- Volume 84, Issue 5, 2003
Volume 84, Issue 5, 2003
- Review
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Association of bovine papillomavirus with the equine sarcoid
More LessThe equine sarcoid, a locally aggressive, fibroblastic skin tumour, is the most common dermatological neoplasm reported in horses; there is no consistently effective therapy. It is widely accepted that bovine papillomavirus (BPV) types 1 and 2 are associated with the pathogenesis of sarcoid disease. Most sarcoids appear to contain detectable viral DNA and RNA and are also known to express the BPV types 1 and 2 major transforming protein, E5, but appear not to produce infectious virions. While the mode of transmission of infection has not been elucidated, viral gene expression, in particular of E5, may contribute to virus persistence and disease pathogenesis by downregulating MHC class I expression. Here, the pathology and epidemiology of the sarcoid and its association with BPV is reviewed; the transforming functions of the BPV oncoproteins and their possible role in sarcoid pathogenesis are discussed; and the practical implications of BPV infection for diagnostic and therapeutic purposes are considered.
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- Animal
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- RNA viruses
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Expansion of human γ/δ T cells in vitro is differentially regulated by the measles virus glycoproteins
More LessImpaired proliferative response of lymphocytes after mitogenic stimulation ex vivo is a key feature of the generalized immunosuppression induced by measles virus (MV). Compelling evidence suggests that negative signalling by the MV glycoprotein (gp) complex and the surface of uninfected lymphocytes is essential for this effect. So far, the inhibitory activity of this complex applied to all lymphocyte subpopulations irrespective of the mode of stimulation and could not be overcome by external stimulation. This study shows that the isopentenyl pyrophosphate (IPP)/IL-2-stimulated expansion of human γ/δ T cell receptor (TCR) T cells from peripheral blood mononuclear cells (PBMCs) is inhibited efficiently when the MV gp complex is expressed on the surface of persistently MV-infected T or monocytic cells. In contrast, persistently infected B cells or infected human dendritic cells (DCs) do not interfere with expansion of γ/δ TCR T cells from PBMCs. These particular two cell populations, however, efficiently inhibit IPP/IL-2-stimulated expansion of γ/δ TCR T cells from purified T cells and this is reverted by resubstitution with monocytes. As revealed by filter experiments, cocultivation with B cells and DCs empower monocytes, at least partially by soluble mediators, to provide membrane contact-dependent costimulatory signals that neutralize the inhibitory effect of the MV gp complex. Thus, γ/δ TCR T cells are sensitive to MV gp-mediated inhibition; however, this is overcome efficiently by signals delivered from monocytes conditioned by B cells and DCs.
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CD46- and CD150-independent endothelial cell infection with wild-type measles viruses
More LessMeasles virus (MV) infects endothelial cells of the skin, the brain and other organs during acute or persistent infections. Endothelial cells are supposed to play an important role in virus spread from the blood stream to surrounding tissues. CD46 and CD150 (signalling lymphocytic activation molecule, SLAM) have been described as cellular receptors for certain MV strains. We found that human umbilical vein and brain microvascular endothelial cells (HUVECs and HBMECs) were CD46-positive, but did not express SLAM. Wild-type MV strains, which do not use CD46 as a receptor at the surface of transfected Chinese hamster ovary cells, infected HUVECs and HBMECs to varying extents in a strain-dependent way. This infection was not inhibited by antibodies to CD46. These data suggest the presence of an additional unidentified receptor for MV uptake and spread in human endothelial cells.
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Genetic variability of Crimean-Congo haemorrhagic fever virus in Russia and Central Asia
Hyalomma marginatum ticks (449 pools, 4787 ticks in total) collected in European Russia and Dermacentor niveus ticks (100 pools, 1100 ticks in total) collected in Kazakhstan were screened by ELISA for the presence of Crimean-Congo haemorrhagic fever virus (CCHFV). Virus antigen was found in 10·2 and 3·0 % of the pools, respectively. RT-PCR was used to recover partial sequences of the CCHFV small (S) genome segment from seven pools of antigen-positive H. marginatum ticks, one pool of D. niveus ticks, four CCFH cases and four laboratory virus strains. Additionally, the entire S genome segments of the CCHFV strains STV/HU29223 (isolated from a patient in European Russia) and TI10145 (isolated from H. asiaticum in Uzbekistan) were amplified, cloned and sequenced. Phylogenetic analysis placed all CCHFV sequences from Russia in a single, well-supported clade (nucleotide sequence diversity up to 3·2 %). Virus sequences from H. marginatum were closely related or identical to those recovered from patients in the same regions of southern Russia. Newly described CCHFV strains from Central Asian countries fell into two genetic lineages. The first lineage was novel and included closely related virus sequences from Kazakhstan and Tajikistan (nucleotide sequence diversity up to 3·2 %). In contrast, a newly described CCHFV strain from Uzbekistan, strain TI10145, clustered on the phylogenetic trees with strains from China.
