- Volume 91, Issue 1, 2010
Volume 91, Issue 1, 2010
- Review
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Functions of Tat: the versatile protein of human immunodeficiency virus type 1
More LessHuman immunodeficiency virus type 1 (HIV-1) Tat is a multifunctional protein that contributes to several pathological symptoms of HIV-1 infection as well as playing a critical role in virus replication. Tat is a robust transactivating protein that induces a variety of effects by altering the expression levels of cellular and viral genes. The functions of Tat are therefore primarily related to its role in modulation of gene expression. In this review the functions of HIV-1 Tat that have been well documented, as well as a number of novel functions that have been proposed for this protein, are discussed. Since some of the functions of Tat vary in different cell types in a concentration-dependent manner and because Tat sometimes exerts the same activity through different pathways, study of this protein has at times yielded conflicting and controversial results. Due to its pivotal role in viral replication and in disease pathogenesis, Tat and the cellular pathways targeted by Tat are potential targets for new anti-HIV drugs.
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Time – the emerging dimension of plant virus studies
More LessRecent research has revealed that some plant viruses, like many animal viruses, have measurably evolving populations. Most of these viruses have single-stranded positive-sense RNA genomes, but a few have single-stranded DNA genomes. The studies show that extant populations of these viral species are only decades to centuries old. The genera in which they are placed have diverged since agriculture was invented and spread around the world during the Holocene period. We suggest that this is not mere coincidence but evidence that the conditions generated by agriculture during this era have favoured particular viruses. There is also evidence, albeit less certain, that some plant viruses, including a few shown to have measurably evolving populations, have much more ancient origins. We discuss the possible reasons for this clear discordance between short- and long-term evolutionary rate estimates and how it might result from a large timescale dependence of the evolutionary rates. We also discuss briefly why it is useful to know the rates of evolution of plant viruses.
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- Animal
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- RNA viruses
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Genomics and evolution of Aedes-borne flaviviruses
We analysed the complete coding sequences of all recognized species of Aedes-borne flavivirus, including previously uncharacterized viruses within the yellow fever virus (YFV), Spondweni virus (SPOV) and dengue virus (DENV) groups. Two major phylogenetic lineages were revealed: one included the YFV and Entebbe bat virus groups, and the other included the DENV, SPOV and Culex-borne flavivirus groups. This analysis supported previous evidence that Culex-borne flaviviruses have evolved from ancestral Aedes-borne viruses. However, the topology at the junction between these lineages remains complex and may be refined by the discovery of viruses related to the Kedougou virus. Additionally, viral evolution was found to be associated with the appearance of new biological characteristics; mutations that may modify the envelope protein structure were identified for seven viruses within the YFV group, and an expansion of host–vector range was identified in the two major evolutionary lineages, which in turn may facilitate the emergence of mosquito-borne flaviviruses.
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Genetic characterization of early isolates of Japanese encephalitis virus: genotype II has been circulating since at least 1951
More LessJapanese encephalitis virus (JEV) consists of five genotypes (GI–V). Phylogenetic characterization of 16 JEV strains isolated from the ‘USSR’, Japan and Korea during the 1930–1970s revealed that 15 strains fell into GIII, confirming that GIII was the predominant genotype of JEV in Japan and Korea between 1935 (isolation of the prototype strain; a GIII virus) and the 1990s (when GI supplanted GIII). One of the Korean isolates fell into GII, demonstrating that GII has been circulating for at least 19 years longer than previously thought. Formerly, GII was associated with endemic disease and this genotype had never been isolated north of Southern Thailand. Additionally, the northern border of GIII prevalence was extended from Japan to the ‘USSR’.
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Involvement of the Fcγ receptor IIA cytoplasmic domain in antibody-dependent enhancement of dengue virus infection
More LessSub-neutralizing concentrations of antibody to dengue virus (DENV) enhance DENV infection of Fcγ receptor-expressing cells. This phenomenon, referred to as antibody-dependent enhancement (ADE), has been hypothesized to be responsible for the severe form of DENV infection, including dengue haemorrhagic fever and dengue shock syndrome. To analyse further the mechanisms of ADE in vitro, this study introduced a series of cytoplasmic mutants into human FcγRIIA. The mutated FcγRIIA was then expressed on COS-7 cells to see whether these mutants could enhance DENV infection. Wild-type FcγRIIA enhanced DENV infection, consistent with previous reports using FcγR-positive monocytes. Disruption of the immune tyrosine activation motif (ITAM) in the cytoplasmic domain of FcγRIIA or removing the sequences between the two ITAM regions eliminated ADE. These findings suggest that the specific structure of the FcγRIIA cytoplasmic domain is essential for the ability of FcγRIIA to mediate ADE.
