- Volume 92, Issue 6, 2011
Volume 92, Issue 6, 2011
- Animal
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- RNA
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Differential inhibition of dengue virus infection in mammalian and mosquito cells by iota-carrageenan
More LessThe antiviral activity against dengue virus-2 (DENV-2) of carrageenans reported here has shown a differential susceptibility of C6/36 HT and Vero cells, taken as models of mosquito and mammalian cells, depending on the structural class of polysaccharides: all polysaccharides blocked DENV-2 infection in monkey Vero cells, but only iota-carrageenans were virus inhibitors in mosquito cells. However, iota-carrageenans were less effective in mosquito cells in comparison with mammalian cells with effective concentration 50 % (EC50) values in C6/36 HT cells 4.9–17.5-fold higher than in Vero cells, as determined by virus yield reduction assay. The mode of action of iota-carrageenan in both cell types was strikingly different: in Vero cells the inhibitory activity was exerted only at the initiation of the cycle, affecting virion binding, whereas in mosquito cells DENV-2 adsorption was not affected and comparable levels of inhibition were obtained if the compound was added to cells together with the virus, after 8 h of infection or by cell pre-treatment before infection. Furthermore, iota-carrageenans induced a subtle alteration in mosquito cells, detected by cell proliferation and protein synthesis analyses, suggesting that a probable cellular target may be responsible for the refractory state of mosquito cells to DENV-2 infection produced by this class of polysulfates. The failure of iota-carrageenan to block DENV-2 adsorption to mosquito cells appeared to be related to the low presence of adequate heparan sulfate (HS) in C6/36 HT cell surface and is indicative of a differential participation of HS residues for DENV-2 entry in both types of cells.
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High levels of HCV core+1 antibodies in HCV patients with hepatocellular carcinoma
The core region of the hepatitis C virus (HCV) genome possesses an overlapping ORF that has been shown to encode a protein, known as the alternate reading frame protein (ARFP), F or core+1. The biological role of this protein remains elusive, as it appears to be non-essential for virus replication. However, a number of independent studies have shown that the ARFP/F/core+1 protein elicits humoral and cellular immune responses in HCV-infected individuals and interacts with important cellular proteins. To assess the significance of the core+1 humoral response in HCV-infected patients, we examined the prevalence of anti-core+1 antibodies in sera from patients with hepatocellular carcinoma (HCC) in comparison with chronically HCV-infected individuals without HCC. We produced two HCV core+1 histidine-tagged recombinant proteins for genotypes 1a (aa 11–160) and 1b (aa 11–144), as well as a non-tagged highly purified recombinant core+1/S protein (aa 85–144) of HCV-1b. Using an in-house ELISA, we tested the prevalence of core+1 antibodies in 45 patients with HCC in comparison with 47 chronically HCV-infected patients without HCC and 77 negative-control sera. More than 50 % of the serum samples from HCC patients reacted with all core+1 antigens, whereas <26 % of the sera from the non-HCC HCV-infected individuals tested positive. No core+1-specific reactivity was detected in any of the control samples. In conclusion, the high occurrence of anti-core+1 antibodies in the serum of HCC patients suggests a role for the ARFP/F/core+1 protein in the pathogenesis of HCC.
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Characterization and purification of recombinant bovine viral diarrhea virus particles with epitope-tagged envelope proteins
More LessBovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. The lipid membrane of the virions is supposed to contain the three glycosylated envelope proteins Erns, E1 and E2, but detailed studies of virus assembly are complicated because no efficient purification method for pestiviruses has been described so far. In this study, we generated infectious BVDV with N-terminally FLAG-tagged Erns or E2 proteins, respectively. The expression of the epitope-tagged Erns and E2 proteins could be shown by immunofluorescence and Western blot experiments. Furthermore, an affinity tag purification protocol for the isolation and concentration of infectious BVDV was established. In the preparation with a titre of 108.75 TCID50 ml−1, spherical particles with a diameter of 43–58 nm (mean diameter: 48 nm) could be detected by negative staining electron microscopy, and immunogold labelling located both Erns and E2 proteins at the virus membrane.