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Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids
More LessLa Crosse virus (LACV), a member of the family Bunyaviridae, is the primary cause of paediatric encephalitis in the United States. In this study, a functional RNA polymerase (L) gene of LACV was cloned and a reverse genetics system established. A reporter minireplicon mimicking the viral genome was constructed by flanking the Renilla luciferase gene with the 3′ and 5′ noncoding regions of the genomic M segment. These noncoding regions serve as promoters for the viral polymerase. Both L and nucleocapsid (N) genes were expressed by means of T7 RNA polymerase, which was provided by the recombinant T7-expressing modified vaccinia virus Ankara. Renilla reporter activity in transfected cells reflected reconstitution of recombinant nucleocapsids by functional L and N gene products. Time-course experiments revealed a rapid increase in minireplicon activity from 10 to 18 h after the onset of L and N expression. Minireplicon activity was found to be dependent on the correct ratio of L to N plasmids, with too much of either construct resulting in downregulation. Furthermore, a specific inhibitory effect of LACV NSs protein on minireplicon activity was found. In passaging experiments using parental helper virions, it was demonstrated that the recombinant nucleocapsids are a useful model for transcription, replication and packaging of LACV.
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Nucleotide variation in Sabin type 2 poliovirus from an immunodeficient patient with poliomyelitis
The molecular and antigenic properties of a Sabin-like type 2 poliovirus, isolated from the stool samples of a 2-year-old agammaglobulinaemic child who developed paralysis 1 year after receiving the third dose of oral poliovirus vaccine, were analysed. The virus revealed 0·88 % genome variation in the VP1 region compared with the standard reference strain, compatible with replication of the virus in the intestine over approximately 1 year. The typical mutations in the 5′NCR and VP1 associated with reversion to neurovirulence for Sabin type 2 poliovirus were found. Despite this, the virus was characterized by both PCR and ELISA tests as Sabin-like and showed temperature sensitivity and neurovirulence in transgenic mice typical of the Sabin type 2 vaccine strain. Gammaglobulin replacement therapy led rapidly to virus clearance, which, when combined with treatment with the antiviral drug pleconaril, stopped virus excretion; no further virus shedding occurred. This is the first case of poliomyelitis and long-term excretion from an immunodeficient patient to be reported in Italy through the active ‘Acute Flaccid Paralysis' surveillance system.
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Evolution of the genome of Human enterovirus B: incongruence between phylogenies of the VP1 and 3CD regions indicates frequent recombination within the species
More LessEnteroviruses show a high degree of sequence variation both between and within serotypes due to the lack of proofreading of the viral RNA-dependent RNA polymerase. In addition, recombination is known to occur not only within but also between different serotypes. We have previously shown that capsid coding sequences of coxsackievirus B4 (CVB4) cluster in several coexisting genotypes (intergenotypic nucleotide difference of 12 % or more) whereas a single lineage of echovirus 30 (EV30) has been prevailing and evolving throughout the last two decades. In the major capsid gene, VP1, clustering of both nucleotide and amino acid sequences correlates with serotype. We have now determined a 501 nucleotide sequence in the non-structural 3CD region of CVB4 and EV30 field strains. Phylogenetic analysis revealed that sequences of Human enterovirus B (HEV-B) were segregated in the 3CD region into three distinct clusters without the VP1-associated serotype/genotype correlation. One of the clusters comprised the E2 strain of CVB4, the EV30 prototype and five other CVB4 field strains whereas the other two clusters, in addition to CVB4 and EV30 strains, also included other HEV-B serotypes. We believe that intertypic recombination is the most likely explanation for the observed incongruence. Similarity analysis based on complete genomes of the CVB4 and EV30 prototypes and the CVB4 E2 strain revealed that a putative recombination spot was mapped within the 2B gene. The incongruence observed in the two genomic domains (P1 and P3) suggests a certain degree of independent evolution, which may be explained by interserotypic recombination within an enterovirus species. It is thus difficult to exclude recombination in the history of any given strain.
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Caspase-mediated cleavage of the feline calicivirus capsid protein
More LessFeline calicivirus (FCV) is responsible for an acute upper respiratory tract disease in cats. The FCV capsid protein is synthesized as a precursor (76 kDa) that is post-translationally processed into the mature 62 kDa capsid protein by removal of the N-terminal 124 amino acids. Our previous studies have also detected a 40 kDa protein, related to the FCV capsid protein, produced during infection. Here we demonstrate that cleavage of the FCV capsid protein, during infection of cells in culture, was prevented by caspase inhibitors. In addition, caspase-2, -3 and -7 were activated during FCV infection, as shown by pro-form processing, an increase in N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin cleavage activity and in situ poly(ADP-ribose) polymerase cleavage. Caspase activation coincided with the induction of apoptosis and capsid cleavage to the 40 kDa fragment. An in vitro cleavage assay, using recombinant human caspases and in vitro-derived FCV capsid protein, revealed that caspase-2, and to a lesser extent caspase-6, cleaved the capsid protein to generate a 40 kDa fragment. Taken together, these results suggest that FCV triggers apoptosis within infected cells and that caspase-induced capsid cleavage occurs concomitantly with apoptosis. The possible role of capsid cleavage in the pathogenesis of FCV infection is discussed.
