Small RNA Response to Infection of the Insect-Specific Lammi Virus and Hanko Virus in an Aedes albopictus Cell Line

RNA interference (RNAi)-mediated antiviral immunity is believed to be the primary defense against viral infection in mosquitoes. The production of virus-specific small RNA has been demonstrated in mosquitoes and mosquito-derived cell lines for viruses in all of the major arbovirus families. However, many if not all mosquitoes are infected with a group of viruses known as insect-specific viruses (ISVs), and little is known about the mosquito immune response to this group of viruses. Therefore, in this study, we sequenced small RNA from an Aedes albopictus-derived cell line infected with either Lammi virus (LamV) or Hanko virus (HakV). These viruses belong to two distinct phylogenetic groups of insect-specific flaviviruses (ISFVs). The results revealed that both viruses elicited a strong virus-derived small interfering RNA (vsiRNA) response that increased over time and that targeted the whole viral genome, with a few predominant hotspots observed. Furthermore, only the LamV-infected cells produced virus-derived Piwi-like RNAs (vpiRNAs); however, they were mainly derived from the antisense genome and did not show the typical ping-pong signatures. HakV, which is more distantly related to the dual-host flaviviruses than LamV, may lack certain unknown sequence elements or structures required for vpiRNA production. Our findings increase the understanding of mosquito innate immunity and ISFVs' effects on their host

Transcriptome Analysis of an Aedes albopictus Cell Line Single- and Dual-Infected with Lammi Virus and WNV

Understanding the flavivirus infection process in mosquito hosts is important and fundamental in the search for novel control strategies that target the mosquitoes' ability to carry and transmit pathogenic arboviruses. A group of viruses known as insect-specific viruses (ISVs) has been shown to interfere with the infection and replication of a secondary arbovirus infection in mosquitoes and mosquito-derived cell lines. However, the molecular mechanisms behind this interference are unknown. Therefore, in the present study, we infected the Aedes albopictus cell line U4.4 with either the West Nile virus (WNV), the insect-specific Lammi virus (LamV) or an infection scheme whereby cells were pre-infected with LamV 24 h prior to WNV challenge. The qPCR analysis showed that the dual-infected U4.4 cells had a reduced number of WNV RNA copies compared to WNV-only infected cells. The transcriptome profiles of the different infection groups showed a variety of genes with altered expression. WNV-infected cells had an up-regulation of a broad range of immune-related genes, while in LamV-infected cells, many genes related to stress, such as different heat-shock proteins, were up-regulated. The transcriptome profile of the dual-infected cells was a mix of up- and down-regulated genes triggered by both viruses. Furthermore, we observed an up-regulation of signal peptidase complex (SPC) proteins in all infection groups. These SPC proteins have shown importance for flavivirus assembly and secretion and could be potential targets for gene modification in strategies for the interruption of flavivirus transmission by mosquitoes

Congenital tremor and splay leg in piglets - insights into the virome, local cytokine response, and histology

Background: Atypical porcine pestivirus (APPV) is a neurotropic virus associated with congenital tremor type A-II. A few experimental studies also indicate an association between APPV and splay leg. The overarching aim of the present study was to provide insights into the virome, local cytokine response, and histology of the CNS in piglets with signs of congenital tremor or splay leg.Results: Characterization of the cytokine profile and virome of the brain in piglets with signs of congenital tremor revealed an APPV-associated upregulation of Stimulator of interferon genes (STING). The upregulation of STING was associated with an increased expression of the gene encoding IFN-alpha but no differential expression was recorded for the genes encoding CXCL8, IFN-beta, IFN-gamma, IL-1 beta, IL-6, or IL-10. No viral agents or cytokine upregulation could be detected in the spinal cord of piglets with signs of splay leg or in the brain of piglets without an APPV-infection. The histopathological examination showed no lesions in the CNS that could be attributed to the APPV-infection, as no difference between sick and healthy piglets could be seen.Conclusion: The results from this study provide evidence of an APPV-induced antiviral cytokine response but found no lesions related to the infection nor any support for a common causative agent

Identification and molecular characterization of highly divergent RNA viruses in cattle, Uganda

