Whole genome sequencing and phylogenetic characterisation of rabies virus strains from Moldova and north-eastern Romania.

BACKGROUND: Rabies is the oldest fatal zoonotic disease recognised as a neglected tropical disease and is caused by an RNA virus belonging to the genus Lyssavirus, family Rhabdoviridae. METHODOLOGY/PRINCIPAL FINDINGS: A deep molecular analysis was conducted on full-length nucleoprotein (N) gene and whole genome sequences of rabies virus from 37 animal brain samples collected between 2012 and 2017 to study the circulation of rabies virus (RABV) variants. The overall aim was to better understand their distribution in Moldova and north-eastern Romania. Both Sanger and high throughput sequencing on Ion Torrent and Illumina platforms were performed. Phylogenetic analysis of the RABV sequences from both Moldova and Romania revealed that all the samples (irrespective of the year of isolation and the species) belonged to a single phylogenetic group: north-eastern Europe (NEE), clustering into three assigned lineages: RO#5, RO#6 and RO#7. CONCLUSIONS/SIGNIFICANCE: High throughput sequencing of RABV samples from domestic and wild animals was performed for the first time for both countries, providing new insights into virus evolution and epidemiology in this less studied region, expanding our understanding of the disease.

Multiple independent introductions of highly pathogenic avian influenza H5 viruses during the 2020-2021 epizootic in France.

During winter 2020-2021, France and other European countries were severely affected by highly pathogenic avian influenza H5 viruses of the Gs/GD/96 lineage, clade 2.3.4.4b. In total, 519 cases occurred, mainly in domestic waterfowl farms in Southwestern France. Analysis of viral genomic sequences indicated that 3 subtypes of HPAI H5 viruses were detected (H5N1, H5N3, H5N8), but most French viruses belonged to the H5N8 subtype genotype A, as Europe. Phylogenetic analyses of HPAI H5N8 viruses revealed that the French sequences were distributed in 9 genogroups, suggesting 9 independent introductions of H5N8 from wild birds, in addition to the 2 introductions of H5N1 and H5N3.

Concomitant NA and NS deletion on avian Influenza H3N1 virus associated with hen mortality in France in 2019.

An H3N1 avian influenza virus was detected in a laying hens farm in May 2019 which had experienced 25% mortality in Northern France. The complete sequencing of this virus showed that all segment sequences belonged to the Eurasian lineage and were phylogenetically very close to many of the Belgian H3N1 viruses detected in 2019. The French virus presented two genetic particularities with NA and NS deletions that could be related to virus adaptation from wild to domestic birds and could increase virulence, respectively. Molecular data of H3N1 viruses suggest that these two deletions occurred at two different times.

Infectious Bronchitis Coronavirus: Genome Evolution in Vaccinated and Non-Vaccinated SPF Chickens.

Infectious Bronchitis virus (IBV) continues to cause significant economic losses for the chicken industry despite the use of many live IBV vaccines around the world. Several authors have suggested that vaccine-induced partial protection may contribute to the emergence of new IBV strains. In order to study this hypothesis, three passages of a challenge IBV were made in SPF chickens sham inoculated or vaccinated at day of age using a live vaccine heterologous to the challenge virus. All birds that were challenged with vaccine heterologous virus were positive for viral RNA. NGS analysis of viral RNA in the unvaccinated group showed a rapid selection of seven genetic variants, finally modifying the consensus genome of the viral population. Among them, five were non-synonymous, modifying one position in NSP 8, one in NSP 13, and three in the Spike protein. In the vaccinated group, one genetic variant was selected over the three passages. This synonymous modification was absent from the unvaccinated group. Under these conditions, the genome population of an IBV challenge virus evolved rapidly in both heterologous vaccinated and non-vaccinated birds, while the genetic changes that were selected and the locations of these were very different between the two groups.

Genome Evolution of Two Genetically Homogeneous Infectious Bursal Disease Virus Strains During Passages in vitro and ex vivo in the Presence of a Mutagenic Nucleoside Analog.

