- Volume 81, Issue 7, 2000
Volume 81, Issue 7, 2000
- Review Article
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- Animal: RNA Viruses
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Mutational analysis of hepatitis C virus NS3-associated helicase
More LessNonstructural protein 3 (NS3) of hepatitis C virus contains a bipartite structure consisting of an N-terminal serine protease and a C-terminal DEXH box helicase. To investigate the roles of individual amino acid residues in the overall mechanism of unwinding, a mutational–functional analysis was performed based on a molecular model of the NS3 helicase domain bound to ssDNA, which has largely been confirmed by a recently published crystal structure of the NS3 helicase–ssDNA complex. Three full-length mutated NS3 proteins containing Tyr(392)Ala, Val(432)Gly and Trp(501)Ala single substitutions, respectively, together with a Tyr(392)Ala/Trp(501)Ala double-substituted protein were expressed in Escherichia coli and purified to homogeneity. All individually mutated forms showed a reduction in duplex unwinding activity, single-stranded polynucleotide binding capacity and polynucleotide-stimulated ATPase activity compared to wild-type, though to different extents. Simultaneous replacement of both Tyr392 and Trp501 with Ala completely abolished all these enzymatic functions. On the other hand, the introduced amino acid substitutions had no influence on NS3 intrinsic ATPase activity and proteolytic efficiency. The results obtained with Trp(501)Ala and Val(432)Gly single-substituted enzymes are in agreement with a recently proposed model for NS3 unwinding activity. The mutant phenotype of the Tyr(392)Ala and Tyr(392)Ala/Trp(501)Ala enzymes, however, represents a completely novel finding.
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Dengue virus type 1 DNA vaccine induces protective immune responses in rhesus macaques
A candidate DNA vaccine expressing dengue virus type 1 pre-membrane and envelope proteins was used to immunize rhesus macaques. Monkeys were immunized intramuscularly (i.m.) or intradermally (i.d.) by three or four 1 mg doses of vaccine, respectively. Monkeys that were inoculated i.m. seroconverted more quickly and had higher antibody levels than those that were inoculated i.d. The sera exhibited virus-neutralizing activity, which declined over time. Four of the eight i.m.-inoculated monkeys were protected completely from developing viraemia when challenged 4 months after the last dose with homologous dengue virus. The other four monkeys had reduced viraemia compared with the control immunized monkeys. The i.d.-inoculated monkeys showed no reduction in viraemia when challenged with the virus. All vaccinated monkeys showed an anamnestic antibody response, indicating that they had established immunological memory. Vaccine-induced antibody had an avidity index similar to that of antibody induced by virus infection; however, no clear correlation was apparent between antibody avidity and virus neutralization titres.
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Germinal centre localization of bovine viral diarrhoea virus in persistently infected animals
More LessImmunohistochemical analysis of peripheral lymph nodes from gnotobiotic calves persistently infected with bovine viral diarrhoea virus (BVDV) revealed extensive deposition of Erns and localization of the viral genome in the light zone of germinal centres. Viral antigen co-localized with immunoglobulin in the germinal centres and was shown to be extracellular. Despite the presence of viral antigen in germinal centres, circulating anti-BVDV antibody was not detected. These findings provide evidence that calves persistently infected with BVDV, in the absence of adventitious infection, can generate a B cell response to the persisting virus. The nature of the tolerance in calves persistently infected with BVDV is discussed in light of these findings.
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The complete sequence of hepatitis E virus genotype 4 reveals an alternative strategy for translation of open reading frames 2 and 3
More LessIsolates of hepatitis E virus (HEV) have recently been described from China that are distinct from Burmese, Mexican and US viruses and constitute a novel genotype (genotype 4). Here, the complete genomic sequence of a representative isolate of genotype 4 HEV, amplified directly from the stool of an acutely infected patient, is presented. Analysis of the entire sequence confirms our previous conclusion, based upon partial sequence data, that these Chinese isolates belong to a novel genotype. Typical of genetic variation in HEV, most nucleotide substitutions occur in the third base of the codon and do not affect the amino acid sequence. The genotype 4 virus is unusual in that a single nucleotide insertion in the ORF 3 region changes the initiation of ORF 3, and perhaps also ORF 2. The consequences of these changes are discussed.
