- Volume 74, Issue 3, 1993
Volume 74, Issue 3, 1993
- Animal
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Ribonucleotide reductase-deficient mutants of pseudorabies virus are avirulent for pigs and induce partial protective immunity
More LessWe have mutagenized and mapped the gene encoding the large subunit of ribonucleotide reductase (RR1) in pseudorabies virus (PRV; synonyms Aujeszky's disease virus, suid herpesvirus type 1). PRV strains carrying an oligonucleotide that leads to termination of translation of the RR1 gene are avirulent for mice. We subsequently constructed a PRV strain carrying a deletion in the RR1 gene and also a PRV strain carrying both the deletion in the RR1 gene and a deletion in the glycoprotein g1 gene, which is a marker for PRV virulence. Both PRV strains were assayed for virulence and immunogenicity in pigs, the natural host for PRV. In contrast to a marker-rescued PRV strain, these RR1-deleted mutants were avirulent, were shed in very low titres in the oropharyngeal fluid by the animals, and induced low titres of neutralizing antibodies. However, protection against clinical signs after infection with virulent PRV was induced by both RR1-deleted mutants. The relative importance of viral RR and thymidine kinase enzymes for deoxynucleotide synthesis in viral replication is discussed. In addition, we discuss the potential use of RR as a target for anti-herpesviral drugs and the use of PRV strains, deleted for the RR1 gene, as vaccine strains.
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Epstein–Barr virus (EBV) nuclear antigen 6 induces expression of the EBV latent membrane protein and an activated phenotype in Raji cells
More LessEpstein–Barr virus (EBV) nuclear antigen (EBNA) 6 (also known as 3c) is a latent nuclear protein with an M r of about 160K which is invariably expressed in EBV-immortalized B cells. It includes a putative basic leucine zipper domain; as such it is a good candidate for a regulator of viral gene expression. More than 75% of the EBNA 6 coding sequence is deleted from viral genomes carried in the Burkitt's lymphoma (BL) tumour-derived cell line, Raji. Thus although Raji cells express normal levels of the remaining five EBNAs and low levels of latent membrane protein (LMP), EBNA 6 protein is completely absent. In this study we have established Raji clones stably expressing EBNA 6 after cotransfection of an EBNA 6 gene under the control of the simian virus 40 early promoter with a selectable marker. Analysis of these clones has revealed that EBNA 6 induces a significant increase in the expression of LMP. In addition the cells have undergone a number of morphological and phenotypic changes consistent with blast-activation of normal B lymphocytes. The Raji cells expressing EBNA 6 show ruffling of the cell membrane and the development of a polarity defined by multiple villous (‘spiky’) projections at one end of the cell. This morphological change is associated with a dramatic increase in the expression of the cytoskeletal protein, vimentin. The EBV-associated B cell activation marker CD23 (blast 2) is induced to high levels although other activation markers such as CD30 and CD39 are unaffected. All these changes appear to be independent of the precise levels of EBNA 6 protein expressed. EBNA 2 has been shown previously to trans-activate the LMP gene and in the control Raji cells, EBNA 6-positive Raji cells and in B lymphoblastoid cells similar levels of EBNA 2 are expressed. Our findings are therefore most consistent with a model in which EBNA 6 either augments or complements the action of EBNA 2 in the induction of LMP and the cascade of gene expression which leads to B cell activation and immortalization by EBV.
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Construction and properties of a turkey herpesvirus recombinant expressing the Marek's disease virus homologue of glycoprotein B of herpes simplex virus
More LessA herpesvirus of turkeys (HVT) recombinant containing a 3.9 kbp fragment of Marek's disease virus (MDV) DNA encoding MDV glycoprotein B (gB), stably integrated into the thymidine kinase (TK) gene of HVT, has been constructed. The replication of the recombinant in chick embryo fibroblasts (CEF) was comparable to that of wild-type HVT. The recombinant expressed authentic MDV gB and its processed forms (110K, 65K and 48K) in CEF as shown by immunoblotting using an MDV-specific anti-peptide serum. Northern blot analysis showed that MDV gB mRNA was transcribed from MDV promoter sequences flanking the MDV gB open reading frame and also from the HVT TK promoter. However, the level of replication of the recombinant in vivo appeared to be lower than wild-type HVT as shown by the titres of HVT antibodies, determined by ELISA. Pathogenicity tests showed that the recombinant was safe and did not cause microscopic or gross Marek's disease lesions or other abnormalities. The results suggest that HVT has potential as a vector for recombinant vaccines.
