- Volume 93, Issue 3, 2012
Volume 93, Issue 3, 2012
- Review
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The polio-eradication programme and issues of the end game
More LessPoliovirus causes paralytic poliomyelitis, an ancient disease of humans that became a major public-health issue in the 20th century. The primary site of infection is the gut, where virus replication is entirely harmless; the two very effective vaccines developed in the 1950s (oral polio vaccine, or OPV, and inactivated polio vaccine, or IPV) induce humoral immunity, which prevents viraemic spread and disease. The success of vaccination in middle-income and developing countries encouraged the World Health Organization to commit itself to an eradication programme, which has made great advances. The features of the infection, including its largely silent nature and the ability of the live vaccine (OPV) to evolve and change in vaccine recipients and their contacts, make eradication particularly challenging. Understanding the pathogenesis and virology of the infection is of major significance as the programme reaches its conclusion.
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- Animal
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- RNA viruses
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Echovirus 11 infection induces dramatic changes in the actin cytoskeleton of polarized Caco-2 cells
More LessBinding of echovirus 11 strain 207 (EV11-207) to Caco-2 monolayers results in rapid transfer of the virus to tight junctions prior to uptake. Using a confocal microscopy based-method, this study quantified the spatiotemporal distribution of actin during the time course of infection by EV11-207 in Caco-2 polarized cells. It was found that binding of EV11-207 to the apical surface resulted in rapid rearrangement of the actin cytoskeleton, concomitant with transport of the virus particles to tight junctions. By interfering with the actin network dynamics, the virus remained trapped at the cell surface, leading to abortion of infection. In addition, it was observed that at 4 h post-infection, concomitant with the detection of virus replication, actin filament was depolymerized and degraded. Finally, it was shown that the mechanisms leading to loss of actin were independent of viral genome synthesis, indicating a potential role for the viral protein synthesis seen in late infection. These data confirmed a previous study on the requirement for an intact actin cytoskeleton for EV11-207 to infect cells and reinforce the notion of actin cytoskeleton subversion by picornaviruses during infection in polarized epithelial cells.
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Foot-and-mouth disease virus epitope dominance in the antibody response of vaccinated animals
More LessFive neutralizing antigenic sites have been identified on the surface of serotype O foot-and-mouth disease virus (FMDV). A set of mAb neutralization-escape mutant viruses was used for the first time to evaluate the relative use of known binding sites by polyclonal antibodies from three target species: cattle, sheep and pigs. Antibodies to all five neutralizing antigenic sites were detected in all three species, with most antibodies directed against antigenic site 2, followed by antigenic site 1. In 76 % of cattle, 65 % of sheep and 58 % of pigs, most antibodies were directed against site 2. Antibodies specific to antigenic sites 3, 4 and 5 were found to be minor constituents in the sera of each of the target species. This implies that antigenic site 2 is a dominant neutralization immunogenic site in serotype O FMDV and may therefore be a good candidate for designing novel vaccines.
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Infection of human alveolar macrophages by human coronavirus strain 229E
Human coronavirus strain 229E (HCoV-229E) commonly causes upper respiratory tract infections. However, lower respiratory tract infections can occur in some individuals, indicating that cells in the distal lung are susceptible to HCoV-229E. This study determined the virus susceptibility of primary cultures of human alveolar epithelial cells and alveolar macrophages (AMs). Fluorescent antibody staining indicated that HCoV-229E could readily infect AMs, but no evidence was found for infection in differentiated alveolar epithelial type II cells and only a very low level of infection in type II cells transitioning to the type I-like cell phenotype. However, a human bronchial epithelial cell line (16HBE) was readily infected. The innate immune response of AMs to HCoV-229E infection was evaluated for cytokine production and interferon (IFN) gene expression. AMs secreted significant amounts of tumour necrosis factor alpha (TNF-α), regulated on activation normal T-cell expressed and secreted (RANTES/CCL5) and macrophage inflammatory protein 1β (MIP-1β/CCL4) in response to HCoV-229E infection, but these cells exhibited no detectable increase in IFN-β or interleukin-29 in mRNA levels. AMs from smokers had reduced secretion of TNF-α compared with non-smokers in response to HCoV-229E infection. Surfactant protein A (SP-A) and SP-D are part of the innate immune system in the distal lung. Both surfactant proteins bound to HCoV-229E, and pre-treatment of HCoV-229E with SP-A or SP-D inhibited infection of 16HBE cells. In contrast, there was a modest reduction in infection in AMs by SP-A, but not by SP-D. In summary, AMs are an important target for HCoV-229E, and they can mount a pro-inflammatory innate immune response to infection.
