- Volume 93, Issue 6, 2012
Volume 93, Issue 6, 2012
- Review
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Deployment of the human immunodeficiency virus type 1 protein arsenal: combating the host to enhance viral transcription and providing targets for therapeutic development
More LessDespite the success of highly active antiretroviral therapy in combating human immunodeficiency virus type 1 (HIV-1) infection, the virus still persists in viral reservoirs, often in a state of transcriptional silence. This review focuses on the HIV-1 protein and regulatory machinery and how expanding knowledge of the function of individual HIV-1-coded proteins has provided valuable insights into understanding HIV transcriptional regulation in selected susceptible cell types. Historically, Tat has been the most studied primary transactivator protein, but emerging knowledge of HIV-1 transcriptional regulation in cells of the monocyte–macrophage lineage has more recently established that a number of the HIV-1 accessory proteins like Vpr may directly or indirectly regulate the transcriptional process. The viral proteins Nef and matrix play important roles in modulating the cellular activation pathways to facilitate viral replication. These observations highlight the cross talk between the HIV-1 transcriptional machinery and cellular activation pathways. The review also discusses the proposed transcriptional regulation mechanisms that intersect with the pathways regulated by microRNAs and how development of the knowledge of chromatin biology has enhanced our understanding of key protein–protein and protein–DNA interactions that form the HIV-1 transcriptome. Finally, we discuss the potential pharmacological approaches to target viral persistence and enhance effective transcription to purge the virus in cellular reservoirs, especially within the central nervous system, and the novel therapeutics that are currently in various stages of development to achieve a much superior prognosis for the HIV-1-infected population.
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- Animal
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- RNA viruses
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Full-length sequences of 11 hepatitis C virus genotype 2 isolates representing five subtypes and six unclassified lineages with unique geographical distributions and genetic variation patterns
More LessIn this study, we characterized full-length hepatitis C virus (HCV) genome sequences for 11 genotype 2 isolates. They were isolated from the sera of 11 patients residing in Canada, of whom four had an African origin. Full-length genomes, each with 18–25 overlapping fragments, were obtained by PCR amplification. Five isolates represent the first complete genomes of subtypes 2d, 2e, 2j, 2m and 2r, while the other six correspond to variants that do not group within any assigned subtypes. These sequences had lengths of 9508–9825 nt and each contained a single ORF encoding 3012–3106 aa. Predicted amino acids were carefully inspected and unique variation patterns were recognized, especially for a 2e isolate, QC64. Phylogenetic analysis of complete genome sequences provides evidence that there are a total of 16 subtypes, of which 11 have been described here. Co-analysis with 68 partial NS5B sequences also differentiated 18 assigned subtypes, 2a–2r, and eight additional lineages within genotype 2, which is consistent with the analysis of complete genome sequences. The data from this study will now allow 10 assigned subtypes and six additional lineages of HCV genotype 2 to have their full-length genomes defined. Further analysis with 2021 genotype 2 sequences available in the HCV database indicated that the geographical distribution of these subtypes is consistent with an African origin, with particular subtypes having spread to Asia and the Americas.
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Mutation analysis of the cross-reactive epitopes of Japanese encephalitis virus envelope glycoprotein
More LessGroup and serocomplex cross-reactive epitopes have been identified in the envelope (E) protein of several flaviviruses and have proven critical in vaccine and diagnostic antigen development. Here, we performed site-directed mutagenesis across the E gene of a recombinant expression plasmid that encodes the Japanese encephalitis virus (JEV) premembrane (prM) and E proteins and produces JEV virus-like particles (VLPs). Mutations were introduced at I135 and E138 in domain I; W101, G104, G106 and L107 in domain II; and T305, E306, K312, A315, S329, S331, G332 and D389 in domain III. None of the mutant JEV VLPs demonstrated reduced activity to the five JEV type-specific mAbs tested. Substitutions at W101, especially W101G, reduced reactivity dramatically with all of the flavivirus group cross-reactive mAbs. The group and JEV serocomplex cross-reactive mAbs examined recognized five and six different overlapping epitopes, respectively. Among five group cross-reactive epitopes, amino acids located in domains I, II and III were involved in one, five and three epitopes, respectively. Recognition by six JEV serocomplex cross-reactive mAbs was reduced by amino acid substitutions in domains II and III. These results suggest that amino acid residues located in the fusion loop of E domain II are the most critical for recognition by group cross-reactive mAbs, followed by residues of domains III and I. The amino acid residues of both domains II and III of the E protein were shown to be important in the binding of JEV serocomplex cross-reactive mAbs.
