- Volume 90, Issue 8, 2009
Volume 90, Issue 8, 2009
- Review
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Tick-borne encephalitis virus – a review of an emerging zoonosis
More LessDuring the last 30 years, there has been a continued increase in human cases of tick-borne encephalitis (TBE) in Europe, a disease caused by tick-borne encephalitis virus (TBEV). TBEV is endemic in an area ranging from northern China and Japan, through far-eastern Russia to Europe, and is maintained in cycles involving Ixodid ticks (Ixodes ricinus and Ixodes persulcatus) and wild vertebrate hosts. The virus causes a potentially fatal neurological infection, with thousands of cases reported annually throughout Europe. TBE has a significant mortality rate depending upon the strain of virus or may cause long-term neurological/neuropsychiatric sequelae in people affected. In this review, we comprehensively reviewed TBEV, its epidemiology and pathogenesis, the clinical manifestations of TBE, along with vaccination and prevention. We also discuss the factors which may have influenced an apparent increase in the number of reported human cases each year, despite the availability of effective vaccines.
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Human immunodeficiency virus type 1 Vpr: functions and molecular interactions
More LessHuman immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is an accessory protein that interacts with a number of cellular and viral proteins. The functions of many of these interactions in the pathogenesis of HIV-1 have been identified. Deletion of the vpr gene reduces the virulence of HIV-1 dramatically, indicating the importance of this protein for the virus. This review describes the current findings on several established functions of HIV-1 Vpr and some possible roles proposed for this protein. Because Vpr exploits cellular proteins and pathways to influence the biology of HIV-1, understanding the functions of Vpr usually involves the study of cellular pathways. Several functions of Vpr are attributed to the virion-incorporated protein, but some of them are attributed to the expression of Vpr in HIV-1-infected cells. The structure of Vpr may be key to understanding the variety of its interactions. Due to the critical role of Vpr in HIV-1 pathogenicity, study of the interactions between Vpr and cellular proteins may help us to understand the mechanism(s) of HIV-1 pathogenicity.
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- Animal
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- RNA viruses
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HBZ is an immunogenic protein, but not a target antigen for human T-cell leukemia virus type 1-specific cytotoxic T lymphocytes
Recently, HBZ has been reported to play an important role in the proliferation of adult T-cell leukaemia (ATL) cells and might be a target of novel therapy for ATL. To develop a novel immunotherapy for ATL, we verified the feasibility of cellular immunotherapy targeting HBZ. We established an HBZ-specific and HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) clone. Detailed study using this CTL clone clearly showed that HBZ is certainly an immunogenic protein recognizable by human CTLs; however, HBZ-specific CTLs could not lyse ATL cells. Failure of HBZ-specific CTLs to recognize human T-cell leukemia virus type 1 (HTLV-1)-infected cells might be due to a low level of HBZ protein expression in ATL cells and resistance of HTLV-1-infected cells to CTL-mediated cytotoxicity. Although HBZ plays an important role in the proliferation of HTLV-1-infected cells, it may also provide a novel mechanism that allows them to evade immune recognition.
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Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes
Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV–H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.
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Complete genomic sequences for hepatitis C virus subtypes 4b, 4c, 4d, 4g, 4k, 4l, 4m, 4n, 4o, 4p, 4q, 4r and 4t
More LessIn this study, we characterized the full-length genomic sequences of 13 distinct hepatitis C virus (HCV) genotype 4 isolates/subtypes: QC264/4b, QC381/4c, QC382/4d, QC193/4g, QC383/4k, QC274/4l, QC249/4m, QC97/4n, QC93/4o, QC139/4p, QC262/4q, QC384/4r and QC155/4t. These were amplified, using RT-PCR, from the sera of patients now residing in Canada, 11 of which were African immigrants. The resulting genomes varied between 9421 and 9475 nt in length and each contains a single ORF of 9018–9069 nt. The sequences showed nucleotide similarities of 77.3–84.3 % in comparison with subtypes 4a (GenBank accession no. Y11604) and 4f (EF589160) and 70.6–72.8 % in comparison with genotype 1 (M62321/1a, M58335/1b, D14853/1c, and 1?/AJ851228) reference sequences. These similarities were often higher than those currently defined by HCV classification criteria for subtype (75.0–80.0 %) and genotype (67.0–70.0 %) division, respectively. Further analyses of the complete and partial E1 and partial NS5B sequences confirmed these 13 ‘provisionally assigned subtypes’.
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Dengue virus infection promotes translocation of high mobility group box 1 protein from the nucleus to the cytosol in dendritic cells, upregulates cytokine production and modulates virus replication
High mobility group box 1 (HMGB1) protein functions in regulation of transcription, cellular activation and pro-inflammatory responses. However, the potential role of HMGB1 during viral infection has not been investigated. This study attempted to elucidate whether the HMGB1-mediated inflammatory response contributes to the pathogenesis of dengue virus (DENV) infection. Our data showed that HMGB1 was released at low DENV infection levels (m.o.i. of 1) under non-necrotic conditions by human dendritic cells (DCs). When DENV-infected DCs were co-cultured with autologous T cells, there was increased production of HMGB1 by both cell types. HMGB1 regulated tumour necrosis factor alpha, interleukin (IL)-6, IL-8 and alpha interferon secretion in DENV-infected DCs. Additionally, increased HMGB1 production was associated with reduced DENV replication titres in DCs. These results suggest that HMGB1 production influences DENV infection in susceptible hosts.