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Expression of reporter genes from the defective RNA CD-61 of the coronavirus infectious bronchitis virus
The defective RNA (D-RNA) CD-61, derived from the Beaudette strain of the avian coronavirus infectious bronchitis virus (IBV), was used as an RNA vector for the expression of two reporter genes, luciferase and chloramphenicol acetyltransferase (CAT). D-RNAs expressing the CAT gene were demonstrated to be capable of producing CAT protein in a helper-dependent expression system to about 1·6 μg per 106 cells. The reporter genes were expressed from two different sites within the CD-61 sequence and expression was not affected by interruption of the CD-61-specific ORF. Expression of the reporter genes was under the control of a transcription-associated sequence (TAS) derived from the Beaudette gene 5, normally used for the transcription of IBV subgenomic mRNA 5. The Beaudette gene 5 TAS is composed of two tandem repeats of the IBV canonical consensus sequence involved in the acquisition of a leader sequence during the discontinuous transcription of IBV subgenomic mRNAs. It is demonstrated that only one canonical sequence is required for expression of mRNA 5 or for the expression of an mRNA from a D-RNA and that either sequence can function as an acceptor site for acquisition of the leader sequence.
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Replication-competent foot-and-mouth disease virus RNAs lacking capsid coding sequences
More LessRNA transcripts were prepared from plasmids encoding an infectious cDNA of foot-and-mouth disease virus (FMDV) or derivatives in which the leader (Lab and Lb) and capsid protein coding sequences were deleted or replaced by sequences encoding chloramphenicol acetyltransferase (CAT). The transcripts were electroporated into BHK cells and the expression of CAT and the FMDV 3C protease was monitored. Detection of CAT and 3C was dependent on the ability of the transcript to replicate. All of the Lb coding sequence and 94% of P1 (the capsid protein precursor) coding sequence could be deleted without any apparent effect on the ability of the RNA to replicate. Thus, no cis-acting replication element is present within this region of the FMDV genome. Trans-encapsidation of these FMDV replicons was very inefficient, which may explain the lack of production of defective-interfering particles in FMDV-infected cells.
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Caspases are not involved in the cleavage of translation initiation factor eIF4GI during picornavirus infection
More LessInfection of cells by many picornaviruses results in the rapid inhibition of cellular protein synthesis due to cleavage of the translation initiation factor eIF4G. The poliovirus (PV) 2A and foot-and-mouth disease virus (FMDV) L proteases are each sufficient to mediate this cleavage, but the cleavage mechanism may be indirect, involving an unidentified cellular protease(s). eIF4G is also targetted for cleavage by caspase-3 during apoptosis. Here, it is shown that caspase inhibitors do not inhibit the cleavage of eIF4GI during PV or FMDV infection. Similarly, in transient-expression studies, the cleavage of eIF4GI induced by PV 2A or FMDV L was unaffected by these inhibitors. Furthermore, the cleavage of eIF4GI was observed in PV-infected MCF-7 cells lacking caspase-3. These data, and the fact that induction of apoptosis yields different eIF4GI cleavage fragments, indicate that caspases do not have a major role in the cleavage of eIF4GI during PV or FMDV infection.
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Antigenic properties of human parechovirus 1
More LessHuman parechoviruses 1 and 2 (HPEV1 and HPEV2, respectively), formerly known as echoviruses 22 and 23, have been assigned to a novel picornavirus genus on the basis of their distinct molecular and biological properties. To study the immunological characteristics of HPEV1 capsid proteins, antigenic analysis was carried out by a peptide scanning technique, which can be used to identify the immunogenic peptide sequences of a protein. Partially overlapping peptides, representing the capsid of HPEV1, were synthesized using a 12 aa window in a three residue shift and reactivity of rabbit and murine HPEV1 antisera against these peptides were tested. Using this method, an antigenic site in the VP0 polypeptide, recognized by both rabbit and murine antisera, was identified. The sequence of this region was conserved among HPEV1 clinical isolates obtained from Finland and the United States. Antiserum against this peptide region showed neutralizing activity against HPEV1 in cell culture. Because the C-terminal region of HPEV1 VP1 contains a functional RGD motif, the antigenicity of this region was also tested. By using the corresponding peptide antiserum, neutralization of HPEV1 was observed. Cross-neutralization between HPEV1 and coxsackievirus A9, an enterovirus with a similar RGD motif in VP1, was also detected.
