- Volume 98, Issue 5, 2017
Volume 98, Issue 5, 2017
- Review
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Rift Valley fever virus: strategies for maintenance, survival and vertical transmission in mosquitoes
Rift Valley fever virus (RVFV) is a mosquito-borne arbovirus causing severe disease in humans and ruminants. Spread of RVFV out of Africa has raised concerns that it could emerge in Europe or the USA. Virus persistence is dependent on successful infection of, replication in, and transmission to susceptible vertebrate and invertebrate hosts, modulated by virus–host and vector–virus interactions. The principal accepted theory for the long-term maintenance of RVFV involves vertical transmission (VT) of virus to mosquito progeny, with the virus surviving long inter-epizootic periods within the egg. This VT hypothesis, however, is yet to be comprehensively proven. Here, evidence for and against the VT of RVFV is reviewed along with the identification of factors limiting its detection in natural and experimental data. The observations of VT for other arboviruses in the genera Alphavirus, Flavivirus and Orthobunyavirus are discussed within the context of RVFV. The review concludes that VT of RVFV is likely but that current data are insufficient to irrefutably prove this hypothesis.
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- ICTV Virus Taxonomy Profiles
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ICTV Virus Taxonomy Profile: Corticoviridae
More LessThe Corticoviridae is a family of icosahedral, internal-membrane-containing viruses with double-stranded circular DNA genomes of approximately 10 kb. Only one species, Pseudoalteromonas virus PM2, has been recognized. Pseudoalteromonas virus PM2 infects Gram-negative bacteria and was isolated from seawater in 1968. Pseudoalteromonas virus PM2 is the first bacterial virus in which the presence of lipids in the virion has been demonstrated. Viral lipids are acquired selectively during virion assembly from the host cytoplasmic membrane. The outer protein capsid is an icosahedron with a pseudo T=21 symmetry. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Corticoviridae, which is available at www.ictv.global/report/corticoviridae.
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ICTV Virus Taxonomy Profile: Iridoviridae
The Iridoviridae is a family of large, icosahedral viruses with double-stranded DNA genomes ranging in size from 103 to 220 kbp. Members of the subfamily Alphairidovirinae infect ectothermic vertebrates (bony fish, amphibians and reptiles), whereas members of the subfamily Betairidovirinae mainly infect insects and crustaceans. Infections can be either covert or patent, and in vertebrates they can lead to high levels of mortality among commercially and ecologically important fish and amphibians. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Iridoviridae, which is available at www.ictv.global/report/iridoviridae.
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- Microbiology Society Prize Lecture
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Influenza: a world of discoveries, outbreaks and controversy
More LessWorking in an area such as influenza is a free ticket into science communication, a pathway aided amply by the amazing evolutionary powers of the virus; regular outbreaks keep the media engaged and the audience keen. Everyone has heard of flu, and they probably already have an opinion: ‘I don’t take the vaccine, it gives me the flu anyway.’ ‘Didn’t the government waste loads of money on that Tamiflu drug that doesn’t work?’ ‘I’ve never had flu because I eat a banana every day and sleep with a boiled onion when I’ve sat next to someone on the train who was coughing.’ Such muddled messages and folklore fallacies could be very damaging unless we as scientists stand up and correct them. In addition, there are wider ethical debates around sharing data from clinical trials and the acceptable limits of scientific research to which we must all contribute.
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- Animal
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- Negative-strand RNA viruses
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Identification of cross-reacting T-cell epitopes in structural and non-structural proteins of swine and pandemic H1N1 influenza A virus strains in pigs
Heterologous protection against swine influenza viruses (SwIVs) of different lineages is an important concern for the pig industry. Cross-protection between ‘avian-like’ H1N1 and 2009 pandemic H1N1 lineages has been observed previously, indicating the involvement of cross-reacting T-cells. Here, reverse vaccinology was applied to identify cross-reacting MHC class I T-cell epitopes from two different SwIV H1 lineages in pigs. In silico prediction followed by in vitro and in vivo testing was used to identify SLA-1*0702 T-cell epitopes in heterologous SwIV-infected pigs. Following viral infection, tetramer specific T-cell populations were identified. The majority of the identified T-cell epitopes were conserved between the examined lineages, suggesting that targeting cross-reactive T-cell epitopes could be used to improve vaccines against SwIV in SLA-1*0702-positive pigs.
