- Volume 79, Issue 12, 1998
Volume 79, Issue 12, 1998
- Articles
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Selection of human anti-human immunodeficiency virus type 1 envelope single-chain antibodies from a peripheral blood cell-based phage repertoire
Monoclonal antibodies play an important role in the development of diagnostic assays. Instead of using hybridoma technology to isolate human immunodeficiency virus type 1-specific antibodies, a phage-displayed antibody library was generated from a small number (107) of peripheral blood lymphocytes from a seropositive donor. Two families of singlechain antibodies (scFvs) were selected by biopanning with the envelope precursor gp160. ELISA and competition in the BlAcore system revealed that one antibody family recognized a conformation-sensitive epitope within gp120, while the other antibody family was gp41-specific. The latter group had sequence similarity to antibodies recognizing the cluster III epitope of gp41. Binding of scFvs to gp160 could be inhibited with the donor’s serum antibodies, indicating that antibodies with a similar specificity were circulating in the donor’s blood. Competition experiments suggested that the epitope of the anti-gp41 antibodies was recognized by a broad range of patients’ sera: 21 out of 22 sera from North American and all 20 sera from African seropositive patients inhibited binding of scFvs. In contrast, three sera from this panel did not react with the epitope of the anti-gp120 antibodies. These data indicate that, because of the conserved nature of its epitope, the anti-gp41 antibody will be suitable for diagnostic applications.
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Characteristics of primary infection of a European human immunodeficiency virus type 1 clade B isolate in chimpanzees
The aim of the study was to select, from a panel of candidate European human immunodeficiency virus type 1 (HIV-1) clade B primary virus isolates, one isolate based on replication properties in chimpanzee peripheral blood mononuclear cells (PBMC). Secondly, to evaluate the in vivo kinetics of primary infection of the selected isolate at two different doses in two mature, outbred chimpanzees (Pan troglodytes). Four different low passage, human PBMC-cultured ‘primary’ HIV-1 isolates with European clade B consensus sequence were compared for their ability to replicate in vitro in chimpanzee versus human PBMC. The isolate which yielded the highest titre and most vigorous cytopathic effect in chimpanzee PBMC was evaluated for coreceptor usage and chosen for evaluation in vivo. Only the HIV-1Han2 isolate replicated in chimpanzee PBMC in vitro at detectable levels. This isolate was demonstrated to utilize CCR4, CCR5 and CXCR4 co-receptors and could be inhibited by β-chemokines. Infection of chimpanzees was demonstrated by viral RNAand DNAPCR analysis, both in plasma as well as in PBMC and lymph node cells as early as 3 weeks after inoculation. Antibodies developed within 6 weeks and continued to increase to a maximum titre of approximately 12800, thereafter remaining in this range over the follow-up period of 2 years. Compared to cell line-adapted HIV-1 isolates there were slight but no dramatic differences in the kinetics of infection of chimpanzees with this particular primary isolate.
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The effect of the cellular stress response on human T-lymphotropic virus type I envelope protein expression
More LessIn this report the influence of the cellular stress response in mediating changes in human T-lympho-tropic virus type I (HTLV-I) viral envelope (Env) protein metabolism is determined. Previously, we reported that induction of the cellular stress response enhanced HTLV-I-mediated syncytia formation following induction of the cellular stress response in persistently infected lymphocytes. In this study, we show that the increase in HTLV-I-mediated syncytia formation following stress response induction is a result of increased cell surface expression of viral Env protein (gp46). Cellular stress in MT 2.6 cells did not alter the turnover of intracellular Env protein (gp68) as no changes in viral protein half-life were demonstrated as compared to non-stressed cells. However, Env expression in stressed cells treated with a protein synthesis inhibitor (cycloheximide) indicates the effect is mediated through increased translation of viral Env protein.
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Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures
More LessRabies virus nucleoprotein (N) was produced in insect cells using the baculovirus expression system described by Préhaud et al. (Virology 178, 486–497, 1990). The protein was either purified on a CsCl gradient, resulting in a mixture of nucleo-capsid-like structures and beaded rings, as observed by electron microscopy, or on a glycerol gradient that resulted in a preparation of the rings only. The rings and nucleocapsid-like structures had the same morphological characteristics as viral nucleocapsids. N in these structures is an 84 Å long and thin molecule that is spaced at around 34 Å along the length of the nucleocapsid, identical in shape and spacing as the nucleoprotein in nucleo-capsids of rabies virus and very similar to those of vesicular stomatitis virus. The recombinant nucleocapsids contained RNA with a stoichiometry similar to that found in viral nucleocapsids. The RNA bound in the beaded rings was a subset of the insect cellular RNA. One of the RNA species was partially sequenced and, although a positive identification could not be made, could correspond to a tRNA. With respect to sensitivity to trypsin and RNase digestion, the recombinant and viral nucleocapsids behaved similar. Trypsin cleaved a 17 kDa fragment from the carboxy terminus of N with only a very small effect on the morphology of the nucleocapsids. RNase A completely digested the resident RNA in both viral and recombinant nucleocapsids into fragments of 4–5 nt long, again with no effect on the morphology of the nucleocapsids. Thus, when the RNA is cleaved, the structure must be maintained by protein-protein contacts. Experiments to remove the resident RNA from viral and recombinant rabies virus nucleocapsids failed, whereas the same methods used to eliminate the RNA from vesicular stomatitis virus nucleocapsids was successful.