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Oligonucleotide array analysis of Toll-like receptors and associated signalling genes in Venezuelan equine encephalitis virus-infected mouse brain
More LessVenezuelan equine encephalitis (VEE) is an emerging infectious disease. VEE virus (VEEV) may cause lethal infection of the central nervous system in horses and humans. The mechanisms underlying the host immune response to VEEV infection in the brain are not fully understood. Toll-like receptors (TLRs) recognize conserved microbial sequences and induce specific biological responses in the form of proinflammatory cytokine induction. TLR expression in blood following VEEV infection has been reported in non-human primates and TLRs are also upregulated in the brains of mice infected with other alphaviruses. In this study, mice (3–5 weeks old) were infected with V3000, a neurovirulent strain of VEEV, and gene expression of TLRs and their associated signalling molecules was evaluated. VEEV infection resulted in upregulation of TLR 1, 2, 3, 7 and 9, chemokines, inflammatory cytokines, interferon (IFN), IFN regulatory factors and genes involved in signal transduction such as Mcp1, Cxcl10, IL12α/β, IFN-β, IRF-1, IRF-7, Jun, Fos, MyD88, Nfkb, Cd14 and Cd86. These results demonstrate the upregulation of TLRs and associated signalling genes following VEEV infection of the brain, with important implications for how VEEV induces inflammation and neurodegeneration.
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Virulence variation among isolates of western equine encephalitis virus in an outbred mouse model
Little is known about viral determinants of virulence associated with western equine encephalitis virus (WEEV). Here, we have analysed six North American WEEV isolates in an outbred CD1 mouse model. Full genome sequence analyses showed ≤2.7 % divergence among the six WEEV isolates. However, the percentage mortality and mean time to death (MTD) varied significantly when mice received subcutaneous injections of 103 p.f.u. of each virus. Two WEEV strains, McMillan (McM) and Imperial 181 (IMP), were the most divergent of the six in genome sequence; McM caused 100 % mortality by 5 days post-infection, whereas IMP caused no mortality. McM had significantly higher titres in the brain than IMP. Similar differences in virulence were observed when McM and IMP were administered by aerosol, intranasal or intravenous routes. McM was 100 % lethal with an MTD of 1.9 days when 103 p.f.u. of each virus was administered by intracerebral inoculation; in contrast, IMP caused no mortality. The presence of IMP in the brains after infection by different routes and the lack of observed mortality confirmed that IMP is neuroinvasive but not neurovirulent. Based on morbidity, mortality, MTD, severity of brain lesions, virus distribution patterns, routes of infection and differences in infection of cultured cells, McM and IMP were identified as high- and low-virulence isolates, respectively.
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Comparison of poliovirus recombinants: accumulation of point mutations provides further advantages
The roles of recombination and accumulation of point mutations in the origin of new poliovirus (PV) characteristics have been hypothesized, but it is not known which are essential to evolution. We studied phenotypic differences between recombinant PV strains isolated from successive stool specimens of an oral PV vaccine recipient. The studied strains included three PV2/PV1 recombinants with increasing numbers of mutations in the VP1 gene, two of the three with an amino acid change I→T in the DE-loop of VP1, their putative PV1 parent and strains Sabin 1 and 2. Growth of these viruses was examined in three cell lines: colorectal adenocarcinoma, neuroblastoma and HeLa. The main observation was a higher growth rate between 4 and 6 h post-infection of the two recombinants with the I→T substitution. All recombinants grew at a higher rate than parental strains in the exponential phase of the replication cycle. In a temperature sensitivity test, the I→T-substituted recombinants replicated equally well at an elevated temperature. Complete genome sequencing of the three recombinants revealed 12 (3), 19 (3) and 27 (3) nucleotide (amino acid) differences from Sabin. Mutations were located in regions defining attenuation, temperature sensitivity, antigenicity and the cis-acting replicating element. The recombination site was in the 5′ end of 3D. In a competition assay, the most mutated recombinant beat parental Sabin in all three cell lines, strongly suggesting that this virus has an advantage. Two independent intertypic recombinants, PV3/PV1 and PV3/PV2, also showed similar growth advantages, but they also contained several point mutations. Thus, our data defend the hypothesis that accumulation of certain advantageous mutations plays a key role in gaining increased fitness.