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Characterization of Japanese swine and human hepatitis E virus isolates of genotype IV with 99 % identity over the entire genome
The full-length genomic sequences were determined of Japanese swine and human hepatitis E virus (HEV) isolates (swJ13-1 and HE-JA1, respectively) with 100 % identity in the partial sequence of open reading frame (ORF) 2 (ORF2, 412 nt). swJ13-1 was isolated from a 4-month-old farm pig born in Hokkaido, Japan, in 2002 and HE-JA1 was recovered from a 55-year-old patient who lived in Hokkaido and who had contracted sporadic acute hepatitis E in 1997. Both isolates consisted of 7240 nt, excluding the poly(A) tail, and contained three ORFs (ORFs 1–3) that encoded proteins of 1707, 674 and 114 aa. The overall nucleotide sequence identity between them was 99·0 % and the deduced amino acid sequence identities of ORFs 1–3 were 99·8, 100 and 100 %, respectively. The high degree of genomic similarity observed between swine and human HEV isolates in a restricted area of Japan further supports the finding that sporadic hepatitis E in Japan is a zoonosis.
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Hepatitis C virus RNA replication is resistant to tumour necrosis factor-α
More LessIt was demonstrated using self-replicating hepatitis C virus (HCV) RNAs that both types of interferons (IFNs) (in particular IFN-α and IFN-γ) are potent inhibitors of HCV replication in Huh-7 cells. Because IFN-γ and tumour necrosis factor (TNF)-α trigger a partially overlapping set of antiviral defence mechanisms, it is tempting to speculate that TNF-α also inhibits HCV replication. However, this study shows that TNF-α does not affect HCV protein and RNA synthesis, nor does it synergistically enhance the inhibitory effect of IFN-γ. Taken together, these results demonstrate that HCV replication in Huh-7 cells is highly resistant to TNF-α. It is, therefore, unlikely that the increased production of TNF-α, which is seen in many hepatitis C patients, contributes to HCV clearance by inducing antiviral defence mechanisms in infected hepatocytes.
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A stable full-length yellow fever virus cDNA clone and the role of conserved RNA elements in flavivirus replication
Yellow fever virus (YF) is the prototype member of the Flavivirus genus. Here, we report the successful construction of a full-length infectious cDNA clone of the vaccine strain YF-17D. YF cDNA was cloned into a low-copy-number plasmid backbone and stably maintained in several E. coli strains. Transcribed RNAs had a specific infectivity of 105–106 p.f.u. (μg RNA)−1, and the resulting virus exhibited growth kinetics, plaque morphology and proteolytic processing similar to the parental virus in cell culture. This clone was used to analyse the importance of conserved flavivirus RNA sequences and the 3′ stem–loop structure in virus replication. The conserved sequences 5′CS and CS1, as well as the 3′ stem–loop structure, were found to be essential for virus replication in cell culture, whereas the conserved sequence CS2 and the region containing YF-specific repeated sequences were dispensable. This infectious clone will aid future studies on YF replication and pathogenesis, as well as facilitate the development of YF-17D-based recombinant vaccines.
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The envelope glycoprotein E2 is a determinant of cell culture tropism in ruminant pestiviruses
Bovine viral diarrhoea virus (BVDV) isolates infect cultured Madin–Darby bovine kidney (MDBK) cells as efficiently as sheep kidney cells. In contrast, border disease virus (BDV) propagates poorly in MDBK cells but infects sheep cells very efficiently. The envelope glycoprotein E2 has been shown to be essential for virus infectivity. To explore the potential role of E2 in pestivirus host range in cell cultures, we engineered a chimeric BVDV with the E2 coding region from BDV. As expected, the BVDV–E2bdv chimera retained the ability of BDV to multiply in sheep cells but experienced a remarkable reduction in its ability to propagate and form plaques in MDBK, a phenotype that is characteristic of the E2 donor, BDV31 virus. Control chimeric BVDV bearing a type II E2 demonstrated that the heterologous E2 does not impair replication in MDBK or lamb cells. These results establish a role for E2 in determining the tropism of a pestivirus in cell culture.
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Time-course induction of apoptosis by wild-type and attenuated strains of rubella virus
More LessThe time-course of rubella virus (RV)-induced apoptosis was studied in RK13 cells. DEVD-specific caspase activity assay and Western blotting for caspase-3 were used to determine the time-course of caspase activation and demonstrated that RV-induced apoptotic changes occur as early as 12 h post-infection (p.i.). Caspase activity followed a cyclic pattern, as seen with apoptotic-inducing drugs, with maximum activity detected at 72 h p.i. Apoptosis caused by wild-type (RN) and attenuated vaccine (Cendehill) strains of RV was compared by TUNEL staining, counting dead floating cells and DNA fragmentation analysis. Although the amount of apoptosis due to the wild-type strain was marginally greater, this was probably due to its faster growth rate.
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Construction and characterization of pentacistronic retrovirus vectors
More LessThe picornavirus foot-and-mouth disease virus 2A sequence was combined with three different internal ribosome entry segments to construct and characterize three independent pentacistronic retroviruses of different sizes. Efficient co-expression of the five proteins was successful and titres obtained for these pentacistronic virus vectors (final genome size ∼7·9 kb) were comparable to those of vector systems with shorter genomes. Other vectors constructed that exceeded the genome length of the wild-type virus suffered frequent deletions.