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Generation and genetic stability of tick-borne encephalitis virus mutants dependent on processing by the foot-and-mouth disease virus 3C protease
More LessMature protein C of tick-borne encephalitis virus (TBEV) is cleaved from the polyprotein precursor by the viral NS2B/3 protease (NS2B/3pro). We showed previously that replacement of the NS2B/3pro cleavage site at the C terminus of protein C by the foot-and-mouth disease virus (FMDV) 2A StopGo sequence leads to the production of infectious virions. Here, we show that infectious virions can also be produced from a TBEV mutant bearing an inactivated 2A sequence through the expression of the FMDV 3C protease (3Cpro) either in cis or in trans (from a TBEV replicon). Cleavage at the C terminus of protein C depended on the catalytic activity of 3Cpro as well as on the presence of an optimized 3Cpro cleavage site. Passage of the TBEV mutants bearing a 3Cpro cleavage site either in the absence of 3Cpro or in the presence of a catalytically inactive 3Cpro led to the appearance of revertants in which protein C cleavage by NS2B/3pro had been regained. In three different revertants, a cleavage site for NS2B/3pro, namely RR*C, was now present, leading to an elongated protein C. Furthermore, two revertants acquired additional mutations in the C terminus of protein C, eliminating two basic residues. Although these latter mutants showed wild-type levels of early RNA synthesis, their foci were smaller and an accumulation of protein C in the cytoplasm was observed. These findings suggest a role of the positive charge of the C terminus of protein C for budding of the nucleocapsid and further support the notion that TBEV protein C is a multifunctional protein.
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Rubella virus-like replicon particles: analysis of encapsidation determinants and non-structural roles of capsid protein in early post-entry replication
More LessRubella virus (RUBV) contains a plus-strand RNA genome with two ORFs, one encoding the non-structural replicase proteins (NS-ORF) and the second encoding the virion structural proteins (SP-ORF). This study describes development and use of a trans-encapsidation system for the assembly of infectious RUBV-like replicon particles (VRPs) containing RUBV replicons (self replicating genomes with the SP-ORF replaced with a reporter gene). First, this system was used to map signals within the RUBV genome that mediate packaging of viral RNA. Mutations within a proposed packaging signal did not significantly affect relative packaging efficiency. The insertion of various fragments derived from the RUBV genome into Sindbis virus replicons revealed that there are several regions within the RUBV genome capable of enhancing encapsidation of heterologous replicon RNAs. Secondly, the trans-encapsidation system was used to analyse the effect of alterations within the capsid protein (CP) on release of VRPs and subsequent initiation of replication in newly infected cells. Deletion of the N-terminal eight amino acids of the CP reduced VRP titre significantly, which could be partially complemented by native CP provided in trans, indicating that this mutation affected an entry or post-entry event in the replication cycle. To test this hypothesis, the trans-encapsidation system was used to demonstrate the rescue of a lethal deletion within P150, one of the virus replicase proteins, by CP contained within the virus particle. This novel finding substantiated the functional role of CP in early post-entry replication.
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A naturally occurring human/hepatitis E recombinant virus predominates in serum but not in faeces of a chronic hepatitis E patient and has a growth advantage in cell culture
Hepatitis E virus is the aetiological agent of acute hepatitis E, a self-limiting disease prevalent in developing countries. Molecular analysis of viral genomic RNA from a chronically infected patient confirmed the recent discovery that chronic infection correlated with extensive diversification of the virus quasispecies: the hypervariable region of some virus genomes in this USA patient contained large continuous deletions and a minor proportion of genomes in faeces and serum had acquired a mammalian sequence that encoded 39 aa of S19 ribosomal protein fused to the virus non-structural protein. Genomes with this insert were selected during virus passage in cultured cells to become the predominant species, suggesting that the inserted sequence promoted virus growth. The results demonstrated that hepatitis E virus can mutate dramatically during a prolonged infection and suggests it may be important to prevent or cure chronic infections before new variants with unpredictable properties arise.
