- Volume 75, Issue 1, 1994
Volume 75, Issue 1, 1994
- Review Article
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- Bacterial
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Nucleotide sequence determination, characterization and purification of the single-stranded DNA-binding protein and major coat protein of filamentous phage ϕLf of Xanthomonas campestris pv. campestris
More LessϕLf is a filamentous bacteriophage of Xanthomonas campestris pv. campestris, which can integrate its genome into the host chromosome. The nucleotide sequence of an EcoRV-SphI fragment (1018 bp) from the ϕLf replicative form DNA was determined. Four contiguous open reading frames (ORFs), orf98-orf43-orf38-orf42, were revealed. ORFs 98 and 42 were identified as the genes encoding a single-stranded DNA-binding protein (Sbp) and a major coat protein, respectively. Sbp was purified and found to bind with a high affinity to ssDNA prepared from ϕLf phage particles. The major coat protein showed sequence features similar to those of the typical major coat proteins of other filamentous phages. However, it appears to be synthesized as a mature product, similar to the situation with Pf3 but different from that with other filamentous phage major coat proteins which are synthesized as pre-coats and are subject to post-translational processing. The M rs, estimated by SDS-PAGE, of both the Sbp (10·9K) and the major coat protein (4·1K) coincide with the values deduced from the nucleotide sequences. ORFs 43 and 38 are proposed to be the genes encoding two minor coat proteins. The order of these four genes is similar to that found in the Escherichia coli filamentous phages.
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- Animal
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Similar genetic susceptibility in iatrogenic and sporadic Creutzfeldt-Jakob disease
More LessCreutzfeldt-Jakob disease (CJD) is one of the transmissible spongiform encephalopathies (TSEs). In all TSEs host susceptibility is an important factor in the development of clinical disease. The prion protein (PrP) gene appears to confer the main component of this susceptibility. The appearance of spontaneous neurodegeneration in PrP transgenic mice carrying a human mutation has raised the possibility that the origin of sporadic CJD is solely genetic. We studied PrP codon 129 polymorphism in 23 of the 25 CJD cases in France related to human growth hormone (hGH) therapy. They constitute the largest and most homogeneous hGH- related iatrogenic CJD population yet analysed. All these CJD cases were homozygous at codon 129, compared with only 50% in the healthy control group (P< 0·00002). These iatrogenic cases also displayed a genotype frequency distribution similar to that observed in sporadic CJD. These results underline the importance of the PrP gene and especially the homozygous codon 129 genotype in determining the risk of developing CJD after contamination by a TSE agent. They also suggest that highly susceptible individuals may exist and raise the possibility that sporadic CJD may have an environmental origin.
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Complex structure of the nuclear translocation signal of influenza virus polymerase PA subunit
More LessThe protein regions involved in the nuclear translocation of the influenza virus PA polymerase subunit have been identified by deletion analysis of the protein expressed from a recombinant simian virus 40. Two regions seem to play a role in the process: region I (amino acids 124 to 139) and region II (amino acids 186 to 247). A nucleoplasmin-like nuclear translocation signal (NLS) has been identified in region I and an additional NLS appears to be present in region II, although no consensus targeting sequence can be detected. Alteration in any of the regions identified by short deletions completely prevented nuclear transport, whereas elimination of the regions I or II by large amino- or carboxy-terminal deletions did not prevent nuclear targeting of the truncated protein. In addition, a point mutation at position 154 completely eliminated nuclear transport. A β-galactosidase fusion protein containing the 280 amino acid terminal region of the PA protein was partially transported to the nucleus and mutant PA proteins with a cytoplasmic phenotype could not be rescued by superinfection with influenza virus. These results suggest that the PA protein contains a functional nuclear targeting region which is required in influenza virus infection, with two independent NLSs, one in region I and the other in region II.
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A small percentage of influenza virus M1 protein contains zinc but zinc does not influence in vitro M1-RNA interaction
More LessA peptide containing the CCHH motif, the putative zinc-binding sequence of influenza virus M1 protein, was found to bind zinc in a one-to-one complex with the characteristics of a typical zinc-binding peptide. Intact influenza virus also contained zinc and we show that this zinc is bound to the M1 protein in the virus. However, only a small proportion of M1 contained zinc: 4% in virus and 6 to 9% in isolated protein. One strain, B/Yamagata/16/88, consistently contained more zinc: 15 to 20% both in virus and in isolated protein. We also determined the RNA binding and transcription inhibition activities of various M1 proteins and found that the zinc content of M1 had no influence on either activity. We suggest that the zinc in M1 has a structural role in the virion other than nucleic acid binding.