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Natural variation of the nef gene in human immunodeficiency virus type 2 infections in Portugal
Human immunodeficiency virus type 2 (HIV-2) infections cause severe immunodeficiency in humans, although HIV-2 is associated frequently with reduced virulence and pathogenicity compared to HIV-1. Genetic determinants that play a role in HIV pathogenesis are relatively poorly understood but nef has been implicated in inducing a more pathogenic phenotype in vivo. However, relatively little is known about the role of nef in HIV-2 pathogenesis. To address this, the genetic composition of 44 nef alleles from 37 HIV-2-infected individuals in Portugal, encompassing a wide spectrum of disease associations, CD4 counts and virus load, has been assessed. All nef alleles were subtype A, with no evidence of gross deletions, truncations or disruptions in the nef-encoding sequence; all were full-length and intact. HIV-2 long terminal repeat sequences were conserved and also indicated subtype A infections. Detailed analysis of motifs that mediate nef function in HIV-1 and simian immunodeficiency virus, such as CD4 downregulation and putative SH2/SH3 interactions, revealed significant natural variation. In particular, the central P104xxPLR motif exhibited wide interpatient variation, ranging from an HIV-1-like tetra-proline structure (PxxP)3 to a disrupted minimal core motif (P104xxQLR). The P107→Q substitution was associated with an asymptomatic phenotype (Fisher's exact test, P=0·026) and low virus loads. These data indicate that discrete differences in the nef gene sequence rather than gross structural changes are more likely to play a role in HIV-2 pathogenesis mediated via specific functional interactions.
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Phylogenetic analysis of feline immunodeficiency virus in Central Europe: a prerequisite for vaccination and molecular diagnostics
More LessFeline immunodeficiency virus (FIV) is a worldwide-occurring lentivirus that severely impairs the immune function of infected domestic cats. Due to structural and biological similarities, FIV represents a promising model for human immunodeficiency virus (HIV) and AIDS. A major obstacle in developing vaccines against lentiviruses is their high mutation rate. Furthermore, mutations in target sequences provide a pitfall for molecular diagnostics. It is therefore important to determine the genetic diversity of lentiviruses in any region where vaccination or implementation of new diagnostic techniques are planned. This study presents a phylogenetic analysis of 30 FIV strains derived from Central Europe. In order to improve the reliability of genotyping, DNA from two different proviral genes was amplified and comparative phylogenetic trees were inferred. The highly coincident results point to the existence of extensive virus variation with the presence of at least two highly divergent subtypes of FIV in Austria and Germany.
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Evaluation of the δ subunit of bovine adaptor protein complex 3 as a receptor for bovine leukaemia virus
More LessA candidate gene of the bovine leukaemia virus (BLV) receptor (BLVR) was cloned previously and predicted to encode a transmembrane protein. Subsequent cloning of related genes from other organisms indicated that the candidate gene is related, but unique, to a gene family of the δ subunit of the adaptor protein (AP) complex 3, AP-3. Therefore, bovine cDNAs (boAP3δ) that are highly homologous to the candidate gene were cloned and sequenced. The nucleotide sequences suggested that the boAP3δ cDNA encodes the δ subunit of boAP3 without transmembrane domains. Part of the AP3δ cDNA isolated from the lymph node, spleen and MDBK cells, from which the BLVR candidate cDNA was derived, has almost the same nucleotide sequences as the boAP3δ cDNA. A boAP3δ protein tagged with green fluorescent protein was localized in the cytoplasm and incorporated into AP-3 in bovine cells. Unlike the previous report about the candidate gene, the boAP3δ gene introduced into murine NIH 3T3 cells did not increase the susceptibility of the cells to BLV infection. Many small insertions and deletions of nucleotides could generate the predicted transmembrane and cytoplasmic regions of the BLVR protein from the prototypic boAP3δ gene.
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A first full outer capsid protein sequence data-set in the Orbivirus genus (family Reoviridae): cloning, sequencing, expression and analysis of a complete set of full-length outer capsid VP2 genes of the nine African horsesickness virus serotypes
More LessThe outer capsid protein VP2 of African horsesickness virus (AHSV) is a major protective antigen. We have cloned full-length VP2 genes from the reference strains of each of the nine AHSV serotypes. Baculovirus recombinants expressing the cloned VP2 genes of serotypes 1, 2, 4, 6, 7 and 8 were constructed, confirming that they all have full open reading frames. This work completes the cloning and expression of the first full set of AHSV VP2 genes. The clones of VP2 genes of serotypes 1, 2, 5, 7 and 8 were sequenced and their amino acid sequences were deduced. Our sequencing data, together with that of the published VP2 genes of serotypes 3, 4, 6 and 9, were used to generate the first complete sequence analysis of all the (sero)types for a species of the Orbivirus genus. Multiple alignment of the VP2 protein sequences showed that homology between all nine AHSV serotypes varied between 47·6 % and 71·4 %, indicating that VP2 is the most variable AHSV protein. Phylogenetic analysis grouped together the AHSV VP2s of serotypes that cross-react serologically. Low identity between serotypes was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralization. The data presented here impact on the development of new vaccines, the identification and characterization of antigenic regions, the development of more rapid molecular methods for serotype identification and the generation of comprehensive databases to support the diagnosis, epidemiology and surveillance of AHS.