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Biochemical characterization of the (nucleoside-2′O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppAC n and GpppAC n
More LessThe flavivirus RNA genome contains a conserved cap-1 structure, 7MeGpppA2′OMeG, at the 5′ end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)- and the (nucleoside-2′O)-MTases (2′O-MTase), reside in a single domain of non-structural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 2′O-MTase activity of NS5MTase of dengue virus (NS5MTaseDV) using purified, short, capped RNA substrates (7MeGpppAC n or GpppAC n ). NS5MTaseDV methylated both types of substrate exclusively at the 2′O position. The efficiency of 2′O-methylation did not depend on the methylation of the N7 position. Using 7MeGpppAC n and GpppAC n substrates of increasing chain lengths, it was found that both NS5MTaseDV 2′O activity and substrate binding increased before reaching a plateau at n=5. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K m values for 7MeGpppAC5 and the co-substrate S-adenosyl-l-methionine (AdoMet) were determined (0.39 and 3.26 μM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 2′O-MTase activity of NS5MTaseDV showed a low turnover of 3.25×10−4 s−1. Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTaseDV, which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC50 0.34 μM) and sinefungin (IC50 0.63 μM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays.
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Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein
The cellular DEAD-box protein DDX3 was recently shown to be essential for hepatitis C virus (HCV) replication. Prior to that, we had reported that HCV core binds to DDX3 in yeast-two hybrid and transient transfection assays. Here, we confirm by co-immunoprecipitation that this interaction occurs in cells replicating the JFH1 virus. Consistent with this result, immunofluorescence staining of infected cells revealed a dramatic redistribution of cytoplasmic DDX3 by core protein to the virus assembly sites around lipid droplets. Given this close association of DDX3 with core and lipid droplets, and its involvement in virus replication, we investigated the importance of this host factor in the virus life cycle. Mutagenesis studies located a single amino acid in the N-terminal domain of JFH1 core that when changed to alanine significantly abrogated this interaction. Surprisingly, this mutation did not alter infectious virus production and RNA replication, indicating that the core–DDX3 interaction is dispensable in the HCV life cycle. Consistent with previous studies, siRNA-led knockdown of DDX3 lowered virus production and RNA replication levels of both WT JFH1 and the mutant virus unable to bind DDX3. Thus, our study shows for the first time that the requirement of DDX3 for HCV replication is unrelated to its interaction with the viral core protein.
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Expression of the NS3 protease of cytopathogenic bovine viral diarrhea virus results in the induction of apoptosis but does not block activation of the beta interferon promoter
Bovine viral diarrhea virus (BVDV; genus Pestivirus) can exist as two biotypes, cytopathogenic (CP) and non-cytopathogenic (NCP). The CP form differs from NCP by the continual expression of free non-structural protein 3 (NS3). CP BVDV infection of cultured cells induces apoptosis, whereas NCP BVDV infection has been reported to block the induction of beta interferon (IFN-β). To investigate the viral mechanisms underlying these effects, NS3 or NS2–3 proteins of NCP and CP BVDV biotypes, together with the cognate NS3 co-factor NS4A, were expressed in cells, and their effect on apoptosis and induction of IFN-β was investigated. Expression of NS3/4A resulted in increased activity of caspase-9 and caspase-3, indicating induction of the intrinsic apoptosis pathway. Mutational analysis revealed that a protease-inactive NS3/4A was unable to induce apoptosis, suggesting that NS3 protease activity is required for initiation of apoptosis during CP BVDV infection. The ability of NS2–3 to modulate activation of the IFN-β promoter was also investigated. These studies confirmed that, unlike the related hepatitis C virus and GB virus-B, BVDV proteases are unable to inhibit TLR3- and RIG-I-dependent activation of the IFN-β promoter. These data suggest that BVDV NS3/4A is responsible for regulating the levels of cellular apoptosis and provide new insights regarding the viral elements associated with CP biotype pathogenesis.
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Comprehensive full-length sequence analyses of human parechoviruses: diversity and recombination
Human parechoviruses (HPeVs) are highly prevalent pathogens among very young children. Although originally classified into two serologically distinct types, HPeV1 and -2, recent analyses of variants collected worldwide have revealed the existence of 12 further types classified genetically by sequence comparisons of complete genome sequences or the capsid (VP1) gene. To investigate the nature of HPeV evolution, its population dynamics and recombination breakpoints, this study generated 18 full-length genomic sequences of the most commonly circulating genotypes, HPeV1 and -3, collected over a time span of 14 years from The Netherlands. By inclusion of previously published full-length sequences, 35 sequences were analysed in total. Analysis of contemporary strains of HPeV1 and those most similar to the prototype strain (Harris) showed that HPeV1 variants fall into two genetically distinct clusters that are much more divergent from each other than those observed within other HPeV types. Future classification criteria for HPeVs may require modification to accommodate the occurrence of variants with intermediate degrees of diversity within types. Recombination was frequently observed among HPeV1, -4, -5 and -6, but was much more restricted among HPeV3 strains. Favoured sites for recombination were found to flank the capsid region, and further sites were found within the non-structural region, P2. In contrast to other HPeV types, the majority of the HPeV3 sequences remained monophyletic across the genome, a possible reflection of its lower diversity and potentially more recent emergence than other HPeV types, or biological and/or epidemiological constraints that limit opportunities for co-infections with potential recombination partners.