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Basic amino acids in the N-terminal half of the PB2 subunit of influenza virus RNA polymerase are involved in both transcription and replication
More LessThe PB2 subunit of influenza virus RNA polymerase is known to be involved in the initiation of transcription of the virus genome via cap binding. However, other specific roles of PB2 for viral RNA synthesis are not well understood. Here, we demonstrate that basic residues, 124R, 142R, 143R, 268R and 331K/332R, in the N-terminal half of PB2 are important for the polymerase activity. Notably, R124A mutation remarkably reduced the synthesis of mRNA, cRNA and vRNA in vivo, which was in good agreement with the data obtained in vitro. Cross-linking studies suggested that a reduction of the polymerase activity in the R124A mutant was due to a significant decrease in binding to the viral RNA promoter. In the three-dimensional structure of the polymerase, 124R is visible through the NTP tunnel and is located close to the polymerase active site. We propose that 124R plays a key role in promoter binding during RNA synthesis.
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Truncated forms of the PA protein containing only the C-terminal domains are associated with the ribonucleoprotein complex within H1N1 influenza virus particles
More LessWe have examined the expression profile of the influenza virus PA protein in pH1N1/2009 virus-infected cells. Immunoblotting analysis of virus-infected MDCK cells revealed the presence of full-length PA protein from 8 h post-infection, together with the simultaneous appearance of PA protein species of approximately 50, 35/39 and 20/25 kDa (collectively referred to as PA*). PA* was also detected in H1N1/WSN-virus-infected cells, indicating that its presence was not virus-specific, and it was also observed in virus-infected A549 and chick embryo fibroblast (CEF) cells, indicating that its presence was not cell-type-specific. PA* was detected in cells expressing the recombinant PA protein, indicating that the PA* formation occurred in the absence of virus infection. These data collectively indicated that PA* formation is an intrinsic property of PA gene expression. The association of PA* with purified influenza virus particles was demonstrated by immunoblotting, and a protease protection assay provided evidence that PA* was packaged into virus particles. The ribonucleoprotein (RNP) complex was isolated from purified influenza virus particles using glycerol gradient centrifugation, which demonstrated that PA* was associated with the RNP complex. To the best of our knowledge, this is the first report to demonstrate that PA protein species containing only segments of the C-terminal domain form during influenza virus infection. Furthermore, these truncated PA protein species are subsequently packaged into virus particles as part of the functional RNP complex.
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Avian influenza virus A H7N9 infects multiple mononuclear cell types in peripheral blood and induces dysregulated cytokine responses and apoptosis in infected monocytes
Most patients with avian influenza A H7N9 virus (H7N9) infection suffer from severe illness, accompanied by dysregulated cytokine/chemokine response, delayed viral clearance and impaired neutralizing antibody response. Here, we evaluated the role of peripheral blood mononuclear cells (PBMCs) in the pathogenesis of H7N9 infection using an ex vivo infection model. H7N9 infected a significantly higher percentage of PBMCs (23.9 %) than those of avian influenza A H5N1 virus (H5N1) (12.3 %) and pandemic H1N1 virus (pH1N1) (5.5 %) (P <0.01). H7N9 infected significantly more B and T lymphocytes than H5N1. When compared with pH1N1, H7N9-infected PBMCs had significantly higher mRNA levels of proinflammatory cytokines and type I interferons (IFNs) at 6 h post-infection (p.i.), but significantly lower levels of IFN-γ and IP-10 at 12 h p.i. Among the PBMCs, CD14+ monocytes were most permissive to H7N9 infection. The percentage of infected CD14+ monocytes was significantly higher for H7N9 than that of pH1N1, but not significantly different from that of H5N1. H7N9-infected monocytes showed higher expression of MIP-1α, MIP-1β and RANTES than that of pH1N1 at 6 h p.i. H7N9- but not pH1N1-infected monocytes died rapidly via apoptosis. Furthermore, pH1N1- but not H7N9-infected monocytes showed increased expression of the monocyte activation and differentiation markers. Unlike pH1N1, H7N9 showed similar PBMC/monocyte cytokine/chemokine expression profile, monocyte cell death and expression of activation/differentiation markers to H5N1. Besides proinflammatory cytokine activation leading to a cytokine storm, impaired IFN-γ production, rapid monocytic death and lack of monocyte differentiation may affect the ability of H7N9-infected innate immune cells to recruit protective adaptive immunity.