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Contribution of Taq polymerase-induced errors to the estimation of RNA virus diversity
M. A. Bracho, A. Moya and E. BarrioThe genetic diversity of a vesicular stomatitis virus population was analysed by RT-PCR, cloning and sequencing of two ~ 500 nucleotide regions of the virus genome. PCR amplifications were performed in parallel experiments with both Taq and Pfu DNA polymerases, and important differences were observed. Between 10 and 22 mutations were detected when virus populations were analysed by Taq amplification (20 clones from each region), whereas amplification of the same samples with Pfu revealed between 0 and 5 mutations. PCR fidelity assays, performed under the same PCR conditions as those used in the population analysis, showed that the Taq error-rate estimate of 0·27 × 10 4 misincorpor- ations per bp per cycle was within the range estimated elsewhere from PCR amplification of recombinant plasmids (0·27–0·85 × 10 4 errors per bp per cycle) or from functional assays (0·2–2 × 10 4 errors per bp per cycle). The error rate of Taq was found to be 9·3 times higher than the error rate of Pfu with DNA as a template, and about 10 times higher with cDNAs obtained by reverse transcription of viral RNA templates from natural populations. In the present study, we discuss (i) the implications of Taq errors on the analysis of genetic variability, based on both the frequency and nature (replacement vs synonymous) of the observed substitutions and (ii) the sample size required to assess the genetic variability in a virus population generated by a single infection.
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Characterization of three co-circulating genotypes of the small hydrophobic protein gene of mumps virus
More LessEighteen virus isolates and 22 serum samples collected between 1971 and 1997 from patients with mumps were genotyped by PCR with specific primer pairs for the A, C and D genotypes of the small hydrophobic (SH) protein gene. All serum samples were subjected to nucleotide sequence analysis of the gene, and the deduced 57-amino- acid sequences were aligned with previously published sequences from the USA, Canada, Portugal, the UK, France, Germany, Switzerland, Denmark, Sweden, Russia, China and Japan. The existence of six genotypes of the SH protein gene, named A to F, was confirmed. In the Stockholm area, co-circulation of genotypes A, C and D at different times was found. There was a striking difference in genotype between the virus isolates and the serum samples. The 18 virus isolates represented genotypes C and D, whereas the 22 serum samples contained genotype A. In most cases, the amino acid sequences of the 22 genotype A specimens were identical to the previously described SBL-1 strain of genotype A. Genotypes C and D were always associated with meningitis, and in some cases parotitis, whereas infection with genotype A most often resulted in parotitis and seldom in meningitis.
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Genetic and antigenic analysis of the G attachment protein of bovine respiratory syncytial virus strains
More LessAntigenic and genetic studies of bovine respiratory syncytial virus (BRSV) were made on isolates obtained from three continents over 27 years. Antigenic variation between eight isolates was initially determined using protein G-specific monoclonal antibodies. Four distinct reaction patterns were observed, two of which corresponded to the previously established subgroups A and AB. A third pattern was produced by five Scandinavian strains and a fourth was observed from a single Dutch isolate. The genetic diversity of 27 strains of BRSV was investigated by comparative nucleotide sequence analysis of a 731 nucleotide fragment in the G protein gene. Nine of the BRSV strains were analysed by direct sequencing of RT-PCR amplicons whereas sequences of 18 BRSV and three human respiratory syncytial virus (HRSV) strains were obtained from GenBank. The analysis revealed similarities of 88–100% among BRSV strains and 38–41 % between BRSV and HRSV. A phylogenetic tree created for BRSV revealed two main branches, one of which divided into five further lineages, each representing a geographic cluster. A correlation was evident between the positions of some strains in the phylogenetic tree and their antigenic pattern. For HRSV strains, a genetic similarity of only 62% allowed the distinction of two antigenic subgroups, A and B, a pattern which was not seen for BRSV. This study showed that genetic analysis was an accurate method for discriminating BRSV strains and that these viruses should be regarded as a single genetic and antigenic group, within which variants can be distinguished.
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Multiple genetic reassortment of avian and human influenza A viruses in European pigs, resulting in the emergence of an H1N2 virus of novel genotype
More LessNovel H1N2 influenza A viruses which were first detected in pigs in Great Britain in 1994 were examined antigenicallyand genetically to determine their origins and establish the potential mechanisms for genetic reassortment. The haemagglutinin (HA) of all swine H1N2 viruses examined was most closely related to, but clearly distinguishable both anti- genically and genetically from, the HA of human H1N1 viruses which circulated in the human population during the early 1980s. Phylogenetic analysis of the HA gene revealed that the swine H1N2 viruses formed a distinct branch on the human lineage and were probably introduced to pigs shortly after 1980. Following apparent transfer to pigs the HA gene underwent genetic variation resulting in the establishment and cocirculation of genetically and antigenically heterogeneous virus populations. Genetic analyses of the other RNA segments of all swine H1N2 viruses indicated that the neuraminidase gene was most closely related to those of early ‘human-like ’ swine H3N2 viruses, whilst the RNA segments encoding PB2, PB1, PA, NP, M and NS were related most closely to those of avian viruses, which have been circulating recently in pigs in Northern Europe. The potential mechanisms and probable progenitor strains for genetic reassortment are discussed, but we propose that the swine H1N2 viruses examined originated following multiple genetic reassortment, initially involving human H1N1 and ‘human-like ’ swine H3N2 viruses, followed by reassortment with ‘avian-like ’ swine H1N1 virus. These findings suggest multiple reassortment and replication of influenza viruses may occur in pigs many years before their detection as clinical entities.