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Vitronectin receptors, αv integrins, are recognized by several non-RGD-containing echoviruses in a continuous laboratory cell line and also in primary human Langerhans' islets and endothelial cells
More LessPreviously published data suggest that the RGD-recognizing integrin, αvβ3, known as the vitronectin receptor, acts as a cellular receptor for RGD-containing enteroviruses, coxsackievirus A9 (CAV-9) and echovirus 9 (E-9), in several continuous cell lines as well as in primary human Langerhans' islets. As this receptor is also capable of binding the ligands by a non-RGD-dependent mechanism, we investigated whether vitronectin receptors, αv integrins, might act as receptors for other echoviruses that do not have the RGD motif. Blocking experiments with polyclonal anti-αvβ3 antibody showed that both primary human islets and a continuous laboratory cell line of green monkey kidney origin (GMK) are protected similarly from the adverse effects of several non-RGD-containing echovirus (E-7, -11, -25, -30, -32) infections. In contrast, corresponding studies on primary human endothelial cells showed that the receptor works only for E-25, E-30, E-32 and CAV-9. The inhibitory effect of the antibody was not restricted to prototype strains of echoviruses, as GMK cells infected with several field isolates of the corresponding serotypes were also protected from virus-induced cytopathic effects. Co-localization of virus particles with the receptor molecules in both GMK and primary human endothelial cells was demonstrated by live-cell stainings and confocal microscopy. Remarkably, in spite of similar virus–receptor co-localization and a comparable protective effect of the αvβ3 antibody, the entry pathways of the studied virus strains seemed to be divergent.
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Characterization of phylogenetically diverse astroviruses of marine mammals
Astroviruses are small, non-enveloped, positive-stranded RNA viruses. Previously studied mammalian astroviruses have been associated with diarrhoeal disease. Knowledge of astrovirus diversity is very limited, with only six officially recognized astrovirus species from mammalian hosts and, in addition, one human and some bat astroviruses were recently described. We used consensus PCR techniques for initial identification of five astroviruses of marine mammals: three from California sea lions (Zalophus californianus), one from a Steller sea lion (Eumetopias jubatus) and one from a bottlenose dolphin (Tursiops truncatus). Bayesian and maximum-likelihood phylogenetic analysis found that these viruses showed significant diversity at a level consistent with novel species. Astroviruses that we identified from marine mammals were found across the mamastrovirus tree and did not form a monophyletic group. Recombination analysis found that a recombination event may have occurred between a human and a California sea lion astrovirus, suggesting that both lineages may have been capable of infecting the same host at one point. The diversity found amongst marine mammal astroviruses and their similarity to terrestrial astroviruses suggests that the marine environment plays an important role in astrovirus ecology.
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Capsid gene divergence in rabbit hemorrhagic disease virus
More LessThe emergence and rapid global spread of rabbit hemorrhagic disease virus (RHDV) remains enigmatic despite two decades of study, largely due to the difficulties associated with modelling substitution processes of the RNA genome for phylogenetic inference. We used maximum-likelihood and Bayesian methods to investigate rates of molecular evolution in the capsid gene, finding evidence of positive selection and of variable substitution rates between nucleotide sites and between lineages. The maximum-likelihood and Bayesian analyses produced fully congruent topologies; however, strong support for older nodes of the phylogeny was only obtained from the Bayesian analyses that utilized the additional information of collection dates for RHDV isolates spanning 22 years. These dates also allowed calibration of the RHDV phylogenetic tree in a calendar year timescale and estimation of dates for the most recent common ancestors of virulent and benign strains. These dates suggested the divergence of RHDV approximately 20 years prior to the first report of haemorrhagic disease in rabbits.
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Molecular evolutionary dynamics of Ross River virus and implications for vaccine efficacy
More LessRoss River virus (RRV) is a mosquito-borne member of the genus Alphavirus that causes epidemic polyarthritis in humans, costing the Australian health system at least US$10 million annually. Recent progress in RRV vaccine development requires accurate assessment of RRV genetic diversity and evolution, particularly as they may affect the utility of future vaccination. In this study, we provide novel RRV genome sequences and investigate the evolutionary dynamics of RRV from time-structured E2 gene datasets. Our analysis indicates that, although RRV evolves at a similar rate to other alphaviruses (mean evolutionary rate of approx. 8×10−4 nucleotide substitutions per site year−1), the relative genetic diversity of RRV has been continuously low through time, possibly as a result of purifying selection imposed by replication in a wide range of natural host and vector species. Together, these findings suggest that vaccination against RRV is unlikely to result in the rapid antigenic evolution that could compromise the future efficacy of current RRV vaccines.
