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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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