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Amino acids 473V and 598P of PB1 from an avian-origin influenza A virus contribute to polymerase activity, especially in mammalian cells
It has been reported that the avian-origin influenza A virus PB1 protein (avian PB1) enhances influenza A virus polymerase activity in mammalian cells when it replaces the human-origin PB1 protein (human PB1). Characterization of the amino acid residues that contribute to this enhancement is needed. In this study, it was found that PB1 from an avian-origin influenza A virus [A/Cambodia/P0322095/2005, H5N1 (Cam)] could enhance the polymerase activity of an attenuated human isolated virus, A/WSN/33, carrying the PB2 K627E mutation (WSN627E) in vitro. Furthermore, 473V and 598P in the Cam PB1 were identified as the residues responsible for this enhanced activity. The results from recombinant virus experiments demonstrated the contribution of PB1 amino acids 473V and 598P to polymerase activity in mammalian cells and in mice. Interestingly, 473V is conserved in pH1N1 viruses from the 2009 pandemic. Substitution of 473V by leucine in pH1N1 PB1 led to a decreased viral polymerase activity and a lower growth rate in mammalian cells, suggesting that the PB1 473V also plays a role in maintaining efficient virus replication of the pH1N1 virus. Thus, it was concluded that two amino acids in avian-origin PB1, 473V and 598P, contribute to the polymerase activity of the H5N1 virus, especially in mammalian cells, and that 473V in PB1 also contributes to efficient replication of the pH1N1 strain.
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Reintroduction of H5N1 highly pathogenic avian influenza virus by migratory water birds, causing poultry outbreaks in the 2010–2011 winter season in Japan
Yoshihiro Sakoda, Hiroshi Ito, Yuko Uchida, Masatoshi Okamatsu, Naoki Yamamoto, Kosuke Soda, Naoki Nomura, Saya Kuribayashi, Shintaro Shichinohe, Yuji Sunden, Takashi Umemura, Tatsufumi Usui, Hiroichi Ozaki, Tsuyoshi Yamaguchi, Toshiyuki Murase, Toshihiro Ito, Takehiko Saito, Ayato Takada and Hiroshi KidaH5N1 highly pathogenic avian influenza virus (HPAIV) was reintroduced and caused outbreaks in chickens in the 2010–2011 winter season in Japan, which had been free from highly pathogenic avian influenza (HPAI) since 2007 when HPAI outbreaks occurred and were controlled. On 14 October 2010 at Lake Ohnuma, Wakkanai, the northernmost part of Hokkaido, Japan, H5N1 HPAIVs were isolated from faecal samples of ducks flying from their nesting lakes in Siberia. Since then, in Japan, H5N1 HPAIVs have been isolated from 63 wild birds in 17 prefectures and caused HPAI outbreaks in 24 chicken farms in nine prefectures by the end of March in 2011. Each of these isolates was genetically closely related to the HPAIV isolates at Lake Ohnuma, and those in China, Mongolia, Russia and Korea, belonging to genetic clade 2.3.2.1. In addition, these isolates were genetically classified into three groups, suggesting that the viruses were transmitted by migratory water birds through at least three different routes from their northern territory to Japan. These isolates were antigenic variants, which is consistent with selection in poultry under the immunological pressure induced by vaccination. To prevent the perpetuation of viruses in the lakes where water birds nest in summer in Siberia, prompt eradication of HPAIVs in poultry is urgently needed in Asian countries where HPAI has not been controlled.