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Cellular kinase inhibitors that suppress enterovirus replication have a conserved target in viral protein 3A similar to that of enviroxime
More LessPreviously, we identified a cellular kinase inhibitor, GW5074, that inhibits poliovirus (PV) and enterovirus 71 replication strongly, although its target has remained unknown. To identify the target of GW5074, we searched for cellular kinase inhibitors that have anti-enterovirus activity similar or related to that of GW5074. With this aim, we performed screenings to identify cellular kinase inhibitors that could inhibit PV replication cooperatively with GW5074 or synthetically in the absence of GW5074. We identified MEK1/2 inhibitors (SL327 and U0126), an EGFR inhibitor (AG1478) and a phosphatidylinositol 3-kinase inhibitor (wortmannin) as compounds with a cooperative inhibitory effect with GW5074, and an Akt1/2 inhibitor (Akt inhibitor VIII) as a compound with a synthetic inhibitory effect with MEK1/2 inhibitors and AG1478. Individual treatment with the identified kinase inhibitors did not affect PV replication significantly, but combined treatment with MEK1/2 inhibitor, AG1478 and Akt1/2 inhibitor suppressed the replication synthetically. The effect of AG1478 in this synthetic inhibition was compensated by other receptor tyrosine kinase inhibitors (IGF-1R inhibitor II and Flt3 inhibitor II). We isolated mutants resistant to Flt3 inhibitor II and GW5074 and found that these mutants had cross-resistance to each treatment. These mutants had a common mutation in viral protein 3A that results in an amino acid change at position 70 (Ala to Thr), a mutation that was previously identified in mutants resistant to a potent anti-enterovirus compound, enviroxime. These results suggest that cellular kinase inhibitors and enviroxime have a conserved target in viral protein 3A to suppress enterovirus replication.
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ORF3 protein of hepatitis E virus is essential for virion release from infected cells
The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein remains unclear. To elucidate the role of the ORF3 protein in the virus life cycle, an infectious cDNA clone (pJE03-1760F/wt) that can replicate efficiently in PLC/PRF/5 and A549 cells and release progeny into the culture medium was used to generate a derivative ORF3-deficient (ΔORF3) mutant whose third in-frame AUG codon of ORF3 was mutated to GCA. The ΔORF3 mutant in the culture medium of mutant RNA-transfected PLC/PRF/5 cells was able to infect and replicate within PLC/PRF/5 and A549 cells as efficiently as the wild-type pJE03-1760F/wt virus. However, less than 1/100 of the number of progeny was detectable in the culture medium of ΔORF3 mutant-infected PLC/PRF/5 cells compared with wild-type-infected PLC/PRF/5 cells, and the HEV RNA level in the culture medium of ΔORF3 mutant-infected A549 cells was below or near the limit of detection. An immunocapture PCR assay revealed that the ORF3 protein is present on the surface of cell-culture-generated wild-type HEV but not on the ΔORF3 mutant. Wild-type HEV in the culture supernatant peaked at a sucrose density of 1.15–1.16 g ml−1, in contrast with the ΔORF3 mutant in culture supernatant, which banded at 1.27–1.28 g ml−1, similar to HEV in cell lysate and faecal HEV. These results suggest that the ORF3 protein is responsible for virion egress from infected cells and is present on the surface of released HEV particles, which may be associated with lipids.
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Formulation of bovine respiratory syncytial virus fusion protein with CpG oligodeoxynucleotide, cationic host defence peptide and polyphosphazene enhances humoral and cellular responses and induces a protective type 1 immune response in mice
More LessRespiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease in children and calves; however, RSV vaccine development has been slow due to early observations that formalin-inactivated vaccines induced Th2-type immune responses and led to disease enhancement upon subsequent exposure. Hence, there is a need for novel adjuvants that will promote a protective Th1-type or balanced immune response against RSV. CpG oligodeoxynucleotides (ODNs), indolicidin, and polyphosphazene were examined for their ability to enhance antigen-specific immune responses and influence the Th-bias when co-formulated with a recombinant truncated bovine RSV (BRSV) fusion protein (ΔF). Mice immunized with ΔF co-formulated with CpG ODN, indolicidin, and polyphosphazene (ΔF/CpG/indol/PP) developed higher levels of ΔF-specific serum IgG, IgG1 and IgG2a antibodies when compared with ΔF alone, and displayed an increase in the frequency of gamma interferon-secreting cells and decreased interleukin (IL)-5 production by in vitro restimulated splenocytes, characteristic of a Th1 immune response. These results were observed in both C57BL/6 and BALB/c strains of mice. When evaluated in a BRSV challenge model, mice immunized with ΔF/CpG/indol/PP developed significantly higher levels of BRSV-neutralizing serum antibodies than mice immunized with the ΔF protein alone, and displayed significantly less pulmonary IL-4, IL-5, IL-13 and eotaxin and reduced eosinophilia after challenge. These results suggest that co-formulation of ΔF with CpG ODN, host defence peptide and polyphosphazene may result in a safe and effective vaccine for the prevention of BRSV and may have implications for the development of novel human RSV vaccines.