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West Nile virus-induced disruption of the blood–brain barrier in mice is characterized by the degradation of the junctional complex proteins and increase in multiple matrix metalloproteinases
More LessWest Nile virus (WNV) encephalitis is characterized by neuroinflammation, neuronal loss and blood–brain barrier (BBB) disruption. However, the mechanisms associated with the BBB disruption are unclear. Complex interactions between the tight junction proteins (TJP) and the adherens junction proteins (AJP) of the brain microvascular endothelial cells are responsible for maintaining the BBB integrity. Herein, we characterized the relationship between the BBB disruption and expression kinetics of key TJP, AJP and matrix metalloproteinases (MMPs) in the mice brain. A dramatic increase in the BBB permeability and extravasation of IgG was observed at later time points of the central nervous system (CNS) infection and did not precede virus–CNS entry. WNV-infected mice exhibited significant reduction in the protein levels of the TJP ZO-1, claudin-1, occludin and JAM-A, and AJP β-catenin and vascular endothelial cadherin, which correlated with increased levels of MMP-1, -3 and -9 and infiltrated leukocytes in the brain. Further, intracranial inoculation of WNV also demonstrated increased extravasation of IgG in the brain, suggesting the role of virus replication in the CNS in BBB disruption. These data suggest that altered expression of junction proteins is a pathological event associated with WNV infection and may explain the molecular basis of BBB disruption. We propose that WNV initially enters CNS without altering the BBB integrity and later virus replication in the brain initiates BBB disruption, allowing enhanced infiltration of immune cells and contribute to virus neuroinvasion via the ‘Trojan-horse’ route. These data further implicate roles of multiple MMPs in the BBB disruption and strategies to interrupt this process may influence the WNV disease outcome.
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Recombinant nucleocapsid-like particles from dengue-2 induce functional serotype-specific cell-mediated immunity in mice
The interplay of different inflammatory cytokines induced during dengue virus infection plays a role in either protection or increased disease severity. In this sense, vaccine strategies incorporating whole virus are able to elicit both functional and pathological responses. Therefore, an ideal tetravalent vaccine candidate against dengue should be focused on serotype-specific sequences. In the present work, a new formulation of nucleocapsid-like particles (NLPs) obtained from the recombinant dengue-2 capsid protein was evaluated in mice to determine the level of protection against homologous and heterologous viral challenge and to measure the cytotoxicity and cytokine-secretion profiles induced upon heterologous viral stimulation. As a result, a significant protection rate was achieved after challenge with lethal dengue-2 virus, which was dependent on CD4+ and CD8+ cells. In turn, no protection was observed after heterologous challenge. In accordance, in vitro-stimulated spleen cells from mice immunized with NLPs from the four dengue serotypes showed a serotype-specific response of gamma interferon- and tumour necrosis factor alpha-secreting cells. A similar pattern was detected when spleen cells from dengue-immunized animals were stimulated with the capsid protein. Taking these data together, we can assert that NLPs constitute an attractive vaccine candidate against dengue. They induce a functional immune response mediated by CD4+ and CD8+ cells in mice, which is protective against viral challenge. In turn, they are potentially safe due to two important facts: induction of serotype specific cell-mediated immunity and lack of induction of antiviral antibodies. Further studies in non-human primates or humanized mice should be carried out to elucidate the usefulness of the NLPs as a potential vaccine candidate against dengue disease.