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- Articles
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Biological Characterization of Structural Components of Adenovirus type 12
E. Norrby and J. AnkerstSUMMARYTwo products with a large mass and five different soluble components of adenovirus type 12 have been identified. The former were virus particles and empty capsids, which both carried group-specific complement-fixing (CF) antigen and were active as complete haemagglutinins (HAs). Their buoyant densities in CsCl were 1·330 to 1·340 and 1·295 to 1·305, respectively. The soluble components were identified as hexons, pentons, isolated fibres, dimers of fibres and isolated vertex capsomeres. Hexons were trypsin-resistant, ther-mostable and carried group-specific CF antigen; ultrastructurally they resembled hexons of other human adenoviruses. Pentons were active as an incomplete HA, causing agglutination in the presence of heterologous antisera against representative members of all three subgroups of adenoviruses. Furthermore, they were trypsin-sensitive and thermolabile. Isolated fibres represented another incomplete HA, exhibiting agglutinating activity only in the presence of antisera against members of subgroup III, e.g. type 2. This component was also identified as the dominating type-specific CF antigen. The length of fibres was estimated as 28 to 32 nm. by electron microscopy. The dimers of fibres represented a complete HA, the activity of which became apparent only after other material was removed. Both isolated fibres and their dimers were trypsin-resistant and thermostable. The assumed pentons and fibre dimers occurred in too low concentration to allow ultrastructural identi-fication. Isolated vertex capsomeres were demonstrated by a haemaggluti- nation enhancement antibody consumption test. They were completely destroyed by trypsin treatment and exhibited a moderate thermostability. Electron microscopy confirmed the presence of capsomere-like structures. These structures occasionally displayed a five-sided contour, thereby differing from hexons.
The sequence of elution of different components from an anion exchanger was: isolated vertex capsomeres, hexons + pentons, fibres and dimers of fibres. In zonal centrifugation experiments the components sedimented at decreasing rates in the following order: hexons, pentons+isolated vertex capsomeres, dimers of fibres and isolated fibres. Probably on account of the morphological characteristics of the various components they presented a different pattern of elution in exclusion chromatography. Dimers of fibres, pentons and even isolated fibres were eluted before hexons. The position of the peak activity of type 12 fibres in the elution diagram was similar to that of types 1, 2 and 5 fibres.
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- Animal
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Anti-glycoprotein B monoclonal antibody protects T cell-depleted mice against herpes simplex virus infection by inhibition of virus replication at the inoculated mucous membranes
More LessA monoclonal antibody (MAb 2c) specific for glycoprotein B of herpes simplex virus (HSV) mediated clearance of the virus from the infected mucous membranes. Young adult C57BL/6J mice were inoculated intravaginally with HSV type 1 and injected intraperitoneally either 24 and 72 h or 65 and 265 h post-inoculation with a polyclonal immune serum or the MAb 2c, both adjusted to the same neutralizing capacity. Immunization with the polyclonal immune serum did not alter the duration of virus shedding from the genital mucous membranes although a lethal outcome of infection was clearly prevented. Immunization with the MAb, however, resulted in a rapid clearance of the virus from the genital tract thus completely inhibiting genital inflammation and lethality. The same effects were achieved in mice depleted in vivo of CD4+ T cells although peripheral virus replication continued longer in these mice. In mice depleted of both CD4+ and CD8+ T cells the polyclonal immune serum was no longer able to protect against lethality, and virus replication in the mucous membranes persisted until the mice died. In contrast, after treatment with the MAb peripheral infection was quickly eliminated and all mice survived. These findings indicate that clearance of the virus from the primary site of replication can be mediated by humoral immunity. The relevance of this observation for vaccination against HSV is discussed.
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- Articles
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Depressors of Interferon Synthesis: Further Studies on the Production, Action and Properties of the So-called Enhancer
More LessSUMMARYMode of action, production and some properties of a viral growth-enhancing factor (enhancer) which was detected in allantoic fluids of eggs infected with parainfluenza type 1 virus were investigated. The factor markedly depressed interferon synthesis induced by u.v.-irradiated Newcastle disease virus in chick embryo cells, but did not inhibit the action of exogenous interferon. It had no effect on the multiplication of Newcastle disease virus in chick embryo cells. The depression of interferon synthesis by the factor was unlikely, therefore, to be due to an inhibition of the early steps of virus cell interaction. On the basis of its biological properties the factor should be termed ‘interferon depressor’. The appearance of the factor in allantoic fluids of eggs infected with parainfluenza type 1 virus could be determined by selectively destroying the interferon also contained in the materials. The activity of the factor reached a peak 24 hr after virus inoculation and remained stationary thereafter. Present data on the chemical nature of the factor are compatible with the view that it is a carbohydrate.
