Unrevealed genetic diversity of GII Norovirus in the swine population of North East Italy.

Noroviruses (NoVs) are one of the major causative agents of non-bacterial gastroenteritis in humans worldwide. NoVs, belonging to Caliciviridae, are classified into ten genogroups (G) and eight P-groups based on major capsid protein (VP1) and of the RNA-dependent-RNA-polymerase (RdRp), respectively. In swine, the main genogroup and P-group identified are GII and GII.P; which can infect humans too. To date, only one case of GIIP.11 have been identified in swine in Italy while the circulation of other P-types is currently unknown. In the present study, 225 swine faecal samples were collected from 74 swine herds in Veneto region through on-farm monitoring. NoV circulation was particularly high in older pigs. The phylogenetic analysis showed the co-circulation of NoVs belonging to two different P-types: GII.P11 and GII.P18, here described for the first time in Italy, presenting an extensive genetic diversity, never described before worldwide. Distinct NoV genetic subgroups and unique amino acid mutations were identified for each P-type for the first time. This study demonstrated the co-circulation of diverse swine NoVs subgroups in Italy, raising questions on the origin of such diversity and suggesting that continuous monitoring of swine NoVs is needed to track the emergence of potentially zoonotic viruses by recombination events.

A Gene Constellation in Avian Influenza A (H7N9) Viruses May Have Facilitated the Fifth Wave Outbreak in China.

The 2016-2017 epidemic of influenza A (H7N9) virus in China prompted concern that a genetic change may underlie increased virulence. Based on an evolutionary analysis of H7N9 viruses from all five outbreak waves, we find that additional subclades of the H7 and N9 genes have emerged. Our analysis indicates that H7N9 viruses inherited NP genes from co-circulating H7N9 instead of H9N2 viruses. Genotypic diversity among H7N9 viruses increased following wave I, peaked during wave III, and rapidly deceased thereafter with minimal diversity in wave V, suggesting that the viruses entered a relatively stable evolutionary stage. The ZJ11 genotype caused the majority of human infections in wave V. We suggest that the largest outbreak of wave V may be due to a constellation of genes rather than a single mutation. Therefore, continuous surveillance is necessary to minimize the threat of H7N9 viruses.

Metagenomics reshapes the concepts of RNA virus evolution by revealing extensive horizontal virus transfer.

Virus metagenomics is a young research filed but it has already transformed our understanding of virus diversity and evolution, and illuminated at a new level the connections between virus evolution and the evolution and ecology of the hosts. In this review article, we examine the new picture of the evolution of RNA viruses, the dominant component of the eukaryotic virome, that is emerging from metagenomic data analysis. The major expansion of many groups of RNA viruses through metagenomics allowed the construction of substantially improved phylogenetic trees for the conserved virus genes, primarily, the RNA-dependent RNA polymerases (RdRp). In particular, a new superfamily of widespread, small positive-strand RNA viruses was delineated that unites tombus-like and noda-like viruses. Comparison of the genome architectures of RNA viruses discovered by metagenomics and by traditional methods reveals an extent of gene module shuffling among diverse virus genomes that far exceeds the previous appreciation of this evolutionary phenomenon. Most dramatically, inclusion of the metagenomic data in phylogenetic analyses of the RdRp resulted in the identification of numerous, strongly supported groups that encompass RNA viruses from diverse hosts including different groups of protists, animals and plants. Notwithstanding potential caveats, in particular, incomplete and uneven sampling of eukaryotic taxa, these highly unexpected findings reveal horizontal virus transfer (HVT) between diverse hosts as the central aspect of RNA virus evolution. The vast and diverse virome of invertebrates, particularly nematodes and arthropods, appears to be the reservoir, from which the viromes of plants and vertebrates evolved via multiple HVT events.

RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases.

Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.

Hepatitis C virus of subtype 2l in Marseille, southeastern France.