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Two domains of the Borna disease virus p40 protein are required for interaction with the p23 protein
More LessBorna disease virus (BDV) has five major open reading frames, which encode the proteins p40, p23, gp18, p57 and p190. By analogy with other negative-strand RNA viruses, p40 is a putative nucleoprotein and p23 is a putative phospho- protein. These proteins are known to form complexes with each other and with the polymerase protein in other viruses. In this paper, it is shown that BDV p40 and p23 can form complexes with each other in infected cells. Furthermore, the amino acids of p40 that are necessary for formation of this complex have been mapped.
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Compatibility of plasmids expressing different antigens in a single DNA vaccine formulation
More LessOne anticipated advantage of DNA immunization is the potential to create multivalent vaccines. We have examined the effects of mixing plasmids into single formulations using plasmids expressing four different membrane bound glycoproteins from bovine herpesvirus-1 (BHV-1), bovine para- influenzavirus-3 (bPI3) and human influenza virus (HINF). Plasmids were delivered by intradermal injection into the tails of mice and the types of responses generated were clearly affected by the expressed antigen. Plasmids expressing glycoproteins B and D of BHV-1, and the haemagglutinin/ neuraminidase of bPI3, generated responses with a predominance of IgG1, suggestive of a Th2 type of response. In contrast, the plasmid expressing HINF haemagglutinin induced an antibody response biased towards IgG2a, indicating a Th1 type of response. In most instances the mixing of plasmids had only slight effects on the magnitude or bias of the responses to the individual components. However, under certain conditions we found that addition of a second plasmid converted an IgG2a biased response to a response with primarily IgG1 antibody. The reverse situation (i.e. an IgG1 conversion to IgG2a), however, was not found. These findings have important implications for the development of multivalent vaccines.
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An RNA virus can adapt to the multiplicity of infection
More LessRNA viruses evolve as complex distributions of mutants termed viral quasispecies. For this reason it is relevant to explore those environmental parameters that favour the selective advantage of some viral subpopulations over others. In the present study we provide direct evidence that the relative fitness of two competing viral subpopulations may depend on the multiplicity of infection (m.o.i.). Two closely related subpopulations of foot-and-mouth disease virus (FMDV) of serotype C, which differed in their history of cytolytic passages in BHK-21 cells, were subjected to growth-competition experiments in BHK-21 cells. One of the populations, termed S, was found to have a selective advantage over the other population, termed L, only when the competition passages were carried out at low m.o.i. In contrast, both populations, L and S, coexisted during serial passages carried out at high m.o.i. No differences between S and L were detected in assays of inhibition of infectivity by synthetic peptides, in cell binding-competition experiments, or in virulence for BHK-21 cells. However, FMDV S displayed increased heparin binding compared with L, and L higher virulence for Chinese hamster ovary (CHO) cells than S. These results with FMDV suggest that small differences in the interaction of the virus with the host cell may contribute to an m.o.i.-dependent selective advantage of one viral subpopulation over a closely related subpopulation. Therefore, different viral mutants from quasispecies replicating in vivo may be selected depending on the number of variant viruses relative to the number of susceptible cells.
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Porcine cells persistently infected with classical swine fever virus protected from pestivirus-induced cytopathic effect
More LessCytopathogenicity of classical swine fever virus (CSFV) depends on the presence of defective particles containing a subgenomic (sg) RNA with a defined deletion. In a previous report we described the spontaneous generation of this sg RNA and therefore of cytopathogenic (cp) CSFV in porcine kidney cell cultures persistently infected with CSFV. Frequently, some cells survived the CPE and could be further propagated. They remained positive for viral antigen and continued to shed complete virus and in most cases also defective virus particles. SK- 6 cells that had survived the CPE (CPEsurv cells) were used to investigate these findings further. In contrast to persistently infected cells that had not experienced a CPE, CPEsurv cells were protected from the CPE when superinfected with cp CSFV or with cp bovine viral diarrhoea virus. Similarly, cells which were rescued and further propagated after acute infection with cp CSFV also proved to be protected from the CSFV-induced CPE. When either virus obtained from CPEsurv cells that had spontaneously lost the sg RNA or virus from which defective particles had been removed was used to establish persistently infected cells, these cells were also protected from the CPE after superinfection with cp CSFV. These findings suggest that the virus contained in CPEsurv cells confers on the host cell the ability to resist the CSFV-induced CPE. However, when naive cells were infected with supernatants from CPEsurv cells that contained defective virus particles, the CPE reappeared within three to five virus passages, indicating that the sg RNA retained its cytopathogenic potential.
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A pneumo-virulent United States isolate of porcine reproductive and respiratory syndrome virus induces apoptosis in bystander cells both in vitro and in vivo
More LessEvidence of apoptosis was detected for the United States porcine reproductive and respiratory syndrome virus (PRRSV) in ATCC CRL11171 cells inoculated with strain ATCC VR2385 and in the tissues of pigs infected with the same strain. Apoptosis was detected by agarose gel electrophoresis, transmission electron microscopy and terminal deoxytransferase dUTP nick end labelling (TUNEL) techniques. By electron microscopy and double-labelling techniques, apoptosis was detected primarily in uninfected bystander cells in the continuous cell line rather than the PRRSV-infected cells. In the lungs, the apoptotic cells were predominantly alveolar and pulmonary intravascular macrophages, and mononuclear cells in the alveolar septa. In the lymph nodes, the apoptotic cells were predominantly tingible body macrophages and mononuclear cells. The induction of apoptosis in a large number of mononuclear cells in the lungs and lymph nodes appears to be a mechanism of PRRSV pathogenesis and might be an explanation for a dramatic reduction in the number of alveolar macrophages and circulating lymphocytes and monocytes in PRRSV-infected pigs.