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Detection of mosquito-only flaviviruses in Europe
Mattia Calzolari, Líbia Zé-Zé, Daniel Růžek, Ana Vázquez, Claire Jeffries, Francesco Defilippo, Hugo Costa Osório, Patrik Kilian, Santiago Ruíz, Anthony R. Fooks, Giulia Maioli, Fátima Amaro, Martin Tlustý, Jordi Figuerola, Jolyon M. Medlock, Paolo Bonilauri, Maria João Alves, Oldřich Šebesta, Antonio Tenorio, Alexander G. C. Vaux, Romeo Bellini, Ivan Gelbič, Maria Paz Sánchez-Seco, Nicholas Johnson and Michele DottoriThe genus Flavivirus, family Flaviviridae, includes a number of important arthropod-transmitted human pathogens such as dengue viruses, West Nile virus, Japanese encephalitis virus and yellow fever virus. In addition, the genus includes flaviviruses without a known vertebrate reservoir, which have been detected only in insects, particularly in mosquitoes, such as cell fusing agent virus, Kamiti River virus, Culex flavivirus, Aedes flavivirus, Quang Binh virus, Nakiwogo virus and Calbertado virus. Reports of the detection of these viruses with no recognized pathogenic role in humans are increasing in mosquitoes collected around the world, particularly in those sampled in entomological surveys targeting pathogenic flaviviruses. The presence of six potential flaviviruses, detected from independent European arbovirus surveys undertaken in the Czech Republic, Italy,Portugal, Spain and the UK between 2007 and 2010, is reported in this work. Whilst the Aedes flaviviruses, detected in Italy from Aedes albopictus mosquitoes, had already been isolated in Japan, the remaining five viruses have not been reported previously: one was detected in Italy, Portugal and Spain from Aedes mosquitoes (particularly from Aedes caspius), one in Portugal and Spain from Culex theileri mosquitoes, one in the Czech Republic and Italy from Aedes vexans, one in the Czech Republic from Aedes vexans and the last in the UK from Aedes cinereus. Phylogenetic analysis confirmed the close relationship of these putative viruses to other insect-only flaviviruses.
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Genetic variation and recombination in Aichi virus
Aichi virus (AiV), a member of the genus Kobuvirus in the family Picornaviridae, causes gastroenteritis in humans. It was noted that AiV differs from other picornaviruses in its unusually high C content and a very high degree of genome-ordered RNA secondary structures. However, the genetic variability and mutational restrictions on a full-genome scale have not been studied. In addition to the available five complete AiV genomes, we determined here another five complete coding sequences of AiV sampled in Germany, 2004. Distinctive AiV genetic features included a low incidence of recombination along the genome without obvious hotspots or spared regions and very low rates of synonymous and non-synonymous variation, supporting an absence of AiV serotypes. In addition, the absence of recombination between AiV genotypes A and B suggested the existence of reproductive isolation between taxonomic units below the species level. In contrast to most other picornaviruses, AiV genomes strongly avoided the UpA dinucleotide, while there was no obvious selection against the CpG dinucleotide. AiV genomes also appeared to contain a codon usage bias (CUB) apparent as an effective number of codons of 39.5, which was amongst the most extreme among RNA viruses. A set of sequence scrambling algorithms was developed to determine the origin of CUB in AiV. While in most picornaviruses the genomic dinucleotide content contributed significantly to CUB, in AiV its extreme nucleotide content, i.e. 57 % third codon position C, was the main driving force behind the apparent CUB.
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Role of porcine reproductive and respiratory syndrome virus nucleocapsid protein in induction of interleukin-10 and regulatory T-lymphocytes (Treg)
Porcine reproductive and respiratory syndrome virus (PRRSV) infection induces interleukin (IL)-10 production and increased numbers of PRRSV-specific regulatory T-lymphocytes in infected pigs. In the present study, the roles of the nucleocapsid (N) protein in induction of IL-10 and CD4+CD25+Foxp3+ lymphocytes (Treg) were investigated. Transfection of porcine monocyte-derived dendritic cells (MoDCs) and pulmonary alveolar macrophages (PAMs) with a plasmid encoding N protein resulted in significant upregulation of IL-10 gene expression in the gene-transfected cells. Structural conformation, but not nuclear localization, of the expressed N protein was indicated to be essential for the ability to induce IL-10. Furthermore, the presence of recombinant N proteins in cultured PBMCs increased the number of IL-10-producing lymphocytes. Strong induction of IL-10-producing cells and Treg was observed when using N protein-pulsed MoDCs, suggesting an important role of MoDCs in induction of IL-10 and Treg by the N protein. Neutralization of IL-10 by addition of an anti-IL-10 antibody in the culture system resulted in marked reduction of PRRSV-induced Treg in the cultured PBMCs. Together, the data demonstrate the immunomodulatory properties of the PRRSV N protein and the linkage between IL-10 production and development of PRRSV-induced Treg. Our results reveal an immunomodulatory function of the PRRSV N protein that may contribute to the unique immunological outcome observed following PRRSV infection.