The risk for the emergence of novel viral zoonotic diseases in animals and humans in Uganda is high given its geographical location with high biodiversity. We aimed to identify and characterize viruses in 175 blood samples from cattle selected in Uganda using molecular approaches. We identified 8 viral species belonging to 4 families (Flaviviridae, Peribunyaviridae, Reoviridae and Rhabdoviridae) and 6 genera (Hepacivirus, Pestivirus, Orthobunya-virus, Coltivirus, Dinovernavirus and Ephemerovirus). Four viruses were highly divergent and tetantively named Zikole virus (Family: Flaviviridae), Zeboroti virus (Family: Reoviridae), Zebtine virus (Family: Rhabdoviridae) and Kokolu virus (Family: Rhabdoviridae). In addition, Bovine Hepacivirus, Obodhiang virus, Aedes pseudoscutellaris reovirus and Schmallenberg virus were identified for the first time in Ugandan cattle. We report 8 viral species belonging to 4 viral families including divergent ones in the blood of cattle in Uganda. Hence, cattle may be reservoir hosts for likely emergence of novel viruses with pathogenic potential to cause zoonotic diseases in different species with serious public health implications

Evolutionary genetics of canine respiratory coronavirus and recent introduction into Swedish dogs

Canine respiratory coronavirus (CRCoV) has been identified as a causative agent of canine infectious respiratory disease, an upper respiratory infection affecting dogs. The epidemiology is currently opaque, with an unclear understanding of global prevalence, pathology, and genetic characteristics. In this study, Swedish privatelyowned dogs with characteristic signs of canine infectious respiratory disease (n = 88) were screened for CRCoV and 13 positive samples (14.7%, 8.4–23.7% [95% confidence interval (CI)]) were further sequenced. Sequenced Swedish CRCoV isolates were highly similar despite being isolated from dogs living in geographically distant locations and sampled across 3 years (2013–2015). This is due to a single introduction into Swedish dogs in approximately 2010, as inferred by time structured phylogeny. Unlike other CRCoVs, there was no evidence of recombination in Swedish CRCoV isolates, further supporting a single introduction. Finally, there were low levels of polymorphisms, in the spike genes. Overall, we demonstrate that there is little diversity of CRCoV which is endemic in Swedish dogs

Large-scale analysis of putative plasmids in clinical multidrug-resistant Escherichia coli isolates from Vietnamese patients

IntroductionIn the past decades, extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant (CR) Escherichia coli isolates have been detected in Vietnamese hospitals. The transfer of antimicrobial resistance (AMR) genes carried on plasmids is mainly responsible for the emergence of multidrug-resistant E. coli strains and the spread of AMR genes through horizontal gene transfer. Therefore, it is important to thoroughly study the characteristics of AMR gene-harboring plasmids in clinical multidrug-resistant bacterial isolates.MethodsThe profiles of plasmid assemblies were determined by analyzing previously published whole-genome sequencing data of 751 multidrug-resistant E. coli isolates from Vietnamese hospitals in order to identify the risk of AMR gene horizontal transfer and dissemination.ResultsThe number of putative plasmids in isolates was independent of the sequencing coverage. These putative plasmids originated from various bacterial species, but mostly from the Escherichia genus, particularly E. coli species. Many different AMR genes were detected in plasmid contigs of the studied isolates, and their number was higher in CR isolates than in ESBL-producing isolates. Similarly, the blaKPC-2, blaNDM-5, blaOXA-1, blaOXA-48, and blaOXA-181 β-lactamase genes, associated with resistance to carbapenems, were more frequent in CR strains. Sequence similarity network and genome annotation analyses revealed high conservation of the β-lactamase gene clusters in plasmid contigs that carried the same AMR genes.DiscussionOur study provides evidence of horizontal gene transfer in multidrug-resistant E. coli isolates via conjugative plasmids, thus rapidly accelerating the emergence of resistant bacteria. Besides reducing antibiotic misuse, prevention of plasmid transmission also is essential to limit antibiotic resistance

Development of HBVdb, a knowledge database for Hepatitis B Virus, for the study of drug resistance : integration of sequence analysis tools and application to the polymerase molecular modeling

