/8/34 (H1N1) strains of influenza virus, with or without viral and bacterial neuraminidase treatment. Using this assay, we found that animal sera contain a spectrum of sialoglycoproteins defined by differing abilities to bind influenza A viruses and to resist the viral neuraminidase. Structural analysis of these inhibitors would provide useful information for the development of anti-influenza virus compounds.
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Comparative analysis of VP8* sequences from rotaviruses possessing M37-like VP4 recovered from children with and without diarrhoea
Rotavirus strains belonging to G types 1 to 4 and having a P3 genotype (M3 7-like VP4) were recovered from children with symptomatic and asymptomatic infections. Partial sequences of their VP4 genes were determined in an attempt to characterize these strains further. The genomic regions encoding VP8*, the connecting and putative fusion peptides and three other regions in VP5* were sequenced. The deduced amino acid sequences were compared with rotavirus strains belonging to different P genotypes that had been previously reported. High degrees of identity were found between the VP8* fragment of all human P3 strains (92·7 to 99·7%) suggesting that they belong to the same genotype, regardless of differences in their virulence. Furthermore, based on comparative sequence analysis, we did not identify any amino acid(s) that differ appreciably between symptomatic and asymptomatic strains and could therefore be associated with virulence. The results suggest that the P3 genotype, although frequently associated with asymptomatic infections, may not be the single determining factor in attenuation of symptoms.
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A comparison of the VP7 gene sequences of human and bovine rotaviruses
The sequences of the gene encoding VP7 (the major outer capsid protein) from one bovine and three human rotavirus strains were determined because of their unusual VP7 specificities. Two of the human strains (PA 169 and PA 151) had VP7 serotype 6 specificity whereas the two other strains, recovered from a child (HAL 1166) and a calf (678) belonged to VP7 serotype 8. The serotype 8 strains exhibited a high degree of sequence conservation when compared with each other and with other serotype 8 strains previously sequenced. The serotype 6 human strains shared a greater degree of sequence similarity with previously reported serotype 6 bovine strains than with other rotavirus serotypes; however the degree of sequence similarity among PA 169, PA 151 and the bovine strains was lower than had been previously reported for strains belonging to the same serotype. The demonstration of rotavirus serotypes that are shared between human and animal species supports the concept that interspecies transmission occurs and may play a role in rotavirus evolution.
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The complete sequence of a human astrovirus
More LessWe have determined the complete genomic sequence of human astrovirus serotype 1 isolated in Newcastle upon Tyne. The genome is 6813 nucleotides long and contains three sequential open reading frames (ORFs). The two closest to the 5′ end are linked by a ribosomal frameshifting motif and contain sequence motifs indicative of non-structural virus proteins: serine protease and RNA-dependent RNA polymerase. A nuclear addressing sequence is also located here. The 3′ ORF encodes the virion structural polypeptides as a polyprotein precursor. This genomic organization resembles that of the plant virus family Luteoviridae.
Nucleotide sequence data described in this paper appear in the EMBL database under accession no. Z25771.
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Nucleotide sequence and expression of the spike (S) gene of canine coronavirus and comparison with the S proteins of feline and porcine coronaviruses
More LessWe have cloned, sequenced and expressed the spike (S) gene of canine coronavirus (CCV; strain K378). Its deduced amino acid sequence has revealed features in common with other coronavirus S proteins: a stretch of hydrophobic amino acids at the amino terminus (the putative signal sequence), another hydrophobic region at the carboxy terminus (the membrane anchor), heptad repeats preceding the anchor, and a cysteine-rich region located just downstream from it. Like other representatives of the same antigenic cluster (CCV-Insavc-1 strain, feline infectious peritonitis and enteric corona- viruses, porcine transmissible gastroenteritis and respiratory coronaviruses, and the human coronavirus HCV 229E), the CCV S polypeptide lacks a proteolytic cleavage site present in many other coronavirus S proteins. Pairwise comparisons of the S amino acid sequences within the antigenic cluster demonstrated that the two CCV strains (K378 and Insavc-1) are 93·3% identical, about as similar to each other as they are to the two feline coronaviruses. The porcine sequences are clearly more divergent mainly due to the large differences in the amino-terminal (residues 1 to 300) domains of the proteins; when only the carboxy-terminal parts (residues 301 and on) are considered the homologies between the canine, feline and porcine S polypeptides are generally quite high, with identities ranging from 90·8 % to 96·8 %. The human coronavirus is less related to the other members of the antigenic group. A phylogenetic tree constructed on the basis of the S sequences showed that the two CCVs are evolutionarily more related to the feline than to the porcine viruses. Expression of the CCV S gene using the vaccinia virus T7 RNA polymerase system yielded a protein of the expected M r (approximately 200K) which could be immunoprecipitated with an anti-feline infectious peritonitis virus polyclonal serum and which was indistinguishable from the S protein synthesized in CCV-infected cells.