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The rate of progression to AIDS is independent of virus dose in simian immunodeficiency virus-infected macaques
More LessOf the viral factors that are proposed to influence the rate of progression to AIDS, the role of infectious dose remains unresolved. Intravenous infection of outbred Macaca mulatta with various doses of simian immunodeficiency virus isolate 8980 (SIV8980) revealed an endpoint from which an infectious dose 50 (ID50) was defined. In the six infected animals, the time to develop AIDS was variable with a spectrum of rapid, intermediate and slow progressors. High and sustained plasma viraemia with marked loss of CD4+ T-cells was a distinguishing feature between rapid versus intermediate and slow progressors. Animals that received the highest doses did not develop the highest sustained viral loads, nor did they progress more rapidly to disease. Similarly, animals infected with lower doses did not uniformly develop lower viral loads or progress more slowly to AIDS. Furthermore, compiled data from more than 21 animals infected with different doses of the same virus administered by the same route failed to reveal any correlation of infectious dose with survival. Indeed, host factors of these outbred animals, rather than dose of the initial inoculum, were probably an important factor influencing the rate of disease progression in each individual animal. Comparison of animals infected with SIVB670, from which SIV8980 was derived, revealed marked differences in disease progression. Clearly, although dose did not influence viral loads nor disease progression, the virulence of the initial inoculum was a major determinant of the rate of progression to AIDS.
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The strong positive correlation between effective affinity and infectivity neutralization of highly cross-reactive monoclonal antibody IIB4, which recognizes antigenic site B on influenza A virus haemagglutinin
More LessMonoclonal antibody (MAb) IIB4 displays a rare combination of virus neutralization (VN) activity and broad cross-reactivity with influenza A virus strains of the H3 subtype isolated in a period from 1973 to 1988. The epitope of this antibody has been identified as around HA1 residues 198, 199 and 201. Here we report that residues 155, 159, 188, 189 and 193 also influence the binding of this antibody. We have used this antibody to study the relationship between antibody affinity and VN activity. Using one MAb and a single epitope on the haemagglutinin (HA) of different influenza viruses we found a strong positive correlation between effective affinity and VN activity of MAb IIB4. A 10-fold increase in effective affinity corresponded to the 2000-fold increase in VN titre. It follows from the law of mass action that for an effective affinity K=9×108 l/mol, 50% VN was achieved at approx. 10% occupation of HA spikes with antibody. In contrast, for an effective affinity K=6×107 l/mol, to achieve 50% VN, occupation of up to 98% of HA spikes was required. An effective affinity about K=6×107 l/mol thus represents the limiting value for VN because a further decrease in the affinity cannot be compensated by a higher concentration of antibody.
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Plasmid DNA encoding influenza virus haemagglutinin induces Th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses
Direct intramuscular injection of plasmid DNA can generate immune responses against encoded antigens. However, the relative ability of DNA vaccines to induce cellular and humoral immunity after a single or booster immunization and the persistence of this response have not been fully elucidated. In this study, induction and maintenance of antibody and T cell subtypes with different doses of naked DNA encoding the haemagglutinin (HA) gene of influenza virus were examined and compared to the immune responses and protection induced by respiratory tract infection and immunization with a killed virus vaccine. Like natural infection, immunization with HA DNA induced potent Th1 responses. Spleen cells from mice immunized once with HA DNA in the dose range 10 ng to 100 μg secreted significant levels of IFN-γ, but low or undetectable IL-5, in response to influenza virus in vitro. Furthermore, CD4+ HA-specific Th1 clones were generated from spleens of immunized mice. Although T cell responses waned 12 weeks after a single immunization, antigen-specific Th1 cells persisted in the spleen for at least 6 months after two booster immunizations. In contrast, influenza virus-specific ELISA IgG titres were low after a single immunization and required two booster immunizations to reach significant levels. Furthermore, haemagglutination inhibition (HI) antibodies were weak or undetectable after two immunizations. Nevertheless, two doses of HA DNA conferred almost complete protection against respiratory challenge with live virus. Thus, despite the limited ability to induce antibodies, DNA vaccines confer protective immunity against influenza virus infection, which appears to be mediated by Th1 cells.