Nous avons développé la base HBVdb (http://hbvdb.ibcp.fr) pour permettre aux chercheurs d'étudier les caractéristiques génétiques et la variabilité des séquences du virus de l'hépatite B (VHB), ainsi que la résistance virale aux traitements. HBVdb contient une collection de séquences annotées automatiquement sur la base de génomes de référence annotés manuellement, ce qui assure une nomenclature normalisée pour toutes les entrées de la base. HBVdb est accessible via un site Web dédié avec des outils d'analyses génériques et spécialisés (annotation, génotypage, détection de profils de résistance), et des jeux de données pré-calculés. La polymérase du VHB est la principale cible des traitements anti-VHB. Les analogues de nucléos(t)ides (NA) inhibent l'activité de la transcriptase inverse (RT), mais il existe des mutations de résistance aux NA. Cependant, un autre domaine enzymatique pourrait être une cible potentielle : la RNase H, liée au domaine RT, permettant la dégradation de l'ARN durant la transcription inverse. Pour pallier l'absence d'une structure expérimentale résolue, et grâce à l'analyse de séquences à partir de HBVdb, nous avons construit le modèle par homologie de la RNase H, qui a permis de définir les caractéristiques de cette RNase H de type 1. Enfin pour vérifier des hypothèses émises à partir de ce modèle, et pour le placer dans son contexte, nous avons construit un modèle plus étendu de la polymérase du VHB, qui comprend la les domaines RT et RNase H, et contribue à répondre à la question sur l'existence d'un domaine de connexion les reliant. Nous avons utilisé notre modèle pour analyser les interactions entre le site catalytique de la RT et le ténofovirWe developed HBVdb (http://hbvdb.ibcp.fr) to allow researchers to investigate the geneticcharacteristics and variability of the HBV sequences and viral resistance to treatment. HBVdb contains a collection of computer-annotated sequences based on manually annotated reference genomes. The automatic annotation procedure ensures standardized nomenclature for all HBV entries across the database. HBVdb is accessible through a dedicated website integrating generic and specialized analysis tools (annotation, genotyping, resistance profile detection), and pre- computed datasets. The HBV polymerase is the main target of anti-HBV drugs, nucleos(t)ides analogues (NA), which inhibit the activity of reverse transcriptase (RT), but NA resistance mutations appeared. Nevertheless, another enzymatic domain could be a potential drug target: RNase H domain, linked to RT, and involved in degradation of the RNA during the reverse transcription. To overcome the lack of experimental solved structure, thanks to sequences analysis from HBVdb, we built an homology model of RNase H, which helped to define the features of this type 1 RNase H. Finally, to confirm assumptions from this model and to put it in a more global context, we built an extensive HBV polymerase model, which includes the RT and RNase H domains, and helps to answer the question about the existence of connection domain linking them. We performed analyses on this model, regarding the interactions between the RT catalytic site and the Tenofovir, mapping known resistance mutations and the most variables positions of the HBV polymeras

Développement d'une base de connaissances du virus de l'hépatite B, HBVdb, pour l'étude de la résistance aux traitements : intégration d'outils d'analyses de séquences et application à la modélisation moléculaire de la polymérase