The nucleotide sequence data presented in this paper have been submitted to the EMBL database and assigned the accession number X77047.
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Molecular cloning and nucleotide sequencing of the 3′-terminal genomic RNA of the porcine reproductive and respiratory syndrome virus
More LessThe genomic RNA of a porcine reproductive and respiratory syndrome virus (PRRSV) isolate from the U.S.A., VR 2385 (ATCC), was copied into cDNA after priming with oligo(dT) and cloned into phage lambda. The cDNA clones representing the 3′-terminal genomic RNA of the virus were isolated and sequenced. The genome is a positive-stranded, polyadenylated RNA with an estimated size of 15 kb. Analysis of the resulting sequence identified three complete open reading frames (ORFs) with the potential to encode polypeptides with predicted M rs of 22·2K (ORF 5), 19·1K (ORF 6) and 13·6K (ORF 7). ORF 7, which is closest to the 3′ end, is predicted to encode a highly basic nucleocapsid protein displaying 58 % amino acid identity to the corresponding protein of the Lelystad virus (LV), a European PRRSV isolate. ORFs 6 and 5, preceding ORF 7, are each predicted to encode proteins containing several hydrophobic domains that are thought to be membrane- associated. The VR 2385 ORF 6 protein is the most conserved structural protein. It has 78% amino acid identity to the equivalent LV protein, and ORF 5 shares only 54% of its amino acid sequence. Northern blot analysis revealed a 3′-coterminal nested set of six subgenomic RNAs in VR 2385 virus-infected CRL 11171 cells. Our results indicate that VR 2385, like LV, is a member of the newly proposed arterivirus group. However, the striking genetic variation and the difference in pathogenicity between LV and VR 2385 suggest that the viruses causing PRRS in the U.S.A. and Europe are highly variable and they may represent different genotypes.
The nucleotide sequence data reported in this paper have been deposited with the GenBank database and assigned the accession number U03040.
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Apoptosis induced by infectious bursal disease virus
More LessChicken peripheral blood lymphocytes (PBLs) show morphological and biochemical features of apoptosis (programmed cell death) when infected in vitro with infectious bursal disease virus (IBDV). DNA extracted from IBDV-infected lymphocytes displayed an intense laddering pattern when visualized after agarose gel electrophoresis. IBDV-infected PBLs had significantly higher apoptotic and necrotic indices measured by acridine orange-ethidium bromide staining than did control lymphocytes. Electron micrographs of the IBDV-infected PBLs revealed features typical of apoptosis such as peripheral condensation of chromatin, blebbing of the plasma membrane and fragmentation of the nucleus and of the cell leading to the formation of apoptotic bodies. These findings indicate that IBDV, in addition to causing necrosis, can also induce apoptosis in avian lymphocytes in vitro.
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Sequence and expression of a baculovirus protein with antigenic similarity to telokin
More LessA protein from baculovirus-infected cells reacted with an antibody against the smooth muscle protein telokin. Because of this unusual similarity, the protein, termed telokin-like protein-20 (TLP20), was isolated and characterized. Its M r on denaturing polyacrylamide gels was 28K and the protein contained a high proportion of β structure. A cDNA for TLP20 was isolated and sequenced. The 3′ non-coding sequence contained a region of high identity with the 5′ end of two other baculovirus genes. The 5′ non-coding region contains several baculovirus regulatory elements. Surprisingly, the derived amino acid sequence showed no homologies to telokin. The cDNA was cloned into a bacterial expression vector and the subsequently expressed protein had a slightly lower M r than the native protein, but cross-reacted with telokin antibody. This paper reports the characterization of a new baculovirus protein that shares some antigenic similarities to the smooth muscle protein telokin.