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Genetic analysis of a novel nidovirus from fathead minnows
More LessA bacilliform virus was isolated from diseased fathead minnows (Pimephales promelas). Analysis of the complete genome coding for the polyprotein (pp1ab), spike (S), membrane (M) and nucleocapsid (N) proteins revealed that the virus was most like white bream virus (WBV), another bacilliform virus isolated from white bream (Blicca bjoerkna L.) and the type species of the genus Bafinivirus within the order Nidovirales. In addition to similar gene order and size, alignment of deduced amino acid sequences of the pp1ab, M, N and S proteins of the fathead minnow nidovirus (FHMNV) with those of WBV showed 46, 44, 39 and 15 % identities, respectively. Phylogenetic analysis using the conserved helicase domain of the replicase showed FHMNV was distinct from WBV, yet the closest relative identified to date. Thus, FHMNV appears to represent a second species in the genus Bafinivirus. A PCR assay was developed for the identification of future FHMNV-like isolates.
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Origin and fate of A/H1N1 influenza in Scotland during 2009
The spread of influenza has usually been described by a ‘density’ model, where the largest centres of population drive the epidemic within a country. An alternative model emphasizing the role of air travel has recently been developed. We have examined the relative importance of the two in the context of the 2009 H1N1 influenza epidemic in Scotland. We obtained genome sequences of 70 strains representative of the geographical and temporal distribution of H1N1 influenza during the summer and winter phases of the pandemic in 2009. We analysed these strains, together with another 128 from the rest of the UK and 292 globally distributed strains, using maximum-likelihood phylogenetic and Bayesian phylogeographical methods. This revealed strikingly different epidemic patterns within Scotland in the early and late parts of 2009. The summer epidemic in Scotland was characterized by multiple independent introductions from both international and other UK sources, followed by major local expansion of a single clade that probably originated in Birmingham. The winter phase, in contrast, was more diverse genetically, with several clades of similar size in different locations, some of which had no particularly close phylogenetic affinity to strains sampled from either Scotland or England. Overall there was evidence to support both models, with significant links demonstrated between North American sequences and those from England, and between England and East Asia, indicating that major air-travel routes played an important role in the pattern of spread of the pandemic, both within the UK and globally.
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The neuraminidase and matrix genes of the 2009 pandemic influenza H1N1 virus cooperate functionally to facilitate efficient replication and transmissibility in pigs
The 2009 pandemic H1N1 virus (pH1N1) contains neuraminidase (NA) and matrix (M) genes from Eurasian avian-like swine influenza viruses (SIVs), with the remaining six genes from North American triple-reassortant SIVs. To characterize the role of the pH1N1 NA and M genes in pathogenesis and transmission, their impact was evaluated in the background of an H1N1 triple-reassortant (tr1930) SIV in which the HA (H3) and NA (N2) of influenza A/swine/Texas/4199-2/98 virus were replaced with those from the classical H1N1 A/swine/Iowa/15/30 (1930) virus. The laboratory-adapted 1930 virus did not shed nor transmit in pigs, but tr1930 was able to shed in infected pigs. The NA, M or both genes of the tr1930 virus were then substituted by those of pH1N1. The resulting virus with both NA and M from pH1N1 grew to significantly higher titre in cell cultures than the viruses with single NA or M from pH1N1. In a pig model, only the virus containing both NA and M from pH1N1 was transmitted to and infected sentinels, whereas the viruses with single NA or M from pH1N1 did not. These results demonstrate that the right combination of NA and M genes is critical for the replication and transmissibility of influenza viruses in pigs.