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Genetic analysis of wild-type Dobrava hantavirus in Slovenia: co-existence of two distinct genetic lineages within the same natural focus
Genetic analysis was performed of wild-type (wt) Dobrava hantavirus (DOB) strains from Slovenia, the country where the virus was first discovered and where it was found to cause haemorrhagic fever with renal syndrome (HFRS), with a fatality rate of 12%. Two hundred and sixty mice of the genus Apodemus, trapped in five natural foci of DOB-associated HFRS during 1990–1996, were screened for the presence of anti-hantavirus antibodies and 49 Apodemus flavicollis and four Apodemus agrarius were found to be positive. RT–PCR was used to recover partial sequences of the wt-DOB medium (M) and small (S) genome segments from nine A. flavicollis and one A. agrarius. Sequence comparison and phylogenetic analysis of the Slovenian wt-DOB strains revealed close relatedness of all A. flavicollis-derived virus sequences (nucleotide diversity up to 6% for the M segment and 5% for the S segment) and the geographical clustering of genetic variants. In contrast, the strain harboured by A. agrarius showed a high level of genetic diversity from other Slovenian DOB strains (14%) and clustered together on phylogenetic trees with other DOB strains harboured by A. agrarius from Russia, Estonia and Slovakia. These findings suggest that the DOB variants carried by the two species of Apodemus in Europerepresent two distinct genetic lineages.
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Neither phosphorylation nor the amino-terminal part of rabies virus phosphoprotein is required for its oligomerization
More LessRabies virus (PV strain) phosphoprotein (P) was expressed in bacteria. This recombinant protein binds specifically to the nucleoprotein–RNA complex purified from infected cells. Chemical cross-linking and gel-filtration studies indicated that the P protein forms oligomers. Analytical centrifugation data demonstrated the co-existence of monomeric and oligomeric forms of rabies virus P protein and suggested that there is an equilibrium between these species. As P expressed in bacteria is not phosphorylated, this result indicates that P phosphorylation is not required for its oligomerization. Although an alignment of several rhabdovirus P sequences revealed that the amino-terminal domain of P has a conserved predicted propensity to form helical coiled coils, an amino-terminally truncated form of P protein, lacking the first 52 residues, was also shown to be oligomeric. Therefore, the amino-terminal domain of rabies virus P is not necessary for its oligomerization.
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- Animal: DNA Viruses
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The role of the UL41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection
The UL41 gene product (vhs) of herpes simplex virus (HSV) is packaged in the virion, and mediates host protein synthesis shutoff at the early stage of the virus replication cycle. In order to clarify the role of vhs in virus replication and virulence, we isolated a completely UL41-deficient mutant (the VRΔ41 strain) and its revertant (the VRΔ41R strain). In the mouse encephalitis model, the replication of strain VRΔ41 was inhibited after 2 days post-infection, resulting in low virulence, by γ-ray-sensitive cells such as lymphocytes and/or neutrophils. The result suggested that some cytokines, produced in VRΔ41-inoculated brains, activate and induce the migration of γ-ray-sensitive cells to the infection site. Therefore, cytokines produced by HSV-1-infected human cells were screened, and potent inductions of interleukin (IL)-1β, IL-8 and macrophage inflammatory protein-1α by VRΔ41 infection were observed. Moreover, the VRΔ41 strain showed 20- and 5-fold higher sensitivity to interferon-α and -β compared to the wild-type strain, respectively. These results indicate that one important role of vhs in vivo is evasion from non-specific host defence mechanisms during primary infection through suppression of cytokine production in HSV-infected cells and reduction of the anti-HSV activity of interferon-α and -β.