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Infection with human coronavirus NL63 enhances streptococcal adherence to epithelial cells
Understanding the mechanisms of augmented bacterial pathogenicity in post-viral infections is the first step in the development of an effective therapy. This study assessed the effect of human coronavirus NL63 (HCoV-NL63) on the adherence of bacterial pathogens associated with respiratory tract illnesses. It was shown that HCoV-NL63 infection resulted in an increased adherence of Streptococcus pneumoniae to virus-infected cell lines and fully differentiated primary human airway epithelium cultures. The enhanced binding of bacteria correlated with an increased expression level of the platelet-activating factor receptor (PAF-R), but detailed evaluation of the bacterium–PAF-R interaction revealed a limited relevance of this process.
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Molecular characterization of a new species in the genus Alphacoronavirus associated with mink epizootic catarrhal gastroenteritis
A coronavirus (CoV) previously shown to be associated with catarrhal gastroenteritis in mink (Mustela vison) was identified by electron microscopy in mink faeces from two fur farms in Wisconsin and Minnesota in 1998. A pan-coronavirus and a genus-specific RT-PCR assay were used initially to demonstrate that the newly discovered mink CoVs (MCoVs) were members of the genus Alphacoronavirus. Subsequently, using a random RT-PCR approach, full-genomic sequences were generated that further confirmed that, phylogenetically, the MCoVs belonged to the genus Alphacoronavirus, with closest relatedness to the recently identified but only partially sequenced (fragments of the polymerase, and full-length spike, 3c, envelope, nucleoprotein, membrane, 3x and 7b genes) ferret enteric coronavirus (FRECV) and ferret systemic coronavirus (FRSCV). The molecular data presented in this study provide the first genetic evidence for a new coronavirus associated with epizootic catarrhal gastroenteritis outbreaks in mink and demonstrate that MCoVs possess high genomic variability and relatively low overall nucleotide sequence identities (91.7 %) between contemporary strains. Additionally, the new MCoVs appeared to be phylogenetically distant from human (229E and NL63) and other alphacoronaviruses and did not belong to the species Alphacoronavirus 1. It is proposed that, together with the partially sequenced FRECV and FRSCV, they comprise a new species within the genus Alphacoronavirus.
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Mapping genetic determinants of the cell-culture growth phenotype of enterovirus 71
More LessEnterovirus 71 (EV71) is a member of the species Human enterovirus A within the family Picornaviridae and is a major causative agent of epidemics of hand, foot and mouth disease associated with severe neurological disease. Three EV71 genogroups, designated A, B and C, have been identified, with 75–84 % nucleotide sequence similarity between them. Two strains, EV71-26M (genogroup B) and EV71-6F (genogroup C), were found to have distinct cell-culture growth (26M, rapid; 6F, slow) and plaque-formation (26M, large; 6F, small) phenotypes. To identify the genome regions responsible for the growth phenotypes of the two strains, a series of chimeric viruses was constructed by exchanging the 5′ untranslated region (UTR), P1 structural protein or P2/P3 non-structural protein gene regions plus the 3′UTR using infectious cDNA clones of both virus strains. Analysis of reciprocal virus chimeras revealed that the 5′UTRs of both strains were compatible, but not responsible for the observed phenotypes. Introduction of the EV71-6F P1 region into the EV71-26M clone resulted in a small-plaque and slow-growth phenotype similar to that of EV71-6F, whereas the reciprocal chimera displayed intermediate-growth and intermediate-sized plaque phenotypes. Introduction of the EV71-26M P2–P3–3′UTR regions into the EV71-6F clone resulted in a large-plaque and rapid-growth phenotype identical to that of strain EV71-26M, whereas the reciprocal chimera retained the background strain large-plaque phenotype. These results indicate that, although both the P1 and P2–P3–3′UTR genome regions influence the EV71 growth phenotype in cell culture, phenotype expression is dependent on specific genome-segment combinations and is not reciprocal.