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Differential activation profiles of Crimean–Congo hemorrhagic fever virus- and Dugbe virus-infected antigen-presenting cells
Crimean–Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic, tick-borne member of the family Bunyaviridae and the genus Nairovirus. To better elucidate the pathogenesis of CCHFV, we analysed the host innate immune response induced in antigen-presenting cells (APCs) infected in vitro by CCHFV. Monocyte-derived dendritic cells (DCs) and macrophages (MPs) were both shown to be permissive for CCHFV and to replicate the virus, as monitored by genomic and antigenomic strand quantification. Virus replication was, however, controlled, corroborating an efficient alpha interferon-induced response. The upregulation of CD-83 and CD-86 indicated that CCHFV induced a partial maturation of DCs, which were also shown to activate the secretion of interleukin (IL)-6 and IL-8, but no tumour necrosis factor alpha (TNF-α). On the other hand, in MPs, CCHFV infection elicited a high IL-6 and TNF-α response and a moderate chemokine response. Nevertheless, when we compared these APC responses with those seen after infection with Dugbe virus (DUGV), a mildly pathogenic virus genetically close to CCHFV, we found that, in spite of some similarities, DUGV induced a higher cytokine/chemokine response in MPs. These results suggest that CCHFV is able to inhibit the activation of inflammatory mediators selectively in infection in vitro and that these differences could be relevant in pathogenesis.
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Towards an understanding of the migration of Crimean–Congo hemorrhagic fever virus
More LessCrimean–Congo haemorrhagic fever (CCHF) is a lethal disease caused by Crimean–Congo hemorrhagic fever virus (CCHFV). It is one of the most widespread medically significant tick-borne pathogens, with a distribution that coincides well with the geographical occurrence of its tick vector, Hyalomma marginatum marginatum. Sporadic outbreaks of CCHF have previously been recognized in Asia, Africa, the Middle East and Europe but, in the 21st century, outbreaks have become more frequent in former Yugoslavia, Turkey and Iran. It has been suggested that CCHFV is a migrating pathogen, but it is not clear to what extent. We have, for the first time, analysed the worldwide migration pattern of CCHFV. Our results showed that Turkey may be a donor in Europe, towards both the east and the west, while the United Arab Emirates acted as a donor in the Middle East, and China was found to be the origin for genotype 2. Finally, we showed that migration of CCHFV was unrestricted between Iran and Pakistan. Considering the distribution and coincidence of the tick vector with CCHFV and CCHF, and the fact that the tick vector is present in western Europe, future outbreaks may extend to include hitherto-naïve areas, suggesting that increased surveillance and geographical mapping of this lethal pathogen are needed.
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Genetic relatedness of H6 subtype avian influenza viruses isolated from wild birds and domestic ducks in Korea and their pathogenicity in animals
We report the genetic characterization of H6 avian influenza (AI) viruses isolated from domestic ducks and wild birds in Korea between April 2008 and April 2009. A phylogenetic analysis showed that the H6N1 viruses of wild birds and domestic ducks were of the same genotype (K-1) and were similar to the H6N1 virus isolated from a live poultry market in 2003, as six of the eight gene segments of those viruses had a common source. However, the H6N2 viruses of domestic poultry were separated into four genotypes (K-2a, K-2b, K-2c and K-2d) by at least a triple reassortment between influenza viruses of low pathogenicity from Korean poultry (H9N2 and H3N2) and viruses from aquatic birds. In an experimental infection of animals, certain H6 AI viruses replicated well in chickens and mice without pre-adaptation, indicating that H6 virus pathogenicity has the potential to be altered due to multiple reassortments, and that these reassortments could result in interspecies transmission to mammals.
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Thogoto virus ML protein is a potent inhibitor of the interferon regulatory factor-7 transcription factor
More LessThe tick-transmitted orthomyxovirus Thogoto virus (THOV) encodes the ML protein acting as a viral suppressor of the host interferon (IFN) system. Here, we describe that type I IFN is strongly induced in primary mouse embryo fibroblasts as well as plasmacytoid dendritic cells upon infection with a THOV mutant lacking the ML gene. However, wild-type THOV encoding ML suppresses induction of IFN by preventing the activation of members of the IFN regulatory factor (IRF) family. We found that reporter gene expression dependent on IRF3 and IRF7 was strongly inhibited by ML. Further experiments revealed that ML interacts with IRF7 and prevents dimerization of the transcription factor and its association with the coactivator TRAF6. Interestingly, another IRF7 activation step, nuclear translocation, is not affected by ML. Our data elucidate ML protein as a virulence factor with an IRF-specific IFN-antagonistic spectrum.
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Regulation of Marburg virus (MARV) budding by Nedd4.1: a different WW domain of Nedd4.1 is critical for binding to MARV and Ebola virus VP40
More LessThe VP40 matrix protein of Marburg virus (MARV) has been shown to be the driving force behind MARV budding, a process in which the PPPY L-domain motif of VP40 plays a critical role. Here, we report that Vps4B and Nedd4.1 play critical roles in MARV VP40-mediated budding. We showed that unidentified activities of the Nedd4.1 HECT domain, along with its E3 ubiquitin ligase activity, may be required for MARV budding. Moreover, we showed that the first WW domain of Nedd4.1, WW1, is critical for binding to MARV VP40, indicating that MARV VP40 and Ebola virus VP40 are recognized by a different WW domain of Nedd4.1. This is the first report showing that the viral L-domains containing PPxY have specificities for binding to WW domains. Our findings provide new insights into MARV budding, which may contribute to the development of novel anti-MARV therapeutic strategies.
