- Volume 91, Issue 1, 2010

Recent 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.

Japanese 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’.

Sub-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.

The 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.

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.

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.

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.

Previously 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.

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.

The 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.

Ross 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.

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.

Crimean–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.

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.

The 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.

The 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.

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.

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.