- Volume 98, Issue 4, 2017

Members of the family Secoviridae are non-enveloped viruses with mono- or bipartite (RNA-1 and RNA-2) linear positive-sense ssRNA genomes with the size of the RNAs combined ranging from 9 to 13.7 kb. They are related to picornaviruses and are classified in the order Picornavirales. The majority of known members infect dicotyledonous plants and many are important plant pathogens (e.g. grapevine fanleaf virus and rice tungro spherical virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the family Secoviridae available at www.ictv.global/report/secoviridae.

Rotavirus A is one of the main causative agents of diarrhoea in lactating and weaned pigs worldwide. Its impact in the swine industry is well documented. However, in Chile, the current epidemiological status of rotavirus on porcine farms is unknown. This study evaluated the current epidemiologic status of rotavirus A infection in Chile using on-farm detection techniques, electrophoretic confirmation, genotyping and phylogenetic clustering by analysis of partial sequences of VP4 and VP7 genes. Rotavirus A was detected in four out of five farms with an overall prevalence of 17.7 % in diarrhoeic pigs. The average age of diarrhoea onset was at 32±6.2 days, corresponding to weaning pigs, and rotavirus was not detected in lactating piglets. Molecular characterization indicated that genotypes G5, G3, P[7] and P[13] are currently the most widely represented on these pigs farms. The phylogenetic analysis showed that farms shared similar G types (VP7), which might denote a common origin. Meanwhile, [P] types (VP4) showed considerable genetic diversity, and this might represent a high rate of reassortment of this genetic segment in rotavirus circulating in the researched area. These findings demonstrate the importance of considering both the geographical and production factors to accurately determine rotavirus prevalence status at the national level, and have relevant implications in determining effective strategies for rotavirus infection control on porcine farms.

The endemicity of avian influenza viruses (AIVs) among Egyptian poultry represents a public health risk. Co-circulation of low pathogenic AIV H9N2 subtype with highly pathogenic AIV H5N1 subtype in Egyptian farms provides a possibility to generate novel reassortant viruses. Here, the genetic characteristics of surface glycoproteins of 59 Egyptian H9N2 viruses, isolated between 2013 and 2015, were analysed. To elucidate the potential of genetic reassortment, 10 H9N2 isolates were selected based on different avian hosts (chickens, ducks, pigeons and quails) and phylogenetic analyses of their full genome sequences were conducted. Additionally, we performed antigenic analysis to further investigate the antigenic evolution of H9N2 viruses isolated during 2011–2015. Different viral characteristics including receptor-binding affinity and drug resistance of representative Egyptian H9N2 viruses were further investigated. The surface glycoproteins of current Egyptian H9N2 viruses were closely related to viruses of the G1-like lineage isolated from Egypt. Several genetic markers that enhance virulence in poultry and transmission to humans were detected. Analysis of the full genome of 10 H9N2 isolates indicated that two pigeon isolates inherited five internal genes from Eurasian AIVs circulating in wild birds. Antigenic conservation of different Egyptian H9N2 isolates from chickens, pigeons and ducks was observed, whereas quail isolates showed antigenic drift. The Egyptian H9N2 viruses preferentially bound to the human-like receptor rather than to the avian-like receptor. Our results suggest that the endemic H9N2 viruses in Egypt contain elements that may favour avian-to-human transmission and thus represent a public health risk.

Viruses of the genus Henipavirus of the family Paramyxoviridae are zoonotic pathogens, which have emerged in Southeast Asia, Australia and Africa. Nipah virus (NiV) and Hendra virus are highly virulent pathogens transmitted from bats to animals and humans, while the henipavirus Cedar virus seems to be non-pathogenic in infection studies. The full replication cycle of the Paramyxoviridae occurs in the host cell’s cytoplasm, where viral assembly is orchestrated by the matrix (M) protein. Unexpectedly, the NiV-M protein traffics through the nucleus as an essential step to engage the plasma membrane in preparation for viral budding/release. Comparative studies were performed to assess whether M protein nuclear localization is a common feature of the henipaviruses, including the recently sequenced (although not yet isolated) Ghanaian bat henipavirus (Kumasi virus, GH-M74a virus) and Mojiang virus. Live-cell confocal microscopy revealed that nuclear translocation of GFP-fused M protein is conserved between henipaviruses in both human- and bat-derived cell lines. However, the efficiency of M protein nuclear localization and virus-like particle budding competency varied. Additionally, Cedar virus-, Kumasi virus- and Mojiang virus-M proteins were mutated in a bipartite nuclear localization signal, indicating that a key lysine residue is essential for nuclear import, export and induction of budding events, as previously reported for NiV-M. The results of this study suggest that the M proteins of henipaviruses may utilize a similar nucleocytoplasmic trafficking pathway as an essential step during viral replication in both humans and bats.