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HLA-C is necessary for optimal human immunodeficiency virus type 1 infection of human peripheral blood CD4 lymphocytes
The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on their infectability by human immunodeficiency virus (HIV) has been suggested previously. This was tested by exploiting the peculiar specificity of monoclonal antibody (mAb) L31 for HLA-C open conformers to show that normal levels of Env-driven fusion were restored in HLA-C transfectants of a major histocompatibility complex-deleted (fusion-incompetent) cell line. The physiological relevance of this finding is now confirmed in this report, where small interfering RNA (siRNA) technology was used to silence HLA-C expression in peripheral blood lymphocytes (PBLs) from 11 healthy donors. Infectability by HIV (strains IIIB and Bal and primary isolates) was significantly reduced (P=0.016) in silenced cells compared with cells that maintained HLA-C expression in 10 of the
11 PBL donors. Normal infectability was resumed, together with HLA-C expression, when the effect of siRNA interference waned after several days in culture. Additional confirmation of the HLA-C effect was obtained in several assays employing HLA-C-positive and -negative cell lines, a number of HIV strains and also pseudoviruses. In particular, viruses pseudotyped with env genes from HIV strains AC10 and QH0692.42 were assayed on siRNA-silenced lymphocytes from three healthy donors: the differences in infection with pseudoviruses were even higher than those observed in infections with normal viruses.
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Neutralization of feline immunodeficiency virus by antibodies targeting the V5 loop of Env
Neutralizing antibodies (NAbs) play a vital role in vaccine-induced protection against infection with feline immunodeficiency virus (FIV). However, little is known about the appropriate presentation of neutralization epitopes in order to induce NAbs effectively; the majority of the antibodies that are induced are directed against non-neutralizing epitopes. Here, we demonstrate that a subtype B strain of FIV, designated NG4, escapes autologous NAbs, but may be rendered neutralization-sensitive following the insertion of two amino acids, KT, at positions 556–557 in the fifth hypervariable (V5) loop of the envelope glycoprotein. Consistent with the contribution of this motif to virus neutralization, an additional three subtype B strains retaining both residues at the same position were also neutralized by the NG4 serum, and serum from an unrelated cat (TOT1) targeted the same sequence in V5. Moreover, when the V5 loop of subtype B isolate KNG2, an isolate that was moderately resistant to neutralization by NG4 serum, was mutated to incorporate the KT motif, the virus was rendered sensitive to neutralization. These data suggest that, even in a polyclonal serum derived from FIV-infected cats following natural infection, the primary determinant of virus-neutralizing activity may be represented by a single, dominant epitope in V5.
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High prevalence of neutralizing activity against multiple unrelated human immunodeficiency virus type 1 (HIV-1) subtype B variants in sera from HIV-1 subtype B-infected individuals: evidence for subtype-specific rather than strain-specific neutralizing activity
More LessIt is assumed that an effective human immunodeficiency virus type 1 (HIV-1) vaccine should be capable of eliciting neutralizing antibodies. However, even the best antibodies known to date lack neutralizing ability against a significant proportion of primary HIV-1 variants and, despite great efforts, still no immunogen is available that can elicit humoral immunity which is protective against infection or disease progression. We tested sera from 35 participants in the Amsterdam Cohort Studies on HIV-1 infection, who were all infected with HIV-1 subtype B and therapy-naïve at the time of sampling, for neutralizing activity against a panel of 23 tier 2–3 HIV-1 variants, with a minimum of five HIV-1 variants per subtype (A, B, C and D). Strong cross-clade neutralizing activity was detected in sera from seven individuals. Strikingly, sera from 22 of 35 individuals (63 %) neutralized three or more of the six tier 2–3 HIV-1 subtype B viruses in the panel. There was a strong correlation between neutralization titre and breadth in serum. Indeed, the IC50 of sera with strong cross-clade neutralizing activity was significantly higher than the IC50 of sera with cross-subtype B activity, which, in turn, had a higher IC50 than sera with the lowest neutralization breadth. These results imply that humoral immunity, at least in HIV-1 subtype B-infected individuals, is often subtype-specific rather than strain-specific and that the breadth of neutralization is correlated with the titre of neutralizing activity in serum. Considering the difficulties in designing a vaccine that is capable of eliciting cross-clade neutralizing activity, subtype-specific vaccines may be explored as an interesting alternative.
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Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus
Feline leukemia virus (FeLV), a common, naturally occurring gammaretrovirus in domestic cats, is associated with degenerative diseases of the haematopoietic system, immunodeficiency and neoplasia. FeLV infection causes an important suppression of neutrophil function, leading to opportunistic infections. Recently, a new microbicidal mechanism named NETosis was described in human, bovine and fish neutrophils, as well as in chicken heterophils. The purpose of the present study was to characterize NETosis in feline neutrophils, as well as to evaluate neutrophil function in FeLV naturally infected symptomatic and asymptomatic cats through the phagocytosis process, release of neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) activity. The results showed that feline neutrophils stimulated with protozoa parasites released structures comprising DNA and histones, which were characterized as NETs by immunofluorescence. Quantification of NETs after neutrophil stimulation showed a significant increase in NET release by neutrophils from FeLV− and FeLV+ asymptomatic cats compared with FeLV+ symptomatic cats. Moreover, the number of released NETs and MPO activity in unstimulated neutrophils of FeLV+ symptomatic cats were higher than those in unstimulated neutrophils from FeLV− and FeLV+ asymptomatic cats. This study reports, for the first time, NET release by feline neutrophils, along with the fact that NET induction may be modulated by a viral infection. The results indicate that the NET mechanism appears to be overactivated in FeLV+ cats and that this feature could be considered a marker of disease progression in FeLV infection.