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Isolation of a novel orthobunyavirus from bat flies (Eucampsipoda africana)
The Bunyaviridae family comprises viruses causing diseases of public and veterinary health importance, including viral haemorrhagic and arboviral fevers. We report the isolation, identification and genome characterization of a novel orthobunyavirus, named Wolkberg virus (WBV), from wingless bat fly ectoparasites (Eucampsipoda africana) of Egyptian fruit bats (Rousettus aegyptiacus) in South Africa. Complete genome sequence data of WBV suggests it is most closely related to two bat viruses (Mojuí dos Campos and Kaeng Khoi viruses) and an arbovirus (Nyando virus) previously shown to infect humans. WBV replicates to high titres in VeroE6 and C6-36 cells, characteristic of mosquito-borne arboviruses. These findings expand our knowledge of the diversity of orthobunyaviruses and their insect vector host range.
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Broad-spectrum inhibition of common respiratory RNA viruses by a pyrimidine synthesis inhibitor with involvement of the host antiviral response
Our previous screening of 50 240 structurally diverse compounds led to the identification of 39 influenza A virus infection inhibitors (Kao R.Y., Yang D., Lau L.S., Tsui W.H., Hu L. et al. Nat Biotechnol 2010;28:600–605). Further screening of these compounds against common respiratory viruses led to the discovery of compound FA-613. This inhibitor exhibited low micromolar antiviral activity against various influenza A and B virus strains, including the highly pathogenic influenza A strains H5N1 and H7N9, enterovirus A71, respiratory syncytial virus, human rhinovirus A, SARS- and MERS-coronavirus. No significant cellular toxicity was observed at the effective concentrations. Animal studies showed an improved survival rate in BALB/c mice that received intranasal FA-613 treatments against a lethal dose infection of A/HK/415742Md/2009 (H1N1). Further cell-based assays indicated that FA-613 interfer with the de novo pyrimidine biosynthesis pathway by targeting the dihydroorotate dehydrogenase. Surprisingly, FA-613 lost its antiviral potency in the interferon-deficient Vero cell line, while maintaining its inhibitory activity in an interferon-competent cell line which showed elevated expression of host antiviral genes when infected in the presence of FA-613. Further investigation of the specific connection between pyrimidine synthesis inhibition and the induction of host innate immunity might aid clinical development of this type of drug in antiviral therapies. Therefore, in acute cases of respiratory tract infections, when rapid diagnostics of the causative agent are not readily available, an antiviral drug with properties like FA-613 could prove to be very valuable.
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- Positive-strand RNA Viruses
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Close genetic relatedness of picornaviruses from European and Asian bats
Our investigation of 1004 faecal specimens from European bats for picornaviruses by broadly reactive nested reverse transcription-PCR found picornaviral RNA in 28 samples (2.8 %). Phylogenetic analysis of the partial 3D genomic region suggested that one bat virus belonged to the species Enterovirus G (EV-G, formerly Porcine enterovirus B). Bat infection was supported by relatively high EV-G concentrations of 1.1×106 RNA copies per gram of faeces. All other bat viruses belonged either to the bat-associated genus Mischivirus, or to an unclassified Picornaviridae group distantly related to the genus Sapelovirus. Members of this unclassified sapelovirus-related group had RNA secondary structures in their 3′-nontranslated regions that were typical of enteroviruses and that resembled structures that occur in bat-associated coronaviruses, suggesting ancient recombination events. Based on sequence distances, several picornaviruses from European and Chinese bats were likely conspecific, suggesting connectivity of virus populations. Due to their high mutation rates and their diversity, picornaviruses may be useful tools for studies of bat and virus ecology.
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Identification of a novel epitope in the C terminus of hepatitis C virus-E2 protein that induces potent and cross-reactive neutralizing antibodies
Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis, but an effective vaccine is still not available to prevent infection. Use of neutralizing antibodies could be a potential therapeutic option. In this study, the presence of anti-HCV antibodies in HCV-infected patients was assessed from 50 patients and the presence of neutralizing antibodies was examined using ‘hepatitis C virus-like particles’. Antibodies from two samples exhibited significant inhibitory activity, suggesting that these may neutralize viral infection. Antigenic determinants generating the neutralizing antibodies from these two samples were delineated by epitope mapping using the core, E1 and E2 regions and a stretch of 45 amino acid peptide (E2C45) derived from the C-terminal region of HCV-E2 protein (aa 634–679) was designed. Results suggest that this hitherto uncharacterized region has the potential to generate neutralizing antibodies against HCV and thus be effective in preventing virus entry into liver cells. Computational analysis of the structure of the modelled peptide (E2C45) suggested high conformational entropy for this region. Furthermore, E2C45 peptide-generated antibodies could block virus entry and monoclonal antibodies generated against this peptide could also significantly reduce virus replication in a cell culture system. It is possible that the inhibition could be partly due to a conformational alteration of the CD81-binding region, preventing virus attachment to liver cells. In conclusion, this work focused on the discovery of a novel epitope at the C terminus of E2 that induces potent neutralizing antibodies in HCV-infected patients.