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A novel reassortant canine H3N1 influenza virus between pandemic H1N1 and canine H3N2 influenza viruses in Korea
During recent canine influenza surveillance in South Korea, a novel H3N1 canine influenza virus (CIV) that is a putative reassortant between pandemic H1N1 2009 and H3N2 CIVs was isolated. Genetic analysis of eight genes of the influenza virus revealed that the novel H3N1 isolate presented high similarities (99.1–99.9 %) to pandemic influenza H1N1, except for in the haemagglutinin (HA) gene. The HA gene nucleotide sequence of the novel CIV H3N1 was similar (99.6 %) to that of CIV H3N2 isolated in Korea and China. Dogs infected with the novel H3N1 CIV did not show any notable symptoms, in contrast to dogs infected with H3N2 CIV. Despite no visible clinical signs of disease, nasal shedding of virus was detected and the infected dogs presented mild histopathological changes.
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Plasmacytoid dendritic cells and Toll-like receptor 7-dependent signalling promote efficient protection of mice against highly virulent influenza A virus
More LessTypes I and III interferons (IFNs) elicit protective antiviral immune responses during influenza virus infection. Although many cell types can synthesize IFN in response to virus infection, it remains unclear which IFN sources contribute to antiviral protection in vivo. We found that mice carrying functional alleles of the Mx1 influenza virus resistance gene partially lost resistance to infection with a highly pathogenic H7N7 influenza A virus strain if Toll-like receptor 7 (TLR7) signalling was compromised. This effect was achieved by deleting either the TLR7 gene or the gene encoding the TLR7 adaptor molecule MyD88. A similar decrease of influenza virus resistance was observed when animals were deprived of plasmacytoid dendritic cells (pDCs) at day 1 post-infection. Our results provide in vivo proof that pDCs contribute to the protection of the lung against influenza A virus infections, presumably via signals from TLR7.
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Hazara virus infection is lethal for adult type I interferon receptor-knockout mice and may act as a surrogate for infection with the human-pathogenic Crimean–Congo hemorrhagic fever virus
Hazara virus (HAZV) is closely related to the Crimean–Congo hemorrhagic fever virus (CCHFV). HAZV has not been reported to cause human disease; work with infectious material can be carried out at containment level (CL)-2. By contrast, CCHFV causes a haemorrhagic fever in humans and requires CL-4 facilities. A disease model of HAZV infection in mice deficient in the type I interferon receptor is reported in this study. Dose–response effects were seen with higher doses, resulting in a shorter time to death and earlier detection of viral loads in organs. The lowest dose of 10 p.f.u. was still lethal in over 50 % of the mice. Histopathological findings were identified in the liver, spleen and lymph nodes, with changes similar to a recent mouse model of CCHFV infection. The findings demonstrate that inoculation of mice with HAZV may act as a useful surrogate model for the testing of antiviral agents against CCHFV.
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The innate antiviral factor APOBEC3G targets replication of measles, mumps and respiratory syncytial viruses
The cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme-catalytic polypeptide 3G; A3G) exerts antiviral activity against retroviruses, hepatitis B virus, adeno-associated virus and transposable elements. We assessed whether the negative-strand RNA viruses measles, mumps and respiratory syncytial might be affected by A3G, and found that their infectivity was reduced by 1–2 logs (90–99 %) in A3G overexpressing Vero cells, and in T-cell lines expressing A3G at physiological levels. Viral RNA was co-precipitated with HA-tagged A3G and could be amplified by RT-PCR. Interestingly, A3G reduced viral transcription and protein expression in infected cells by 50–70 %, and caused an increased mutation frequency of 0.95 mutations per 1000 nt in comparison to the background level of 0.22/1000. The observed mutations were not specific for A3G [cytidine to uridine (C→U) or guanine to adenine (G→A) hypermutations], nor specific for ADAR (adenosine deaminase acting on RNA, A→G and U→C transitions, with preference for next neighbour-nucleotides U = A>C>G). In addition, A3G mutants with inactivated catalytic deaminase (H257R and E259Q) were inhibitory, indicating that the deaminase activity is not required for the observed antiviral activity. In combination, impaired transcription and increased mutation frequencies are sufficient to cause the observed reduction in viral infectivity and eliminate virus replication within a few passages in A3G-expressing cells.