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An amino acid change in the non-structural NS2 protein of an influenza A virus mutant is responsible for the generation of defective interfering (DI) particles by amplifying DI RNAs and suppressing complementary RNA synthesis
More LessThe mutated non-structural NS2 protein of an influenza A virus mutant, Wa-182, has been shown to be responsible for the production of defective interfering (DI) particles lacking the PA gene after a single cycle high-multiplicity infection. Using a subclone of Wa-182, A3/e-3, that inherited the Wa-182 phenotype but contained only a marginal amount of DI RNAs derived from the PA gene, we showed that replication of the PA genome RNA was suppressed primarily at the step of complementary RNA (cRNA) synthesis. On the other hand, the small amounts of DI RNA species present in the stock of A3/e-3 were shown to be replicated efficiently. These findings suggested that the suppression of cRNA synthesis of the PA gene was caused by preferential amplification of the DI RNAs. The suppression of PA gene cRNA synthesis subsequently resulted in suppression of both virion RNA synthesis and secondary transcription of the PA gene. Such aberrant replication of the PA gene was found to be attributable to an amino acid change in the NS2 protein at position 32, from isoleucine to threonine. These results suggest that the NS2 protein plays a role in promoting normal replication of the genomic RNAs by preventing the replication of short-length RNA species.
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Modulation of antiviral immune responses by exogenous cytokines: effects of tumour necrosis factor-α, interleukin-1α, interleukin-2 and interferon-γ on the immunogenicity of an inactivated rabies vaccine
In vivo administration of exogenous cytokines may influence elicited immune responses, and hence may change the efficacy of a vaccine. We investigated the effects of tumour necrosis factor-α (TNF-α), interleukin- 1α (IL-1α), interleukin-2 (IL-2) and interferon-γ (IFN-γ) on the immune response elicited by inactivated rabies virus vaccine in a mouse model. Each of the cytokines increased virus-specific IgG responses after primary and after secondary immunization. A single dose of 1·3 ng TNF-α or 1L-1α, when injected shortly before vaccination, only marginally stimulated resistance to challenge infection (four- and seven-fold, respectively) without enhancing virus neutralizing antibody (VNAb) responses. In contrast, a single injection of 103 units of IFN-α or five daily injections of 1·6 μg IL-2 increased vaccine dilutions protecting 50% of mice (PD50 values) 77- to 50-fold, respectively, with a concomitant enhancement of VNAb. At a 1:10000 dilution of a standard inactivated rabies vaccine preparation both ILN-γ and 1L-2 increased protective immunity without enhancing VNAb responses; in non-vaccinated animals this treatment had no effect on resistance to challenge. Combined administration of IFN-y and 1L-2 syner- gistically enhanced VNAb responses. In contrast to the other cytokines tested, 1FN-γ preferentially stimulated virus-specific IgG2a production. It also augmented the vaccine-induced priming of rabies virus-specific spleno- cyte proliferation. These results document that certain cytokines alone or in combination are potent immunological adjuvants which may direct and modulate immunization-induced antiviral immune responses.
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Molecular evolution and epidemiology of dengue-3 viruses
More LessThe nucleic acid sequences of the pre-membrane/ membrane and envelope protein genes of 23 geographically and temporally distinct dengue (DEN)-3 viruses were determined. This was accomplished by reverse transcriptase-PCR amplification of the structural genes followed by automated DNA sequence analysis. Comparison of nucleic acid sequences revealed that similarity among the viruses was greater than 90 %. The similarity among deduced amino acids was between 95 % and 100 %, and in many cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis allowed the generation of phylo-genetic trees, demonstrating that geographically independent evolution of DEN-3 viruses had occurred. The DEN-3 viruses were separated into four genetically distinct subtypes. Subtype I consists of viruses from Indonesia, Malaysia, the Philippines and the South Pacific islands; subtype II consists of viruses from Thailand; subtype III consists of viruses from Sri Lanka, India, Africa and Samoa; subtype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus. Phylogenetic analysis has also contributed to our understanding of the molecular epidemiology and worldwide distribution of DEN-3 viruses.