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In vitro reactivation of latent equid herpesvirus-1 from CD5+/CD8+ leukocytes indirectly by IL-2 or chorionic gonadotrophin
More LessIL-2 and equine chorionic gonadotrophin (eCG) initiated reactivation of equid herpesvirus-1 (EHV-1) from venous lymphocytes at a frequency of 1/10 5. Indirect immunofluoresence showed that > 80% of virus-positive leukocytes were CD5 /CD8 with the remaining 20% being CD5 /CD8 /CD4 . Cocultivation demonstrated that the reactivated virus was infectious. In addition, virus was reactivated in vitro from leukocytes of > 70% of horses by the mitogens phytohaemagglutinin (PHA) and poke- weed mitogen (PWM). Transfer of supernatants showed that IL-2 and eCG acted indirectly by causing the release of other mediators from adherent cells; these mediators then reactivated EHV-1 from T cells. Blocking experiments with anti-IL-2 showed that PWM and PHA acted via IL-2 but that eCG did not. This is the first clear definition of the lymphoid cells that harbour latent EHV-1 in vivo and correlates with current RT-PCR and in situ hybridization of latency-associated transcripts in lymphocytes. This method of reactivation in vitro can be used to detect horses carrying latent EHV-1 in vivo and also has the potential to dissect the sequence of events involved in reactivation in vitro.
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Dendritic cells presenting equine herpesvirus-1 antigens induce protective anti-viral immunity
Equine herpesvirus-1 (EHV-1) causes rhino- pneumonitis, abortion and CNS disorders in horses. Using intranasal inoculation, the mouse model of this disease mimics the major pathogenic and clinical features of the equine disease. The aim of this study was to investigate whether murine dendritic cells (DC) can be infected with EHV-1 and whether they can be used as cellular vaccines for the induction of prophylactic anti-EHV-1 immunity. It was found that the DC lines FSDC, D2SC1, 18 (all H-2d) and 80/1 (H-2k), when incubated with the Ab4 strain of EHV-1, do not change their morphology and phenotype but sustain virus replication and induce proliferation of naive, syngeneic T cells. An even stronger proliferation of T cells was seen when DC were used that had been pre-exposed to heat-inactivated virus. DC lines were therefore pulsed with inactivated virus and were then administered intranasally to either BALB/c or C3H mice on days 25,15 and 5. Control groups received either medium, unpulsed DC or inactivated virus only. Animals were challenged with EHV-1. Whereas mice of control panels showed clinical signs of EHV-1 disease and 27 % died, animals immunized with the pulsed DC lines showed only subtle clinical symptoms, lost significantly less weight, exhibited a reduced virus load in their lungs and CNS and did not succumb to the disease during the observation period. These results show that murine DC can present EHV-1 and initiate a protective anti-viral immunity in vivo.
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Cis-acting elements in the lytic origin of DNA replication of Marek’s disease virus type 1
More LessThe replication origin of Marek ’s disease virus (MDV) type 1 was analysed by using a transient replication assay with plasmids containing various fragments of MDV strain Md5 genomic DNA. Plasmid pMBH, containing the Bam HI-H fragment, showed replication activity in MDV-infected chicken embryonic fibroblasts (CEF). By deletion analysis of pMBH, two regions, the promoter-enhancer region of the MDV pp38 gene and the 132 bp tandem direct repeat, were shown to be required for replication activity. Replication of pMBH was not observed in uninfected CEF, suggesting that a trans-acting factor(s) encoded by the MDV genome was necessary for replication.
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The herpes simplex virus 2 kb latency associated transcript (LAT) leader sequence allows efficient expression of downstream proteins which is enhanced in neuronal cells: possible function of LAT ORFs.
More LessHerpes simplex viruses (HSV)-1 and -2 enter latency in peripheral ganglia during which time a number of latency associated transcripts (LATs) are produced. The most abundant (2 kb) LAT contains ORFs of significant size which could play a role in latency. We hypothesized that the leader sequence of the 2 kb LAT might regulate expression of the LAT ORFs in neurons and might thus enhance some aspect of the latency process. We show that constructs with the HSV-1 2 kb LAT leader sequence in front of a CAT gene are efficiently translated, with enhanced activity in cells of neuronal origin, but that from within a larger LAT-derived RNA from which 2 kb LATs are efficiently spliced, expression levels are low. Thus some further regulation of expression must occur if LAT ORFs are expressed in vivo. Experiments to test any regulatory function of the LAT ORFs in suppressing or stimulating immediate early (IE) gene expression during latency did not, however, show effects on IE promoters in cotransfection assays, nor on an early gene promoter which has recently been shown to be expressed early in reactivation.
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Identification and characterization of the herpes simplex virus type 1 UL51 gene product.
More LessThe products of the herpes simplex virus type 1 UL51 gene were identified as phosphoproteins with apparent molecular masses of 27, 29 and 30 kDa. The proteins were produced at the late stage of infection, in a manner highly dependent on viral DNA synthesis, and were associated with extracellular virions. Immunofluorescence studies localized the UL51 proteins mainly to the cytoplasm, both in infected Vero cells and in transfected COS- 1 cells singly expressing the UL51 gene.
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Identification of the UL4 protein of herpes simplex virus type 1.