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- Animal
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A mutant of herpes simplex virus type 1 in which the UL13 protein kinase gene is disrupted
More LessGene UL13 of herpes simplex virus type 1 (HSV-1) has previously been proposed to encode a protein kinase. An HSV-1 mutant with UL13 inactivated by insertion of the Escherichia coli lacZ gene was constructed. This UL13-lacZ mutant was found to grow to near wild-type (wt) titres in tissue culture. Comparison of silver-stained SDS-PAGE profiles of wt and UL13-lacZ virions demonstrated that the UL13 protein is a readily detectable component of wt virions, located in the tegument and probably equivalent to the previously described species VP18.8. Studies of in vitro phosphorylation with nuclear extracts of virus-infected cells and with detergent-treated virions showed that the UL13 protein is involved in phosphorylation of the tegument protein VP22. Extracts of cells engineered to express UL13, and infected with UL13-lacZ virus, were also capable of VP22 phosphorylation.
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The herpes simplex virus type 1 US11 gene product is a phosphorylated protein found to be non-specifically associated with both ribosomal subunits
Microsequencing of a cyanogen bromide peptide obtained from a basic phosphoprotein co-sedimenting with purified ribosomes extracted from herpes simplex virus type 1-infected human epidermoid carcinoma 2 cells identified this protein as a product of the true late US11 gene. An antibody was raised against a recombinant fusion protein expressed in Escherichia coli from a plasmid carrying 75% of the US11 coding sequence including the carboxy terminus. This antibody was used to prode Western blots carried out under various conditions of one- and two-dimensional electrophoresis. The electrophoretic behaviour of the immunoreactive proteins offered further proof that they were indeed products of the US11 gene. This US11 protein, which has phosphates on multiple serine residues, is brought into the cell by the virion and found to be present within ribosome fractions early after infection. This association with ribosomes is non-specific and due to probable aggregation or oligomerization of this proline-rich basic protein allowing its co-sedimentation with ribosomes during the different subcellular fractionation steps used for the purification of ribosomal subunits.
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Trans-complementation among naturally occurring deletion mutants of hepatitis B virus and integrated viral DNA for the production of viral particles with mutant genomes in hepatoma cell lines
More LessCultured hepatoma cells (HepG2) were cotransfected with two different plasmids carrying a head-to-tail dimer of recombinant hepatitis B virus (HBV) DNA cloned from deletion mutants isolated from the circulation of persistently infected hosts. They were tested for the secretion of viral particles with mutant genome encapsidation. A recombinant plasmid defective in the S gene and one defective in both the C and P genes complemented in trans for the production of viral particles. Mutant genomes from either of the recombinants were encapsidated. Similarly, a recombinant defective in the C gene and another defective in the P gene trans-complemented for the production of viral particles containing mutant genomes. A hepatoma cell line with integrated HBV DNA sequences defective in the C and P genes (PLC/PRF/5) when transfected with a recombinant defective in the S gene produced viral particles with the HBV genome from the transfecting recombinants. These results confirm the expected trans-complementation among the S, C and P genes of HBV, when either episomal or integrated into chromosomes, for the maintenance of defective HBV mutants in persistently infected hosts.
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Characterization of new baculovirus genotypes arising from inoculation of Pieris brassicae with granulosis viruses
More LessPrevious studies have shown that of 15 Artogeia (Pieris) rapae granulosis virus isolates (ArGV1 to ArGV15) only two, ArGV1 and ArGV2, gave a normal dose-mortality response in larvae from an established colony of Pieris brassicae. We report here that at extremely high doses, approaching 10000 times the LD50 for ArGV1 and ArGV2, three other ArGV isolates caused low and irregular levels of mortality in P. brassicae. At similar doses Agrotis segetum GV caused 43% mortality in one infection, but no deaths ensued from other inoculations with this virus. Restriction endonuclease analysis of viral DNA recovered from individual larval cadavers revealed that, in most cases, progeny virus differed from the inoculum and consisted either of ArGV1 or of novel genotypes explicable as recombinants between genomes of the inoculum and of ArGV1. Field-collected P. brassicae inoculated with ArGV8 yielded a similar range of progeny genotypes. Physical maps were constructed for two such recombinants, based on comparative restriction analysis with reference to the published map of ArGV1 and to those of ArGV5 and ArGV8, which are presented. Replication of the inoculum genotype was observed in only two infections. The origin of ArGV1 DNA appearing among progeny from these infections and the relevance of our results to identifying ArGV DNA sequences that modulate pathogenicity for P. brassicae are discussed.