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Moonlighting glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is required for efficient hepatitis C virus and dengue virus infections in human Huh-7.5.1 cells
More LessHijacking of cellular biosynthetic pathways by human enveloped viruses is a shared molecular event essential for the viral lifecycle. In this study, the accumulating evidence of the importance of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the host secretory pathway led us to hypothesize that this moonlighting enzyme could play a key role in the lifecycle steps of two important Flaviviridae members, hepatitis C virus (HCV) and dengue virus (DENV). We used short interfering RNA (siRNA)-mediated knockdown of human GAPDH in Huh-7.5.1 cells– both pre- and post-HCV infection– to demonstrate that GAPDH is a host factor for HCV infection. siRNA-induced GAPDH knockdown performed pre-HCV infection inhibits HCV core production in infected cells and leads to a decrease in infectivity of the HCV-infected cell supernatants. siRNA-induced GAPDH knockdown performed post-HCV infection does not have an effect on HCV core abundance in infected cells, but does lead to a decrease in infectivity of the HCV-infected cell supernatants. Exogenous expression of V5-tagged human GAPDH, pre- and post-infection, increases the infectivity of HCV-infected cell supernatants, suggesting a role for GAPDH during HCV post-replication steps. Interestingly, siRNA-induced GAPDH knockdown in HCV replicon-harbouring cells had no effect on viral RNA replication. Importantly, we confirmed the important role of GAPDH in the HCV lifecycle using Huh-7-derived stable GAPDH-knockdown clones. Finally, siRNA-induced GAPDH knockdown inhibits intracellular DENV-2 E glycoprotein production in infected cells. Collectively, our findings suggest that the moonlighting enzyme, GAPDH, is an important host factor for HCV infection, and they support its potential role in the DENV lifecycle.
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Development and characterization of Sindbis virus with encoded fluorescent RNA aptamer Spinach2 for imaging of replication and immune-mediated changes in intracellular viral RNA
More LessViral RNA studies often rely on in situ hybridization and reverse transcriptase-PCR to provide snapshots of RNA dynamics in infected cells. To facilitate analysis of cellular RNAs, aptamers Spinach and Spinach2 that bind and activate the conditional fluorophore 3, 5-difluoro-4-hydroxybenzylidene imidazolinon have been developed. To determine the feasibility of applying this technology to viral RNA, we have used cDNA clones of the TE strain of Sindbis virus (SINV) to construct multiple viruses containing one or two copies of tRNA-scaffolded Spinach2 after a second subgenomic promoter, TEds-1Sp and TEds-2Sp within the 3′UTR, TE-1UTRSp, or after a second subgenomic promoter and in the 3′UTR, TEds-1Sp+1 UTRSp. TEds-1Sp+1 UTRSp gave the brightest signal and replicated well in cell culture, while TEds-2Sp was the dimmest and replicated poorly. Selection of baby hamster kidney cells infected with TEds-1Sp+1 UTRSp for improved signal intensity identified a virus with a stronger signal and point mutations in the tRNA scaffold. Imaging of SINV in BHK cells showed RNA to be concentrated in filopodia that contacted and transferred RNA to adjacent cells. The effect of treatment with anti-E2 antibody, which effects non-cytolytic clearance of SINV from neurons, on viral RNA was cell-type-dependent. In antibody-treated BHK cells, intracellular viral RNA increased and spread of infection continued. In undifferentiated and differentiated AP7 neuronal cells antibody treatment induced viral RNA clearance. Both viruses with two inserted aptamers were prone to deletion. These studies form the basis for further development of aptamer-labelled viral RNAs that will facilitate functional studies on the dynamics of infection and clearance.