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In vivo and in vitro studies on the antiviral activities of viperin against influenza H1N1 virus infection
Influenza A virus has caused a number of pandemics in past decades, including the recent H1N1-2009 pandemic. Viperin is an interferon (IFN)-inducible protein of innate immunity, and acts as a broad-spectrum antiviral protein. We explored the antiviral activities and mechanisms of viperin during influenza virus (IFV) infection in vitro and in vivo. Wild-type (WT) HeLa and viperin-expressing HeLa cells were infected with influenza A/WSN/33/H1N1 (WSN33) virus, and subjected to virological, light and electron microscopic analyses. Viperin expression reduced virus replication and titres, and restricted viral budding. Young and old viperin-knockout (KO) mice and WT control animals were challenged with influenza WSN33 at lethal doses of 103 and 104 p.f.u. via the intratracheal route. Lungs were subjected to histopathological, virological and molecular studies. Upon lethal IFV challenge, both WT and KO mice revealed similar trends of infection and recovery with similar mortality rates. Viral quantification assay and histopathological evaluation of lungs from different time points showed no significant difference in viral loads and lung damage scores between the two groups of mice. Although the in vitro studies demonstrated the ability of viperin to restrict influenza H1N1 virus replication, the viperin-deficient mouse model indicated that absence of viperin enhanced neither the viral load nor pulmonary damage in the lungs of infected mice. This may be due to the compensation of IFN-stimulated genes in the lungs and/or the influenza non-structural protein 1-mediated IFN antagonism dampening the IFN response, thereby rendering the loss of viperin insignificant. Nevertheless, further investigations that exploit the antiviral mechanisms of viperin as prophylaxis are still warranted.
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Low pathogenic H7 subtype avian influenza viruses isolated from domestic ducks in South Korea and the close association with isolates of wild birds
We characterized low pathogenic avian influenza (LPAI) viruses of the H7 subtype that were isolated from domestic ducks and wild birds in South Korea from 2008 to 2011. A total of 20 H7 viruses were collected from live-bird markets (LBMs), duck farms and wild-bird habitats using avian influenza (AI) surveillance and epidemiological approaches. A phylogenetic analysis of the H7 viruses that were isolated from domestic ducks and wild birds demonstrated that they were separated into 12 genotypes (A–D and Wb-1–8, respectively), indicating genetic diversity. These H7 viruses were related to the recently isolated Eurasian LPAI H7 viruses and various influenza viruses that are circulating in Asia, including southern China and South Korea. The same genotype was not found between domestic poultry and wild-bird isolates; however, most of the H7 viruses in poultry (genotypes B and C) were closely related to the H7 virus isolated from a wild bird (genotype Wb-3). Animal-challenge studies revealed that certain H7 AI viruses replicated well only in chickens or ducks depending on the genotype, indicating that the pathogenicity of H7 viruses has the potential to be altered due to multiple reassortments, and these viruses can potentially expand their host range. Our results are evidence of abundant and frequent reassortment between H7 viruses in poultry and wild birds and emphasize the continuing need to monitor the evolutionary genetics of the influenza virus in poultry and wild birds.
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Infection and pathogenesis of Huaiyangshan virus (a novel tick-borne bunyavirus) in laboratory rodents
A novel tick-borne bunyavirus (Huaiyangshan virus, HYSV), which causes haemorrhagic fever-like disease, has recently been reported in China. So far no animal experiments have been performed to study its pathogenesis. Towards developing an animal model for HYSV fever, newborn and adult mice and rats and golden hamsters were inoculated intracerebrally or intraperitoneally with HYSV. Newborn rats and newborn mice, especially Kunming (KM) mice, appeared highly susceptible. Remarkably, the KM mice that died of the HYSV infection developed large necrotic areas in the liver, while no obvious pathological changes were observed within the other organs. PCR and immunohistochemical analyses of the post-mortem material detected both HYSV antigen and RNA in almost all organs, indicating a systemic infection. Our data demonstrate that HYSV can cause a lethal infection of both newborn mice and newborn rats with apparent pathological damage of the liver. This animal model may help to understand the pathogenesis of the HYSV infection in humans.