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Evolution of structural proteins of feline immunodeficiency virus: molecular epidemiology and evidence of selection for change
More LessThe DNA sequences of structural genes of several U.K. and European isolates of feline immunodeficiency virus (FIV) were determined and compared with those of other worldwide isolates. Phylogenetic analyses of both gag and env sequences demonstrate that a Japanese isolate represents a distinct sequence subgroup, with corrected amino acid distances to the other isolates averaging 23% in env and 8% in gag. Analysis also reveals that an evolutionary radiation of FIV occurred with many isolates diverging at approximately the same time, and that although isolates from similar geographical sources often cluster together, there is evidence of more than one origin for FIV in the U.K., The Netherlands and Italy. Estimation of the numbers of silent and replacement nucleotide substitutions indicates the presence of constraints against amino acid changes in gag and conserved regions of env but suggests that positive selection for protein sequence changes operates in variable regions of env. The possible immunological forces underlying these changes are discussed.
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Molecular characterization of two isolates of human T cell leukaemia virus type II from Italian drug abusers and comparison of genome structure with other isolates
The human T cell leukaemia virus type II (HTLV-II), whose pathogenicity is as yet unclear, was recently found to be associated with intravenous drug abuse in North America and Europe. HTLV-II was isolated from two Italian drug abusers belonging to the same cohort and coinfected with human immunodeficiency virus type 1. Two new isolates, HTLV-II Gu and Va, were established in a culture of BJAB cells, a continuous B cell line (Epstein–Barr virus-negative), and characterized by nucleotide sequence analysis of the long terminal repeat (LTR) and portions of the gag, env and X regions. These sequences were compared to those of the HTLV-II Mo isolate reported in the literature. No major variations were observed in important regulatory elements of LTR nor in the stem-bulge-loop configuration known to be essential for binding of rex protein. The results obtained from the sequence of the 1988 nucleotides examined indicated a 1.6% variability between the Gu and Va isolates and about 6% with respect to Mo. Notable differences were found in the structure of putative open reading frames of the X region when compared to those reported for the Mo isolate. Restriction analysis of proviral DNA of two isolates and comparison with the physical map of the Mo isolate confirmed the existence of genetic heterogeneity in the HTLV-II group and demonstrated that the new isolates Gu and Va belong to the HTLV-IIb subtype. The results of this study show that the new isolates have distinct features with respect to the Mo isolate though all important regulatory elements of the LTR appear to be well conserved.
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Mapping cell-mediated immunodominant domains of the rubella virus structural proteins using recombinant proteins and synthetic peptides
More LessAlthough it is known that rubella-immune individuals have T cells that proliferate in vitro in response to rubella virus (RV), the determinants that evoke this response have not been identified. This study utilized recombinant proteins that express overlapping sequences of the RV structural open reading frame to identify domains of the structural proteins that contain cell-mediated immunodominant sequences. Lysates enriched with RV fusion proteins (RecA-RV-LacZ) were prepared from Escherichia coli transformed with plasmids which contained specific RV cDNA inserts. Approximately 62% of RV-immune individuals gave RV-specific responses to one or more of the RV fusion proteins. Over 10% of immune individuals recognized the capsid sequence C1-C29. Lymphoproliferation data from studies using six overlapping synthetic peptides representing this sequence suggested that as much as 70% of the immune population may recognize this domain. An E1 sequence, E1202-E1283, was recognized by 15% of the RV-immune individuals with the fusion proteins. Five synthetic peptides representing this sequence had an overall response rate of 50%. The sequence C64-C97 failed to evoke any RV-specific responses with the fusion proteins and synthetic peptides representing this sequence were used to verify that the RV fusion proteins and the criteria used to identify RV-specific responses were adequate. These peptides gave a response rate of only 6%. In general, significant responses to specific fusion proteins correlated with high responses (stimulation index ≥ 4.0) to representative synthetic peptides. This study suggests that the recombinant proteins were beneficial in identifying cell-mediated immunodominant domains of the RV structural proteins which could be further characterized with synthetic peptides.
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Expression of the G glycoprotein gene of human respiratory syncytial virus in Salmonella typhimurium
The attachment protein, G, of human respiratory syncytial virus (RSV) is an M r 84K to 90K species which has a high content of N-linked and O-linked carbohydrates. The unglycosylated form of this protein was expressed by inserting a full-length cDNA copy of the mRNA from the A2 strain of RSV into a prokaryotic expression vector under the control of the lambda PL promoter. Salmonella typhimurium cells transformed with the G-containing plasmid synthesized a protein of M r 40000 that specifically reacted with polyclonal and two neutralizing monoclonal antibodies raised against the native RSV G glycoprotein. Recombinant G protein was purified by immunoaffinity chromatography using a neutralizing monoclonal antibody. Cotton rats immunized with the recombinant G protein produced serum antibodies to the G glycoprotein that neutralized RSV in vitro. The study demonstrates that the G protein of RSV can be expressed in bacteria and that at least one neutralizing epitope is not structurally dependent on carbohydrates.