In our previous studies, the reassortant virus containing only the PR8 H1N1 matrix (M) gene in the background of the modified bat influenza Bat09 : mH1mN1 virus could be generated. However, whether M genes from other origins can be rescued in the background of the Bat09 : mH1mN1 virus and whether the resulting novel reassortant virus is virulent remain unknown. Herein, two reassortant viruses were generated in the background of the Bat09 : mH1mN1 virus containing either a North American or a Eurasian swine influenza virus M gene. These two reassortant viruses and the reassortant virus with PR8 M as well as the control Bat09 : mH1mN1 virus replicated efficiently in cultured cells, while the reassortant virus with PR8 M grew to a higher titre than the other three viruses in tested cells. Mouse studies showed that reassortant viruses with either North American or Eurasian swine influenza virus M gene did not enhance virulence, whereas the reassortant virus with PR8 M gene displayed higher pathogenicity when compared to the Bat09 : mH1mN1 virus. This is most likely due to the fact that the PR8 H1N1 virus is a mouse-adapted virus. Furthermore, reassortment potential between the Bat09 : mH1mN1 virus and an H3N2 swine influenza virus (A/swine/Texas/4199-2/1998) was investigated using co-infection of Madin–Darby canine kidney cells, but no reassortant viruses were detected. Taken together, our results indicate that the modified bat influenza virus is most likely incapable of reassortment with influenza A viruses with in vitro co-infection experiments, although reassortant viruses with different M genes can be generated by reverse genetics.

The genus Phlebovirus includes the sandfly fever viruses and tick-transmitted uukuviruses. Sandfly fever group viruses have been isolated from various vertebrate species and from phlebotomines and occasionally alternative arthropods, e.g. mosquitoes, or ceratopogonids of the genus Culicoides. Uukuniemi serogroup viruses have been isolated from various vertebrate species and from ticks. Despite the public health importance of some viruses of the genus, the genomic diversity of phleboviruses that could be incriminated as causative of human or veterinary diseases remains underestimated. Here we describe the nearly complete sequences and genomic characterization of two phleboviruses belonging to the Bujaru antigenic complex: the prototype species and the Munguba virus. Furthermore, six previously unclassified phleboviruses isolated in Brazil were also sequenced and characterized: Ambe, Anhanga, Joa, Uriurana, Urucuri and Tapara viruses. The results of the phylogenetic analysis indicated that these viruses group with viruses of three antigenic complexes (Bujaru, Tapara and frijoles clades), with two unclassified phleboviruses. We also performed genomic reassortment analysis and confirmed that there were no events for the viruses described in this study, but we found a new potential reassortment in Medjerda Valley virus, which contains S and L segments of Arbia virus, and probably a unique M segment, both viruses circulate in the same geographic region, indicating these two isolates represent two distinct viruses. This study provides insights into the genetic diversity, classification and evolution of phleboviruses.

The putatively non-virulent subtype of infectious salmon anaemia virus (ISAV), ISAV-HPR0, is proposed to act as a progenitor and reservoir for all virulent ISAVs and thus represent a potential risk factor for the emergence of infectious salmon anaemia (ISA) disease. Here, we provide the first evidence of genetic and functional evolution from an ISAV-HPR0 variant (FO/07/12) to a low-virulent ISAV virus (FO/121/14) in a Faroese Atlantic salmon marine farm. The FO/121/14 virus infection was not associated with specific clinical signs of ISA and was confined to a single net-pen, while various ISAV-HPR0 subtypes were found circulating in most epidemiologically linked marine and freshwater farms. Sequence analysis of all eight segments revealed that the FO/121/14 virus was identical, apart from a substitution in the fusion (F) gene (Q266L) and a deletion in the haemagglutinin-esterase (HE) gene, to the FO/07/12 variant from a freshwater farm, which supplied smolts exclusively to the FO/121/14-positive net-pen. An immersion challenge with the FO/121/14 virus induced a systemic infection in Atlantic salmon associated with a low mortality and mild clinical signs confirming its low pathogenicity. Our results demonstrate that mutations in the F protein and deletions in the highly polymorphic region (HPR) of the HE protein represent a minimum requirement for ISAV to gain virulence and to switch cell tropism from a localized epithelial infection to a systemic endotheliotropic infection. This documents that ISAV-HPR0 represents a reservoir and risk factor for the emergence of ISA disease.