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Porcine reproductive and respiratory syndrome virus in Ontario, Canada 1999 to 2010: genetic diversity and restriction fragment length polymorphisms
Classification of Ontario porcine reproductive and respiratory syndrome virus (PRRSV) field isolates (n = 505) from 1999 to 2010, based on a global type 2 PRRSV ORF5 phylogenetic framework, revealed genetic diversity comparable to PRRSV in the USA, with sequences assigned to five of nine lineages (1, 2, 5, 8 and 9). Importantly, the tree topology indicated a Canadian ancestry for the highly virulent MN184-related strains that first emerged in 2001 in the USA. Mapping of the RFLP patterns onto the phylogenetic tree revealed numerous examples of different RFLP patterns located within the same phylogenetic cluster. Statistical analysis showed occurrences where similar RFLP patterns masked diverse genetic distances and instances of close genetic proximity with divergent RFLP patterns. Collectively, extensive genetic diversity prevails in type 2 PRRSV in one region of the North American swine industry, and it is not described adequately by RFLP typing, which might have value in differentiating strains at the local farm level.
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Alternative attachment factors and internalization pathways for GIII.2 bovine noroviruses
More LessBovine noroviruses belong to the family Caliciviridae, genus Norovirus. Two genotypes have been described and viruses genetically related to the Jena and Newbury2 strains have been classified into genotypes 1 and 2, respectively. In this study, virus-like particles (VLP) of the previously detected B309 Belgian strain, genetically related to genotype 2 bovine noroviruses, were used to investigate virus–host interactions in vitro. B309 VLP were shown to bind to several bovine cell lines. This binding was not affected by heparinase or chondroitinase treatment but was significantly inhibited by both sodium periodate, α-galactosidase, trypsin and phospholipase C treatment. Cell treatment by neuraminidase also moderately affected this binding. Taken together, these results show that, in addition to a galactosyl residue, sialic acid could also be involved in binding to susceptible cells. In addition, both the cholesterol-dependent pathway and macropinocytosis are used for B309 VLP internalization by Madin–Darby bovine kidney cells. The data increase the knowledge on bovine norovirus cell interactions.
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Insertion of a multibasic cleavage site in the haemagglutinin of human influenza H3N2 virus does not increase pathogenicity in ferrets
A multibasic cleavage site (MBCS) in the haemagglutinin (HA) protein of influenza A virus is a key determinant of pathogenicity in chickens, and distinguishes highly pathogenic avian influenza (HPAI) viruses from low pathogenic avian influenza viruses (LPAI). An MBCS has only been detected in viruses of the H5 and H7 subtypes. Here we investigated the phenotype of a human H3N2 virus with an MBCS in HA. Insertion of an MBCS in the H3N2 virus resulted in cleavage of HA and efficient replication in Madin–Darby canine kidney cells in the absence of exogenous trypsin in vitro, similar to HPAI H5N1 virus. However, studies in ferrets demonstrated that insertion of the MBCS into HA did not result in increased virus shedding, cellular host range, systemic replication or pathogenicity, as compared with wild-type virus. This study indicates that acquisition of an MBCS alone is insufficient to increase pathogenicity of a prototypical seasonal human H3N2 virus.
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Characterization of influenza A viruses isolated from wild waterfowl in Zambia
Although the quest to clarify the role of wild birds in the spread of the highly pathogenic H5N1 avian influenza virus (AIV) has yielded considerable data on AIVs in wild birds worldwide, information regarding the ecology and epidemiology of AIVs in African wild birds is still very limited. During AIV surveillance in Zambia (2008–2009), 12 viruses of distinct subtypes (H3N8, H4N6, H6N2, H9N1 and H11N9) were isolated from wild waterfowl. Phylogenetic analyses demonstrated that all the isolates were of the Eurasian lineage. Whilst some genes were closely related to those of AIVs isolated from wild and domestic birds in South Africa, intimating possible AIV exchange between wild birds and poultry in southern Africa, some gene segments were closely related to those of AIVs isolated in Europe and Asia, thus confirming the inter-regional AIV gene flow among these continents. Analysis of the deduced amino acid sequences of internal proteins revealed that several isolates harboured particular residues predominantly observed in human influenza viruses. Interestingly, the isolates with human-associated residues exhibited higher levels of virus replication in the lungs of infected mice and caused more morbidity as measured by weight loss than an isolate lacking such residues. This study stresses the need for continued monitoring of AIVs in wild and domestic birds in southern Africa to gain a better understanding of the emergence of strains with the potential to infect mammals.