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Comparison of soluble and secreted forms of human parainfluenza virus type 3 glycoproteins expressed from mammalian and insect cells as subunit vaccines
Human parainfluenza virus type 3 (PIV-3) is one of the leading causes of paediatric viral respiratory disease. The PIV-3 genome encodes two envelope glycoproteins, F and HN, which are the major targets for the host antibody response. We have expressed secreted forms of the F and HN proteins and a novel chimeric FHN glycoprotein in insect cells using recombinant baculovirus vectors and secreted forms of the F and FHN glycoproteins in stably transformed Chinese hamster ovary (CHO) cells. Comparison of the mammalian cell- and insect cell-expressed F and FHN proteins by SDS-PAGE showed that the CHO cell-expressed proteins are several kilodaltons larger in size than the baculovirus-produced proteins. A partial characterization of the oligosaccharide structures of the F and FHN proteins revealed that the size difference is due to the different oligosaccharide structures added to these proteins by the two cell lines. The F, HN and FHN proteins were immunoaffinity-purified from the culture medium of baculovirus-infected Sf9 cells and the F and FHN proteins were immunoaffinity-purified from the culture medium of CHO cells. A comparison of the immunogenicity and efficacy of the mammalian cell- and insect cell-produced FHN proteins was tested in cotton rats. The CHO cell- and baculovirus-produced FHN proteins were found to induce similar levels of PIV-3-specific ELISA-positive and neutralizing antibodies and both proteins provided near complete protection when animals were vaccinated with low doses of the FHN protein.
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Protection of cotton rats against human parainfluenza virus type 3 by vaccination with a chimeric FHN subunit glycoprotein
More LessA cotton rat model of experimental human parainfluenza virus type 3 (PIV-3) infection was used to examine the efficacy of FHN, a novel chimeric glycoprotein which contains the extracellular regions of the fusion (F) and haemagglutinin—neuraminidase (HN) glycoproteins of PIV-3. The FHN protein was expressed in insect cells using a baculovirus vector system. FHN vaccination resulted in induction of neutralizing antibodies, was completely protective at doses of 100 ng, and was superior to vaccination with secreted forms F and HN proteins, or mixtures of the F and HN glycoproteins. In addition, FHN immunization induced lymphoproliferative responses in mice which were directed against both the F and HN glycoproteins. Fusion of the F and HN proteins into a single chimeric glycoprotein appeared to enhance the protective immune response compared to that elicited by the individual glycoproteins or mixtures of the two glycoproteins.
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Expression of the HN, F, NP and M proteins of Sendai virus by recombinant vaccinia viruses and their contribution to protective immunity against Sendai virus infections in mice
Recombinant vaccinia viruses (RVVs) expressing each of the haemagglutinin—neuraminidase (HN), fusion (F), nucleocapsid (NP) and matrix (M) proteins of Sendai virus were constructed to investigate their capacities to induce protective immunity against Sendai virus infections. The proteins expressed in cultured cells appeared to be authentic with respect to their antigenicity, electrophoretic mobility, surface expression of the HN and F proteins and, in the case of the HN protein, biological activities. Mice inoculated intranasally with these RVVs developed serum antibodies to the respective Sendai virus proteins, suggesting their in vivo expression. In mice immunized with RVV carrying either the HN or the F gene, growth of the challenging Sendai virus was almost completely suppressed in the lung, indicating their capacities to induce effective protective immunity against Sendai virus infections. In contrast, in mice immunized with RVV carrying the NP or M gene, the challenging virus propagated as well as in the control mice, but the virus titres were significantly lower at the late stage of infection than those in the control mice, suggesting that they can also induce protective immunity especially at the late stage of the challenge infection.
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The structure of the 5′ terminal cap of the respiratory syncytial virus mRNA
More LessThe 5′ terminal cap structure of the mRNAs of human respiratory syncytial virus synthesized in vitro in the presence of S-adenosyl-l-methionine consists of a 7-methyl guanosine linked to an unmethylated guanosine through a 5′-5′ pyrophosphate linkage formed by using the α and β phosphates of GTP. The complete cap structure is m7G (5′)ppp(5′)Gp… which is devoid of ribose 2′-O-methylation. Capping, including methylation, is coupled to transcription. These results constitute the first report of a pneumoviral mRNA cap structure.