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Multiple genomic sequences of hepatitis delta virus are associated with cDNA promoter activity and RNA double rolling-circle replication
More LessTo understand how DNA-dependent RNA polymerase II (pol II) recognizes hepatitis delta virus (HDV) RNA as a template, it is first necessary to identify the HDV sequence that acts as a promoter of pol II-initiated RNA synthesis. Therefore, we isolated the pol II-response element from HDV cDNA and examined the regulation by hepatitis delta antigens (HDAgs). Two HDV cDNA fragments containing bidirectional promoter activity were identified. One was located at nt 1582–1683 (transcription-promoter region 1, TR-P1) and the other at nt 1223–1363 (transcription-internal region 5, TR-I5). The promoter activities of these two regions were enhanced by HDAgs to differing degrees. Next, the role of these sequences in an HDV cDNA-free RNA replication system was characterized by site-directed mutagenesis. Our data showed that: (i) the AUG codon at the HDAg ORF of HDV RNA (nt 1599–1601) that mutates to UAG (amber stop codon) results in loss of dimeric but not monomeric HDV RNA synthesis. (ii) A 5 nt mutation of TR-P1 (P1-m5, nt 1670–1674) abolishes RNA replication completely. Two-nucleotide-mutated RNA (P1-m2, nt 1662–1663) is able to synthesize short RNAs but not monomeric HDV RNA. (iii) A mutation in 5 nt at the TR-I5 region (I5-m5, nt 1351–1355) also abolishes HDV replication. Mutants with 2 nt mutations (I5-m2, nt 1351–1352) or 3 nt mutations (I5-m3, nt 1353–1355) inhibit HDV dimeric but not monomeric RNA synthesis. Furthermore, large HDAg is expressed in cells transfected with I5-m3 and I5-m2 RNAs and that demonstrate the RNA-editing event in the monomeric HDV RNA. These results provide further understanding of the double rolling-circle mechanism in HDV RNA replication.
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Tax1-expressing feline 8C cells are useful to monitor the life cycle of human T-cell leukemia virus type I
Extremely low infectivity has hampered direct (cell-free) infection studies of human T-cell leukemia virus type I (HTLV-I). In order to break through this barrier, we examined the susceptibility of many kinds of cells to HTLV-I and found a feline kidney cell line, 8C, that is highly susceptible to HTLV-I and produced remarkable amounts of infectious progeny viruses. Tax1 protein encoded by HTLV-I is known as a transcription activator for viral and cellular genes. We found that the 8C cells expressing the Tax1 protein (8C/TaxWT cells) can produce more progeny viruses than 8C cells when the cells were exposed to cell-free HTLV-I. A large number of syncytia were also induced in these cells. Here, we propose 8C/TaxWT cells as a useful tool to study the cell-free HTLV-I infection.
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Geographical, genetic and functional diversity of antiretroviral host factor TRIMCyp in cynomolgus macaque (Macaca fascicularis)
The antiretroviral factor tripartite motif protein 5 (TRIM5) gene-derived isoform (TRIMCyp) has been found in at least three species of Old World monkey: rhesus (Macaca mulatta), pig-tailed (Macaca nemestrina) and cynomolgus (Macaca fascicularis) macaques. Although the frequency of TRIMCyp has been well studied in rhesus and pig-tailed macaques, the frequency and prevalence of TRIMCyp in cynomolgus macaques remain to be definitively elucidated. Here, the geographical and genetic diversity of TRIM5α/TRIMCyp in cynomolgus macaques was studied in comparison with their anti-lentiviral activity. It was found that the frequency of TRIMCyp in a population in the Philippines was significantly higher than those in Indonesian and Malaysian populations. Major and minor haplotypes of cynomolgus macaque TRIMCyp with single nucleotide polymorphisms in the cyclophilin A domain were also found. The functional significance of the polymorphism in TRIMCyp was examined, and it was demonstrated that the major haplotype of TRIMCyp suppressed human immunodeficiency virus type 1 (HIV-1) but not HIV-2, whilst the minor haplotype of TRIMCyp suppressed HIV-2 but not HIV-1. The major haplotype of TRIMCyp did not restrict a monkey-tropic HIV-1 clone, NL-DT5R, which contains a capsid with the simian immunodeficiency virus-derived loop between α-helices 4 and 5 and the entire vif gene. These results indicate that polymorphisms of TRIMCyp affect its anti-lentiviral activity. Overall, the results of this study will help our understanding of the genetic background of cynomolgus macaque TRIMCyp, as well as the host factors composing species barriers of primate lentiviruses.