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- DNA viruses
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Impact of deficiency in CCR2 and CX3CR1 receptors on monocytes trafficking in herpes simplex virus encephalitis
More LessThe role played by resident microglia and by the infiltration of peripheral monocytes/macrophages in the innate immune response during herpes simplex virus type 1 (HSV-1) encephalitis was evaluated in mice deficient for the CCR2 and CX3CR1 receptors. CCR2−/−, CX3CR1−/− and C57BL/6 wild-type (WT) male mice were infected intranasally with 7×105 p.f.u. of an HSV-1 clinical strain and monitored for signs of encephalitis and survival. In addition, brain viral DNA load and cytokine levels were evaluated by RT-PCR and magnetic bead-based immunoassay, respectively. The cellular response was assessed by fluorescence-activated cell sorting of blood and brain leukocytes. Infected CX3CR1−/− mice had a significantly lower mean life expectancy than WT mice (P<0.05, log-rank test) and demonstrated an increased infiltration of Ly-6Chigh ‘inflammatory’ macrophages in the brain (P<0.05). Infected CCR2−/− mice had fewer monocytes (P<0.05), with a lower proportion of Ly-6Chigh ‘inflammatory’ monocytes in the blood than the other groups (P<0.05). Brain viral DNA loads were only slightly higher in knockout mice than in WT mice (P-value not significant). These data suggest that CCR2 and especially CX3CR1 receptors are necessary to initiate a proper immune response during HSV encephalitis. More precisely, CCR2 is crucial for the emigration of monocytes from the bone marrow to the blood, whereas CX3CR1 is mostly implicated in the regulation of infiltrating cells from the blood to the site of infection and in the control of the immune homeostasis of the brain.
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Vaxfectin-adjuvanted plasmid DNA vaccine improves protection and immunogenicity in a murine model of genital herpes infection
The herpes simplex type 2 (HSV-2) envelope glycoprotein (gD2) was evaluated as a potential antigen candidate for a plasmid DNA (pDNA)-based HSV-2 vaccine. The pDNA was formulated with Vaxfectin, a cationic lipid-based adjuvant, and tested in a murine HSV-2 lethal challenge model. gD2 was expressed as full-length (FL) and secreted (S) gD2 forms. A 0.1 µg pDNA dose was tested to distinguish treatment conditions for survival and a 100 µg pDNA dose was tested to distinguish treatment conditions for reduction in vaginal and latent HSV-2 copies. Vaxfectin-formulated gD2 pDNA significantly increased serum IgG titres and survival for both FL gD2 and S gD2 compared with gD2 pDNA alone. Mice immunized with FL gD2 formulated with Vaxfectin showed reduction in vaginal and dorsal root ganglia (DRG) HSV-2 copies. The stringency of this protection was further evaluated by testing Vaxfectin-formulated FL gD2 pDNA at a high 500 LD50 inoculum. At this high viral challenge, the 0.1 µg dose of FL gD2 Vaxfectin-formulated pDNA yielded 80 % survival compared with no survival for FL gD2 pDNA alone. Vaxfectin-formulated FL gD2 pDNA, administered at a 100 µg pDNA dose, significantly reduced HSV-2 DNA copy number, compared with FL gD2 DNA alone. In addition, 40 % of mice vaccinated with adjuvanted FL pDNA had no detectable HSV-2 viral genomes in the DRG, whereas all mice vaccinated with gD2 pDNA alone were positive for HSV-2 viral genomes. These results show the potential contribution of Vaxfectin-gD2 pDNA to a future multivalent HSV-2 vaccine.
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Virion endocytosis is a major target for murid herpesvirus-4 neutralization
More LessHerpesviruses consistently transmit from immunocompetent carriers, implying that their neutralization is hard to achieve. Murid herpesvirus-4 (MuHV-4) exploits host IgG Fc receptors to bypass blocks to cell binding, and pH-dependent protein conformation changes to unveil its fusion machinery only after endocytosis. Nevertheless, neutralization remains possible by targeting the virion glycoprotein H (gH)–gL heterodimer, and the neutralizing antibody responses of MuHV-4 carriers are improved by boosting with recombinant gH–gL. We analysed here how gH–gL-directed neutralization works. The MuHV-4 gH–gL binds to heparan sulfate. However, most gH–gL-specific neutralizing antibodies did not block this interaction; neither did they act directly on fusion. Instead, they blocked virion endocytosis and transport to the late endosomes, where membrane fusion normally occurs. The poor endocytosis of gH–gL-neutralized virions was recapitulated precisely by virions genetically lacking gL. Therefore, driving virion uptake appears to be an important function of gH–gL that provides a major target for antibody-mediated neutralization.