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Host specificity of the anti-interferon and anti-apoptosis activities of parainfluenza virus P/C gene products
More LessHuman parainfluenza virus type 1 (HPIV-1) and Sendai virus (SeV) are highly homologous in structure and sequence, whilst maintaining distinct host ranges. These viruses express accessory proteins from their P/C gene that are known to have activities against innate immunity. The accessory proteins expressed from the P/C gene of these viruses are different. In addition to the nested set of C proteins, SeV expresses V protein from edited P mRNA, which is not expressed by HPIV-1. This study evaluated the host specificity and role of the P/C gene products in anti-interferon (IFN) and anti-apoptosis activity by characterizing a recombinant SeV, rSeVhP, in which the SeV P/C gene was replaced with that of HPIV-1. Unlike SeV, rSeVhP infection strongly activated IFN regulatory transcription factor (IRF)-3 and nuclear factor-κB, resulting in an increased level of IFN-β induction compared with SeV in murine cells. In contrast, activation of IRF-3 was not observed in rSeVhP-infected human A549 cells. rSeVhPSV, which expressed SeV V protein from an inserted gene in rSeVhP, induced less IFN-β than rSeVhP, suggesting that V contributes to the suppression of IFN production in murine cells. Furthermore, rSeVhP induced apoptotic cell death in murine but not in A549 cells. These data indicate the functional difference in P/C gene products from SeV and HPIV-1 in antagonizing IFN induction and apoptosis, which is likely to be one of the major factors for pathogenicity in specific hosts.
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Differential RNA silencing suppression activity of NS1 proteins from different influenza A virus strains
More LessThe NS1 gene of influenza A virus encodes a multi-functional protein that plays an important role in counteracting cellular antiviral mechanisms such as the interferon (IFN), protein kinase R and retinoic acid-inducible gene product I pathways. In addition, NS1 has recently been shown to have RNA interference (RNAi) or RNA silencing suppression (RSS) activity. This study analysed the IFN antagonistic activity of NS1 and the RSS activity for several influenza subtypes: H1N1, H3N2, H5N1 and H7N7. It was shown that the various NS1 proteins were capable of inhibiting the activation of an IFN-responsive promoter. However, differential RSS activity was measured among the NS1 variants. The NS1 protein of strain A/WSN/33 (H1N1) was most potent in suppressing short hairpin RNA-mediated gene silencing. In contrast, NS1 proteins of the highly pathogenic H5N1 strains A/VN/1194/04 and A/HK/156/97 were most potent in complementing the RSS function of the human immunodeficiency virus type 1 Tat protein. These results show that the ability of NS1 to suppress RNAi varies among influenza strains and is likely to contribute to differences in viral replication capacity and pathogenicity.
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Analysis of Puumala hantavirus in a bank vole population in northern Finland: evidence for co-circulation of two genetic lineages and frequent reassortment between strains
More LessIn this study, for the first time, two distinct genetic lineages of Puumala virus (PUUV) were found within a small sampling area and within a single host genetic lineage (Ural mtDNA) at Pallasjärvi, northern Finland. Lung tissue samples of 171 bank voles (Myodes glareolus) trapped in September 1998 were screened for the presence of PUUV nucleocapsid antigen and 25 were found to be positive. Partial sequences of the PUUV small (S), medium (M) and large (L) genome segments were recovered from these samples using RT-PCR. Phylogenetic analysis revealed two genetic groups of PUUV sequences that belonged to the Finnish and north Scandinavian lineages. This presented a unique opportunity to study inter-lineage reassortment in PUUV; indeed, 32 % of the studied bank voles appeared to carry reassortant virus genomes. Thus, the frequency of inter-lineage reassortment in PUUV was comparable to that of intra-lineage reassortment observed previously (Razzauti, M., Plyusnina, A., Henttonen, H. & Plyusnin, A. (2008). J Gen Virol 89, 1649–1660). Of six possible reassortant S/M/L combinations, only two were found at Pallasjärvi and, notably, in all reassortants, both S and L segments originated from the same genetic lineage, suggesting a non-random pattern for the reassortment. These findings are discussed in connection to PUUV evolution in Fennoscandia.
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Second-site mutations in Borna disease virus overexpressing viral accessory protein X
More LessThe X protein of Borna disease virus (BDV) is an essential factor that regulates viral polymerase activity and inhibits apoptosis of persistently infected cells. We observed that a BDV mutant which carries an additional X gene replicated well in cell culture only after acquiring second-site mutations that selectively reduced expression of the endogenous X gene. In rat brains, the virus acquired additional mutations which inactivated the ectopic X gene or altered the sequence of X. These results demonstrate that BDV readily acquires mutations if strong selection pressure is applied. They further indicate that fine-tuning of X expression determines viral fitness.