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Canine ASCT1 and ASCT2 are functional receptors for RD-114 virus in dogs
More LessAll domestic cats carry an infectious endogenous retrovirus termed RD-114 virus. Recently, we and others found that several live-attenuated vaccines for dogs were contaminated with infectious RD-114 virus. In this study, we confirmed that the RD-114 virus efficiently infected and proliferated well in canine primary kidney cells, as well as three tested canine cell lines. Further, we identified canine ASCT1 and ASCT2, sodium-dependent neutral amino acid transporters, as RD-114 virus receptors. Canine ASCT2 also acts as a functional receptor for simian retrovirus 2, a pathogenic retrovirus that induces immunodeficiency in rhesus macaques. Identification of the canine receptor for RD-114 virus will help in evaluating the risk from vaccines contaminated by the virus.
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Human T-cell leukemia viruses are highly unstable over a wide range of temperatures
The biological properties of human T-cell leukemia virus type I (HTLV-I) and HTLV type II (HTLV-II) are not well elucidated as cell-free viruses. We established new assay systems to detect the infectivity of cell-free HTLVs and examined the stability of cell-free HTLVs at different temperatures. HTLVs lost infectivity more rapidly than did bovine leukemia virus (BLV), which is genetically related to HTLVs. The half-lives of three HTLV-I strains (two cosmopolitan strains and one Melanesian strain) at 37 °C were approximately 0.6 h, whereas the half-life of a BLV strain was 8.5 h. HTLV-I rapidly lost infectivity unexpectedly at 0 and 4 °C. We examined the stability of vesicular stomatitis virus pseudotypes with HTLV-I, HTLV-II or BLV Env proteins, and the Env proteins of HTLVs were found to be more unstable at 4 and 25 °C than the Env proteins of the BLV. Over the course of the viral life cycle, heat treatment inhibited HTLV-I infection at the phase of attachment to the host cells, and inhibition was more marked upon entry into the cells. The HTLV-I Env surface (SU) protein (gp46) was easily released from virions during incubation at 37 °C. However, this release was inhibited by pre-treatment of the virions with N-ethylmaleimide, suggesting that the inter-subunit bond between gp46 SU and gp21 transmembrane (TM) proteins is rearranged by disulfide bond isomerization. HTLVs are highly unstable over a wide range of temperatures because the disulfide bonds between the SU and TM proteins are labile.
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- DNA viruses
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Interferon treatment suppresses enteric adenovirus infection in a model gastrointestinal cell-culture system
More LessExposure to interferon results in the rapid transcriptional induction of genes, many of which function to create an antiviral environment in potential host cells. For the majority of adenoviruses, replication is unaffected by the actions of interferon. It has previously been shown, using non-gastrointestinal cells, that the species F human adenoviruses are sensitive to the action of interferon. Here, we have developed an enterocyte-like cell-culture model to re-evaluate this question, and determined the effects of interferon on species F adenovirus during infection of gastrointestinal cells. We show that species F adenovirus type 40 is sensitive to the effects of interferon in gastrointestinal-like cells, which may help to explain its fastidious growth in culture.