The rate of eradication of chronic hepatitis C considerably increases with direct-acting antiviral agents, particularly hepatitis C virus (HCV) polymerase inhibitors. While implementing full-length HCV NS5B polymerase sequencing in our clinical microbiology laboratory, we identified atypical HCV sequences, classified as subtype 2l, from 2 patients. HCV-2l NS5B polymerase sequences were detected from 5 and 14 additional patients by screening our laboratory hepatitis virus sequence database and the NCBI GenBank sequence database. Phylogenetic analyses show unambiguously that all HCV-2l sequences are clustered apart from HCV 2 non-l sequences, which compose a second cluster. Mean (±SD) nucleotide identity between near full-length NS5B fragments of subtype 2l was 93.4 ± 0.8% (range: 92.4-95.1). Of note, all HCV-2l sequences obtained in our laboratory and in other centers were from serum samples collected in France. Analysis of the HCV-2l NS5B polymerase amino acid sequences at 30 positions critical for interaction with or resistance to HCV polymerase inhibitors showed specific patterns.

[Epidemic variants of norovirus genotype GII.4 in Nizhny Novgorod in 2006 - 2012].

AIM: Genotyping of noroviruses that had circulated in the territory of Nizhny Novgorod during 6 epidemic seasons (2006 - 2012), detection of dominating genovariants and analysis of their change. MATERIALS AND METHODS: Feces samples from children hospitalized in an intestinal infection department of one of the infectious disease hospitals of Nizhny Novgorod served as material for the study. Noroviruses were detected by reverse transcription polymerase chain reaction. Genotypes and gene variants were determined by analysis of nucleotide sequences of viral genome regions coding capsid protein and RNA-dependent RNA-polymerase. RESULTS: During examination of 6589 children with an acute intestinal infection between July 2006 and June 2012 noroviruses were detected in 17.55% of cases. Nucleotide sequences of capsid and/or polymerase gene regions were determined for 114 norovirus isolates. Genotyping has shown that noroviruses of 8 various genotypes had circulated in the territory of Nizhny Novgorod--GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII. 12, GII.13 with the domination of GII.4 noroviruses for the whole observation period. A dynamic of change of epidemic variants of genotype GII.4 noroviruses that had been accompanied by an increase of frequency of detection of norovirus in children hospitalized with acute intestinal infection similar to global was established. A short-term circulation of GII.4 2006b-NN 2008 norovirus subvariant in spring of 2008 and spread of genotype GII.12 norovirus during 2009, 2010 epidemic season were also shown. CONCLUSION: The data obtained give evidence to the necessity of norovirus circulation monitoring with the aim of early detection of novel virus variants that may determine an increase of norovirus infection morbidity.

[The taxonomy of the Khasan virus (KHAV), a new representative of phlebovirus genera (Bunyaviridae), isolated from the ticks haemaphysalis longicornis (Neumann, 1901) in the Maritime Territory (Russia)].

The Khasan virus (KHAV) was originally isolated in Khasansky District and Maritime Territory in 1971 from the ticks Haemophysalis longicornis (Neumann, 1901) collected from the deers Cervus nippon (Temmink, 1838). Based on the biological properties and virion morphology, KHAV was identified as an unclassified member of the Bunyaviridae family. In order to elucidate the KHAV taxonomy in more detail, viral genome was partially sequenced using the next-generation sequencing technology. According to the phylogenetic analysis conducted for partial sequences of the three genome segments, KHAV was attributed to the genus Phlebovirus. KHAV is phylogenetically mostly related to the viruses of the Uukuniemi group and has up to 62% identity with them. The maximum identity level is observed for sequences of the RNA-dependent-RNA-polymerase (RdRp) gene. The KHAV homology level with the tick-borne Uukuniemi group viruses is 50 to 62%; however, for the mosquito-borne phleboviruses it does not exceed 30%.

De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5.

Hepatitis C virus (HCV) shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The V(max)/K(m) ratios were statistically different between genotype 1 and the other genotypes (p<0.001), mainly due to differences in V(max) values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions.