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Cross-reactivity of the anti-PML antibody PG-M3 with the herpes simplex virus type 1 immediate early protein ICP4
More LessThe PML protein is one of the components of ND10, nuclear matrix-associated structures which undergo rapid disintegration at the onset of herpes simplex virus type 1 (HSV-1) infection. This disruption event has been frequently visualized in immunofluorescence assays using the anti-PML mouse monoclonal antibody PG-M3. This antibody was surprisingly found to also stain nuclear virus replication compartments when employed at higher concentrations. This was shown to be due to an unexpected cross-reactivity of the PG-M3 antibody with the HSV-1 immediate early protein ICP4, a known component of replication compartments. The sequences of ICP4 recognized by PG-M3 were found to map to the extreme amino-terminal end of the protein, which includes a 21 amino acid segment that is partially homologous to the peptide of PML that was used to make PG-M3. These results suggest that PG-M3 may no longer represent an appropriate antibody for use in visualizing the fate of PML and ND10 during HSV-1 infection.
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Human cytomegalovirus UL37 immediate-early regulatory proteins traffic through the secretory apparatus and to mitochondria
More LessThe human cytomegalovirus (HCMV) UL36–38 immediate-early (IE) locus encodes the UL37 exon 1 (pUL37x1) and UL37 (gpUL37) regulatory proteins, which have anti-apoptotic activities. pUL37x1 shares its entire sequence, including a hydrophobic leader and an acidic domain, with the exception of one residue, with the amino terminus of gpUL37. gpUL37 has, in addition, unique N-linked glycosylation, transmembrane and cytosolic domains. A rabbit polyvalent antiserum was generated against residues 27–40 in the shared amino-terminal domain and a mouse polyvalent antiserum was generated against the full-length protein to study trafficking of individual UL37 proteins in human cells that transiently expressed gpUL37 or pUL37x1. Co-localization studies by confocal laser scanning microscopy detected trafficking of gpUL37 and pUL37x1 from the endoplasmic reticulum to the Golgi apparatus in permissive U373 cells and in human diploid fibroblasts (HFF). Trafficking of gpUL37 to the cellular plasma membrane was detected in unfixed HFF cells. FLAG-tagged gpUL37 trafficked similarly through the secretory apparatus to the plasma membrane. By using confocal microscopy and immunoblotting of fractionated cells, gpUL37 and pUL37x1 were found to co-localize with mitochondria in human cells. This unconventional dual trafficking pattern through the secretory apparatus and to mitochondria is novel for herpesvirus IE regulatory proteins.
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Characterization of the Epstein–Barr virus BRRF1 gene, located between early genes BZLF1 and BRLF1
More LessThe switch from latency to a productive cycle in Epstein–Barr virus (EBV)-infected B cells proliferating in vitro is thought to be due to the transcriptional activation of two viral genes, BZLF1 and BRLF1, encoding two transcription factors called EB1 and R respectively. However, a third gene, BRRF1 is contained in the BZLF1/BRLF1 locus, overlapping with BRLF1 but in inverse orientation. We have characterized the 5′ end of the BRRF1 mRNA and the promoter, PNa, at which BRRF1 pre-mRNA is initiated. We show that although a single BRRF1 mRNA species is induced by 12-O-tetradecanoylphorbol 13-acetate/sodium butyrate in several EBV-infected B cell lines, in Akata cells treated with anti-IgG two BRRF1 mRNAs can be detected. Transcription initiated at the BRRF1 promoter was activated by EB1 but not by R, and EB1-binding sites which contribute to the EB1-activated transcription have been mapped to between positions −469 and +1. A 34 kDa protein could be translated from the BRRF1 mRNA both in vitro and in vivo, and was found predominantly in the nucleus of HeLa cells transfected with a BRRF1 expression vector. Thus there are three promoters in the region of the EBV chromatin containing the BZLF1/BRLF1 genes, two of which, PZ and PNa, potentially share regulatory elements.