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Cytokine production by primary human macrophages infected with highly pathogenic H5N1 or pandemic H1N1 2009 influenza viruses
Highly pathogenic H5N1 avian influenza viruses have caused infection in humans, with a high mortality rate, since 1997. While the pathogenesis of this infection is not completely understood, hypercytokinaemia and alveolar macrophages are thought to play a role. To gain further insight into the cytokine-mediated pathogenesis of this infection in humans, we measured various cytokines produced by primary human macrophages infected with H5N1, pandemic H1N1 or seasonal influenza viruses. We found that many cytokines were produced at higher levels on infection with the H5N1 strains tested compared with seasonal influenza viruses. Interestingly, the extent of cytokine induction varied among the H5N1 strains and did not correlate with replicative ability in macrophages. Further, a pandemic H1N1 virus induced higher levels of several cytokines compared with seasonal viruses and some H5N1 strains. Our results demonstrate that high cytokine induction is not a universal feature of all H5N1 viruses.
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PB1-mediated virulence attenuation of H5N1 influenza virus in mice is associated with PB2
H5N1 avian influenza viruses demonstrate different phenotypes, such as pathogenicity after one or serial passages in mammalian hosts or cells. To establish the molecular basis of these phenotypes, we cloned isolates from the lungs of mice infected with human A/Vietnam/1194/2004 (H5N1) influenza virus. Large-plaque isolates were less pathogenic to mice than small-plaque isolates. Genome sequencing revealed that the small-plaque and large-plaque isolates differed in several amino acids. In order to assess their effects on pathogenicity in mice, two amino acid changes common to attenuated isolates, one in PB2 (I63T) and the other in PB1 (T677M), were inserted into a wild-type recombinant virus construct. The PB2 (I63T) or PB1 (T677M) mutations alone did not alter the phenotype of H5N1 virus, whereas recombinant virus with both mutations was less pathogenic than the wild-type recombinant virus. Furthermore, the PB1 (T677M) mutation showed a lower replication efficiency, although it had higher polymerase activity. The recombinant virus with the PB2 (63T) mutation replicated as well as the wild-type recombinant virus. These results suggest that the C terminus of PB1 of H5N1 influenza virus mediates virulence attenuation of H5N1 influenza virus in mice, associating with the N terminus of PB2. However, the role of the N terminus of PB2 in virulence attenuation in mice remains unclear.
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Aguacate virus, a new antigenic complex of the genus Phlebovirus (family Bunyaviridae)
Genomic and antigenic characterization of Aguacate virus, a tentative species of the genus Phlebovirus, and three other unclassified viruses, Armero virus, Durania virus and Ixcanal virus, demonstrate a close relationship to one another. They are distinct from the other nine recognized species within the genus Phlebovirus. We propose to designate them as a new (tenth) serogroup or species (Aguacate virus) within the genus. The four viruses were all isolated from phlebotomine sandflies (Lutzomyia sp.) collected in Central and South America. Aguacate virus appears to be a natural reassortant and serves as one more example of the high frequency of reassortment in this genus.
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A novel hantavirus detected in Yunnan red-backed vole (Eothenomys miletus) in China
Rodents are the major natural reservoir of hantaviruses, which cause two main human zoonoses, haemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome (HPS) in the Americas. Surveillance of hantaviruses in rodents plays an important role in the prevention and control of HFRS and HPS. In this study, small mammals were captured in an HFRS-endemic region, Luxi County within the Yunnan Province of China, from the autumn of 2009 to the spring of 2010, and assessed for the presence of hantaviruses. A high ratio of hantavirus infection was detected in the Yunnan red-backed vole (Eothenomys miletus). Full-length sequences of the small, middle and large segments were determined from one positive sample (designated Luxi hantavirus). Analyses of the coding sequences indicated that this virus represents a distinct hantavirus species within the hantavirus group identified from the Rodentia subfamily Arvicolinae.