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B23/nucleophosmin is involved in regulation of adenovirus chromatin structure at late infection stages, but not in virus replication and transcription
More LessB23/nucleophosmin has been identified in vitro as a stimulatory factor for replication of adenovirus DNA complexed with viral basic core proteins. In the present study, the in vivo function of B23 in the adenovirus life cycle was studied. It was found that both the expression of a decoy mutant derived from adenovirus core protein V that tightly associates with B23 and small interfering RNA-mediated depletion of B23 impeded the production of progeny virions. However, B23 depletion did not significantly affect the replication and transcription of the virus genome. Chromatin immunoprecipitation analyses revealed that B23 depletion significantly increased the association of viral DNA with viral core proteins and cellular histones. These results suggest that B23 is involved in the regulation of association and/or dissociation of core proteins and cellular histones with the virus genome. In addition, these results suggest that proper viral chromatin assembly, regulated in part by B23, is crucial for the maturation of infectious virus particles.
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Attenuation of CD4+ T-cell function by human adenovirus type 35 is mediated by the knob protein
The complement-regulatory protein CD46 is the primary receptor for human adenovirus type 35 (HAdV-35) and can regulate human immune-cell activation. CD4 + T-cells are critical for initiating and maintaining adaptive immunity elicited by infection or vaccination. It was reported previously that HAdV-35 can bind these cells and suppress their activation. The data reported here demonstrate that recombinant trimeric HAdV-35 knob proteins alone can induce CD46 receptor downregulation and inhibit interleukin-2 production and proliferation of human CD4+ T-cells in vitro similarly to mAbs specific to the CD46 region bound by HAdV-35 knobs. A mutant knob protein with increased affinity for CD46 compared with the wild-type knob caused equivalent effects. In contrast, a CD46-binding-deficient mutant knob protein did not inhibit T-cell activation. Thus, the capacity of HAdV-35 to attenuate human CD4 + T-cell activation depends predominantly on knob interactions with CD46 and can occur independently of infection.
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Prevalence and phylogenetic analysis of the current porcine circovirus 2 genotypes after implementation of widespread vaccination programmes in the USA
More LessTo determine the prevalence of porcine circovirus 2 (PCV2) genotypes in the USA during 2010–2011, 5 years after widespread PCV2 vaccination, serum samples from clinically normal pigs that were PCV2 vaccinated (n = 1177), non-vaccinated (n = 378) or of unknown vaccination status (n = 120), and 100 lung samples from pigs diagnosed with PCV-associated disease (PCVAD) were tested. The presence of PCV2, PCV1, PCV1-2a and porcine parvovirus (PPV) DNA was determined by PCR. Determination of the PCV2 genotype was done by differential PCR and sequencing. The prevalence of PCV2a and PCV2b in serum samples was 7.7 % (129/1675) and 8.4 % (141/1675), respectively. PCV2a DNA was only detected in non-vaccinated pigs. For the 100 PCVAD pigs, the prevalence of PCV2a and PCV2b in lung tissues was 13.0 and 65.0 %, respectively. Partial PCV2 ORF2 sequences (9–563 nt) were obtained from 85 PCV2 DNA-positive samples (24 normal pigs and 61 PCVAD cases). Phylogenetic analysis revealed that 12.9 % (11/85) of the sequences belonged to the 2E clade and the PCV2a genotype and 87.1 % (74/85) belonged to the 1B clade and the PCV2b genotype. The alignment of putative PCV2 capsid amino acid sequences revealed possible recombination or mutation between PCV2a and PCV2b genotypes. Chimeric PCV1-2a was not detected in any of the samples and the prevalence rates of PCV1 and PPV were low. Our results suggest PCV2b is more prevalent than PCV2a in PCVAD cases and in vaccinated herds PCV2b circulation is common. The data generated in this study provide novel information on the distribution of PCV2 genotypes in vaccinated pig populations.
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Volumes and issues
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