Herpes simplex virus type 1 gene UL4 is predicted to encode a 199-amino-acid protein with a molecular mass of 21·5 kDa. We report here identification of this protein and its localization in the nuclei of infected cells. Antisera raised against oligopeptides corresponding to the C terminus of the predicted UL4 protein were used for identification of a 25 kDa protein as the product of the UL4 gene. This protein was not detected in cells infected with a UL4 defective mutant virus, but was synthesized by coupled in vitro transcription-translation of the UL4 gene. Synthesis of the 25 kDa protein was blocked by phosphonoacetic acid, an inhibitor of DNA synthesis, indicating that the UL4 gene is expressed with γ kinetics. Subcellular fractionation showed the protein to be localized in the nucleus. It was not detected in virions or light particles.
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Strong conservation of the constitutive activity of the IE1/2 transcriptional control region in wild-type strains of human cytomegalovirus.
More LessThe IE1/2 transcriptional control region of human cytomegalovirus (HCMV) drives the expression of the HCMV major immediate-early genes (UL123–122), which encode proteins crucial for initiation of the virus replicative cycle. Nucleotide sequence polymorphism in this region of the viral genome could account for variations in the replication of HCMV wild-type strains. In order to test this hypothesis, the constitutive transcription-enhancing activity of the IE1/2 transcriptional control region derived from 12 clinical isolates of HCMV was compared. This was done by PCR amplification of the respective elements followed by cloning up-stream of a β -globin reporter gene. After transient expression in various cell types, including human teratocarcinoma cell lines, and quantification of RNA levels, the activating function of this complex cis- element was shown to be strongly conserved. This was mirrored by high nucleotide sequence conservation, even within the so-called modulator region. This strong evolutionary conservation of sequence and of transcription-enhancing function strengthens the assumption that the IE1/2 transcriptional control region plays an essential role in initiation of the HCMV replicative cycle.
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Identification and characterization of the Tupaia herpesvirus DNA polymerase gene.
More LessTupaia herpesviruses (THVs) have been isolated from malignant lymphomas and from degenerating lung or spleen cell cultures of tree shrews (Tupaia spp.), but because of a lack of genetic information the final classification of THVs is still open. In the present work the viral DNA polymerase (DPOL) gene was mapped within the genome of the different THV strains using PCR and degenerate oligonucleotide primers. Nucleotide sequences of the DPOL genes of THV strains 1 to 5 were determined and used for comparative analyses. The transcriptional activity of the THV-2 DPOL gene was confirmed by RT-PCR. It was found that the different THV strains are very closely related to each other. When compared to other herpesviruses the highest amino acid sequence identities detected were with DPOLs of the murine and human cytomegaloviruses. These results justify the conclusion that THVs are members of the subfamily Betaherpes- virinae.
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Sequence analyses of human herpesvirus-8 strains from both African human immunodeficiency virus-negative and -positive childhood endemic Kaposi’s sarcoma show a close relationship with strains identified in febrile children and high variation in the K1 glycoprotein.
More LessHuman herpesvirus-8 (HHV-8) DNA sequences have been identified in all forms of Kaposi’s sarcoma (KS), a cancer found primarily in adult AIDS patients. We have identified HHV-8 strains in a rare human immunodeficiency virus (HlV)-negative form of KS, which is endemic in children in parts of sub-Saharan Africa. This was shown in Zambia, where we also had identified HHV-8 sequences in blood from HIVnegative febrile children without KS. In order to investigate the relationship of these Zambian strains to each other and to those from other forms of KS, we compared them to strains we have characterized from European AIDS KS (Denmark) and all published sequences from all forms of KS. Four distinct genomic regions were examined by PCR and sequencing: ORF26, ORF75, gH and K1. The results showed a distinct grouping of strains from both sets of Zambian children in all genomic regions studied, but which was most pronounced in the K1 glycoprotein gene. This gene was highly variable, encoding up to 25% amino acid sequence variation. In contrast, the Zambian groups were closely related to each other, with only 2% variation. Similar results were found in comparisons to the K1 sequences from HIV-positive febrile infants or KS children. The data raise the possibility that in areas where rare childhood endemic KS occurs, geographical variation in HHV-8 may relate to differences in virulence or transmission.
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Induction of apoptosis in vitro and in vivo by H-1 parvovirus infection.
Apoptosis induced by H-1 parvovirus infection was investigated in C6 rat glioblastoma cells and in newborn rats. Apoptotic changes, such as chromatin condensation, the appearance of apoptotic nuclear bodies and oligonucleosomal DNA ladders, were observed in infected C6 cells 2 days after infection. Inhibitor assay results suggest that a caspase-3-dependent apoptosis activation pathway is induced by H-1 virus infection in C6 cells. Observations made in vivo revealed that the number of apoptotic cells increased in the infected cerebellum, coinciding with known virus infection sites.
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Simultaneous expression of recombinant baculovirus-encoded chicken anaemia virus (CAV) proteins VP1 and VP2 is required for formation of the CAV-specific neutralizing epitope.