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Measles virotherapy in a mouse model of adult T-cell leukaemia/lymphoma
More LessAdult T-cell leukaemia/lymphoma (ATL) is a highly aggressive CD4+ T-cell malignancy caused by human T-cell leukaemia virus type 1. Measles virus (MV) oncolytic therapy has been reported to be efficient in reducing tumour burden in subcutaneous xenograft models of lymphoproliferative disorders such as myeloma, B-cell lymphoma and cutaneous T-cell lymphoma, but its potential to reduce tumour burden in disseminated lymphoproliferative disorders such as ATL remains to be determined. In this study, MV oncolytic therapy was evaluated in the MET-1/NOD/SCID xenograft mouse model of ATL. Treatment with the vaccine-related strain MV-NSE led to a significant reduction in tumour burden. In mice with a high tumour burden, therapy with MV-NSE significantly increased survival beyond any other single treatment tested previously using this model. Interestingly, signs of morbidity (cachexia) in mice treated with MV were not directly associated with tumour burden, but were correlated with the secretion of interleukin-6 by MET-1 cells and host cells. The results suggest that MV therapy could be a promising therapy for generalized lymphoproliferative disease.
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Structural and sequence integrity are essential for the replication of the viroid-like satellite RNA of lucerne transient streak virus
More LessLucerne transient streak virus (LTSV, genus Sobemovirus) supports the replication and encapsidation of a 322 nt untranslated small-circular RNA (scLTSV). Since scLTSV does not code for any proteins or share sequence similarity with its helper virus (LTSV), it is presumed that it uses structural and sequence motifs to signal the helper virus (and host) machinery for its replication and encapsidation. Insertion and deletion mutations were introduced at various locations within the scLTSV molecule. Our results showed that most mutants were not infectious, with only two exceptions, a (−1) nucleotide deletion and a 9 nt, palindromic insertion mutant which preserved the overall rod-like structure of the scLTSV. Sequence analysis of cDNA clones revealed that the palindromic sequence was replicated for up to 12 days of infection, before the sequence reverted back to its wild-type form. Our results indicate that scLTSV has an optimal sequence and secondary structure for replication, movement and/or packaging within the LTSV helper virus.
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Recombination analysis of intermediate human adenovirus type 53 in Japan by complete genome sequence
Human adenovirus type 53 (HAdV-53) has commonly been detected in samples from epidemic keratoconjunctivitis (EKC) patients in Japan since 1996. HAdV-53 is an intermediate virus, containing hexon-chimeric, penton base and fiber structures similar to HAdV-22 and -37, HAdV-37 and HAdV-8, respectively. HAdV-53-like intermediate strains were first isolated from EKC samples in Japan in the 1980s. Here, the complete genome sequences of three such HAdV-53-like intermediate strains (870006C, 880249C and 890357C) and four HAdV-53 strains were determined, and their relationships were analysed. The seven HAdV strains were classified into three groups, 870006C/880249C, 890357C and the four HAdV-53 strains, on the basis of phylogenetic analyses of the partial and complete genome sequences. HAdV strains within the same group showed the highest nucleotide identities (99.87–100.00 %). Like HAdV-53, the hexon loop 1 and 2 regions of 870006C, 880249C and 890357C showed the highest identity with HAdV-22. However, these strains did not show a hexon-chimeric structure similar to HAdV-22 and -37, or a penton base similar to HAdV-37. The fiber genes of 870006C and 880249C were identical to that of HAdV-37, but not HAdV-8. Thus, the three intermediate HAdVs isolated in the 1980s were similar to each other but not to HAdV-53. The recombination breakpoints were inferred by the Recombination Detection Program (rdp) using whole-genome sequences of these seven HAdV and of 12 HAdV-D strains from GenBank. HAdV-53 may have evolved from intermediate HAdVs circulating in the 1980s, and from HAdV-8, -22 and -37, by recombination of sections cut at the putative breakpoints.