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Clonality, expression and methylation patterns of the Epstein-Barr virus genomes in lethal midline granulomas classified as peripheral angiocentric T cell lymphomas
We analysed the terminal repeats of Epstein-Barr virus (EBV) in DNAs isolated from six lethal midline granuloma (LMG) biopsies. A single fused terminal fragment could be detected in each case, indicating that these angiocentric peripheral T cell lymphomas represent clonal proliferations of cells infected with EBV on a single occasion. Using reverse transcriptase-PCR, we detected EBV nuclear antigen (EBNA) 1 and latent membrane protein (LMP) 1, but not EBNA 2 messages in LMG biopsy RNAs. The splicing pattern of the EBNA 1 message was consistent with the usage of a promoter localized in the BamHI F fragment (F promoter). The BamHI W fragment repeats and LMP-coding sequences were highly methylated in all cases. In contrast, the LMP regulatory sequences were found to be hypomethylated or partially methylated, as in LMP-expressing nasopharyngeal carcinomas.
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Detection of multiple ‘Ebnotypes’ in individual Epstein-Barr virus carriers following lymphocyte transformation by virus derived from peripheral blood and oropharynx
Transformation of a B lymphocyte into a lymphoblastoid cell line (LCL) by Epstein-Barr virus (EBV) results in the expression of EBV nuclear antigens (EBNAs) of which the size spectrum (‘Ebnotype’) is characteristic for the transforming virion. Ebnotyping has been used as an epidemiological tool for studies of EBV infection. We compared the occurrence of a single and of multiple Ebnotypes, as defined by EBNAs 1, 2 and 6, in healthy and diseased EBV carriers. Cases from which two or more LCLs could be established from peripheral blood or oropharyngeal cultures were considered informative. The frequency of multiple Ebnotypes was relatively low in healthy individuals and in patients with infectious mononucleosis or with haematological diseases who were awaiting a bone marrow transplant [blood, 11 of 74 patients (15%); oropharynx, 12 of 49 patients (24%)], whereas it was relatively high in recipients of bone marrow or cardiac allografts and one patient with AIDS [blood, 12 of 34 patients (35%); oropharynx, 11 of 16 patients (69%)]. Three patterns of the simultaneous presence of multiple Ebnotypes were distinguished. The first, most frequent, pattern observed predominantly in oropharyngeal cultures of all groups consisted of minority Ebnotypes differing from the majority type by only a single EBNA protein (usually EBNA 1). The second, less frequent, pattern observed in the healthy carriers and the (candidate) transplant recipients consisted of minority Ebnotypes differing from the majority type by two EBNA proteins (mostly EBNAs 1 and 6). The third pattern, characterized by the simultaneous presence of totally different Ebnotypes, was restricted to the (candidate) transplant recipients and the AIDS patient and was more frequently observed in the blood than in the oropharynx. We suggest that the first two patterns result from heterologous recombinations occurring during viral replication at repeat sequences within the EBNA coding regions, whereas the third pattern reflects multiple infections with exogenous viruses.
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Heterogeneity within the Epstein-Barr virus nuclear antigen 2 gene in different strains of Epstein-Barr virus
More LessDNA isolated from biopsies of endemic Burkitt’s lymphoma (BL) from New Guinea was analysed for the presence of Epstein-Barr virus (EBV) sequences using the polymerase chain reaction. Primers were designed to amplify sequences within the Epstein-Barr virus nuclear antigen (EBNA) 1 and 2 genes. These analyses detected the EBNAl sequence in all the biopsies studied. Additional sets of primers directed against the EBNA2 gene were used in order to categorize the EBV strains as A-type or B-type (39% A-type; 50% B-type; 5% A- and B-type; 5% untypeable). These results indicated that DNA sequence heterogeneity within the EBNA2 gene region may exist in different strains of EBV. The extent of DNA sequence heterogeneity among different strains of EBV was determined by sequencing of a region within the EBNA2 gene in a number of different A-type and B-type strains of EBV originating from Africa or New Guinea. The results demonstrated DNA sequence heterogeneity within the EBNA2 gene in different strains of EBV. This heterogeneity was more extensive among A-type strains than B-type strains of EBV.
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Late phase inhibition of murine cytomegalovirus replication by synergistic action of interferon-gamma and tumour necrosis factor
More LessWe have shown previously that the antiviral function of CD4+ T lymphocytes against murine cytomegalovirus (MCMV) is associated with the release of interferon- (IFN-γ). We now demonstrate that IFN-γ and tumour necrosis factor alpha (TNF-α) display synergism in their antiviral activity. As little as 2 ng/ml of IFN-γ and TNF-α reduced the virus yield by about three orders of magnitude. There was no effect on immediate early (IE) and early (E) gene expression as far as the candidate genes IE1, El and those encoding the major DNA- binding protein and the DNA polymerase were concerned. Late gene transcription, assayed by the candidate genes encoding glycoprotein B and the MCMV homologue of ICP 18·5, was blocked and MCMV DNA replication was found to be reduced but not halted. The most prominent finding of the cytokine effect, seen by electron microscopy, was an alteration of nucleocapsid formation. Altogether, the synergism is multifaceted and acts at more than one stage during viral morphogenesis. Because the cytokines clearly do not act at an early stage of infection we conclude that the mode of cytokine activity differs between alpha- and betaherpesviruses.