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High prevalence of four novel astrovirus genotype species identified from rodents in China
Astroviruses cause gastrointestinal and neurological infections in humans and animals. Since astrovirus is genetically diverse and different astrovirus genotypes can be found in the same animal species, astrovirus is a potential zoonotic threat to humans. In this study, we screened for astroviruses in rodents from Hong Kong, Hunan and Guangxi. Astrovirus was detected in 11.9 % (67/562) of rectal swab specimens. Phylogenetic analysis of the ORF1b region, which encodes the RdRp, showed that there were four distinct clusters (clusters A, B, C and D). Whole genome sequencing was performed for 11 representative strains from each of these four clusters. The mean amino acid genetic distances (p-dist) of full-length ORF2 were >0.634 between clusters A, B, C and other known astroviruses. The p-dist between clusters A and B, A and C, and B and C were 0.371–0.375, 0.517–0.549 and 0.524–0.555, respectively. Within cluster C, the p-dist between HN-014 and GX-006 was 0.372. Since strains with p-dist of ≥0.368 in ORF2 are now considered to be of separate genotypes species, cluster A, cluster B, cluster C-HN-014 and cluster C-GX-006 can be classified as novel genotype species. Cluster D was most closely related to the rodent astrovirus previously identified in Hong Kong. Since rodents live in close proximity to humans, interspecies jumping of these novel astroviruses may represent a threat to human health.
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Critical role of RIG-I and MDA5 in early and late stages of Tulane virus infection
More LessHuman noroviruses are a major cause of acute gastroenteritis worldwide, but the lack of a robust cell culture system or small animal model have hampered a better understanding of innate immunity against these viruses. Tulane virus (TV) is the prototype virus of a tentative new genus, Recovirus, in the family Caliciviridae. Its epidemiology and biological properties most closely resemble human norovirus. The host innate immune response to RNA virus infection primarily involves pathogen-sensing toll-like receptors (TLRs) TLR3 and TLR7 and retinoic acid-inducible gene I-like receptor RIG-I and melanoma differentiation associated gene 5 (MDA5). In this study, by using siRNA knockdown, we report that TV infection in LLC-MK2 cells results in an early [3 h post infection (h p.i.), P<0.05] RIG-I-dependent and type I interferon-mediated antiviral response, whereas an MDA5-mediated antiviral effect was observed at later (12 h p.i.; P<0.05) stages of TV replication. Induction of RIG-I and MDA5 was critical for inhibition of TV replication. Furthermore, pre-activation of the RIG-I/MDA5 pathway prevented TV replication (>900-fold decrease; P<0.05), suggesting that RIG-I and MDA5 ligands could be used to develop novel preventive and therapeutic measures against norovirus.
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Japanese encephalitis virus activates autophagy through XBP1 and ATF6 ER stress sensors in neuronal cells
Endoplasmic reticulum (ER) stress and autophagy are key cellular responses to RNA virus infection. Recent studies have shown that Japanese encephalitis virus (JEV)-induced autophagy negatively influences virus replication in mouse neuronal cells and embryonic fibroblasts, and delays virus-induced cell death. Here, we evaluated the role of ER stress pathways in inducing autophagy during JEV infection. We observed that JEV infection of neuronal cells led to activation of all three sensors of ER stress mediated by eIF2α/PERK, IRE1/XBP1 and ATF6. The kinetics of autophagy induction as monitored by levels of SQSTM1 and LC3-II paralleled activation of ER stress. Inhibition of the eIF2α/PERK pathway by siRNA-mediated depletion of proteins and by the PERK inhibitor had no effect on autophagy and JEV replication. However, depletion of XBP1 and ATF6, alone or in combination, prevented autophagy induction and significantly enhanced JEV-induced cell death. JEV-infected cells depleted of XBP1 or ATF6 showed reduced transcription of ER chaperones, ERAD components and autophagy genes, resulting in reduced protein levels of the crucial autophagy effectors ATG3 and BECLIN-1. Conversely, pharmacological induction of ER stress in JEV-infected cells further enhanced autophagy and reduced virus titres. Our study thus demonstrates that a crucial link exists between the ER stress pathways and autophagy in virus-infected cells, and that these processes are highly regulated during virus infection.