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Coding potential and transcript analysis of fowl adenovirus 4: insight into upstream ORFs as common sequence features in adenoviral transcripts
More LessRecombinant fowl adenoviruses (FAdVs) have been successfully used as veterinary vaccine vectors. However, insufficient definitions of the protein-coding and non-coding regions and an incomplete understanding of virus–host interactions limit the progress of next-generation vectors. FAdVs are known to cause several diseases of poultry. Certain isolates of species FAdV-C are the aetiological agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS). In this study, we report the complete 45667 bp genome sequence of FAdV-4 of species FAdV-C. Assessment of the protein-coding potential of FAdV-4 was carried out with the Bio-Dictionary-based Gene Finder together with an evaluation of sequence conservation among species FAdV-A and FAdV-D. On this basis, 46 potentially protein-coding ORFs were identified. Of these, 33 and 13 ORFs were assigned high and low protein-coding potential, respectively. Homologues of the ancestral adenoviral genes were, with few exceptions, assigned high protein-coding potential. ORFs that were unique to the FAdVs were differentiated into high and low protein-coding potential groups. Notable putative genes with high protein-coding capacity included the previously unreported fiber 1, hypothetical 10.3K and hypothetical 10.5K genes. Transcript analysis revealed that several of the small ORFs less than 300 nt in length that were assigned low coding potential contributed to upstream ORFs (uORFs) in important mRNAs, including the ORF22 mRNA. Subsequent analysis of the previously reported transcripts of FAdV-1, FAdV-9, human adenovirus 2 and bovine adenovirus 3 identified widespread uORFs in AdV mRNAs that have the potential to act as important translational regulatory elements.
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ICP27 protein of Marek’s disease virus interacts with SR proteins and inhibits the splicing of cellular telomerase chTERT and viral vIL8 transcripts
More LessAll herpesviruses have a post-transcriptional regulatory protein that prevents precursor mRNA splicing and leads to the shutting off of host protein synthesis. The ICP27 protein of herpes simplex virus 1 (HSV-1) is the prototype of these proteins. Marek’s disease virus (MDV-1), an alphaherpesvirus that induces lymphoma in birds, also has an ICP27 protein that is produced in lytic MDV-1-infected cells. We characterized this protein. We demonstrated ICP27 production in latently infected MSB-1 cells, but only on MDV-1 reactivation. ICP27 was found predominantly in specific structures within the nucleus. The ICP27 of MDV-1 colocalized and interacted with SR proteins. We demonstrated inhibitory effects of MDV-1 ICP27 on the splicing of both the viral vIL8 and cellular chTERT (telomerase reverse transcriptase) genes. Thus, the ICP27 of MDV-1 plays a similar role to the ICP27 of HSV-1 and may be involved in MDV-1 replication and the development of Marek’s disease.
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Single cell detection of latent cytomegalovirus reactivation in host tissue
The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor–GFP reporter mice, we never observed reactivation originating from CX3CR1+ monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b+ and CX3CR1+ subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.
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Roles of stress-activated protein kinases in the replication of Singapore grouper iridovirus and regulation of the inflammatory responses in grouper cells
More LessStress-activated protein kinases (SAPKs), including p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK), are usually activated in response to different environmental stimuli, including virus infection. In the present study, the roles of SAPKs during Singapore grouper iridovirus (SGIV) infection were investigated in fish cells. The results showed that increased phosphorylation of JNK1/2 and p38 MAPK occurred during active replication of SGIV in grouper cell cultures. Moreover, downstream effectors (c-Jun, MAPK-activated protein kinase 2, p53, activator protein 1, Myc and nuclear factor of activated T cells) were activated after SGIV infection, suggesting that SGIV replication activated the JNK and p38 MAPK signalling pathways. Notably, using specific inhibitors, it was found that viral gene transcripts, protein expression and viral titres were not affected by inhibition of p38 MAPK but were suppressed significantly by inhibiting JNK1/2 activation. In addition, transcription of grouper immune genes including interferon regulatory factor 1, interleukin-8 and tumour necrosis factor alpha (TNF-α) were regulated by JNK, whilst only TNF-α was regulated by p38 MAPK. It is proposed that the JNK pathway is important for SGIV replication and modulates the inflammatory responses during virus infection.