Two goose megriviruses (W18 and HN56) were detected and sequenced. Both viruses possessed megrivirus-like genomic features, including unusually long genomes (9840 and 10 101 nt). W18 and HN56 were very similar to duck megrivirus (DMV) in the P2 and P3 regions, but much less similar in the P1 and 2A1 regions. HN56 may be a potential recombinant virus, with a distinct P1 region possibly originating from an unknown picornavirus. W18 may represent a novel type of DMV, showing a P1 sequence identity of 67 %. Similar levels (64–68 %) of P1 sequence identity were also displayed by melegrivirus A with W18 and DMV, demonstrating an equal genetic separation of the capsid region among W18, DMV and melegrivirus A. For the 2A1 region, the divergence among W18, HN56 and DMV was remarkable, involving point mutations and a long insertion/deletion. The present work contributes to the understanding of unique features and phylogeny of megriviruses.

With advances in viral surveillance and next-generation sequencing, highly diverse novel astroviruses (AstVs) and different animal hosts had been discovered in recent years. However, the existence of AstVs in marmots had yet to be shown. Here, we identified two highly divergent strains of AstVs (tentatively named Qinghai Himalayanmarmot AstVs, HHMAstV1 and HHMAstV2), by viral metagenomic analysis in liver tissues isolated from wild Marmota himalayana in China. Overall, 12 of 99 (12.1 %) M . himalayana faecal samples were positive for the presence of genetically diverse AstVs, while only HHMAstV1 and HHMAstV2 were identified in 300 liver samples. The complete genomic sequences of HHMAstV1 and HHMAstV2 were 6681 and 6610 nt in length, respectively, with the typical genomic organization of AstVs. Analysis of the complete ORF 2 sequence showed that these novel AstVs are most closely related to the rabbit AstV, mamastrovirus 23 (with 31.0 and 48.0 % shared amino acid identity, respectively). Phylogenetic analysis of the amino acid sequences of ORF1a, ORF1b and ORF2 indicated that HHMAstV1 and HHMAstV2 form two distinct clusters among the mamastroviruses, and may share a common ancestor with the rabbit-specific mamastrovirus 23. These results suggest that HHMAstV1 and HHMAstV2 are two novel species of the genus Mamastrovirus in the Astroviridae. The remarkable diversity of these novel AstVs will contribute to a greater understanding of the evolution and ecology of AstVs, although additional studies will be needed to understand the clinical significance of these novel AstVs in marmots, as well as in humans.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viruses affecting the swine industry worldwide. MicroRNAs have recently been demonstrated to play vital roles in virus–host interactions. Our previous research on small RNA deep sequencing showed that the expression level of miR-10a increased during the viral life cycle. The present study sought to determine the function of miR-10a and its molecular mechanism during PRRSV infection. In the current study, the result of PRRSV infection inducing miR-10a expression was validated by quantitative reverse transcriptase PCR. Overexpression of miR-10a-5p using its mimics markedly reduced the expression level of intracellular PRRSV ORF7 mRNA and N protein. Simultaneously, overexpression of miR-10a-5p also significantly decreased the expression level of extracellular viral RNA and virus titres in the supernatants. These results demonstrated that miR-10a-5p could suppress the replication of PRRSV. A direct interaction between miR-10a-5p and signal recognition particle 14 (SRP14) was confirmed using bioinformatic prediction and experimental verification. miR-10a-5p could directly target the 3′UTR of pig SRP14 mRNA in a sequence-specific manner and decrease SRP14 expression through translational repression but not mRNA degradation. Further, knockdown of SRP14 by small interfering RNA also inhibits the replication of PRRSV. Collectively, these results suggested that miR-10a-5p inhibits PRRSV replication through suppression of SRP14 expression, which not only provides new insights into virus–host interactions during PRRSV infection but also suggests potential new antiviral strategies against PRRSV infection.