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Identification of the gp67 gene of a baculovirus pathogenic to the spruce budworm, Choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus
More LessThe baculovirus gp67 gene encodes a pH-dependent membrane fusion protein and has been identified in both Autographa califomica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) and Orygia pseudotsugata MNPV (OpMNPV). We have identified a homologous gene in the spruce budworm virus, Choristoneura fumiferana MNPV (CfMNPV). The CfMNPV gp67 gene is 79% identical to AcMNPV gp67 at the level of nucleotide sequence and 82% identical at the level of predicted amino acid sequence. As with OpMNPV and AcMNPV gp67, the CfMNPV gp67 protein is found exclusively in the budded virus phenotype of the baculovirus.
The nucleotide sequence data reported in this paper have been submitted to the GenBank database and given the accession number L124120.
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Genome organization of the DNA-binding protein gene region of Cryptophlebia leucotreta granulosis virus is closely related to that of nuclear polyhedrosis viruses
More LessThe nucleotide sequence and genomic organization of a conserved genome region within the EcoRI G fragment of Cryptophlebia leucotreta granulosis virus (C1GV) is presented. Five open reading frames (ORFs) were identified which were homologous to those of Auto- grapha califomica nuclear polyhedrosis virus (AcMNPV), located upstream of the helicase gene (pl43) at 63·6 to 65·6 map units. The ORFs of C1GV and AcMNPV share nucleotide sequence homologies of about 47 to 53 % and are very similarly arranged. One of the C1GV ORFs potentially encodes a basic DNA- binding protein with an M r of about 7·3K. Its predicted amino acid sequence mainly consists of multiple arginine and serine residues and shows a 52 to 55 % identity to the DNA-binding proteins of AcMNPV and other nuclear polyhedrosis viruses. Its amino acid composition conforms to that of the DNA-binding proteins of Plodia interpunctella granulosis virus and Spodoptera litura granulosis virus.
The nucleotide sequence reported here will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession number X77048.
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Nucleotide sequence, genomic organization and synthesis of infectious transcripts from a full-length clone of artichoke mottle crinkle virus
More LessThe complete nucleotide sequence of the genome of artichoke mottle crinkle virus (AMCV), a member of the tombusvirus group, has been determined. The genome is 4790 nucleotides (nt) in length. A full-length cDNA of the AMCV genome has been cloned in pUC9 downstream of the T7 RNA polymerase promoter. Transcripts were infective when inoculated onto Nicotiana clevelandii and N. benthamiana plants. The AMCV genome contains five open reading frames (ORFs). The first ORF from the 5′ terminus (ORF1) encodes a protein with a predicted M rof 33K. ORF2 extends through the amber termination codon of ORF1 to yield a polypeptide of predicted M r92K and which is the putative RNA- dependent RNA polymerase. ORF3 codes for the coat protein (41K). Two nested ORFs in different reading frames (ORFs 4 and 5) code for a 22K and a 19K polypeptide respectively. Sequence homologies suggest that the 22K protein could be involved in eell-to-cell movement of virus. ORFs 3, 4 and 5 are translated from two 3′ coterminal subgenomic (sg) RNAs, the 5′ termini of which have been mapped. The two sg RNAs are 2155 (sgl) and 934 (sg2) nt in length. ORF3 is expressed from sgl RNA whereas ORFs 4 and 5 are potentially expressed from sg2 RNA. Time course experiments with Cynara scolymus protoplasts indicate that during AMCV infection both positive and negative strands of genomic and sg RNAs are produced and that sg2 RNA is produced before and at a higher level than sgl RNA.