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Herpesvirus papio encodes a functional homologue of the Epstein–Barr virus apoptosis suppressor, BHRF1
More LessThe human tumour virus Epstein–Barr virus (EBV) encodes a 17 kDa protein, BHRF1, which is a member of the Bcl-2 family and has been shown to suppress apoptosis. The role of this gene in the life-cycle of EBV has not been fully elucidated. In order to identify motifs conserved in herpesviruses and possibly shed light on its function we isolated a BHRF1 homologue from herpesvirus papio (cercopithecine herpesvirus-12) a closely related gammaherpesvirus of baboons. The gene, hvpBHRF1, also encodes a 17 kDa protein which shares 64% identity and 79% similarity with EBV BHRF1 at the amino acid level. In biological assays, hvpBHRF1 and BHRF1 conferred similar levels of protection on human keratinocytes induced to apoptose with cis-platin.
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Establishment of a cell line persistently infected with bovine herpesvirus-4 by use of a recombinant virus
More LessBovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, productively infects multiple cell lines of various species and causes cell death. A human rhabdomyosarcoma cell line, RD-4, infected with BHV-4 produced low levels of early and late viral RNAs and infectious virus, but exhibited no cytopathic effect. Using a recombinant BHV-4 containing a neomycin-resistance gene, we established RD-4-derived cell lines persistently infected with BHV-4. The viral genome in these cells was predominantly circular. Because of drug selection, every cell contained a viral genome. In addition, all cells stained with a BHV-4-specific antiserum. Therefore, these cell lines are not carrier cultures. These cells produced infectious virus at all passages tested. Even though cells were selected and maintained at a concentration of geneticin at least 2·5 times that necessary to kill uninfected RD-4 cells, selected cells contained only approximately one viral genome per diploid host cell genome. Persistently infected cells grew more slowly than uninfected cells, even in the absence of drug. The slower growth of these cells suggests that any growth advantage conferred by multiple copies of the neomycin-gene-carrying viral genome might be offset by the detrimental effects of viral gene expression. This situation contrasts with other gammaherpesviruses, which are able to growth-transform cells.
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Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes
Two types of porcine circovirus (PCV) have been isolated and are referred to as PCV1 and PCV2. PCV1 represents an apathogenic virus, whereas PCV2 is associated with post-weaning multisystemic wasting syndrome. The two PCVs are related, since they display about 70% identity based on nucleotide sequences. In order to discriminate between common and type-specific antigens, an immunocytological approach was used following transfections with cloned circovirus DNAs, as well as recombinant proteins expressed by either baculovirus or plasmid vectors. The ORF1-encoded proteins in the two viruses were shown to be antigenically related, whereas the ORF2 proteins were recognized differentially by polyclonal anti-PCV2 antibodies. Furthermore, PEPSCAN analysis performed on overlapping fragments of the genes encoding part of ORF1 and the entire ORF2 and ORF3 led to the identification of five dominant immunoreactive areas, one located on ORF1 and four on ORF2. However, only some ORF2 peptides proved to be immunorelevant epitopes for virus type discrimination. The potential use of ORF2-derived antigens as diagnostic tools is demonstrated.
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Inverse relationship between the expression of the human papillomavirus type 16 transcription factor E2 and virus DNA copy number during the progression of cervical intraepithelial neoplasia
The human papillomavirus type 16 (HPV-16) status of 43 cervical biopsies, which had been characterized histologically as normal, various grades of cervical intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma, was examined by using (i) a novel antibody against the HPV-16 E2 protein, (ii) sensitive HPV-16 DNA in situ hybridization and (iii) microdissection/PCR for the E2 ORF. The data indicate that E2 protein expression is highest in koilocytes in lower-grade CIN (I), but decreases with increasing grade, whereas the detection of HPV DNA is delayed until CIN I/II, rising to the highest levels in carcinoma cells. Co-localization of E2 with HPV-16 DNA-positive cells was most commonly observed in koilocytes in CIN II lesions. PCR analyses of microdissected epithelium from the same or serial sections indicated that E2 ORFs were retained in an intact form in a number of higher-grade CIN lesions and invasive carcinomas.