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- DNA viruses
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Glycoprotein G from pseudorabies virus binds to chemokines with high affinity and inhibits their function
More LessPseudorabies virus (PRV), also known as suid herpesvirus, is the aetiological agent of Aujeszky's disease in swine. In other animals, except higher-order primates, PRV infection is often fatal. The mechanisms of PRV pathogenesis and immune modulation are largely unknown. PRV codes for 11 glycoproteins. Among them, glycoprotein G (gG) is the most abundant PRV protein found in the supernatant of PRV-infected cell cultures. PRV-gG has low amino acid sequence similarity with gG from other animal alphaherpesviruses and its function is unknown. gG from other animal alphaherpesviruses, with the exception of at least equine herpesvirus 4, binds to chemokines. We show here that PRV-gG binds to the human chemokine CL1 and several CC and CXC human chemokines with high affinity. Chemokine-binding activity can be detected in the supernatants of PRV-infected cell cultures, and insertional inactivation of the gene encoding gG from the PRV genome results in loss of chemokine-binding activity. Binding of PRV-gG to chemokines inhibits chemokine-mediated cell migration, suggesting a role for PRV-gG in immune evasion.
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Characterization of microRNAs encoded by the bovine herpesvirus 1 genome
Bovine herpesvirus 1 (BoHV-1) is a ubiquitous and important pathogen of cattle worldwide. This study reports the identification of 10 microRNA (miRNA) genes, Bhv1-mir-B1–Bhv1-mir-B10, encoded by the BoHV-1 genome that were processed into 12 detectable mature miRNAs as determined by ultra-high throughput sequencing bioinformatics analyses of small RNA libraries and expression studies. We found that four of the miRNA genes were present as two copies in the BoHV-1 genome, resulting in a total of 14 miRNA encoding loci. Unique features of the BoHV-1 miRNAs include evidence of bidirectional transcription and a close association of two miRNA genes with the origin of replication, including one miRNA that is encoded within the origin of replication. The miRNA gene Bhv1-mir-B5 was encoded on the opposite DNA strand to the latency associated transcript, potentially giving rise to antisense transcripts originating from this locus. The association of herpesvirus miRNAs with latency appears to be a common feature in the alphaherpesviruses. Analyses of the BoHV-5 genome for putative miRNA gene orthologues identified a high degree of evolutionary conservation for nine of the BoHV-1 miRNA genes. The possible roles for BoHV-1 miRNAs in the regulation of known BoHV-1 transcription units and the genetics of the BoHV-1 genotypes are also discussed.
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Quantitative analysis of Epstein–Barr virus (EBV)-related gene expression in patients with chronic active EBV infection
Chronic active Epstein–Barr virus (CAEBV) infection is a systemic Epstein–Barr virus (EBV)-positive lymphoproliferative disorder characterized by persistent or recurrent infectious mononucleosis-like symptoms in patients with no known immunodeficiency. The detailed pathogenesis of the disease is unknown and no standard treatment regimen has been developed. EBV gene expression was analysed in peripheral blood samples collected from 24 patients with CAEBV infection. The expression levels of six latent and two lytic EBV genes were quantified by real-time RT-PCR. EBV-encoded small RNA 1 and BamHI-A rightward transcripts were abundantly detected in all patients, and latent membrane protein (LMP) 2 was observed in most patients. EBV nuclear antigen (EBNA) 1 and LMP1 were detected less frequently and were expressed at lower levels. EBNA2 and the two lytic genes were not detected in any of the patients. The pattern of latent gene expression was determined to be latency type II. EBNA1 was detected more frequently and at higher levels in the clinically active patients. Quantifying EBV gene expression is useful in clarifying the pathogenesis of CAEBV infection and may provide information regarding a patient's disease prognosis, as well as possible therapeutic interventions.
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A region at the left end of the fowl adenovirus 9 genome that is non-essential in vitro has consequences in vivo
More LessThe regions at the left and right ends of fowl adenovirus (FAdV) genomes are not well-characterized in comparison to those of human adenoviruses. Using a series of deletion mutants, we analysed a 2.4 kb region near the left end of the FAdV-9 genome (nt 400–2782) that contains packaging-signal motifs VI and VII and open reading frames (ORFs) 0, 1, 1A, 1B, 1C and 2. Viable viruses with specific deletions in this region had wild-type characteristics in vitro, as measured by cytopathic effect, plaque morphology, virus titres and growth kinetics. However, one mutant (FAdV-9Δ4), which lacked these ORFs and retained the packaging motifs, did not replicate at wild-type levels in vivo, as judged in infected eggs by virus titres in allantoic fluid and in infected chickens by antibody responses, virus titres in faeces and virus genome copy numbers in tissues. These findings indicate that some of the ORFs in this region, although dispensable in vitro, are important for in vivo replication of FAdV-9.