More LessChicken anaemia virus (CAV) expresses three proteins, VP1, VP2 and VP3, but its capsid contains only the VP1 protein. In this paper, we report that for production of the neutralizing epitope, cosynthesis of (recombinant) VP1 and VP2 has to take place. We show via immunofluorescence that recom- binant-baculovirus-infected Sf9 cells synthesizing VP1 (or VP2) alone react very poorly with CAV- specific neutralizing antibodies. In contrast, Sf9 cells co-infected with VP1- and VP2-recombinant baculo-viruses, or infected with a single recombinant baculovirus co-expressing both VP1 and VP2, react strongly with the neutralizing antibodies. Furthermore, immunoprecipitation assays show that VP1 and VP2 interact directly with each other, which indicates that the non-structural protein VP2 might act as a scaffold protein in virion assembly. Recombinant baculovirus expressing VP1 and VP2 is, therefore, a potential production system for a subunit vaccine against CAV infection.
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The large T antigen of simian virus 40 binds and inactivates p53 but not p73.
More LessThe p73 proteins α and β were identified based on their similarity to the tumour suppressor gene product p53. p53 and the p73 proteins activate transcription from p53-responsive promoters. The large T antigen of simian virus 40 (SV40) forms a specific complex with p53 and inhibits p53-medi- ated transcription. Here we show that the large T antigens from SV40 and JC virus strongly reduce the transcriptional activity of p53 but do not detectably affect the ability of the p73 proteins to transactivate. p53 but not the p73 proteins associate with SV40 T antigen in vitro. Finally, p53 colocalizes with a cytoplasmic mutant of SV40 T antigen, whereas both variants of p73 fail to colocalize with cytoplasmic T antigen. These results indicate that T antigen selectively binds and inactivates p53 but does not detectably affect the p73 proteins.
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Sequence variability in the putative coding region of TT virus: evidence for two rather than several major types.
More LessRecently a new human virus, TT virus (TTV) was identified in the serum of a patient with posttransfusion hepatitis of unknown aetiology. Comparative sequence analysis of a 222 nt fragment of ORF1 of TTV was performed to assess the genomic variability of this virus. Phylogenetic analysis of the nucleotide sequences of 76 TTV isolates collected in 17 countries segregated them into two major groups: TTV 1 and TTV 2. The TTV 1 group comprised two distinct subgroups, which corresponded to previously described TTV subtypes 1a and 1b. The TTV 2 group was separated into four main branches, two of which included sequences previously provisionally attributed as TTV types 2 and 3. Bootstrap resampling, however, did not support the reliability of this grouping, suggesting that the isolates in the TTV 2 group should be considered as subtypes of a single type rather than different TTV types.
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Quasispecies nature of three maize streak virus isolates obtained through different modes of selection from a population used to assess response to infection of maize cultivars.
More LessThree maize streak virus (MSV) isolates were derived from an MSV population used to assess the response to infection of maize cultivars. Isolate SP1 was obtained from this population through short acquisition and inoculation periods (1 and 5 min, respectively), using a single Cicadulina mbila vector. Isolate SP2 was derived from SP1 after transmission to a wild perennial host (Coix lacryma-jobi), on which it was maintained for about 4 years without insect transmission. Isolate N2A, the most pathogenic isolate, was obtained from the initial population after serial passages on almost completely resistant inbred maize lines. The complexity of each isolate was analysed by RFLP analysis and sequencing based on 120 SP1 clones, 36 SP2 clones and 40 N2A clones. All three isolates were composed of different but related clones, consistent with a quasispecies structure. The mutations were distributed throughout the genome. Mutation frequencies, based on all available sequences, were 3·8 × 10 4 for SP1, 10· 5 × 10 4 for SP2 and 6· 9 × 10 4 for N2A. As expected from the bottleneck selection step, the intra-isolate variability of SP1 was relatively low. Comparison between SP1 and SP2 showed that SP1 heterogeneity increased during maintenance on the wild host. Furthermore, the consensus sequences of SP1 and SP2 differed by two non-synonymous substitutions in the complementary sense gene repA. N2A had a relatively low degree of heterogeneity, but was composed of several sub-populations. The results reflect the influence of the mode of selection of MSV isolates on their quasispecies organization, i.e. distribution of variants, and master sequence.
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Ten distinct circular ssDNA components, four of which encode putative replication-associated proteins, are associated with the faba bean necrotic yellows virus genome.
More LessFour further circular ssDNA components (C7–C10), about 1 kb in size and structurally similar to the previously described components (C1–C6) found associated with a Syrian (Sy) isolate of faba bean necrotic yellows virus (FBNYV), have been identified. Similar to C1 and C2, two of the new components (C7 and C9) encode putative replication- associated (Rep) proteins of 33 ·2 and 32·7 kDa, respectively, the former of which is 90% identical to the C10 Rep protein of milk vetch dwarf virus (MDV). C8 encodes a putative protein (17 4 kDa) whose function is unknown, but which is highly conserved between FBNYV and the other nanoviruses MDV, subterranean clover stunt virus and banana bunchy top virus. The putative protein (19·7 kDa) encoded by C10 contains an LXCXE motif, which is also present in the homologues of its relatives, suggesting that they may all interact with plant retinoblastoma-like proteins. Sequence information for seven components of an Egyptian (Eg) FBNYV isolate indicated that six of them share > 96% identity with FBNYV-Sy. However, the C1 Rep protein of FBNYV-Eg was only 63·5% identical to that of FBNYV-Sy, but was 88·3 % identical to the MDV-C2 Rep protein. We conclude that the FBNYV genome consists of seven to ten components, six of which encode non-Rep proteins. All ten components of the FBNYV genome, except the Rep components C2 and C9, had closely related counterparts in the MDV genome. In spite of this similarity, FBNYV and MDV appear to be distinct virus species.
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Sequences of ten circular ssDNA components associated with the milk vetch dwarf virus genome.