Virological surveillance and phylogenetic analysis of the PB2 genes of influenza viruses isolated from wild water birds flying from their nesting lakes in Siberia to Hokkaido, Japan in autumn.

Recent introduction of H5N1 highly pathogenic avian influenza virus (HPAIV) in wild birds from poultry in Eurasia signaled the possibility that this virus may perpetuate in nature. Surveillance of avian influenza especially in migratory birds, therefore, has been conducted to provide information on the viruses brought by them to Hokkaido, Japan, from their nesting lakes in Siberia in autumn. During 2008-2009, 62 influenza viruses of 21 different combinations of hemagglutinin (HA) and neuraminidase (NA) subtypes were isolated. Up to September 2010, no HPAIV has been found, indicating that H5N1 HPAIV has not perpetuated at least dominantly in the lakes where ducks nest in summer in Siberia. The PB2 genes of 54 influenza viruses out of 283 influenza viruses isolated in Hokkaido in 2000-2009 were phylogenetically analysed. None of the genes showed close relation to those of H5N1 HPAIVs that were detected in wild birds found dead in Eurasia on the way back to their northern territory in spring.

A novel virus of the late blight pathogen, Phytophthora infestans, with two RNA segments and a supergroup 1 RNA-dependent RNA polymerase.

Double-stranded RNA representing four distinct electrophoretic patterns was found in a screen of Phytophthora infestans isolates. Two dsRNAs that always appeared together were sequenced. RNA 1, which was 3160 nt plus a poly (A) tail, contained a single deduced ORF with the potential to encode a polyprotein of 977 aa with motifs characteristic of supergroup I viral RdRps. The 2776 nt, polyadenylated RNA2 contained an ORF with a potential to encode a polyprotein of 847 aa including a possible trypsin-like serine protease, and a second putative ORF of unknown function. An alternative form of RNA2, in which a 19-nt stretch was replaced by a 9-nt sequence, was detected in 4 of 17 clones sequenced. Based on genome structure and phylogenetic analysis, this virus did not fit into any known virus family and we tentatively named it Phytophthora infestans RNA virus 1 (PiRV-1).

NgRDR1, an RNA-dependent RNA polymerase isolated from Nicotiana glutinosa, was involved in biotic and abiotic stresses.

The RNA-dependent RNA polymerases (RDRs) play a key role in RNA silencing, heterochromatin formation and natural gene regulation. Here, a novel RDR gene was isolated from Nicotiana glutinosa, designated as NgRDR1. The full-length cDNA of NgRDR1 encodes a 1117-amino acid protein which harbors the five conserved regions in plant RDRs, including the most remarkable motif DbDGD (b is a bulky residue). Amino acid sequence alignment revealed that NgRDR1 exhibited a high degree of identity with other higher plant RDR genes. Five exons were detected in the genomic DNA sequence, and the fourth exon is 151bp, the location and the length of which are conserved among different plant species. From the phylogenetic tree constructed with different kinds of plant RDRs, it is determined that NgRDR1 falls into group I, and is closely associated with the dicotyledons RDRs. The analysis of the 5'-flanking region of NgRDR1 revealed a group of putative cis-acting elements. The results of expression analysis showed that the transcripts of NgRDR1 can be induced by biotic stresses, such as exogenous signaling molecules including salicylic acid (SA), SA analogues, hydrogen peroxide (H(2)O(2)), and methyl jasmonate (MeJA). Furthermore, NgRDR1 expression can be up-regulated by potato virus Y (PVY), tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV), but not by potato virus X (PVX). Besides, different kinds of fungi can also induce NgRDR1 expression. These results indicate that NgRDR1 may play an important role in response to biotic and abiotic stresses.

Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA Polymerase gene families and their expression analysis during reproductive development and stress in rice.