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Detection of polyoma and corona viruses in bats of Canada
Several instances of emerging diseases in humans appear to be caused by the spillover of viruses endemic to bats, either directly or through other animal intermediaries. The objective of this study was to detect, identify and characterize viruses in bats in the province of Manitoba and other regions of Canada. Bats were sampled from three sources: live-trapped Myotis lucifugus from Manitoba, rabies-negative Eptesicus fuscus, M. lucifugus, M. yumanensis, M. septentrionalis, M. californicus, M. evotis, Lasionycteris (L.) noctivagans and Lasiurus (Las.) cinereus, provided by the Centre of Expertise for Rabies of the Canadian Food Inspection Agency (CFIA), and L. noctivagans, Las. cinereus and Las. borealis collected from a wind farm in Manitoba. We attempted to isolate viruses from fresh tissue samples taken from trapped bats in cultured cells of bat, primate, rodent, porcine, ovine and avian origin. We also screened bat tissues by PCR using primers designed to amplify nucleic acids from members of certain families of viruses. We detected RNA of a group 1 coronavirus from M. lucifugus (3 of 31 animals) and DNA from an as-yet undescribed polyomavirus from female M. lucifugus (4 of 31 animals) and M. californicus (pooled tissues from two females).
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- DNA viruses
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A mutation in helicase motif IV of herpes simplex virus type 1 UL5 that results in reduced growth in vitro and lower virulence in a murine infection model is related to the predicted helicase structure
More LessA variant was selected from a clinical isolate of herpes simplex virus type 1 (HSV-1) during a single passage in the presence of a helicase–primase inhibitor (HPI) at eight times the IC50. The variant was approximately 40-fold resistant to the HPI BAY 57-1293 and it showed significantly reduced growth in tissue culture with a concomitant reduction in virulence in a murine infection model. The variant contained a single mutation (Asn342Lys) in the UL5 predicted functional helicase motif IV. The Asn342Lys mutation was transferred to a laboratory strain, PDK cl-1, and the recombinant acquired the expected resistance and reduced growth characteristics. Comparative modelling and docking studies predicted the Asn342 position to be physically distant from the HPI interaction pocket formed by UL5 and UL52 (primase). We suggest that this mutation results in steric/allosteric modification of the HPI-binding pocket, conferring an indirect resistance to the HPI. Slower growth and moderately reduced virulence suggest that this mutation might also interfere with the helicase–primase activity.
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In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)
Fibropapillomatosis (FP) of green turtles has a global distribution and causes debilitating tumours of the skin and internal organs in several species of marine turtles. FP is associated with a presently non-cultivable alphaherpesvirus Chelonid fibropapilloma-associated herpesvirus (CFPHV). Our aims were to employ quantitative PCR targeted to pol DNA of CFPHV to determine (i) if DNA sequesters by tumour size and/or cell type, (ii) whether subculturing of cells is a viable strategy for isolating CFPHV and (iii) whether CFPHV can be induced to a lytic growth cycle in vitro using chemical modulators of replication (CMRs), temperature variation or co-cultivation. Additional objectives included determining whether non-tumour and tumour cells behave differently in vitro and confirming the phenotype of cultured cells using cell-type-specific antigens. CFPHV pol DNA was preferentially concentrated in dermal fibroblasts of skin tumours and the amount of viral DNA per cell was independent of tumour size. Copy number of CFPHV pol DNA per cell rapidly decreased with cell doubling of tumour-derived fibroblasts in culture. Attempts to induce viral replication in known CFPHV-DNA-positive cells using temperature or CMR failed. No significant differences were seen in in vitro morphology or growth characteristics of fibroblasts from tumour cells and paired normal skin, nor from CFPHV pol-DNA-positive intestinal tumour cells. Tumour cells were confirmed as fibroblasts or keratinocytes by positive staining with anti-vimentin and anti-pancytokeratin antibodies, respectively. CFPHV continues to be refractory to in vitro cultivation.
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Glycoprotein N subtypes of human cytomegalovirus induce a strain-specific antibody response during natural infection
More LessHuman cytomegalovirus (HCMV) encodes several highly polymorphic envelope glycoproteins; however, the biological relevance of this polymorphism is unclear. Glycoprotein N (gN) is one member of this polymorphic protein family. Four major gN genotypes (gN1–4) have been identified. We have tested the hypothesis that the gN polymorphism represents a mechanism to evade a neutralizing antiviral antibody response. Four recombinant viruses that differed only in the expression of the gN genotype were constructed on the genetic background of HCMV strain AD169. Exchange of gN genotypes had a minor detectable influence on virus replication, gN expression and gN–gM complex formation. Randomly selected human sera were analysed for neutralizing activity against the recombinant viruses. Of these, 70 % showed no difference in neutralizing titre between the viruses, whereas 30 % showed strain-specific neutralization. Differences in 50 % neutralization titre reached >8-fold. Viruses expressing the gN4 genotype were neutralized significantly better than those expressing the other gN genotypes. Strain specificity, or lack thereof, could not be attributed to the presence or absence of anti-gN antibodies, as all sera contained antibodies reacting with gN (as determined by ELISA). Thus, polymorphism of gN could contribute to evasion of an efficient neutralizing-antibody response and facilitate reinfection in previously seropositive individuals.
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Evaluation of smallpox vaccines using variola neutralization
The search for a ‘third’-generation smallpox vaccine has resulted in the development and characterization of several vaccine candidates. A significant barrier to acceptance is the absence of challenge models showing induction of correlates of protective immunity against variola virus. In this light, virus neutralization provides one of few experimental methods to show specific ‘in vitro’ activity of vaccines against variola virus. Here, we provide characterization of the ability of a modified vaccinia virus Ankara vaccine to induce variola virus-neutralizing antibodies, and we provide comparison with the neutralization elicited by standard Dryvax vaccination.