More LessMilk vetch dwarf virus (MDV) is a member of the proposed genus Nanovirus, and its genome is composed of multiple, circular ssDNA components of about 1 kb. We have cloned and sequenced ten ssDNA components and designated them MDV-C1 to C10. Each DNA component contains one potential major open reading frame, and contains a putative stem-loop structure in the non-coding region. Notably, four components (C1, C2, C3 and C10) encode distinct replication-associated (Rep) proteins of 33 kDa, which show only limited (42–57%) amino acid identity. The six other components encode proteins with calculated molecular masses ranging from 12·7 to 19·7 kDa. Comparison of the sequences with those of other nanoviruses reveals that MDV is closely related to faba bean necrotic yellows virus (FBNYV) and subterranean clover stunt virus (SCSV). Six putative MDV genome products, including one Rep and five non-Rep proteins, show high (70·4–90·9%) amino acid identity to the corresponding six FBNYV proteins, whereas two other Rep proteins encoded by MDV-C2 and C3 are 82·3% and 73·0% identical to those encoded by SCSV-C2 and C6, respectively. These results indicate that MDV, FBNYV and SCSV have diverged from a common origin, which had multiple Rep components. In addition, the putative proteins encoded by MDV-C4 and its homologues contain a consensus retinoblastoma-binding motif, suggesting that they may be involved in controlling the host cell cycle.
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Mutations in the potyvirus helper component protein: effects on interactions with virions and aphid stylets.
Mutations of K → E in the highly conserved ‘KITC’ motif of the potyvirus helper component (HC) protein result in loss of HC function in aphid transmission, presumably because of inability to interact with virions, stylets or both. In this study we show that HC of potato virus C (PVC), a naturally occurring variant of potato virus Y (PVY) that has the K → E mutation, lacks the ability to be retained in stylets, whereas PVY HC is retained. The K → E mutation in either PVC or a site-directed mutant of tobacco etch virus (TEV) did not hinder binding to capsid protein, nor did deletion of the N-terminal 107 aa of TEV HC. An additional mutation, F → L at aa 10 of TEV HC, which renders HC non-functional but does not affect binding to capsid protein, is reported. Collectively, the results suggest that the N-terminal domain is required for interaction of HC with stylets rather than for binding to virions.
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VPg, coat protein and five non-structural proteins of potato A potyvirus bind RNA in a sequence-unspecific manner.
More LessThe potato A potyvirus (PVA)-encoded proteins P1, HC-Pro, P3, CI, VPg, NIaPro, NIb and coat protein (CP) were expressed as 6 × His-tagged recombinant proteins in Escherichia coli and purified to homogeneity. RNA binding was tested using purified proteins in Northwestern and liquid assays. PVA proteins except P3 bound to positive- and negativesense transcripts prepared from the nontranslated 5′ - and 3′ -regions of PVA genomic RNA and to full- length transcripts of PVA RNA. RNA binding by these proteins showed no sequence specificity since they also bound to various non-PVA control RNAs. Binding properties of P1, HC-Pro, CI and NIaPro are consistent with previous studies carried out on a few other potyviruses, but the binding of VPg, NIb and CP to RNA reveal novel interactions between RNA and potyvirus proteins. Furthermore, the RNA- binding properties of all major proteins of a potyvirus have not been reported previously.
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Specificity of resistance to pea seed-borne mosaic potyvirus in transgenic peas expressing the viral replicase (Nlb) gene.
More LessTransgenic pea lines carrying the replicase (NIb) gene of pea seed-borne mosaic potyvirus (PSbMV) were generated and used in experiments to determine the effectiveness of induced resistance upon heterologous isolates. Three pea lines showed inducible resistance in which an initial infection by the homologous isolate (PSbMV-DPD1) was followed by a highly resistant state. Resistance was observed in plants in either the homozygous or hemizygous condition and resulted in no overall yield loss despite the initial infection. Resistance was associated with a loss of both viral and transgene RNA, which is indicative of a mechanism based upon post-transcriptional gene silencing. There was no correlation between the steady-state levels of transgene RNA and ability of the plants to show resistance. To test the specificity of the resistance, plants were also inoculated with the most distantly related sequenced PSbMV isolate, NY. PSbMV-NY varied between experiments in its ability to induce resistance, suggesting that the sequence identity in the NIb gene is borderline for the specificity required for triggering gene silencing. Upon challenge inoculation of virus-free recovered leaves, the specificity of the induced resistance varied between the two isolates and indicated that the virus and transgene additively determined the resistant state. These results suggest that the sequence requirements for triggering gene silencing may differ from those involved in the degradation process.
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Amino acids of alfalfa mosaic virus coat protein that direct formation of unusually long virus particles.
More LessIn contrast to most alfalfa mosaic virus (AMV) strains (YSMV, S, M and 425), AMV strains VRU and 15/64 can form abnormally long virus particles, an ability which has been linked to the coat protein (CP). In order to study this phenomenon, the CP-encoding RNAs 3 of AMV strains VRU and 15/64 were cloned and fully sequenced. Comparative sequence analyses of AMV RNA 3 sequences derived from different strains revealed two non-conservative amino acid substitutions, Ser66 and Leu175, which occur exclusively in the closely related VRU- and 15/64-CPs. When these amino acid alterations were introduced into the CP of AMV strain 425 unusually long virus particles were assembled. This confirms that amino acids Ser66 and Leu175 of the CPs of AMV strains VRU and 15/64 are involved in the formation of tubular virus particles.