BACKGROUND: Important developmental processes in both plants and animals are partly regulated by genes whose expression is modulated at the post-transcriptional level by processes such as RNA interference (RNAi). Dicers, Argonautes and RNA-dependent RNA polymerases (RDR) form the core components that facilitate gene silencing and have been implicated in the initiation and maintenance of the trigger RNA molecules, central to process of RNAi. Investigations in eukaryotes have revealed that these proteins are encoded by variable number of genes with plants showing relatively higher number in each gene family. To date, no systematic expression profiling of these genes in any of the organisms has been reported. RESULTS: In this study, we provide a complete analysis of rice Dicer-like, Argonaute and RDR gene families including gene structure, genomic localization and phylogenetic relatedness among gene family members. We also present microarray-based expression profiling of these genes during 14 stages of reproductive and 5 stages of vegetative development and in response to cold, salt and dehydration stress. We have identified 8 Dicer-like (OsDCLs), 19 Argonaute (OsAGOs) and 5 RNA-dependent RNA polymerase (OsRDRs) genes in rice. Based on phylogeny, each of these genes families have been categorized into four subgroups. Although most of the genes express both in vegetative and reproductive organs, 2 OsDCLs, 14 OsAGOs and 3 OsRDRs were found to express specifically/preferentially during stages of reproductive development. Of these, 2 OsAGOs exhibited preferential up-regulation in seeds. One of the Argonautes (OsAGO2) also showed specific up-regulation in response to cold, salt and dehydration stress. CONCLUSION: This investigation has identified 23 rice genes belonging to DCL, Argonaute and RDR gene families that could potentially be involved in reproductive development-specific gene regulatory mechanisms. These data provide an insight into probable domains of activity of these genes and a basis for further, more detailed investigations aimed at understanding the contribution of individual components of RNA silencing machinery during reproductive phase of plant development.

The Arabidopsis AtNPR1 inversely modulates defense responses against fungal, bacterial, or viral pathogens while conferring hypersensitivity to abiotic stresses in transgenic rice.

The nonexpressor of pathogenesis-related (PR) genes (NPR1) protein plays an important role in mediating defense responses activated by pathogens in Arabidopsis. In rice, a disease-resistance pathway similar to the Arabidopsis NPR1-mediated signaling pathway one has been described. Here, we show that constitutive expression of the Arabidopsis NPR1 (AtNPR1) gene in rice confers resistance against fungal and bacterial pathogens. AtNPR1 exerts its protective effects against fungal pathogens by priming the expression of salicylic acid (SA)-responsive endogenous genes, such as the PR1b, TLP (PR5), PR10, and PBZ1. However, expression of AtNPR1 in rice has negative effects on viral infections. The AtNPR1-expressing rice plants showed a higher susceptibility to infection by the Rice yellow mottle virus (RYMV) which correlated well with a misregulation of RYMV-responsive genes, including expression of the SA-regulated RNA-dependent RNA polymerase 1 gene (OsRDR1). Moreover, AtNPR1 negatively regulates the expression of genes playing a role in the plant response to salt and drought stress (rab21, salT, and dip1), which results in a higher sensitivity of AtNPR1 rice to the two types of abiotic stress. These observations suggest that AtNPR1 has both positive and negative regulatory roles in mediating defense responses against biotic and abiotic stresses.

Nomenclature and functions of RNA-directed RNA polymerases.

There is little relationship between eukaryotic RNA-directed RNA polymerases (RDRs), viral RNA-dependent RNA polymerases (RdRps) and DNA-dependent RNA polymerases, indicating that RDRs evolved as an independent class of enzymes early in evolution. In fungi, plants and several animal systems, RDRs play a key role in RNA-mediated gene silencing [post-transcriptional gene silencing (PTGS) in plants and RNA interference (RNAi) in non-plants] and are indispensable for heterochromatin formation, at least, in Schizosaccharomyces pombe and plants. Recent findings indicate that PTGS, RNAi and heterochromatin formation not only function as host defence mechanisms against invading nucleic acids but are also involved in natural gene regulation. RDRs are required for these processes, initiating a broad interest in this enzyme class.