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Inhibition by iota-carrageenan of the spread of murine cytomegalovirus from the peritoneal cavity to the blood plasma
More LessThe mechanism of iota-carrageenan (ι-CAR)-induced protection against murine cytomegalovirus (MCMV) infection of mice was analysed. The virus titres in the target organs on the fourth day correlated with that in the plasma at 1 h after intraperitoneal inoculation of MCMV, irrespective of ι-CAR treatment. Pretreatment of mice with ι-CAR induced infiltration of polymorphonuclear neutrophils into the peritoneal cavity and inhibition of viral spread from the peritoneal cavity to the plasma. Although the direct relationship of these two phenomena was unclear, the inhibition by ι-CAR of viral spread resulted in protection against MCMV infection of mice.
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Virulence and pathogenesis of non-virulent and virulent strains of pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus
Pseudorabies virus (PRV) expressing the envelope glycoprotein El (E1) of hog cholera virus (HCV) was used as a model to study the potential risks connected with the use of a live herpesvirus vaccine expressing a foreign gene. The gene encoding E1 was inserted into the glycoprotein X (gX) locus of both a virulent PRV strain and a non-virulent PRV strain in which the virulence genes encoding glycoprotein I (gI) and thymidine kinase (TK) had been inactivated. We investigated whether strain M205 (gI −, TK −,gX − , E1+) had a changed cell or host tropism or virulence compared with strain M206 (gI − ,TK − ,gX − ) in pigs, rabbits, hamsters, rats, mice and rhesus monkeys. The insertion of E1 into this non-virulent PRV strain caused no change in cell or host tropism. However, pigs inoculated with M205 shed less virus over a shorter period than pigs inoculated with M206. Theoretically, virulent PRV strains expressing E1 (gX −, E1+) could arise through transfer of the E1 gene of M205 to a virulent PRV strain. Therefore, we inoculated pigs with strain M12 (gX −, E1 − ) or the control strain M104 (gX − ) and compared the virulence and pathogenesis. M12 and M104 were of approximately equal virulence and the pathogenesis of both strains was similar. We concluded that incorporating E1 of HCV into the gX locus of PRV did not change cell or host tropism, nor did it change the virulence of either nonvirulent or virulent PRV.
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Immunogenicity of high expression adenovirus-hepatitis B virus recombinant vaccines in dogs
High yielding adenovirus (Ad)-hepatitis B recombinant (Ad-Hep B) viruses were prepared by insertion of the hepatitis B surface antigen (HBsAg) gene into the early region 3 (E3 region) of Ad4 or Ad7 vectors containing intact or largely deleted E3 regions. Both E3-deleted and non-deleted recombinants produced about six- to eightfold higher amounts of HBsAg than previously reported recombinants. These recombinant viruses were evaluated for immunogenicity in dogs which sustain abortive lung infections by Ad4 and Ad7. Recombinants containing E3 deletions elicited 10- to 12-fold stronger anti- HBs primary responses than previously evaluated low yield recombinants. Further immunizations with heterotypic Ad-Hep B recombinants induced substantial anti-HBs booster responses as well as anti-‘a’ epitope responses. In contrast, recombinant viruses containing intact E3 regions induced only weak or negligible anti- HBs responses, although such viruses induced strong antibody responses to the Ad vectors. The immunogenicity of high-yielding Ad recombinants correlated with temporal expression of HBsAg and thus the dog represents a valuable model for evaluation of immune responses to heterologous proteins that are expressed early and that do not require efficient DNA replication. Recombinants expressing HBsAg late in the infectious cycle require further testing in the fully permissive chimpanzee model. This study establishes that the E3- deleted high yield Ad4 and Ad7 recombinants represent promising live oral hepatitis B vaccine candidates.
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Expression of a foreign epitope on the surface of the adenovirus hexon
More LessTo present short protein sequences to the host immune system a foreign epitope has been expressed on the surface of the adenovirus virion as part of the hexon. As the trimeric hexon constitutes 240 out of the 252 capsomers of the virus, the foreign epitope is repeated 720 times on the virion surface. An eight amino acid sequence from the major antigenic site in the VP1 capsid protein of poliovirus type 3 was engineered into two regions of the adenovirus type 2 hexon. The two loop regions chosen to accommodate the foreign sequences are exposed on the surface of the virion, show sequence variation between serotypes and are the sites of interaction with neutralizing antibodies. Virus with substitutions in loop I had wild-type growth characteristics, whereas virus with substitutions in loop II grew poorly. Adenoviruses with poliovirus sequences in loop I were recognized and efficiently neutralized by antisera specific for the poliovirus sequence; an antiserum raised against the adenovirus with the poliovirus insert specifically recognized the VP1 capsid protein of poliovirus type 3. It is therefore feasible to alter the surface properties of the adenovirus virion and in doing so to manipulate the immune response to this virus.