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Dragonfly cyclovirus, a novel single-stranded DNA virus discovered in dragonflies (Odonata: Anisoptera)
Dragonfly cyclovirus (DfCyV), a new species of ssDNA virus discovered using viral metagenomics in dragonflies (familyLibellulidae) from the Kingdom of Tonga. Metagenomic sequences of DfCyV were similar to viruses of the recently proposed genus Cyclovirus within the family Circoviridae. Specific PCRs resulted in the recovery of 21 DfCyV genomes from three dragonfly species (Pantala flavescens, Tholymis tillarga and Diplacodes bipunctata). The 1741 nt DfCyV genomes share >95 % nucleotide identity and are classified into 11 subtypes representing a single strain. The DfCyV genomes share 48–63 % genome-wide nucleotide identity with cycloviruses identified in human faecal samples. Recombination analysis revealed three recombinant DfCyV genomes, suggesting that recombination plays an important role in cyclovirus evolution. To our knowledge, this is the first report of a circular ssDNA virus identified in insects, and the data may help elucidate evolutionary links among novel Circoviridae recently identified in animals and environmental samples.
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Hepatitis B virus X protein overcomes all-trans retinoic acid-induced cellular senescence by downregulating levels of p16 and p21 via DNA methylation
More LessDespite current molecular evidence suggesting that hepatitis B virus (HBV) X protein (HBx) plays an important role during HBV-mediated hepatocarcinogenesis, the detailed mechanism is still controversial. Here, it was shown that HBx overcomes cellular senescence provoked by all-trans retinoic acid (ATRA) in HepG2 cells, as demonstrated by the impaired induction of irreversible G1 arrest and senescence-associated β-galactosidase activity by ATRA in the presence of HBx. The anti-senescence effect of HBx was also observed in another human hepatoma cell line, Hep3B, but not in Huh-7 cells in which the p16 and p21 proteins are absent. In addition, HBx suppressed ATRA-mediated induction of p16 and p21 in HepG2 cells via promoter hypermethylation, resulting in inactivation of retinoblastoma protein. Furthermore, the ability of HBx to overcome ATRA-induced cellular senescence almost completely disappeared when the levels of p16 and p21 in the HBx-expressing cells became similar to those in the control cells by complementation in the former by exogenous expression, knockdown of their expression in the latter using specific small interfering RNA or treatment with a DNA methylation inhibitor, 5-Aza-2′-deoxycytidine. These results suggest that HBx executes its potential by downregulating levels of p16 and p21 via DNA methylation. As cellular senescence is a tumour-suppression process, the present study provides a new strategy by which HBV promotes hepatocarcinogenesis.
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Metagenomic identification of a novel anellovirus in Pacific harbor seal (Phoca vitulina richardsii) lung samples and its detection in samples from multiple years
To investigate viral pathogens potentially involved in a mortality event of 21 Pacific harbor seals (Phoca vitulina richardsii) in California in 2000, viral metagenomics was performed directly on lung samples from five individuals. Metagenomics revealed a novel seal anellovirus (SealAV), which clusters phylogenetically with anelloviruses from California sea lions and domestic cats. Using specific PCR, SealAV was identified in lung tissue from two of five animals involved in the 2000 mortality event, as well as one of 20 harbor seal samples examined post-mortem in 2008. The identification of SealAV in multiple years demonstrates that this virus is persistent in the harbor seal population. SealAV is the second anellovirus reported in the lungs of pinnipeds, suggesting that anellovirus infections may be common amongst marine mammals and that more research is needed to understand the roles of these viruses in marine mammal health and disease.