Several RNA interactions are thought to play a role in the regulation of the hepatitis C virus (HCV) life cycle. Most of these interactions involve the 5BSL3.2 domain and therefore occur at the 3′ end of the viral genomic RNA. A long-range interaction has also been described between 5BSL3.2 and the 5′ untranslated region (UTR). Another interaction involves the SLVI stem loop of the core coding region and the 5′UTR. We aimed to analyse the role of this SLVI domain, which likely interferes with others interactions. By evaluating RNA stability, translation and RNA synthesis, we showed that the SLVI stem loop extensively modulates the effect of the interactions mediated by the 5BSL3.2 domain and strongly affects the IIId/5BSL3.2 interaction. Numerous interactions in HCV genomic RNA have been described in the UTRs and the coding sequence but their roles are poorly understood. We showed that the SLVI domain located in the core coding sequence plays an important role in the translation of the polyprotein, but also in the modulation of long-range RNA interactions centred on the 5BSL3.2 domain. The SLVI domain has been absent from most studies, especially from the extensively used subgenomic replicon; our data highlight the importance of this domain in the studies of these long-range interactions in the HCV life cycle.

Enterovirus 71 (EV71) is a causative agent of hand, foot and mouth disease (HFMD). EV71 causes fever, rash, diarrhoea and, in some cases, acute encephalopathy/encephalitis, which can be fatal. No specific treatment is currently available for EV71 infection. Here, we conducted a cDNA library screen and identified acid β-glucosidase 1 (GBA1; also known as β-glucocerebrosidase) as an EV71 resistance factor. The anti-EV71 function of GBA1 was verified by gene transduction and knockdown experiments. Cerezyme, a molecular drug used to treat Gaucher’s disease and having recombinant human GBA1 as the active ingredient, protected against EV71 infection. The anti-EV71 activity of GBA1 was bimodal: endogenous GBA1 restricted cell surface expression levels of scavenger receptor class B, member 2 (SCARB2), also known as lysosomal integral membrane protein 2 (LIMP-2), and exogenous recombinant GBA1 interfered with EV71 to interact with SCARB2 outside the cell. Thus, our findings suggest that GBA1 may represent a novel molecular target for the treatment of EV71 infection.

Viral covert mortality disease (VCMD) has caused serious losses to shrimp aquaculture in China in recent years and the ridgetail white prawn Exopalaemon carinicauda has been suspected to be one important factor in perpetuating the high prevalence of covert mortality nodavirus (CMNV) infections due to its perennial presence in shrimp farming ponds and water from natural habitats. Experiments were carried out to determine the possibility of vertical transmission of CMNV in E. carinicauda in this study. CMNV infection in gonads, fertilized eggs and larvae was investigated by using the methods of reverse transcription nested PCR (nRT-PCR), in situ hybrization (ISH) and transmission electron microscopy (TEM). The ovarian tissue and testis tissue of artificially infected parental E. carinicauda were proved to be CMNV-positive by nRT-PCR. Fertilized eggs were also found to be CMNV-positive by nRT-PCR whether the fertilized eggs originated from the CMNV-positive female broodstock mated with the CMNV-negative male broodstock, or they originated from the CMNV-negative female broodstock mated with the CMNV-positive males. The results of ISH indicated that the positive signals were evident in the oocytes within ovarian tissue and nauplii. By TEM analysis, CMNV virions were observed in oogonia, oocytes, spermatocytes, fertilized eggs and nauplii. The presence of CMNV in fertilized eggs and larvae indicates that CMNV can transmit vertically via sperm and oocytes in E. carinicauda, which highlights the high probability of vertical transmission of CMNV in the main species of cultured shrimp and prawns.