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- DNA viruses
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Human papillomavirus type 16-specific T cell responses and their association with recurrence of cervical disease following treatment
More LessHuman papillomavirus type 16 (HPV-16) L1- and E7-specific T cell responses were measured in 58 women with abnormal cervical cytology in a prospective study. On recruitment, patients responded most frequently and with the highest numbers of responding cells to the L1 region aa 311–345 and this response was significantly associated with the presence of cervical disease (P=0·041). Responses to the L1 peptide aa 281–295 were significantly higher in patients with CIN III than in those with HPV/CIN I or CIN II lesions (P=0·027). The E7 region aa 70–98 was the most immunogenic in patients with squamous intraepithelial lesions of the cervix (SIL) but the responses detected were not significantly higher than in patients without SIL. Following treatment, the T cell response profiles of patient groups did not change significantly. However, on analysis of the responses of individual patients with and without recurrent disease on follow-up, significant differences were found. Recurrence of disease was associated with T cell responses to the E7 region aa 70–98 at the patient's first clinic visit (P=0·017). Recurrence of disease was also accompanied by an increase in the total number of L1-specific short-term T cell lines (STLs) at follow-up, whereas absence of disease was accompanied by a decrease in L1-specific STLs. The data also suggested a possible link between E7 70–98-specific responses and acquisition of disease by patients who were previously disease-free. Further studies are warranted to determine whether this response could be useful as a marker of recurrent disease in some patients.
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Capsaicin-induced reactivation of latent herpes simplex virus type 1 in sensory neurons in culture
More LessHerpes simplex virus type 1 (HSV-1) produces a life-long latent infection in neurons of the peripheral nervous system, primarily in the trigeminal and dorsal root ganglia. Neurons of these ganglia express high levels of the capsaicin receptor, also known as the vanilloid receptor-1 (VR-1). VR-1 is a non-selective ion channel, found on sensory neurons, that primarily fluxes Ca2+ ions in response to various stimuli, including physiologically acidic conditions, heat greater than 45 °C and noxious compounds such as capsaicin. Using an in vitro neuronal model to study HSV-1 latency and reactivation, we found that agonists of the VR-1 channel – capsaicin and heat – resulted in reactivation of latent HSV-1. Capsaicin-induced reactivation of HSV-1 latently infected neurons was dose-dependent. Additionally, activation of VR-1 at its optimal temperature of 46 °C caused a significant increase in virus titres, which could be attenuated with the VR-1 antagonist, capsazepine. VR-1 activation that resulted in HSV-1 reactivation was calcium-dependent, since the calcium chelator BAPTA significantly reduced reactivation following treatment with caspsaicin and forskolin. Taken together, these results suggest that activation of the VR-1 channel, often associated with increases in intracellular calcium, results in HSV-1 reactivation in sensory neurons.
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Herpes simplex virus infection of murine sensory ganglia induces proliferation of neuronal satellite cells
More LessHerpes simplex virus (HSV) is a virtually ubiquitous human pathogen that, following cutaneous infection, latently infects neurons of sensory ganglia. Satellite cells (SCs) ensheath and provide metabolic support for these neurons, and could potentially participate in controlling HSV disease. Although SCs are restrictive for HSV replication, hypercellularity of non-neuronal cells in ganglia is prominent during HSV infection in animal models. SCs proliferate in response to trauma, e.g. nerve cut or crush, but it is not known if proliferation occurs in response to viral infection. To address this issue, cell proliferation, measured by bromodeoxyuridine (BrdU) uptake, and immune infiltrate, measured by CD45 labelling, were examined during acute infection in a mouse model. Because SCs do not express CD45, the BrdU+ CD45− cell subset represents the proliferating SC population. We report that during acute ganglionic HSV infection there is a substantial increase in SC numbers. We suggest that SC proliferation in response to HSV infection may occur in order to facilitate neuronal survival.
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Analysis of the role of the membrane-spanning and cytoplasmic tail domains of herpes simplex virus type 1 glycoprotein D in membrane fusion
More LessGlycoprotein D (gD) of herpes simplex virus type 1 is a type 1 membrane protein in the virus envelope that binds to receptor molecules on the cell surface and which induces cell–cell fusion when co-expressed with gB, gH and gL. A chimeric gD molecule in which the membrane anchor and cytoplasmic tail domains were replaced with analogous regions from the human CD8 molecule was as competent as wild-type gD at mediating membrane fusion and virus entry. However, when gD was tethered to the membrane by means of a glycosylphosphatidylinositol (gpi)-anchor sequence, which binds only to the outer leaflet of the lipid bilayer, it was unable to function in cell–cell fusion assays. This chimera was incorporated into virions as efficiently as wild-type gD and yet virus particles containing gpi-linked gD entered cells more slowly than virions containing wild-type gD in their envelopes, suggesting that gD must be anchored in both leaflets of a lipid bilayer for it to function in both cell fusion and virus entry.