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Capripoxvirus G-protein-coupled chemokine receptor: a host-range gene suitable for virus animal origin discrimination
The genus Capripoxvirus within the family Poxviridae comprises three closely related viruses, namely goat pox, sheep pox and lumpy skin disease viruses. This nomenclature is based on the animal species from which the virus was first isolated, respectively, goat, sheep and cattle. Since capripoxviruses are serologically identical, their specific identification relies exclusively on the use of molecular tools. We describe here the suitability of the G-protein-coupled chemokine receptor (GPCR) gene for use in host-range grouping of capripoxviruses. The analysis of 58 capripoxviruses showed three tight genetic clusters consisting of goat pox, sheep pox and lumpy skin disease viruses. However, a few discrepancies exist with the classical virus–host origin nomenclature: a virus isolated from sheep is grouped in the goat poxvirus clade and vice versa. Intra-group diversity was further observed for the goat pox and lumpy skin disease virus isolates. Despite the presence of nine vaccine strains, no genetic determinants of virulence were identified on the GPCR gene. For sheep poxviruses, the addition or deletion of 21 nucleic acids (7 aa) was consistently observed in the 5′ terminal part of the gene. Specific signatures for each cluster were also identified. Prediction of the capripoxvirus GPCR topology, and its comparison with other known mammalian GPCRs and viral homologues, revealed not only a classical GPCR profile in the last three-quarters of the protein but also unique features such as a longer N-terminal end with a proximal hydrophobic α-helix and a shorter serine-rich C-tail.
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Comparison of 12 turkey hemorrhagic enteritis virus isolates allows prediction of genetic factors affecting virulence
Turkey hemorrhagic enteritis virus (THEV) is a member of the genus Siadenovirus and causes disease in turkey poults characterized by splenomegaly, bloody diarrhoea and death. The mechanism responsible for intestinal lesion formation and mortality is not known, although there is strong evidence that it is immune-mediated. All strains of THEV are serologically indistinguishable, although there are naturally occurring avirulent strains of THEV that replicate efficiently in turkeys without the intestinal haemorrhage or mortality associated with more virulent strains. The purpose of this study was to determine which viral genes are involved in virulence. The full-length genome of an avirulent vaccine strain was sequenced and compared with the genome of a virulent field isolate from Israel that was sequenced in 1998. Comparison of the two 26.3 kb genomes revealed 49 nucleotide differences resulting in 14 putative amino acid changes within viral proteins. Sequencing of the regions surrounding the 14 missense mutations revealed variations in ORF1, E3 and the fiber (fib) knob domain in five additional strains with varying degrees of virulence. Complete sequences of these genes were determined in a total of 11 different strains of THEV. All strains had at least one missense mutation in ORF1, and all but two of the strains had one missense mutation in E3. At least one missense mutation was found in the fiber knob domain in six out of seven virulent strains. Sequence variation of ORF1, E3 and fib in strains of THEV with different phenotypes strongly indicates that these genes are the key factors affecting virulence.
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Antibody responses to 26 skin human papillomavirus types in the Netherlands, Italy and Australia
Solar UV radiation is the main risk factor for cutaneous squamous cell carcinoma (SCC), but infections with skin human papillomavirus (HPV) types have also been linked to the development of SCC. Little is known about the natural history of these infections and whether the seroprevalence of skin HPV types is affected by ambient or individual levels of sun exposure. This study investigated this by analysing sera for antibodies to 26 skin HPV types from five phylogenetic genera obtained from 807 healthy individuals from the Netherlands, Italy and Australia, countries with strong differences in sunlight intensity. Overall HPV seroprevalence was similar across the three countries (50–57 % for β-HPV types, 40–48 % for γ-HPV types), and the most frequent β-HPV and γ-HPV types were the same in all countries. The highest seroprevalences for 24 of the 26 skin HPV types were observed in Italy (14 types) and Australia (ten types). Seroprevalence among men was generally higher than among women, and the male sex was significantly associated with both β-HPV [odds ratio (OR) 2.81, 95 % confidence interval (CI) 1.64–4.82] and γ-HPV (OR 2.42, 95 % CI 1.40–4.18) antibodies in Australia. The only measure of sun sensitivity or UV exposure significantly associated with skin HPV seroprevalence was found for weekend sun exposure in Australia and β-HPV antibodies. It was concluded that type spectra and HPV seroprevalence are similar in countries with different sunlight intensity, and that levels of UV exposure do not play a strong role in the development of skin HPV antibodies in this study population.
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Characterization of seven novel human papillomavirus types isolated from cutaneous tissue, but also present in mucosal lesions
More LessSeven novel human papillomavirus (HPV) types were isolated and characterized. HPV 94 is related most closely to HPV 10 and belongs to the genus Alphapapillomavirus, whereas HPV 98, HPV 99, HPV 100, HPV 104, HPV 105 and HPV 113 all belong to the genus Betapapillomavirus. These HPV types were isolated from and demonstrated in cutaneous tissue, but HPV 98, HPV 100, HPV 104 and HPV 113 were also detected in malignant oesophageal and oral lesions. The general prevalence of these HPV types in lesions is infrequent.