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Identification of epitopes in cucumber mosaic virus using a phage-displayed random peptide library.
More LessAntigenic sites in the cucumber mosaic virus (CMV) coat protein (CP) have been identified using a polyclonal antiserum prepared against glutaral- dehyde-fixed virions. Antibodies were used to screen a random peptide library of heptamers displayed on the surface of a bacteriophage. Eight of 36 (22%) sequenced phage clones had inserts resembling a putative virion surface domain of the CMV CP. This region has the sequence LETDEL, corresponding to amino acids 194–199 in the Fny- CMV CP. The binding of phage clones to Fny-CMV antiserum was inhibited by a synthetic peptide representing this region. Six of 36 (17%) phage clones contained sequences corresponding to a C- terminal sequence in the Fny-CMV CP, which is thought to be internal in assembled virions. This sequence, EHQRIPTSGV, represents amino acids 206–215 and all but the P residue were observed in at least one clone. Four of 36 (11%) sequenced phage clones carried sequences that matched a portion of the sequence RLLLPDSV, corresponding to amino acids 89–96 in the Fny-CMV CP. This region was also identified as the antigenic site recognized by a monoclonal antibody (MAb23C10E4). Eleven percent of the phage (4 of 36) contained sequences matching at least three amino acids of the N-terminal region in the CMV CP. The positions of the antigenic sites seen in this study are consistent with a predicted structure for the CMV CP.
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Molecular characterization of Fiji disease fijivirus genome segment 9.
More LessThis is the first report of sequence from Fiji disease fijivirus (FDV), the type member of the genus Fijivirus of the family Reovi ridae. FDV genome segment (S9) comprised 1843 nt and contained two non-overlapping ORFs, separated by a 57 nt intergenic region. S9 ORF 1 comprised 1008 ntand encoded a 335-amino-acid polypeptide (predicted molecular mass 38·6 kDa), while ORF 2 comprised 627 nt and encoded a 208-amino-acid polypeptide (predicted molecular mass 23·8 kDa). The 5′ and 3′ non-coding regions were 49 and 102 nt, respectively. The S9 terminal sequences were 5′ AAGU-UUUU------UGUC 3′, and located immediately adjacent to these sequences were 12 bp imperfect inverted repeats. The entire S9 ORF 1 and the hydrophilic regions of S9 ORF 2 were each expressed as a fusion protein with the maltose-binding protein in Escherichia coli. Antibodies produced against the ORF 1 fusion protein reacted strongly with a protein of approximately 39 kDa present in both crude extracts of FDV-infected sugarcane and partially purified FDV preparations. In contrast, antibodies raised against the modified ORF 2 fusion protein did not react with any proteins in the same samples. Further, polyclonal antibodies produced against partially purified FDV reacted with the ORF 1, but not the modified ORF 2, fusion protein. These results indicate that FDV S9 ORF 1 encodes a major structural protein, while ORF 2 probably encodes a non-structural protein.
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Regulation of pathogenicity in hop stunt viroid-related group II citrus viroids.
More LessNucleotide sequences were determined for two hop stunt viroid-related Group II citrus viroids characterized as either a cachexia disease non-pathogenic variant (CVd-IIa) or a pathogenic variant (CVd-IIb). Sequence identity between the two variants of 95·6% indicated a conserved genome with the principal region of nucleotide difference clustered in the variable (V) domain. Full-length viroid RT-PCR cDNA products were cloned into plasmid SP72. Viroid cDNA clones as well as derived RNA transcripts were transmissible to citron (Citrus medica L.) and Luffa aegyptiaca Mill. To determine the locus of cachexia pathogenicity as well as symptom expression in Luffa, chimeric viroid cDNA clones were constructed from segments of either the left terminal, pathogenic and conserved (T1-P-C) domains or the conserved, variable and right terminal (C-V- T2) domains of CVd-IIa or CVd-IIb in reciprocal exchanges. Symptoms induced by the various chimeric constructs on the two bioassay hosts reflected the differential response observed with CVd-IIa and -IIb. Constructs with the C-V-T2 domains region from clone-IIa induced severe symptoms on Luffa typical of CVd-IIa, but were non-symptomatic on mandarin as a bioassay host for the cachexia disease. Constructs with the same region (C-V-T2) from the clone-IIb genome induced only mild symptoms on Luffa, but produced a severe reaction on mandarin, as observed for CVd-IIb. Specific site- directed mutations were introduced into the V domain of the CVd-IIa clone to construct viroid cDNA clones with either partial or complete conversions to the CVd-IIb sequence. With the introduction of six site-specific changes into the V domain of the clone- IIa genome, cachexia pathogenicity was acquired as well as a moderation of severe symptoms on Luffa.
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The shortest known prion protein gene allele occurs in goats, has only three octapeptide repeats and is non-pathogenic.
More LessThe prion protein (PrP) gene modulates the incidence and incubation periods of transmissible spongiform encephalopathies of sheep, goats, mice and man. Here, a new caprine PrP allele encoding the shortest naturally occurring PrP protein so far described is reported. This variant contains only three instead of the usual five copies of a short peptide repeat [Pro-Gln/His-Gly-Gly-Gly-(Gly)-Trp- Gly-Gln] characteristic of PrP, with an additional Trp to Gly substitution in codon 102. Fifteen out of 111 genotyped goats carried the novel PrP allele and 14 survived without signs of disease for at least 4 years. One goat heterozygous for the polymorphism was challenged experimentally with SSBP/1-scrapie and succumbed after an unusually long incubation period.
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