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Cloning and expression of the complement receptor glycoprotein C from Herpesvirus simiae (herpes B virus): protection from complement-mediated cell lysis
More LessSimian herpes B virus (SHBV) is the herpes simplex virus (HSV) homologue for the species Macaca. Unlike in its natural host, and unlike other animal herpesviruses, SHBV causes high mortality in accidentally infected humans. SHBV-infected cells, like those infected with HSV-1 and equine herpesvirus types 1 and 4, express complement C3 receptor activity. To study immunoregulatory functions involved in susceptibility/resistance against interspecies transmission, the SHBV glycoprotein C (gCSHBV) gene (encoding 467 aa) was isolated. Sequence analysis revealed amino acid identity with gC proteins from HSV-2 (46·9 %), HSV-1 (44·5 %) and pseudorabies virus (21·2 %). Highly conserved cysteine residues were also noted. Similar to gCHSV-2, gCSHBV is less glycosylated than gCHSV-1, resulting in a molecular mass of 65 kDa if expressed in replication-deficient vaccinia virus Ankara. Stable transfectants expressing full-length gCSHBV on the cell surface induced C3 receptor activity and were substantially protected from complement-mediated lysis; no protection was observed with control constructs. This suggests that expression of the gC homologues on infected cell surfaces might also contribute to the survival of infected cells in addition to decreased virion inactivation. Interestingly, soluble gCSHBV isolated from protein-free culture supernatants did not interfere with the binding of the alternative complement pathway activator properdin to C3b, which is similar to our findings with gCHSV-2 and could be attributed to major differences in the amino-terminal portion of the protein with extended deletions in both gCSHBV and gCHSV-2. Binding of recombinant gCSHBV to polysulphates was observed. This, together with the heparin-sensitivity of the gCSHBV–C3 interaction on the infected cell surface, suggests a role in adherence to heparan sulphate, similar to the gC proteins of other herpesviruses.
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Orf virus-encoded interleukin-10 inhibits maturation, antigen presentation and migration of murine dendritic cells
More LessOrf virus (ORFV) belongs to the genus Parapoxvirus and induces cutaneous pustular lesions in sheep, goats and humans. ORFV is unusual in that it has the ability to reinfect its host and this suggests that the generation of immunological memory has been impaired, thus exposing the host to subsequent infection. The discovery that ORFV encodes an IL-10-like virokine raises the question of whether this factor adversely affects the cells that initiate the acquired immune response. We examined the effect of ORFV-IL-10 on immature murine bone marrow-derived dendritic cells (BMDC). Immature BMDC are activated on exposure to antigen and undergo maturation. This process is characterized by increased expression of CD80, CD86 and MHC class II and reduced antigen uptake. We found that the maturation of BMDC is impaired in cells treated with ORFV-IL-10 prior to antigen exposure and this was exemplified by the reduced expression of the cell-surface markers described above. We have also shown that the activation of a haemagglutinin peptide (HAT)-specific T cell hybridoma by dendritic cell-mediated presentation of HAT and heat-inactivated influenza virus AP8/34 was markedly reduced following exposure to ORFV-IL-10. Finally, we examined the effect of ORFV-IL-10 on Langerhans' cell (LC) migration using cultured murine skin explant tissue and showed that this virokine impaired the spontaneous migration of LC from the epidermis and induced changes in LC morphology. Our findings suggest that ORFV-IL-10 has the capacity to impair the initiation of an acquired immune response and hence inhibit the generation of immunological memory necessary for immunity on subsequent exposure.
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Relatedness and heterogeneity at the near-terminal end of the genome of a parapoxvirus bovis 1 strain (B177) compared with parapoxvirus ovis (Orf virus)
More LessThe present study provides for the first time an extended investigation of individual genes located at the near-terminal right end of the genome of parapoxvirus bovis 1, Bovine papular stomatitis virus (BPSV) strain B177 and Orf virus (ORFV). Comparison of the respective DNA sequences of ORFV strain D1701 (9·9 kbp) and BPSV B177 (7·7 kbp) revealed a very similar organization of closely related genes transcribed in a rightward orientation. The most salient findings of this study were: (i) the absence of the ORFV-specific vascular endothelial growth factor (VEGF-E) gene in the BPSV isolate; (ii) the presence of an interleukin-10 (IL-10) orthologue; and (iii) the detection of three new genes encoding ankyrin-repeat-containing polypeptides. These results not only contribute to potential improvements of future molecular differentiation between the parapoxvirus species, but also shed new light on different pathobiologies among parapoxviruses.
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Two novel spliced genes in human cytomegalovirus
Two novel spliced genes (UL131A and UL128) flanking UL130 were predicted from sequence comparisons between human cytomegalovirus (HCMV) and its closest known relative, chimpanzee cytomegalovirus (CCMV), and the splicing patterns were confirmed by mRNA mapping experiments. Both genes were transcribed with late kinetics and shared a polyadenylation site. Comparisons with wild-type HCMV in infected human tissues showed that three of five isolates passaged in cell culture contained disruptions of UL128, one was frameshifted in UL131A and one exhibited a deletion affecting UL131A and UL130. CCMV and the Colburn strain of simian cytomegalovirus, which have been passaged in cell culture, also exhibit disruptions of UL128. These observations indicate that expression of either one of UL128 and UL131A is deleterious to growth of primate cytomegaloviruses in cell culture. Although the functions of these genes are unknown, sequence comparisons suggest that UL128 encodes a β-chemokine.