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Biochemical analysis of infected cell polypeptide (ICP)0, ICP4, UL7 and UL23 incorporated into extracellular herpes simplex virus type 1 virions
More LessHerpes simplex virus type 1 (HSV-1) capsids assemble in the nucleus but acquire their teguments from various cellular compartments. Unfortunately, little is known about their exact arrangement and when they coat the newly produced capsids. The complexity of the virions is further highlighted by our recent proteomics analysis that detected the presence of several novel or controversial components in extracellular HSV-1 virions. The present study probes the localization and linkage to the virus particles of some of these incorporated proteins. We confirm the recently reported tight association of infected cell polypeptide (ICP)0 with the capsid and show that this property extends to ICP4. We also confirm our proteomics data and show biochemically that UL7 and UL23 are indeed mature virion tegument components that, unlike ICP0 and ICP4, are salt-extractable. Interestingly, treatment with N-ethylmaleimide, which covalently modifies reduced cysteines, strongly prevented the release of UL7 and UL23 by salts, but did not perturb the interactions of ICP0 and ICP4 with the virus particles. This hitheir at distinct biochemical properties of the virion constituents and the selective implication of reduced cysteines in their organization and dynamics. Finally, the data revealed, by two independent means, the presence of ICP0 and ICP4 on intranuclear capsids, consistent with the possibility that they may at least partially be recruited to the virus particles early on. These findings add significantly to our understanding of HSV-1 virion assembly and to the debate about the incorporation of ICP0 and ICP4 in virus particles.
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Identification of a novel single-stranded, circular DNA virus from bovine stool
We report the identification of a novel single-stranded, circular DNA virus isolated from bovine stool. The virus, named bovine stool-associated circular DNA virus (BoSCV), has a genome comprising 2600 bases of circular ssDNA, with two putative ORFs encoding replicase and capsid proteins, arranged inversely. The stem–loop structure was located between the 3′ ends of the two putative ORFs, as in chimpanzee stool-associated circular virus (ChimpSCV) and unlike other circular DNA viruses, including members of the families Circoviridae, Nanoviridae and Geminiviridae. BoSCV was also genetically similar to ChimpSCV, with approximately 30 % identity in the replicase and capsid proteins. A phylogenetic analysis based on the replicase protein showed that BoSCV and ChimpSCV are in the same clade. A field survey using BoSCV-specific PCRs targeting ORF1 detected BoSCV and BoSCV-like sequences in bovine and porcine stool samples. BoSCV appears to belong to a new genus of circular DNA viruses.
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Interactions between hepatitis B virus and aflatoxin B1: effects on p53 induction in HepaRG cells
Infection by hepatitis B virus (HBV) and dietary exposure to aflatoxin B1 (AFB1) are the main risk factors for the development of chronic liver disease and hepatocellular carcinoma (HCC). How these factors cooperate is still largely unknown. AFB1 activation leads to DNA adduction and mutagenesis, with a specific mutation at codon 249 in TP53 (p.R249S). So far, only limited studies have addressed the effects of AFB1 on HBV replication. We have analysed the effects of both risk factors on p53 induction during HBV infection in HepaRG, a cell line with hepatocyte-like morphology that metabolizes AFB1 and supports HBV infection. Exposure to AFB1 up to 5 µM induced a downregulation of HBV replication after 48 h, as measured by a decrease in viral antigens in the culture medium (HBsAg, HBeAg and large envelope protein) and in intracellular levels of HBV transcripts, DNA and HBsAg. Conversely, HBV infection did not significantly modify AFB1-DNA adduct formation or repair as assessed by immunodot-blot assay, and the induction of p53 in response to AFB1 was similar in infected and non-infected HepaRG cells. Overall, our results suggest that AFB1 exposure decreases HBV replication, whereas DNA damage by AFB1 and subsequent p53 induction is not affected by the presence of the virus. Thus, in HepaRG cell line, AFB1 and HBV do not cooperate to increase DNA damage by AFB1. Further studies on the effects of both factors in a context of chronicity are needed to better understand synergistic effects.