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The complete nucleotide sequence of fowl adenovirus type 8
Davor Ojkic and Éva NagyThe fowl adenovirus type 8 (FAdV-8) genome was sequenced and found to be 45063 nucleotides in length, the longest adenovirus (AdV) genome for which the complete nucleotide sequence has been determined so far. No regions homologous to early regions 1, 3 and 4 (E1, E3 and E4) of mastadenoviruses were recognized. Gene homologues for early region 2 (E2) proteins, intermediate protein IVa2 and late proteins were found by their similarities to protein sequences from other AdVs. However, sequences homologous to intermediate protein IX and late protein V could not be identified. Sequences for virus-associated RNA could also not be recognized. Two regions of repeated sequences were found on the FAdV-8 genome. The shorter repeat region contained five identical and contiguous direct repeats that were each 33 bp long, while the longer repeat region was made of 13 identical and contiguous, 135 bp long repeated subunits.
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- Insect
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Expression of a Tranosema rostrale polydnavirus gene in the spruce budworm, Choristoneura fumiferana
More LessThe endoparasitic wasp Tranosema rostrale (Ichneumonidae) transmits a polydnavirus (PDV) to its host, Choristoneura fumiferana, during oviposition. Unlike most other PDVs examined, the virus of T. rostrale (TrPDV) does not appear to play an important role in suppressing the host cellular immune response. However, it inhibits host metamorphosis. In the present study, TrPDV gene expression was examined in parasitized and virus-injected last-instar caterpillars. Northern analysis with viral DNA as a probe revealed only one detectable mRNA, of about 650 bp. The corresponding cDNA, termed TrV1, was cloned and sequenced and found to encode a protein of 103 amino acids which, following cleavage of the putative signal peptide, has a predicted molecular mass of 9·3 kDa. This protein displays limited similarity to the VHv1.4 cysteine-rich protein from the PDV of Campoletis sonorensis, mostly within the signal peptide region. By using a TrV1-specific probe, the TrV1 gene was localized to segment G of the TrPDV genome. The cuticle and fat body were identified as the principal sites of TrV1 transcription, with little transcription observed in haemocytes and midgut. Western analysis of proteins extracted from selected tissues of parasitized insects suggested that the TrV1 protein is secreted in the haemolymph. As observed for other PDVs, injection of TrPDV did not suppress transcription of the gene that encodes juvenile hormone esterase, the activity of which is inhibited by the virus. We speculate that the TrV1 protein may play a role in the inhibition of C. fumiferana metamorphosis.
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Filamentous actin is required for lepidopteran nucleopolyhedrovirus progeny production
More LessAutographa californica M nucleopolyhedrovirus (AcMNPV) is the prototypical member of the Nucleopolyhedrosis genus of the Baculoviridae, a family of large, double-stranded DNA viruses that are highly diverse. Nucleocapsid morphogenesis of AcMNPV and others in the Nucleopolyhedrovirus genus takes place within the nuclei of infected host cells. Previously, we showed that filamentous actin (F-actin) is essential for this process to occur in AcMNPV-infected cells, an unprecedented finding for a DNA virus that replicates within the nucleus. Because of the fundamental importance of this requirement to our understanding of virus–host interactions, and because of the diversity of viruses included within the Nucleopolyhedrovirus genus, we were compelled to determine whether the replication of other nucleopolyhedroviruses was also F-actin dependent. We report here that progeny virus production of six other lepidopteran nucleopolyhedroviruses, representing both phylogenetic groups I and II within the genus, is also F-actin dependent. The six viruses studied (Spodoptera frugiperda MNPV, Bombyx mori NPV, Orgyia pseudotsugata MNPV, Lymantria dispar MNPV, Anticarsia gemmatalis MNPV and Helicoverpa zea SNPV) were unable to produce progeny in the presence of either cytochalasin D or latrunculin A, two actin-binding agents that interfere with F-actin-dependent processes but differ in their modes of action. F-actin-dependent progeny morphogenesis, therefore, appears to be a characteristic common among viruses in this genus that have lepidopteran hosts.