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Assembly and export determine the intracellular distribution of hepatitis B virus core protein subunits
More LessLittle is known about the parameters and factors that determine the intracellular distribution of the hepatitis B virus core protein (HBc). In order to study HBc in living cells, HBc was tagged with green fluorescent protein (GFP). Being assembly-incompetent, the GFP-fusion protein was distributed equally throughout the cell. Mutational inactivation of known serine-phosphorylation sites within the C-terminal region led to predominantly intranuclear localization. Phosphorylation of these targets, presumably by an SR domain protein kinase, resulted in a predominantly cytoplasmic localization, which suggests active cytoplasmic export or retention. The phosphoserine itself, and not its negative charge, appears essential for the underlying mechanism. In addition, the arginine-rich, protamine-like domain surrounding these phosphorylation sites does not function as the dominant nuclear-localization signal, as had been assumed previously, because neither deleting nor altering these sequences led to a change in intracellular HBc subunit distribution. Restoring the capability of the fusion protein to form capsids by co-assembly with assembly-competent, sterically uncompromised HBc subunits provided a second assay that gave insight into the effects resulting from capsid formation. Assembly was found to be the dominant factor in the cytoplasmic retention of the GFP–HBc fusion protein. Furthermore, the stability of these empty capsids was influenced by the cell-cycle inhibitor nocodazole. Thus, the intracellular distribution of HBc is dominated by cytoplasmic assembly, which is supported by the active nuclear export of HBc subunits, and modulated during the cell cycle by the instability of capsids.
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The genetic backbone modulates the phenotype of hepatitis B surface antigen mutants
More LessHepatitis B virus (HBV) vaccine and diagnostic escape mutants are a growing concern. The principle target of detection, hepatitis B surface antigen (HBsAg), encoded by S, is completely overlapped by the reverse transcriptase encoding P. With the increased incidence of nucleos(t)ide analogue resistance altering P, the concurrent impact on S must be assessed. HBV DNA from 59 HBsAg-positive plasma samples was sequenced across the polymerase/surface region and the amino acid sequence of HBsAg was inferred. ELISAs were formatted containing individually bound monoclonal antibodies directed against three discrete epitopes on HBsAg. Similar point mutations occurring in different genotypes were shown to influence epitope conformation differently, indicating that the genetic backbone is a major factor in predicting phenotype. C-terminal changes associated with antiviral resistance were found to modulate epitope profiles of HBsAg. Treatment options which may promote drug resistance should be avoided to both protect antiviral treatment and prevent facilitation of vaccine and diagnostic escape mutants.
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Novel circular DNA viruses in stool samples of wild-living chimpanzees
Viral particles in stool samples from wild-living chimpanzees were analysed using random PCR amplification and sequencing. Sequences encoding proteins distantly related to the replicase protein of single-stranded circular DNA viruses were identified. Inverse PCR was used to amplify and sequence multiple small circular DNA viral genomes. The viral genomes were related in size and genome organization to vertebrate circoviruses and plant geminiviruses but with a different location for the stem–loop structure involved in rolling circle DNA replication. The replicase genes of these viruses were most closely related to those of the much smaller (∼1 kb) plant nanovirus circular DNA chromosomes. Because the viruses have characteristics of both animal and plant viruses, we named them chimpanzee stool-associated circular viruses (ChiSCV). Further metagenomic studies of animal samples will greatly increase our knowledge of viral diversity and evolution.
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- Plant
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Sequence diversity, population genetics and potential recombination events in grapevine rupestris stem pitting-associated virus in Pacific North-West vineyards
More LessGrapevine rupestris stem pitting-associated virus (GRSPaV; genus Foveavirus, family Flexiviridae) is present in many grape-growing regions of the world. A total of 84 full-length coat protein (CP) sequences and 57 sequences representing the helicase-encoding region (HR) of the RNA-dependent RNA polymerase were obtained from wine grape cultivars grown in the Pacific North-West (PNW) of the United States and their molecular diversity was compared with corresponding sequences previously reported from other grape-growing regions. In pairwise comparisons, the CP sequences from PNW isolates showed identities between 80 and 100 % at the nucleotide level and the HR sequences showed identities between 79 and 100 %. A global phylogenetic analysis of the CP and HR sequences revealed segregation of GRSPaV isolates into four major lineages with isolates from PNW distributed in all four lineages, indicating a lack of clustering by geographical origin. Scion cultivars grafted onto rootstock were found to contain mixtures of more genetic variants belonging to different lineages than own-rooted cultivars. Assessment of population genetic parameters found that the CP was more variable than the HR region. The discordant gene phylogenies obtained for some CP and HR sequences and the identification of potential recombination events involving parents from different lineages provided strong evolutionary evidence for genetic diversity among GRSPaV isolates. These results underscore the highly variable nature of the virus with implications for grapevine health status and distribution of virus-tested planting materials. This study also contributes to an increased understanding of molecular population genetics of viruses infecting deciduous woody perennials.