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SMAD proteins of oligodendroglial cells regulate transcription of JC virus early and late genes coordinately with the Tat protein of human immunodeficiency virus type 1
JC virus (JCV) is the aetiological agent of progressive multifocal leukoencephalopathy (PML), a fatal, demyelinating disease of the brain affecting people with AIDS. Although immunosuppression is involved in infection of the brain by JCV, a direct influence of human immunodeficiency virus type 1 (HIV-1) has also been established. The Tat protein of HIV-1 has been implicated in activation of the cytokine transforming growth factor (TGF)-β in HIV-1-infected cells and in stimulating JCV gene transcription and DNA replication in oligodendroglia, the primary central nervous system cell type infected by JCV in PML. This study demonstrated that Tat can cooperate with SMAD proteins, the intracellular effectors of TGF-β, at the JCV DNA control region (CR) to stimulate JCV gene transcription. Tat stimulated JCV early gene transcription in KG-1 oligodendroglial cells when expressed via transfection or added exogenously. Using chromatin immunoprecipitation, it was shown that exogenous Tat enhanced binding of SMAD2, -3 and -4 and their binding partner Fast1 to the JCV CR in living cells. When SMAD2, -3 and -4 were expressed together, Tat, expressed from plasmid pTat, stimulated transcription from both early and late gene promoters, with the early promoter exhibiting stimulation of >100-fold. Tat, SMAD4 and JCV large T-antigen were all visualized in oligodendroglial cells at the border of an active PML lesion in the cerebral frontal lobe. These results revealed a positive reinforcement system in which the SMAD mediators of the TGF-β system act cooperatively with Tat to stimulate JCV gene transcription.
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Pathogenesis of Autographa californica multiple nucleopolyhedrovirus in fifth-instar Anticarsia gemmatalis larvae
We have investigated infection and pathogenesis of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in Anticarsia gemmatalis (velvetbean caterpillar) larvae using a lacZ recombinant virus (AcMNPV-hsp70/lacZ) to track the temporal progression of infection in the midgut intestine and haemocoel. A. gemmatalis was highly resistant to fatal infection by occlusion bodies (OBs; LD50>5.5×105 OB) and budded virus (BV; LD50>3×105 BV) administered via oral and systemic routes, respectively. Orally administered occlusion-derived virus (ODV) efficiently attached and fused to midgut cells; however, high levels of infection-induced apoptosis limited infection in the midgut. Transcriptional analysis of AcMNPV genes expressed in the midgut of OB-inoculated A. gemmatalis larvae showed high levels of mRNA encoding the major capsid protein VP39 in the absence of immediate-early transactivator 1 (ie-1) expression. In the midgut, virus was efficiently transferred from infected midgut epithelial cells to nearby tracheolar cells and circulating haemocytes to initiate systemic infection in the haemocoel. However, haemocoelic BV did not efficiently disseminate infection and only cuticular epidermal cells displayed high levels of viral infection. Flow cytometry analysis of haemocytes isolated from BV-inoculated A. gemmatalis larvae showed low-level expression of the BV envelope protein GP64 on the cell surface, suggesting that A. gemmatalis haemocytes have a limited capacity for amplifying virus. These results show that AcMNPV is not an effective biological control agent for limiting crop damage caused by A. gemmatalis larvae.
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Chlorella viruses prevent multiple infections by depolarizing the host membrane
Previous experiments established that when the unicellular green alga Chlorella NC64A is inoculated with two viruses, usually only one virus replicates in a single cell. That is, the viruses mutually exclude one another. In the current study, we explore the possibility that virus-induced host membrane depolarization, at least partially caused by a virus-encoded K+ channel (Kcv), is involved in this mutual exclusion. Two chlorella viruses, PBCV-1 and NY-2A, were chosen for the study because (i) they can be distinguished by real-time PCR and (ii) they exhibit differential sensitivity to Cs+, a well-known K+ channel blocker. PBCV-1-induced host membrane depolarization, Kcv channel activity and plaque formation are only slightly affected by Cs+, whereas all three NY-2A-induced events are strongly inhibited by Cs+. The addition of one virus 5–15 min before the other results primarily in replication of the first virus. However, if virus NY-2A-induced membrane depolarization of the host is blocked by Cs+, PBCV-1 is not excluded. We conclude that virus-induced membrane depolarization is at least partially responsible for the exclusion phenomenon.