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- Small DNA Viruses
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Usefulness of humanized cDNA-uPA/SCID mice for the study of hepatitis B virus and hepatitis C virus virology
Urokinase-type plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice transplanted with human hepatocytes are permissive for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. However, one of the problems affecting uPA transgenic mice is the expansion of mouse hepatocyte colonies due to homologous recombination of the uPA gene. In this study, we attempted to infect HBV and HCV in humanized cDNA-uPA/SCID mice, a novel uPA transgenic mouse model designed to overcome this disadvantage. Three hundred and eighty-six uPA/SCID and 493 cDNA-uPA/SCID mice were transplanted with human hepatocytes and then injected with either HBV- or HCV-positive human serum samples or HBV-transfected cell culture medium. Twelve weeks after human hepatocyte transplantation, the mouse serum concentration of human albumin, which is correlated with the degree of repopulation by human hepatocytes, was significantly higher in cDNA-uPA/SCID mice compared with uPA/SCID mice. HBV-infected cDNA-uPA/SCID mice showed significantly greater and more persistent viraemia, and similar virological effects by entecavir treatment were achieved in both systems. HCV-infected cDNA-uPA/SCID mice developed more frequent and significantly higher viraemia compared with uPA/SCID mice. The present study using a large number of mice showed that cDNA-uPA/SCID mice transplanted with human hepatocytes developed high and long-term persistent viraemia following HBV and HCV infection, and a higher survival rate was observed in cDNA-uPA/SCID compared with uPA/SCID mice. These mice may be a useful animal model for the study of HBV and HCV virology and the analysis of the effect of antiviral drugs.
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Molecular epidemiology of hepatitis B virus infection in Tanzania
Despite the significant public health problems associated with hepatitis B virus (HBV) in sub-Saharan Africa, many countries in this region do not have systematic HBV surveillance or genetic information on HBV circulating locally. Here, we report on the genetic characterization of 772 HBV strains from Tanzania. Phylogenetic analysis of the S-gene sequences showed prevalence of HBV genotype A (HBV/A, n=671, 86.9 %), followed by genotypes D (HBV/D, n=95, 12.3 %) and E (HBV/E, n=6, 0.8 %). All HBV/A sequences were further classified into subtype A1, while the HBV/D sequences were assigned to a new cluster. Among the Tanzanian sequences, 84 % of HBV/A1 and 94 % of HBV/D were unique. The Tanzanian and global HBV/A1 sequences were compared and were completely intermixed in the phylogenetic tree, with the Tanzanian sequences frequently generating long terminal branches, indicating a long history of HBV/A1 infections in the country. The time to the most recent common ancestor was estimated to be 188 years ago [95 % highest posterior density (HPD): 132 to 265 years] for HBV/A1 and 127 years ago (95 % HPD: 79 to 192 years) for HBV/D. The Bayesian skyline plot showed that the number of transmissions ‘exploded’ exponentially between 1960–1970 for HBV/A1 and 1970–1990 for HBV/D, with the effective population of HBV/A1 having expanded twice as much as that of HBV/D. The data suggest that Tanzania is at least a part of the geographic origin of the HBV/A1 subtype. A recent increase in the transmission rate and significant HBV genetic diversity should be taken into consideration when devising public health interventions to control HBV infections in Tanzania.
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- Large DNA Viruses
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Human cytomegalovirus microRNAs are carried by virions and dense bodies and are delivered to target cells
Human cytomegalovirus (HCMV) infection results in the production of virions, dense bodies (DBs) and non-infectious enveloped particles, all of which incorporate proteins and RNAs that can be transferred to host cells. Here, we investigated whether virions and DBs also carry microRNAs (miRNAs) and assessed their delivery and functionality in cells. Human lung fibroblasts (MRC-5) were infected with the HCMV strain AD169, and conditioned cell culture medium was collected and centrifuged. The pellets were treated with RNase-ONE, and the virions and DBs were purified with a potassium tartrate–glycerol gradient and dialysed. The virions and DBs were incubated with micrococcal nuclease, DNA and RNA were extracted and then analysed with TaqMan PCR assays, while the proteins were examined with Western blots. To assess the delivery of miRNAs to cells and their functionality, virions and DBs were irradiated with UV light. The purity of the virions and DBs was confirmed by typical morphology, the presence of the structural protein pp65 and the HCMV genome, the ability to infect MRC-5 cells and the absence of the host genome. RNA analysis revealed the presence of 14 HCMV-encoded miRNAs (UL22A-5p, US25-1-5p, UL22A-3p, US5-2-3p, UL112-3p, US25-2-3p, US25-2-5p, US33-3p, US5-1, UL36-5p, US4-5p, UL36-3p, UL70-5p and US25-1-3p), HCMV immediate-early mRNA and long non-coding RNA2.7, moreover, two host-encoded miRNAs (hsa-miR-218-5p and hsa-miR-21-5p) and beta-2-microglobulin RNA. UV-irradiated virions and DBs delivered viral miRNAs (US25-1-5p and UL112-3p) to the host cells, and miR-US25-1-5p was functional in a luciferase reporter assay. We conclude that virions and DBs carry miRNAs that are biologically functional and can be delivered to cells, which may affect cellular processes.
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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)