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JC virus promoter/enhancers contain TATA box-associated Spi-B-binding sites that support early viral gene expression in primary astrocytes
More LessJC virus (JCV) is the aetiological agent of the demyelinating disease progressive multifocal leukoencephalopathy, an AIDS defining illness and serious complication of mAb therapies. Initial infection probably occurs in childhood. In the working model of dissemination, virus persists in the kidney and lymphoid tissues until immune suppression/modulation causes reactivation and trafficking to the brain where JCV replicates in oligodendrocytes. JCV infection is regulated through binding of host factors such as Spi-B to, and sequence variation in the non-coding control region (NCCR). Although NCCR sequences differ between sites of persistence and pathogenesis, evidence suggests that the virus that initiates infection in the brain disseminates via B-cells derived from latently infected haematopoietic precursors in the bone marrow. Spi-B binds adjacent to TATA boxes in the promoter/enhancer of the PML-associated JCV Mad-1 and Mad-4 viruses but not the non-pathogenic, kidney-associated archetype. The Spi-B-binding site of Mad-1/Mad-4 differs from that of archetype by a single nucleotide, AAAAGGGAAGGGA to AAAAGGGAAGGTA. Point mutation of the Mad-1 Spi-B site reduced early viral protein large T-antigen expression by up to fourfold. Strikingly, the reverse mutation in the archetype NCCR increased large T-antigen expression by 10-fold. Interestingly, Spi-B protein binds the NCCR sequence flanking the viral promoter/enhancer, but these sites are not essential for early viral gene expression. The effect of mutating Spi-B-binding sites within the JCV promoter/enhancer on early viral gene expression strongly suggests a role for Spi-B binding to the viral promoter/enhancer in the activation of early viral gene expression.
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- Plant
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Differences in the mechanism of inoculation between a semi-persistent and a non-persistent aphid-transmitted plant virus
More LessInoculation of the semi-persistent cauliflower mosaic virus (CaMV, genus Caulimovirus) is associated with successive brief (5–10 s) intracellular stylet punctures (pd) when aphids probe in epidermal and mesophyll cells. In contrast to non-persistent viruses, there is no evidence for which of the pd subphases (II-1, II-2 and II-3) is involved in the inoculation of CaMV. Experiments were conducted using the electrical penetration graph (EPG) technique to investigate which particular subphases of the pd are associated with the inoculation of CaMV to turnip by its aphid vector Brevicoryne brassicae. In addition, the same aphid species/test plant combination was used to compare the role of the pd subphases in the inoculation of the non-persistent turnip mosaic virus (TuMV, genus Potyvirus). Inoculation of TuMV was found to be related to subphase II-1, confirming earlier results, but CaMV inoculation appeared to be related exclusively to subphase II-2 instead. The mechanism of CaMV inoculation and the possible nature of subphase II-2 are discussed in the scope of our findings.
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- Other agents
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Characterization of intracellular localization of PrPSc in prion-infected cells using a mAb that recognizes the region consisting of aa 119–127 of mouse PrP
More LessGeneration of an abnormal isoform of the prion protein (PrPSc) is a key aspect of the propagation of prions. Elucidation of the intracellular localization of PrPSc in prion-infected cells facilitates the understanding of the cellular mechanism of prion propagation. However, technical improvement in PrPSc-specific detection is required for precise analysis. Here, we show that the mAb 132, which recognizes the region adjacent to the most amyloidogenic region of PrP, is useful for PrPSc-specific detection by immunofluorescence assay in cells pre-treated with guanidine thiocyanate. Extensive analysis of the intracellular localization of PrPSc in prion-infected cells using mAb 132 revealed the presence of PrPSc throughout endocytic compartments. In particular, some of the granular PrPSc signals that were clustered at peri-nuclear regions appeared to be localized in an endocytic recycling compartment through which exogenously loaded transferrin, shiga and cholera toxin B subunits were transported. The granular PrPSc signals at peri-nuclear regions were dispersed to the peripheral regions including the plasma membrane during incubation at 20 °C, at which temperature transport from the plasma membrane to peri-nuclear regions was impaired. Conversely, dispersed PrPSc signals appeared to return to peri-nuclear regions within 30 min during subsequent incubation at 37 °C, following which PrPSc at peri-nuclear regions appeared to redisperse again to peripheral regions over the next 30 min incubation. These results suggest that PrPSc is dynamically transported along with the membrane trafficking machinery of cells and that at least some PrPSc circulates between peri-nuclear and peripheral regions including the plasma membrane via an endocytic recycling pathway.
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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)