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Effect of citrus hosts on the generation, maintenance and evolutionary fate of genetic variability of citrus exocortis viroid
More LessCitrus exocortis viroid (CEVd) populations are composed of closely related haplotypes whose frequencies in the population result from the equilibrium between mutation, selection and genetic drift. The genetic diversity of CEVd populations infecting different citrus hosts was studied by comparing populations recovered from infected trifoliate orange and sour orange seedling trees after 10 years of evolution, with the ancestral population maintained for the same period in the original host, Etrog citron. Furthermore, populations isolated from these trifoliate orange and sour orange trees were transmitted back to Etrog citron plants and the evolution of their mutant spectra was studied. The results indicate that (i) the amount and composition of the within-plant genetic diversity generated varies between these two hosts and is markedly different from that which is characteristic of the original Etrog citron host and (ii) the genetic diversity found after transmitting back to Etrog citron is indistinguishable from that which is characteristic of the ancestral Etrog citron population, regardless of the citrus plant from which the evolved populations were isolated. The relationship between the CEVd populations from Etrog citron and trifoliate orange, both sensitive hosts, and those from sour orange, which is a tolerant host, is discussed.
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Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells
More LessDysfunction of the endoplasmic reticulum associated protein degradation/proteasome system is believed to contribute to the initiation or aggravation of neurodegenerative disorders associated with protein misfolding, and there is some evidence to suggest that proteasome dysfunctions might be implicated in prion disease. This study investigated the effect of proteasome inhibitors on the biogenesis of both the cellular (PrPC) and abnormal (PrPSc) forms of prion protein in CAD neuronal cells, a newly introduced prion cell system. In uninfected cells, proteasome impairment altered the intracellular distribution of PrPC, leading to a strong accumulation in the Golgi apparatus. Moreover, a detergent-insoluble and weakly protease-resistant PrP species of 26 kDa, termed PrP26K, accumulated in the cells, whether they were prion-infected or not. However, no evidence was found that, in infected cells, this PrP26K species converts into the highly proteinase K-resistant PrPSc. In the infected cultures, proteasome inhibition caused an increased intracellular aggregation of PrPSc that was deposited into large aggresomes. These findings strengthen the view that, in neuronal cells expressing wild-type PrPC from the natural promoter, proteasomal impairment may affect both the process of PrPC biosynthesis and the subcellular sites of PrPSc accumulation, despite the fact that these two effects could essentially be disconnected.
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Volumes and issues
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Volume 105 (2024)
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Volume 104 (2023)
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Volume 103 (2022)
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Volume 102 (2021)
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Volume 101 (2020)
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Volume 100 (2019)
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Volume 99 (2018)
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Volume 98 (2017)
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Volume 97 (2016)
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Volume 96 (2015)
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Volume 95 (2014)
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Volume 94 (2013)
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Volume 93 (2012)
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Volume 92 (2011)
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Volume 91 (2010)
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Volume 90 (2009)
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Volume 89 (2008)
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Volume 88 (2007)
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Volume 87 (2006)
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Volume 86 (2005)
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Volume 85 (2004)
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Volume 84 (2003)
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Volume 83 (2002)
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Volume 82 (2001)
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Volume 81 (2000)
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Volume 80 (1999)
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Volume 79 (1998)
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Volume 78 (1997)
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Volume 77 (1996)
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Volume 76 (1995)
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Volume 75 (1994)
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Volume 74 (1993)
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Volume 73 (1992)
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Volume 72 (1991)
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Volume 71 (1990)
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Volume 70 (1989)
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Volume 69 (1988)
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Volume 68 (1987)
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Volume 67 (1986)
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Volume 66 (1985)
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Volume 65 (1984)
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Volume 64 (1983)
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Volume 63 (1982)
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Volume 62 (1982)
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Volume 61 (1982)
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Volume 60 (1982)
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Volume 59 (1982)
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Volume 58 (1982)
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Volume 57 (1981)
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Volume 56 (1981)
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Volume 55 (1981)
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Volume 54 (1981)
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Volume 53 (1981)
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Volume 52 (1981)
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Volume 51 (1980)
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Volume 50 (1980)
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Volume 49 (1980)
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Volume 48 (1980)
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Volume 47 (1980)
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Volume 46 (1980)
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Volume 45 (1979)
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Volume 44 (1979)
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Volume 43 (1979)
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Volume 42 (1979)
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Volume 41 (1978)
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Volume 40 (1978)
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Volume 39 (1978)
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Volume 38 (1978)
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Volume 37 (1977)
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Volume 36 (1977)
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Volume 35 (1977)
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Volume 34 (1977)
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Volume 33 (1976)
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Volume 32 (1976)
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Volume 31 (1976)
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Volume 30 (1976)
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Volume 29 (1975)
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Volume 28 (1975)
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Volume 27 (1975)
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Volume 26 (1975)
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Volume 25 (1974)
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Volume 24 (1974)
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Volume 23 (1974)
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Volume 22 (1974)
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Volume 21 (1973)
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Volume 20 (1973)
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Volume 19 (1973)
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Volume 18 (1973)
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Volume 17 (1972)
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Volume 16 (1972)
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Volume 15 (1972)
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Volume 14 (1972)
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Volume 13 (1971)
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Volume 12 (1971)
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Volume 11 (1971)
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Volume 10 (1971)
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Volume 9 (1970)
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Volume 8 (1970)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)