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Human herpesvirus-8 (Kaposi's sarcoma-associated virus) ORF50 increases in vitro cell susceptibility to human immunodeficiency virus type 1 infection
ORF50, an immediate-early gene of human herpesvirus-8 (HHV-8), encodes a transactivating protein necessary for virus reactivation and lytic replication. ORF50 was reported recently to synergize with human immunodeficiency virus type 1 (HIV-1) tat at a post-transcriptional level. To study the effects of these molecular interactions on HIV replication and biology, cellular clones stably transformed with ORF50 were obtained by transfection of cell lines of different origin. These clones were infected subsequently with HIV. Experiments showed that ORF50 enhances HIV replication in T and B cells (Jurkat and BC-3 cells) and induces susceptibility and transient permissiveness in non-susceptible glial (A172) cells. Upregulation of viral receptors and co-receptors did not account for increased sensitivity to HIV infection and therefore the action of ORF50 might be modulated by the intracellular environment. Interestingly, non-susceptible cells transformed with ORF50 showed transient production of HIV particles that could spread to adjacent cells by direct contact. These findings show that HHV-8 ORF50 has an enhancing effect on HIV replication in vitro and suggest that the two viruses might interact in co-infected patients.
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Unique Epstein–Barr virus (EBV) latent gene expression, EBNA promoter usage and EBNA promoter methylation status in chronic active EBV infection
More LessChronic active Epstein–Barr virus infection (CAEBV) has been considered to be a non-neoplastic T-cell lymphoproliferative disease associated with Epstein–Barr virus (EBV) infection. In EBV-associated diseases, the cell phenotype-dependent differences in EBV latent gene expression may reflect the strategy of the virus in relation to latent infection. We previously reported that EBV latent gene expression was restricted; EBV nuclear antigen 1 (EBNA1) transcripts were consistently detected in all spleen samples from five CAEBV patients, but EBNA2 transcripts were detected in only one sample. EBV latent gene expression is controlled by distinct usage of three EBNA promoters (Cp, Wp and Qp). In this study, we examined the EBNA promoter usage by RT-PCR and the methylation status in the Cp and Wp regions using bisulfite PCR analysis in spleen samples from CAEBV patients. EBNA1 transcripts were unexpectedly initiated not from Qp but from Cp in all samples in spite of the restricted form of latency. Furthermore, while Cp was active, Cp was heavily methylated, indicating that CAEBV has unique EBV latent gene expression, EBNA promoter usage and EBNA promoter methylation status, in part due to unique splicing of Cp-initiated transcripts and an activation mechanism in hypermethylated Cp.
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Ovary development and polydnavirus morphogenesis in the parasitic wasp Chelonus inanitus. I. Ovary morphogenesis, amplification of viral DNA and ecdysteroid titres
More LessPolydnaviruses are unique symbiotic viruses that are replicated in the calyx cells of the ovary of some parasitic wasps. They have a segmented genome of circular double-stranded DNA and are injected along with the wasp's egg into the host, where they are essential for successful parasitism. Polydnaviruses replicate from integrated proviral DNA, and after excision of viral segments, flanking DNA is rejoined. Little is known about ovarian morphogenesis, the mode of amplification of the viral DNA and the involvement of ecdysteroids. Here we have analysed these parameters in the course of pupal–adult development in the braconid wasp Chelonus inanitus. Immediately after pupation, ovarian cells proliferated and calyx cells began to differentiate; at this stage ecdysteroids, in particular 20-hydroxyecdysone, were highest. Thereafter, calyx cells began to increase in size and DNA content and eventually became gigantic. Amplification of non-viral DNA (actin) and viral DNA in its integrated and excised form and of corresponding rejoined flanking regions was measured by quantitative real-time PCR. In the early phase of calyx cell differentiation, copy numbers of actin and integrated viral DNA increased to a similar extent. This, along with the increase in nuclear volume and DNA content in the absence of extensive cell proliferation, suggested polyploidization of the early stage calyx cells. In the following phase, integrated viral DNA was selectively and intensively amplified and eventually excised and circularized. As copy numbers of excised circular viral DNA and rejoined flanking DNA reached similarly high levels, excised viral DNA appeared not to replicate. After adult eclosion, amplification of viral DNA declined.
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Ovary development and polydnavirus morphogenesis in the parasitic wasp Chelonus inanitus. II. Ultrastructural analysis of calyx cell development, virion formation and release
More LessPolydnaviruses are unique symbiotic viruses that are formed only in calyx cells in the ovary of parasitic wasps in the families Braconidae and Ichneumonidae; accordingly, two genera, Bracovirus and Ichnovirus are recognized. We have presented a detailed ultrastructural analysis of ovary and calyx cell differentiation and virion morphogenesis, together with the first data on virion release in a bracovirus. Differentiation of the ovary into germarium/vitellarium and the calyx region begins immediately after pupation. In the periphery and central part of the calyx, some cells and their nuclei begin to enlarge and the DNA content increases. The calyx cell nuclei then further increase and become highly lobulated, nuclear pores become very abundant and the cytoplasm is rich in ribosomes. This suggests synthesis and import of viral envelope proteins as viral envelopes appear in the nuclei shortly later. The appearance of viral envelopes is accompanied by a swelling of the nucleus and a change in electron density. Thereafter, the calyx cells reach the final stage with a highly swollen nucleus containing virogenic stroma and mature virions with nucleocapsids. Up to this stage, the DNA content of nuclei increases 120-fold and the volume 45-fold. The mature calyx cells are positioned in the vicinity of the oviduct lumen; for release of virions first the nuclear and then the plasma membrane disintegrate. On the border of the oviduct lumen, cells of an epithelial layer become phagocytic and remove debris, leading to a calyx fluid that contains only densely packed virions.