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Induction of neutralizing antibodies by varicella-zoster virus gpII glycoprotein expressed from recombinant vaccinia virus
More LessThe gpII glycoprotein of varicella-zoster virus (VZV) was produced in CV1 cells via vaccinia virus recombinants. Two different DNA constructs were expressed: the first one encodes the complete gpII protein (gpII s+a+) and the second a truncated species lacking the membrane anchorage domain (gpII s+a-). To achieve expression both coding sequences had to be engineered at the 5′ end by substituting the unusually short (24 bp) natural signal sequence by a more conventional one encoding 29 amino acids. Recombinant gpII proteins were detected in vaccinia virus-infected cells by ELISA and immunoprecipitation. Both forms of recombinant gpII proteins were produced as glycosylated single-chain molecules of respectively 110K and 90K. Upon reduction these were only partially converted into subunits. A rabbit infected with the vaccinia virus recombinant expressing the complete gpII produced antibodies which recognized VZV antigens and neutralized VZV infectivity in vitro, independent of complement.
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The glycoprotein B homologue of human herpesvirus 6
More LessThe gene for the homologue of herpesvirus glycoprotein B (gB) has been identified in the genome of human herpesvirus 6 (HHV-6), strain U1102, and the nucleotide sequence was determined. The open reading frame encodes a protein of 830 amino acids (93.2K) with the characteristics of a transmembrane glycoprotein and close similarity to the gp58/116 complex of human cytomegalovirus (HCMV). Monoclonal antibodies 2D10 and 2B9 have been shown previously to react with an HHV-6 glycoprotein of apparent M r 112K, and its proteolytic cleavage products of M r 64K and 58K. We show that both monoclonal antibodies detect prokaryotically expressed carboxy-terminal fragments of the HHV-6 gB homologue. This indicates that the HHV-6 gB homologue is probably processed by proteolytic cleavage similar to its equivalents in HCMV and various other herpesviruses.
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Replication-defective recombinant adenovirus expressing the Epstein—Barr virus (EBV) envelope glycoprotein gp340/220 induces protective immunity against EBV-induced lymphomas in the cottontop tamarin
More LessA replication-defective recombinant adenovirus (Ad) expressing the full length Epstein—Barr virus (EBV) major envelope glycoprotein gp340/220 was tested for its ability to protect against EBV-induced lymphoma in the cottontop tamarin. Antibody responses against Ad capsid proteins and EBV gp340/220 were observed but these antibodies did not neutralize EBV in vitro. However, all immunized animals were protected against challenge following three intramuscular doses of the recombinant Ad. These data indicate that the recombinant Ad is potentially a useful vector for vaccination.
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Analysis of Epstein—Barr virus gene transcription in lymphoma induced by the virus in the cottontop tamarin by construction of a cDNA library with RNA extracted from a tumour biopsy
More LessInoculation of the cottontop tamarin with Epstein—Barr virus (EBV) gives rise to the development of mono- and/or oligoclonal large cell malignant lymphoma. A cDNA library was generated with the RNA extracted from an EBV-induced tamarin lymphoma biopsy in order to study the transcripts expressed in the tumour tissue. Fifteen EBV-specific cDNA clones were localized in the corresponding viral genomic fragments. Among them, two correspond to the EBNA-2 gene, and two others to the latent membrane protein gene. The majority of the cDNA clones were localized in the BamHI A fragment which has not been associated with latent expression. Furthermore, cDNAs were also found from the BamHI D and I fragments. Sequence analysis of the cDNAs localized in BamHI A showed that they correspond to a rightward transcript in the BALF-3 region, with the one clone that was sequenced containing four exons and three introns. The above results were confirmed by testing three different biopsies with the rapid amplification of cDNA ends-PCR method.
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Susceptibility of human cells to Puumala virus infection
Nephropathia epidemica involves several organs including kidney, lung, liver and brain. To investigate the susceptibility of putative target cells to the agent responsible, Puumala virus, we screened established human cell lines of lung (WI-38, A-427, CCD-11Lu), kidney (A-704), liver (Hep G2), pharynx (Detroit 562), submaxillary gland (A-253) and neural (SK-N-MC, SH-SY5Y) origin as well as primary human kidney glomerular cells, endothelial cells and peripheral blood monocytes/macrophages. Propagation of the Sotkamo strain of Puumala virus was also tested in the primary kidney, spleen and lung cells of bank voles (the natural host of the virus). All of the primary cells and most of the established cell lines expressed viral protein, synthesized viral RNA and secreted infectious virus, except the neural SK-N-MC and SH-SY5Y cells. None of the tested cell types except the primary bank vole kidney cells could propagate the virus as efficiently as the Vero E6 cells. The observed host cell range is wide and consistent with a multiorgan involvement of Puumala virus. No cytopathic effects were seen in any of the infected cell cultures.