Rodent models have been used extensively to study West Nile virus (WNV) infection because they develop severe neurological symptoms similar to those observed in human WNV neuroinvasive disease. Most of this research has focused on old lineage (L) 1 strains, while information about pathogenicity is lacking for the most recent L1 and L2 strains, as well as for newly defined lineages. In this study, 4-week-old Swiss mice were inoculated with a collection of 12 WNV isolates, comprising 10 old and recent L1 and L2 strains, the putative L6 strain from Malaysia and the proposed L7 strain Koutango (KOU). The intraperitoneal inoculation of 10-fold dilutions of each strain allowed the characterization of the isolates in terms of LD50, median survival times, ID50, replication in neural and extraneural tissues and antibody production. Based on these results, we classified the isolates in three groups: high virulence (all L1a strains, recent L2 strains and KOU), moderate virulence (B956 strain) and low virulence (Kunjin and Malaysian isolates). We determined that the inoculation of a single dose of 1000 p.f.u. would be sufficient to classify WNV strains by pathotype. We confirmed the enhanced virulence of the KOU strain with a high capacity to cause rapid systemic infection. We also corroborated that differences in pathogenicity among strains do not correlate with phylogenetic lineage or geographic origin, and confirmed that recent European and African WNV strains belonging to L1 and L2 are highly virulent and do not differ in their pathotype profile compared to the prototype NY99 strain.

Foot-and-mouth disease virus is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell cap-dependent protein synthesis within infected cells. This results from the Lpro-dependent cleavage of the cellular translation initiation factor eIF4G. Lpro also releases itself from the virus capsid precursor by cleaving the L/P1 junction. Using site-directed mutagenesis of the Lpro coding sequence, we have investigated the role of 51 separate amino acid residues in the functions of this protein. These selected residues either are highly conserved or are charged and exposed on the protein surface. Using transient expression assays, within BHK-21 cells, it was found that residues around the active site (W52, L53 and A149) of Lpro and others located elsewhere (K38, K39, R44, H138 and W159) are involved in the induction of eIF4G cleavage but not in the processing of the L/P1 junction. Modified viruses, encoding such amino acid substitutions within Lpro, can replicate in BHK-21 cells but did not grow well in primary bovine thyroid cells. This study characterizes mutant viruses that are deficient in blocking host cell responses to infection (e.g. interferon induction) and can assist in the rational design of antiviral agents targeting this process and in the production of attenuated viruses.

Persistent human papillomavirus (HPV) infection is a key event in HPV-induced carcinogenesis. As part of the prospective Finnish Family HPV Study, we analysed the physical state and viral copy numbers of HPV16 in asymptomatic oral infections that either persisted or cleared during the 6-year follow-up. The persister group comprised 14 women and 7 men with 51 and 21 HPV16-positive brush samples. The clearance group included 41 women and 13 men, with 64 and 24 samples, respectively. Physical state and viral DNA load were assessed by using quantitative PCR for HPV16 E2 and E6 genes. E2/E6 ratio was calculated and HPV16 was classified as episomal, mixed or integrated with values of 0.93–1.08, <0.93 and 0, respectively. In both genders, the physical state of HPV16 was significantly different between the cases and controls (P<0.001). HPV16 was episomal in all men and 66 % (27/41) of women who cleared their infection. HPV16 was mixed and/or integrated in71 % and 57 %of the women and men persisters, respectively. The mean HPV16 copy number per 50 ng genomic DNA was nearly 5.5-fold higher in the women than in the men clearance group (P=0.011). Only in men, HPV16 copy numbers were higher in persisters than in the clearance group (P=0.039). To conclude, in both genders, persistent oral HPV16 infections were associated with the mixed or integrated form of HPV16, while in the clearance groups, episomal HPV16 predominated. This indicates that HPV16 integration is a common event even in asymptomatic oral infections, which might predispose the infected subjects to progressive disease.

This study is focused on the identification of the faecal virome of healthy chickens raised in high-density, export-driven poultry farms in Brazil. Following high-throughput sequencing, a total of 7743 de novo-assembled contigs were constructed and compared with known nucleotide/amino acid sequences from the GenBank database. Analyses with blastx revealed that 279 contigs (4 %) were related to sequences of eukaryotic viruses. Viral genome sequences (total or partial) indicative of members of recognized viral families, including Adenoviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae and Reoviridae, were identified, some of those representing novel genotypes. In addition, a range of circular replication-associated protein encoding DNA viruses were also identified. The characterization of the faecal virome of healthy chickens described here not only provides a description of the viruses encountered in such niche but should also represent a baseline for future studies comparing viral populations in healthy and diseased chicken flocks. Moreover, it may also be relevant for human health, since chickens represent a significant proportion of the animal protein consumed worldwide.