We developed HBVdb (http://hbvdb.ibcp.fr) to allow researchers to investigate the geneticcharacteristics and variability of the HBV sequences and viral resistance to treatment. HBVdb contains a collection of computer-annotated sequences based on manually annotated reference genomes. The automatic annotation procedure ensures standardized nomenclature for all HBV entries across the database. HBVdb is accessible through a dedicated website integrating generic and specialized analysis tools (annotation, genotyping, resistance profile detection), and pre- computed datasets. The HBV polymerase is the main target of anti-HBV drugs, nucleos(t)ides analogues (NA), which inhibit the activity of reverse transcriptase (RT), but NA resistance mutations appeared. Nevertheless, another enzymatic domain could be a potential drug target: RNase H domain, linked to RT, and involved in degradation of the RNA during the reverse transcription. To overcome the lack of experimental solved structure, thanks to sequences analysis from HBVdb, we built an homology model of RNase H, which helped to define the features of this type 1 RNase H. Finally, to confirm assumptions from this model and to put it in a more global context, we built an extensive HBV polymerase model, which includes the RT and RNase H domains, and helps to answer the question about the existence of connection domain linking them. We performed analyses on this model, regarding the interactions between the RT catalytic site and the Tenofovir, mapping known resistance mutations and the most variables positions of the HBV polymeraseNous avons développé la base HBVdb (http://hbvdb.ibcp.fr) pour permettre aux chercheurs d'étudier les caractéristiques génétiques et la variabilité des séquences du virus de l'hépatite B (VHB), ainsi que la résistance virale aux traitements. HBVdb contient une collection de séquences annotées automatiquement sur la base de génomes de référence annotés manuellement, ce qui assure une nomenclature normalisée pour toutes les entrées de la base. HBVdb est accessible via un site Web dédié avec des outils d'analyses génériques et spécialisés (annotation, génotypage, détection de profils de résistance), et des jeux de données pré-calculés. La polymérase du VHB est la principale cible des traitements anti-VHB. Les analogues de nucléos(t)ides (NA) inhibent l'activité de la transcriptase inverse (RT), mais il existe des mutations de résistance aux NA. Cependant, un autre domaine enzymatique pourrait être une cible potentielle : la RNase H, liée au domaine RT, permettant la dégradation de l'ARN durant la transcription inverse. Pour pallier l'absence d'une structure expérimentale résolue, et grâce à l'analyse de séquences à partir de HBVdb, nous avons construit le modèle par homologie de la RNase H, qui a permis de définir les caractéristiques de cette RNase H de type 1. Enfin pour vérifier des hypothèses émises à partir de ce modèle, et pour le placer dans son contexte, nous avons construit un modèle plus étendu de la polymérase du VHB, qui comprend la les domaines RT et RNase H, et contribue à répondre à la question sur l'existence d'un domaine de connexion les reliant. Nous avons utilisé notre modèle pour analyser les interactions entre le site catalytique de la RT et le ténofovi

Screening for atypical porcine pestivirus in Swedish boar semen used for artificial insemination and a characterisation of the seminal RNA microbiome including the virome

Background This study aimed to characterise the RNA microbiome, including the virome of extended semen from Swedish breeding boars, with particular focus on Atypical porcine pestivirus (APPV). This neurotropic virus, associated with congenital tremor type A-II in piglets, was recently demonstrated to induce the disease through insemination with semen from infected boars. Results From 124 Artificial Insemination (AI) doses from Swedish breeding boars, APPV was detected in one dose in addition to a sparse seminal RNA virome, characterised by retroviruses, phages, and some fecal-associated contaminants. The detected seminal microbiome was large and characterized by Gram-negative bacteria from the phylum Proteobacteria, mainly consisting of apathogenic or opportunistic bacteria. The proportion of bacteria with a pathogenic potential was low, and no antimicrobial resistance genes (ARGs) were detected in the datasets. Conclusion Overall, the results indicate a good health status among Swedish breeding boars. The detection of APPV in semen raises the question of whether routine screening for APPV in breeding boars should be instigated

Four novel picornaviruses detected in Magellanic Penguins (Spheniscus magellanicus) in Chile

Members of the Picornaviridae family comprise a significant burden on the poultry industry, causing diseases such as gastroenteritis and hepatitis. However, with the advent of metagenomics, a number of picornaviruses have now been revealed in apparently healthy wild birds. In this study, we identified four novel viruses belonging to the family Picornaviridae in healthy Magellanic penguins, a near threatened species. All samples were subsequently screened by RT-PCR for these new viruses, and approximately 20% of the penguins were infected with at least one of these viruses. The viruses were distantly related to members of the genera Hepatovirus, Tremovirus, Gruhelivirus and Crahelvirus. Further, they had more than 60% amino acid divergence from other picornaviruses, and therefore likely constitute novel genera. Our results demonstrate the vast undersampling of wild birds for viruses, and we expect the discovery of numerous avian viruses that are related to hepatoviruses and tremoviruses in the future