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- Plant
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Multiple infection, recombination and genome relationships among begomovirus isolates found in cotton and other plants in Pakistan
Begomoviruses occur in many plant species in Pakistan and are associated with an epidemic of cotton leaf curl disease that has developed since 1985. PCR analysis with primer pairs specific for each of four already sequenced types of DNA-A of cotton leaf curl virus (CLCuV-PK types a, 26, 72b and 804a), or for okra yellow vein mosaic virus (OYVMV), indicated that many individual naturally infected plants of cotton and other malvaceous species contained two or three begomovirus sequences. Similarly, sequence differences among overlapping fragments of begomovirus DNA-A, amplified from individual naturally infected plants, indicated much multiple infection in malvaceous and non-malvaceous species. Some cotton plants contained DNA-A sequences typical of begomoviruses from non-malvaceous species, and some non-malvaceous plants contained sequences typical of CLCuV-PK. Some DNA-A sequences were chimaeric; they each included elements typical of different types of CLCuV-PK, or of different malvaceous and/or non-malvaceous begomoviruses. Often an apparent recombination site occurred at the origin of replication. No complete CLCuV-PK DNA-A sequence was found in malvaceous or non-malvaceous species collected in Pakistan outside the area of the cotton leaf curl epidemic but chimaeric sequences, including a part that was typical of CLCuV-PK DNA-A, did occur there. We suggest that recombination among such pre-existing sequences was crucial for the emergence of CLCuV-PK. Recombination, following multiple infection, could also explain the network of relationships among many of the begomoviruses found in the Indian subcontinent, and their evolutionary divergence, as a group, from begomoviruses causing similar diseases in other geographical regions.
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Limitations on the use of fused green fluorescent protein to investigate structure–function relationships for the cauliflower mosaic virus movement protein
More LessTo investigate the process of tubule formation for the cauliflower mosaic virus movement protein (CaMV MP), the green fluorescent protein (GFP) was fused to the MP to provide a vital marker for MP location after expression in insect cells. In contrast to the long tubular structures seen previously following baculovirus-based expression of the wild-type MP, the fusion protein produced only aggregates of fluorescing material in the cytoplasm. However, by co-expressing wild-type MP and GFP–MP, or by engineering their co-accumulation by introducing a foot-and-mouth disease virus 2A cleavage sequence between GFP and MP, long GFP-fluorescing tubules were formed. The experiments suggest that the presence of GFP at the N or C terminus of the tubule-forming domain of the CaMV MP places steric constraints upon the aggregation of the MP into a tubule but that this can be overcome by providing wild-type protein for inclusion in the aggregate.
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Potato leafroll virus protein P1 contains a serine proteinase domain
X. Li, M. D. Ryan and J. W. LambThe multi-domain potato leafroll virus replicase protein P1 was expressed in insect cells from the polyhedrin promoter of Autographa californica nucleopolyhedrovirus. Using antisera raised against P1, it was shown that P1 was cleaved near the VPg in insect cells in a manner similar to that in plant cells, to produce a ∼27 kDa C-terminal fragment. Furthermore, it was shown that the proposed serine proteinase-like domain within P1 is responsible for this processing and that this can occur in a trans (intermolecular) reaction. Four conserved residues within the serine proteinase domain that are essential for catalysis have been identified, consistent with the proposal that this domain comprises a serine proteinase.
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Sugarcane yellow leaf virus: a novel member of the Luteoviridae that probably arose by inter-species recombination
More LessThe 5895 nucleotide long single-stranded RNA genome of Sugarcane yellow leaf virus Florida isolate (SCYLV-F) includes six major ORFs. All but the first of these are homologous to genes of known function encoded by viruses of the three newly defined genera in the Luteoviridae (‘luteovirids’), i.e. poleroviruses, luccccteoviruses and the enamoviruses. SCYLV-F ORFs 1 and 2 are most closely related to their polerovirus counterparts, whereas SCYLV-F ORFs 3 and 4 are most closely related to counterparts in luteovirus genomes, and SCYLV-F ORF5 is most closely related to the read-through protein gene of the only known enamovirus. These differences in affinity result from inter-species recombination. Two recombination sites in the genome of SCYLV-F map to the same genomic locations as previously described recombinations involving other luteovirids. A fourth type of luteovirid, Soybean dwarf virus, has already been described. Our analyses indicate that SCYLV-F represents a distinct fifth type.
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