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Polydnavirus particle proteins with similarities to molecular chaperones, heat-shock protein 70 and calreticulin
More LessMultipartite nucleic acid-containing virus-like particles, known as polydnaviruses, are special structures produced by female parasitoid wasps to deliver wasp components into the body of their host at oviposition. The particles confer protection for the developing parasitoid by passive and active means. Although several genes expressed from the circular DNA of these particles have been identified from various host–parasitoid systems, there is not much known about the structural proteins of these particles. Here we report on two genes encoding Cotesia rubecula particle proteins with similarities to molecular chaperones, calreticulin and heat-shock protein 70.
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Transduction of cultured fish cells with recombinant baculoviruses
More LessFive fish cell lines were tested for their ability to be transduced by Ac-CAlacZ, a recombinant baculovirus that is capable of expressing a β-galactosidase reporter gene from the CAG promoter (consisting of a cytomegalovirus enhancer element, a chicken actin promoter and rabbit β-globin termination sequences). TO (Tilapia ovary), EPC (carp), CHH-1 (Chum salmon heart fibroblast) and CHSE-214 (chinook salmon embryo) cells were transducible, as demonstrated by an in situ β-galactosidase assay, whereas RTG-2 (rainbow trout gonad) cells were not. The EPC cell line was used for more detailed studies on baculovirus transduction. The transduction frequency was found to be higher at 28 °C than at 21 °C. Addition of the histone deacetylase inhibitor sodium butyrate increased the number of blue cells detected 5- to 7-fold. The m.o.i. was positively correlated with transduction frequency, although the relationship did not appear to be strictly linear, as has been observed with mammalian cells. The temperature at which baculoviruses were adsorbed to EPC cells did not affect levels of β-galactosidase expression. We also examined expression levels of β-galactosidase in EPC cells after infection with a baculovirus construct that overexpresses the vesicular stomatitis virus G protein and displays it on the virion surface. Expression levels with this virus were approximately 15-fold higher than were observed with Ac-CAlacZ.
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- Other Agents
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Disease-associated PrP in the enteric nervous system of scrapie-affected Suffolk sheep
Disease-associated prion protein (PrPd) in the enteric nervous system (ENS) of 20- to 24-month-old Suffolk sheep in the late subclinical and early clinical phase of scrapie was studied. Sites in the alimentary tract extending from the forestomachs and abomasum to the colon from scrapie-affected sheep (PrPARQ/ARQ) and scrapie-resistant sheep (PrPARR/ARQ and PrPARR/ARR) were examined. PrPd was found only in scrapie-affected sheep and was most prominent in the ENS when abundant deposits of PrPd were also present in adjacent lymphoid nodules. Immunolabelling with the nerve fibre markers PgP 9.5 and neuron-specific enolase and the satellite cell marker glial fibrillary acidic protein revealed the extensive ganglionated networks of the myenteric and submucosal plexi. Fewer nerve fibres were present in the lamina propria, T-cell dominated interfollicular areas and dome regions of Peyer's patches. A substantial network of nerve fibres was detected in many lymphoid nodules of both the scrapie-affected and scrapie-resistant sheep. Nerve fibres were also detected within the capsule of lymphoid nodules. Electron microscopy revealed the presence of nerves in the lymphoid nodules, showing a close association with follicular dendritic cells, lymphocytes and tingible body macrophages. In demonstrating that lymphoid nodules in the Peyer's patches of scrapie-affected sheep possess a substantial network of nerve fibres, the present study shows that nodules provide close contact between nerve fibres and cell populations known to contain abundant PrPd, including follicular dendritic cells and tingible body macrophages, and that gut-associated lymphoid nodules in sheep may represent an important site for neuroinvasion.
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Distinct profiles of PrPd immunoreactivity in the brain of scrapie- and BSE-infected sheep: implications for differential cell targeting and PrP processing
More LessPrevious studies have shown that the patterns of disease-specific prion protein (PrPd) accumulation in the brain (the ‘PrPd profile’) of scrapie-affected sheep are mainly influenced by the source of scrapie agent. We have now extended those studies to investigate the effect of different PrP antibodies on the PrPd profile of scrapie- and bovine spongiform encephalopathy (BSE)-affected sheep. Immunohistochemical examination of brains of 20 sheep was performed with four different PrP antibodies (P4, 521.7, 505.2 and R486), and the animals were allocated to four groups of five sheep each depending on the transmissible spongiform encephalopathy (TSE) agent source (two natural scrapie sources, SSBP/1 and BSE). Although the PrPd profiles depended on the antibody used, the four TSE sources could always be differentiated. Natural Suffolk scrapie showed the highest levels of glia-associated PrPd, natural Welsh Mountain scrapie uniquely had consistent vascular PrPd plaques, SSBP/1 produced the highest intracellular accumulations of PrPd and BSE led to moderate accumulation of all PrPd patterns except for vascular plaques. The variations in PrPd profile between TSE sources appeared to be the result of variations in cell tropism and in PrP processing. These processing differences are possibly associated with changes in PrPd conformation, and are manifest as differences in intracellular truncation and in release to the extracellular space of the abnormal protein. Moreover, variations in PrPd conformation would appear to be also influenced by the cell type supporting infection, arguing that it is modulated by the interaction between the infectious agent and the host.
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