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Helicoverpa armigera nucleopolyhedrovirus occlusion-derived virus-associated protein, HA100, affects oral infectivity in vivo but not virus replication in vitro
ORF100 (ha100) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has been reported as one of the unique genes of group II alphabaculoviruses encoding a protein located in the occlusion-derived virus (ODV) envelope and nucleocapsid. The protein consists of 510 aa with a predicted mass of 58.1 kDa and is a homologue of poly(ADP–ribose) glycohydrolase in eukaryotes. Western blot analysis detected a 60 kDa band in HearNPV-infected HzAM1 cells starting at 18 h post-infection. Transient expression of GFP-fused HA100 in HzAM1 cells resulted in cytoplasmic localization of the protein, but after superinfection with HearNPV, GFP-fused HA100 was localized in the nucleus. To study the function of HA100 further, an ha100-null virus was constructed using bacmid technology. Viral one-step growth curve analyses showed that the ha100-null virus had similar budded virus production kinetics to that of the parental virus. Electron microscopy revealed that deletion of HA100 did not alter the morphology of ODVs or occlusion bodies (OBs). However, bioassays in larvae showed that the 50 % lethal concentration (LC50) value of HA100-null OBs was significantly higher than that of parental OBs; the median lethal time (LT50) of ha100-null OBs was about 24 h later than control virus. These results indicate that HA100 is not essential for virus replication in vitro. However, it significantly affects the oral infectivity of OBs in host insects, suggesting that the association HA100 with the ODV contributes to the infectivity of OBs in vivo.
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- Plant
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- RNA
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Interactions between a luteovirus and the GroEL chaperonin protein of the symbiotic bacterium Buchnera aphidicola of aphids
More LessLuteoviruses and poleroviruses are important plant viruses transmitted exclusively by aphids in a circulative manner via the aphid haemolymph. A chaperonin protein, GroEL, synthesized in aphids by a symbiotic bacterium, Buchnera aphidicola, is hypothesized to bind to virus particles in the haemolymph, thereby promoting transmission. To investigate this hypothesis, the GroEL-binding site for barley yellow dwarf virus (BYDV) was determined in vitro, and the abundance of GroEL protein in different aphid tissues was investigated. Virus binding to a peptide library representing the full GroEL molecule revealed a single binding site that coincides with the site that anchors two GroEL rings to form the native GroEL tetradecamer. In the functional form of the GroEL protein, virus binding would compete with the formation of the two GroEL rings. Using a mAb raised against a Buchnera-specific GroEL epitope, GroEL was detected in Buchnera cells by immunoblotting and immunocytochemistry, but not in the aphid haemolymph, fat body or gut. From the prediction here that GroEL–virus interactions are probably severely limited by competition with other GroEL molecules, and the evidence that GroEL is not available to interact with virus particles in vivo, it is concluded that GroEL–virus interactions are unlikely to contribute to virus transmission by aphids.
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- Other Agents
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Emergence of multiple prion strains from single isolates of ovine scrapie
More LessThe infectious agent associated with prion diseases such as ovine scrapie shows strain diversity. Ovine prion strains have typically been identified by their transmission properties in wild-type mice. However, strain typing of ovine scrapie isolates in wild-type mice may not reveal properties of the infectious prion agent as they exist in the original host. This could be circumvented if ovine scrapie isolates are passaged in ovine prion protein (PrP)-transgenic mice. This study used incubation time, lesion profile, immunohistochemistry of the disease-associated PrP (PrPSc) and molecular profile to compare the range of ovine prion strains that emerged from sheep scrapie isolates following serial passage in wild-type and ovine PrP transgenic mice. It was found that a diverse range of ovine prion strains emerged from homozygous ARQ and VRQ scrapie isolates passaged in wild-type and ovine PrP transgenic mice. However, strain-specific PrPSc deposition and PrP27–30 molecular profile patterns were identified in ovine PrP transgenic mice that were not detected in wild-type mice. Significantly, it was established that the individual mouse brain selected for transmission during prion strain typing had a significant influence on strain definition. Serial passage of short- and long-incubation-time animals from the same group of scrapie-inoculated mice revealed different prion strain phenotypes. These observations are consistent with the possibility that some scrapie isolates contain more than one prion strain.
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