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Molecular evidence that epizootic Venezuelan equine encephalitis (VEE) I-AB viruses are not evolutionary derivatives of enzootic VEE subtype I-E or II viruses
More LessEnzootic strains of Venezuelan equine encephalitis (VEE) virus occur in the United States (Florida), Mexico, Central America and South America. Epizootic VEE first occurred in North and Central America in a widespread outbreak between 1969 and 1972. To investigate the likelihood that this epizootic VEE virus, identified as VEE antigenic subtype I-AB, evolved from enzootic viruses extant in the region, we cloned and sequenced the 26S mRNA region of the genomes of the Florida VEE subtype II virus, strain Everglades Fe3-7c, and the Middle American subtype I-E virus, strain Mena II. This region of the genome encodes the viral structural proteins. The sequences of the 26S mRNA regions of the Everglades and Mena virus genomes differed from that of the reference epizootic VEE subtype I-AB virus, Trinidad donkey strain, by 453 and 887 nucleotides and by 66 and 131 amino acids, respectively. These data confirm previous reports demonstrating significant antigenic and genetic distance between VEE I-AB virus and viruses of subtypes I-E and II. It is unlikely that the epizootic VEE I-AB virus responsible for the 1969 outbreak originated from mutation of enzootic VEE viruses in North or Middle America.
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Characterization of virus inclusion bodies in bluetongue virus-infected cells
More LessA combined qualitative and quantitative approach has been used to examine the role of virus inclusion bodies (VIBs) in the morphogenesis of bluetongue virus (BTV). VIBs were detected as early as 4 h post-infection (p.i.), and their number and profile areas increased significantly between 12 and 16 h, and 20 and 28 h p.i. respectively. Core- and virus-like particles were found within and at the periphery of the VIB matrix, respectively, and their numerical density (number per area of VIB matrix) decreased during the course of infection whereas the numerical density of virus particles in the cytoplasm increased. Virus-like particles had a diameter of 57 ± 8 nm and core-like particles appeared to fall into two size ranges, 32 ± 3 nm and 38 ± 3 nm in diameter. Both pre- and post-embedding immunoelectron microscopy procedures were used to localize BTV structural and non-structural proteins within the VIBs. The VIB matrix was labelled with antibodies to structural proteins VP5 and VP7 and non-structural proteins NS1 and NS2. Cores within VIBs contained proteins VP5, VP7 and NS1 but not VP2. Virus-like particles at the periphery of VIBs contained VP2, VP5, VP7 and NS1. The results suggest that BTV particles are synthesized, assembled and released from the perimeter of VIBs and not from within the matrix. Cores embedded in the VIBs are likely to have been trapped there during expansion of the matrix during replication.
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Expression of hepatitis A virus poly(U) polymerase in the periplasmic space of Escherichia coli
More LessA cDNA containing almost all of the hepatitis A virus (HAV) P3 sequences was expressed as a fusion protein with Protein A. A novel poly(U) polymerase activity was detected in the periplasmic space of Escherichia coli cells transformed with this plasmid, and this activity showed many of the expected properties of a picornavirus 3Dpol. A number of HAV-specific polypeptides were detected in these cells, and it is unclear which of these was responsible for the polymerase activity.
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Influenza virus naked RNA can be expressed upon transfection into cells co-expressing the three subunits of the polymerase and the nucleoprotein from simian virus 40 recombinant viruses
More LessThe functionality of the influenza virus polymerase subunits and the nucleoprotein expressed from simian virus 40 (SV40) recombinants has been tested by their ability to direct the in vivo expression of influenza virus-like RNAs. These RNAs, which contained either the chloramphenicol acetyltransferase (CAT) or haemagglutinin (HA) genes, were synthesized and reconstituted in vitro into viral ribonucleoproteins with a polymerase/nucleoprotein mixture purified from influenza virus-infected cells. Only the coinfection with SV40 recombinant viruses expressing the three polymerase subunits and the nucleoprotein allowed the expression of the transfecting CAT or HA RNAs, confirming that this set of viral genes is the minimal requirement for viral gene expression. Unexpectedly, transfection of the corresponding naked RNAs into SV40 recombinant-infected cells was as effective in directing the synthesis of CAT or HA proteins as the standard reconstituted ribonucleoprotein transfection. These results may be important for the genetic analysis of trans-acting factors involved in influenza virus transcription and replication and may open the way to rescuing influenza viruses in the absence of a helper virus.