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Sequence and structural analysis of murine adenovirus type 1 hexon
More LessThe nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank database and assigned accession number M81889.
The genomic region encoding the major capsid protein (hexon) of murine adenovirus type 1 (MAV-1) has been isolated and sequenced. The sequence predicts a 908 residue MAV-1 hexon protein and is flanked by a portion of the upstream pVI gene and the downstream endoproteinase gene. The order of these genes and their location in the middle of the genome are the same as those found in other adenoviruses sequenced to date. Multiple sequence alignment with the other five known hexon protein sequences reveals an overall residue identity of 51% and residue conservation of 66%. In comparison with human adenovirus type 2 (Ad2), MAV-1 hexon has major deletions between residues 141 to 170, 270 to 284 and 446 to 455. Since these regions in the Ad2 hexon are partially exposed on the outer surface of the virion, they may represent type-specific antigenic determinants. The MAV-1 hexon sequence has been modelled using the known three-dimensional structure of the Ad2 hexon. The variable regions in which the mutations, deletions and insertions occur are located in the l1 and 12 loops of the molecule that form the protruding hexon towers on the external surface of the virion.
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Production of mink enteritis parvovirus empty capsids by expression in a baculovirus vector system: a recombinant vaccine for mink enteritis parvovirus in mink
More LessThe VP-2 gene of mink enteritis parvovirus (MEV) was amplified by the polymerase chain reaction using MEV DNA isolated from the faeces of a naturally infected mink. Subsequently the VP-2 gene was cloned into a baculovirus expression vector. Recombinant baculo-viruses were isolated and the MEV VP-2 gene product was characterized after expression in Sf9 insect cells. The MEV VP-2 product had the same size as that reported for the wild-type MEV VP-2 protein and was recognized by convalescent sera from MEV-infected mink and a panel of monoclonal antibodies reactive to MEV. Furthermore, the VP-2 protein was able to form parvovirus-like particles, which had haemagglutinating properties comparable with the wild-type MEV. The cloned VP-2 gene was sequenced and only five nucleotide differences were found after alignment with the known sequences of the MEV type 1 and type 2 isolates. Surprisingly, the VP-2 gene encoded a valine and a tyrosine at amino acid positions 232 and 234, identical to the situation found in MEV type 1, but at position 300 there was a valine which is a determinant of MEV type 2. Immunization of mink with approximately 40000 haemagglutinating units of recombinant MEV VP-2 induced a measurable antibody response as tested by haemagglutination inhibition. Furthermore, the immunized mink did not excrete virus and did not develop clinical disease upon challenge with a virulent isolate of MEV.
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Immune response to human papillomavirus type 16 E6 gene in a live vaccinia vector
L. Gao, B. Chain, C. Sinclair, L. Crawford, J. Zhou, J. Morris, X. Zhu and H. StaussImmunization of mice with a recombinant vaccinia virus expressing the human papillomavirus type 16 (HPV-16) E6 gene elicits specific antibody, proliferative and cytotoxic T lymphocyte responses. T and B cell epitopes were mapped by using synthetic peptides. This study provides the background to future investigation aimed at developing prophylactic and therapeutic vaccines against HPV-16 infection and cervical cancer.
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Delayed-type hypersensitivity response to human papillomavirus type 16 E6 protein in a mouse model
More LessA mouse model incorporating the epitheliotropic nature of human papillomavirus (HPV) infections has been used to study an immune response to HPV type 16 (HPV-16) E6 protein in vivo. Using a transplantation technique, a novel immortal keratinocyte cell line expressing the E6 protein has been grafted onto syngeneic mice to re-form a differentiated epithelium overlying a granulation tissue bed. By this approach the presentation of viral antigens to the immune system can be modelled in a way analogous to the natural infection. Here we report a delayed-type hypersensitivity (DTH) reaction in grafted mice challenged intradermally with a recombinant vaccinia virus expressing the HPV-16 E6 protein. The specificity of the response was confirmed by the absence of a DTH reaction to challenge with virus expressing either HPV-16 E7 or L1 protein.
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