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Pathogenicity of Alternanthera mosaic virus is affected by determinants in RNA-dependent RNA polymerase and by reduced efficacy of silencing suppression in a movement-competent TGB1
Four biologically active cDNA clones were derived from the Alternanthera mosaic virus (AltMV; genus Potexvirus) isolate, AltMV-SP, which differ in symptoms in infected Nicotiana benthamiana plants. Two clones induced necrosis and plant death; a mixture of all four clones induced milder symptoms than AltMV-SP. Replication of all clones was enhanced by a minimum of fourfold at 15 °C. A mixture of clones 4-7 (severe) and 3-1 (mild) was indistinguishable from AltMV-SP, but the ratio of 4-7 to 3-1 differed at 25 and 15 °C. RNA copy numbers of mixed infections were always below those of 4-7 alone. Determinants of symptom severity were identified in both Pol and TGB1; the mildest (4-1) and most severe (3-7) clones differed at three residues in the ‘core’ Pol domain [R(1110)P, K(1121)R, R(1255)K] and one [S(1535)P] in the C-terminal Pol domain of RNA-dependent RNA polymerase, and one in TGB1 [P(88)L]. Pol [P1110,R1121,K1255]+TGB1L(88)] always induced systemic necrosis at 15 °C. Gene exchanges of Pol and TGB1 each affected replication and symptom expression, with TGB1P(88) significantly reducing silencing suppression. The difference in silencing suppression between TGB1P(88) and TGB1L(88) was confirmed by an agroinfiltration assay. Further, co-expression of TGB1P(88) and TGB1L(88) resulted in interference in the suppression of silencing by TGB1L(88). Yeast two-hybrid analysis confirmed that TGB1P(88) and TGB1L(88) interact. These results identify a TGB1 residue that significantly affects replication and silencing suppression, but maintains full movement functions.
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Single amino acid changes in the turnip mosaic virus viral genome-linked protein (VPg) confer virulence towards Arabidopsis thaliana mutants knocked out for eukaryotic initiation factors eIF(iso)4E and eIF(iso)4G
Previous resistance analyses of Arabidopsis thaliana mutants knocked out for eukaryotic translation initiation factors showed that disruption of the At-eIF(iso)4E or both the At-eIF(iso)4G1 and At-eIF(iso)4G2 genes resulted in resistance against turnip mosaic virus (TuMV). This study selected TuMV virulent variants that overcame this resistance and showed that two independent mutations in the region coding for the viral genome-linked protein (VPg) were sufficient to restore TuMV virulence in At-eIF(iso)4E and At-eIF(iso)4G1×At-eIF(iso)4G2 knockout plants. As a VPg–eIF(iso)4E interaction has been shown previously to be critical for TuMV infection, a systematic analysis of the interactions between A. thaliana eIF4Es and VPgs of virulent and avirulent TuMVs was performed. The results suggest that virulent TuMV variants may use an eIF4F-independent pathway.
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Selective modification of rice (Oryza sativa) gene expression by rice stripe virus infection
Rice stripe disease, caused by rice stripe virus (RSV), is one of the major virus diseases in east Asia. Rice plants infected with RSV usually show symptoms such as chlorosis, weakness, necrosis in newly emerged leaves and stunting. To reveal rice cellular systems influenced by RSV infection, temporal changes in the transcriptome of RSV-infected plants were monitored by a customized rice oligoarray system. The transcriptome changes in RSV-infected plants indicated that protein-synthesis machineries and energy production in the mitochondrion were activated by RSV infection, whereas energy production in the chloroplast and synthesis of cell-structure components were suppressed. The transcription of genes related to host-defence systems under hormone signals and those for gene silencing were not activated at the early infection phase. Together with concurrent observation of virus concentration and symptom development, such transcriptome changes in RSV-infected plants suggest that different sets of various host genes are regulated depending on the development of disease symptoms and the accumulation of RSV.
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- Phage
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Specific hydrophobic residues in the α4 helix of λCII are crucial for maintaining its tetrameric structure and directing the lysogenic choice
More LessThe CII protein of the temperate bacteriophage λ is the decision-making factor that determines the viral lytic/lysogenic choice. It is a homotetrameric transcription activator that recognizes and binds specific direct repeat sequences TTGCN6TTGC in the λ genome. The quaternary structure of CII is held by a four-helix bundle. It is known that the tetrameric organization of CII is necessary for its activity, but the molecular mechanism behind this requirement is not known. By specific site-directed mutagenesis of hydrophobic residues in the α4 helix of CII that constitutes the four-helix bundle, we found that residues leu70, val74 and leu78 were crucial for maintaining the tetrameric structure of the protein. When any of these residues was substituted by a polar one, CII lost its activity and failed to promote lysogeny. This loss of activity was accompanied by the inability of CII to form tetramers, to bind DNA or to activate transcription.
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Volumes and issues
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Volume 105 (2024)
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