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Partial characterization of the lettuce infectious yellows virus genomic RNAs, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous RNA plant viruses
More LessPurified virions of lettuce infectious yellows virus (LIYV), a tentative member of the closterovirus group, contained two RNAs of approximately 8500 and 7300 nucleotides (RNAs 1 and 2 respectively) and a single coat protein species with M r of approximately 28000. LIYV-infected plants contained multiple dsRNAs. The two largest were the correct size for the replicative forms of LIYV virion RNAs 1 and 2. To assess the relationships between LIYV RNAs 1 and 2, cDNAs corresponding to the virion RNAs were cloned. Northern blot hybridization analysis showed no detectable sequence homology between these RNAs. A partial amino acid sequence obtained from purified LIYV coat protein was found to align in the most upstream of four complete open reading frames (ORFs) identified in a LIYV RNA 2 cDNA clone. The identity of this ORF was confirmed as the LIYV coat protein gene by immunological analysis of the gene product expressed in vitro and in Escherichia coli. Computer analysis of the LIYV coat protein amino acid sequence indicated that it belongs to a large family of proteins forming filamentous capsids of RNA plant viruses. The LIYV coat protein appears to be most closely related to the coat proteins of two clostero-viruses, beet yellows virus and citrus tristeza virus.
The nucleotide sequence reported in this paper has been submitted to the GenBank database and assigned the accession number U05242.
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Nucleotide sequences of apple stem pitting virus and of the coat protein gene of a similar virus from pear associated with vein yellows disease and their relationship with potex- and carlaviruses
More LessThe nucleotide sequence (9306 nucleotides) of cDNA clones of apple stem pitting virus (ASPV) obtained from a double-stranded RNA template, extracted from diseased plant tissue, was determined. The genome is composed of five open reading frames (ORFs) encoding putative proteins with M rs of 247083, 25147, 12832, 7429 and 43712, and has a poly(A) tail. Using two oligonucleotides designed from the ASPV sequence information a 1598 bp fragment from near the 3′ terminus of the viral RNA, containing the coat protein of M r 43 766, was amplified from vein yellows (VY)-infected pear plants by PCR. The sequence determined showed eight nucleotide changes resulting in five amino acid substitutions compared with the sequence of ASPV. When compared to potex-, carla-, clostero- and capillo- viruses, the ASPV genome organization appeared to be most closely related to that of potexviruses, but with a larger coat protein of M r 44K (ORF5). The predicted coat protein size was confirmed by immunoblot analysis. The results show that ASPV does not fall into subgroup A of the closteroviruses but that it probably belongs in an as yet undefined group of viruses. They also suggest that the virus associated with VY is a strain of ASPV.
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Avocado sunblotch disease: a persistent viroid infection in which variants are associated with differential symptoms
More LessVariants of avocado sunblotch viroid (ASBVd) of between 247 and 250 nucleotides in length have been recovered from diseased avocado tissues. The sunblotch syndrome covers a complex pattern of disease symptoms which are associated with infection by variants of ASBVd. The viroid species are designated ASBVd-B, ASBVd-V and ASBVd-Sc from their association with bleached, variegated or symptomless carrier tissues respectively. Host-viroid interactions and structural relationships among the variants suggest a transition in sunblotch disease from a severe acute to a persistent mild form of infection.
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Nucleotide sequence of the coat protein genes of strawberry latent ringspot virus: lack of homology to the nepoviruses and comoviruses
More LessThe sequence of the 3′-terminal 2424 nucleotides of RNA-2 of the flowering cherry strain of strawberry latent ringspot virus (SLRV) was determined from cDNA clones. The sequence contains a reading frame in the virus-sense strand of 2070 nucleotides, a 3′ untranslated region of 552 nucleotides and a 3′-terminal poly(A) tract. The positions of the two coat proteins of SLRV within the reading frame were determined from sequence data obtained by N-terminal sequencing using Edman degradation. The larger coat protein with an M r of 43K is located 5′ of the smaller coat protein of 27K, and the two proteins are apparently cleaved at a Ser-Gly bond. Although there are numerous similarities between SLRV and the nepoviruses and comoviruses, there is no significant homology between the SLRV coat proteins and the coat proteins of either group. Furthermore, the hydropathy profiles of the SLRV coat proteins are unlike those of either group. No comparisons could be made with the fabaviruses owing to lack of sequencing information. This lack of homology suggests that SLRV is more distantly related to the nepoviruses and comoviruses than has been considered previously.
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