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Nucleotide sequence of one component of the banana bunchy top virus genome contains a putative replicase gene
More LessOne DNA component of the banana bunchy top virus (BBTV) genome was cloned and sequenced. This component is present as a circular, ssDNA in the virions and consists of 1111 nucleotides. It contains one large open reading frame (ORF) of 858 nucleotides in the virion sense; this ORF encodes a putative replicase based on the presence of a dNTP-binding motif (GGEGKT). Two smaller ORFs (249 and 366 nucleotides), in the complementary orientation, could not be assigned any obvious function. Neither of these ORFs had significant sequence homology with any known DNA plant virus gene or gene product. Computer analysis of this component predicted a strong stem-loop structure in the virion sense putative untranslated region; a nonanucleotide sequence in the loop was nearly identical to the nonanucleotide invariant loop sequence of geminiviruses and coconut foliar decay virus. There is strong evidence that the genome of BBTV consists of more than one component because no ORF was found that would encode a protein the size of the BBTV coat protein. BBTV has some characteristics in common with geminiviruses but cannot be classified as one. Rather, BBTV probably belongs to an undescribed plant virus group which could also include subterranean clover stunt virus and coconut foliar decay virus.
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3′-Terminal sequence of the plum pox virus PS and ŏ6 isolates: evidence for RNA recombination within the potyvirus group
More LessThe sequence of the 3′-terminal 1768 nucleotides of the PS and ŏo6 isolates of plum pox virus (PPV) has been determined and compared with that of the equivalent regions of other PPV isolates sequenced previously. The sequenced region is part of the PPV open reading frame encoding the last 186 amino acids of the NIb protein and the coat protein (CP, 330 amino acids), followed by a non-coding region of 220 nucleotides and a poly(A) tail. PPV-PS and PPV-ŏo6, just like PPV-El Amar, show rather high levels of nucleotide diversity in the sequence encoding the C-terminal region of the NIb protein (19.4 to 31%) and the N terminus of CP (22.8 to 41.1%) when compared with PPV-Rankovic, PPV-D and PPV-NAT, whereas the level of diversity in the rest of the CP sequence and the 3′ non-coding region is low (8 to 10.8% and 5.5 to 7.7%, respectively). However, the first 429 sequenced nucleotides of PPV-ŏo6 are very similar to those of the PPV-Rankovic, PPV-D and PPV-NAT isolates, whereas the rest of the sequence clearly resembles PPV-PS. Thus, PPV-ŏo6 seems to be the result of a natural recombination event between two wild strains of PPV. To our knowledge this is the first evidence of homologous RNA recombination (a process which could play an important role in the evolution of RNA viruses) within the potyvirus group.
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Evidence that cowpea aphid-borne mosaic and blackeye cowpea mosaic viruses are two different potyviruses
More LessThe immunoreactivity of a panel of polyclonal anti-bodies and monoclonal antibodies (MAbs) raised against African isolates of potyviruses from cowpea and African yam bean was examined in ELISAs. A serological study including reference isolates followed by further characterization in differential hosts resulted in separation of the potyviruses into two distinct serogroups, one containing blackeye cowpea mosaic virus (BlCMV) and the other containing cowpea aphid-borne mosaic virus (CAMV). Using biotin-labelled MAbs, the BlCMV isolates were further subdivided into two serotypes and the CAMV isolates into five serotypes. Because both BlCMV and CAMV induce a very similar mosaic disease in cowpea, different ELISA procedures using mixed MAbs were evaluated and a single protocol was developed which allowed reliable diagnosis of both viruses.
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Multiplication of tomato spotted wilt virus in its insect vector, Frankliniella occidentalis
More LessThe accumulation of two proteins, the nucleocapsid (N) protein and a non-structural (NSs) protein both encoded by the S RNA of tomato spotted wilt virus (TSWV), was followed in larvae during development and in adults of Frankliniella occidentalis after ingesting the virus for short periods on infected plants. The amounts of both proteins increased, as shown by ELISA and Western blot analysis, within 2 days above the levels ingested, indicating multiplication of TSWV in these insects. Accumulation of these proteins and of virus particles was further confirmed by in situ immunolabelling of the salivary glands and other tissues of adult thrips. The accumulation of large amounts of N and NSs protein, the occurrence of several vesicles with virus particles in the salivary glands and the massive numbers of virus particles in the salivary gland ducts demonstrate that the salivary glands are a major site of TSWV replication. The occurrence of virus particles in the salivary vesicles is indicative of the involvement of the Golgi apparatus in the maturation of the virus particles and its transport to the salivary ducts.
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