The avibirnavirus infectious bursal disease virus (IBDV) is responsible for a highly contagious and sometimes lethal disease of chickens (Gallus gallus). IBDV genetic variation is well-described for both field and live-attenuated vaccine strains, however, the dynamics and selection pressures behind this genetic evolution remain poorly documented. Here, genetically homogeneous virus stocks were generated using reverse genetics for a very virulent strain, rvv, and a vaccine-related strain, rCu-1. These viruses were serially passaged at controlled multiplicities of infection in several biological systems, including primary chickens B cells, the main cell type targeted by IBDV in vivo. Passages were also performed in the absence or presence of a strong selective pressure using the antiviral nucleoside analog 7-deaza-2'-C-methyladenosine (7DMA). Next Generation Sequencing (NGS) of viral genomes after the last passage in each biological system revealed that (i) a higher viral diversity was generated in segment A than in segment B, regardless 7DMA treatment and viral strain, (ii) diversity in segment B was increased by 7DMA treatment in both viruses, (iii) passaging of IBDV in primary chicken B cells, regardless of 7DMA treatment, did not select cell-culture adapted variants of rvv, preserving its capsid protein (VP2) properties, (iv) mutations in coding and non-coding regions of rCu-1 segment A could potentially associate to higher viral fitness, and (v) a specific selection, upon 7DMA addition, of a Thr329Ala substitution occurred in the viral polymerase VP1. The latter change, together with Ala270Thr change in VP2, proved to be associated with viral attenuation in vivo. These results identify genome sequences that are important for IBDV evolution in response to selection pressures. Such information will help tailor better strategies for controlling IBDV infection in chickens.

Genomic relatedness of a canine Lactococcus garvieae to human, animal and environmental isolates.

Lactococcus (L.) garvieae is a zoonotic fish pathogen that can also cause bacteraemia and endocarditis in humans and has been isolated from healthy or diseased domestic animals. Nevertheless L. garvieae is more an opportunistic, than a primary pathogen since most affected humans have predisposing conditions and comorbidities. L. garvieae is also present in other animal species, most frequently cattle, but also sheep, goats, water buffaloes, and pigs, and much more rarely dogs, cats, horses, camel, turtle, snake and crocodile. The purpose of this study was to genomically (i) confirm the identification by MALDI-TOF MS® of a L. garvieae from the nasal discharge of a dog with chronic respiratory disorders and (ii) compare this canine isolate with human and animal L. garvieae isolates. According to the BLAST analysis after Whole Genome Sequencing, this canine isolate was more than 99% identical to 3 L. garvieae and belonged to a new Multi-Locus Sequence Type (ST45). MLST and whole genomes-based phylogenetic analysis were performed on the canine isolate and the 40 genomes available in Genbank. The canine L. garvieae was most closely related to an Australian camel and an Indian fish L. garvieae and more distantly to human L. garvieae. Twenty-five of the 29 putative virulence-associated genes searched for were detected, but not the 16 capsule-encoding genes. The heterogeneity of the L. garvieae species is reflected by the diversity of the MLSTypes and virulotypes identified and by the phylogenetic analysis.

Molecular characterization of encephalomyocarditis virus strains isolated from an African elephant and rats in a French zoo.

In November 2013, a fatal encephalomyocarditis virus (EMCV) case in a captive African elephant (Loxodonta africana) occurred at the Réserve Africaine de Sigean, a zoo in the south of France. Here we report the molecular characterization of the EMCV strains isolated from samples collected from the dead elephant and from 3 rats (Rattus rattus) captured in the zoo at the same time. The EMCV infection was confirmed by reverse-transcription real-time PCR (RT-rtPCR) and genome sequencing. Complete genome sequencing and sequence alignment indicated that the elephant's EMCV strain was 98.1-99.9% identical to the rat EMCV isolates at the nucleotide sequence level. Phylogenetic analysis of the ORF, P1, VP1, and 3D sequences revealed that the elephant and rat strains clustered into lineage A of the EMCV 1 group. To our knowledge, molecular characterization of EMCV in France and Europe has not been reported previously in a captive elephant. The full genome analyses of EMCV isolated from an elephant and rats in the same outbreak emphasizes the role of rodents in EMCV introduction and circulation in zoos.

Differences in Toxic Response Induced by Three Variants of the Diarrheic Shellfish Poisoning Phycotoxins in Human Intestinal Epithelial Caco-2 Cells.

Diarrheic shellfish poisoning (DSP) is caused by the consumption of shellfish contaminated with a group of phycotoxins that includes okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2). These toxins are inhibitors of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), but show distinct levels of toxicity. Aside from a difference in protein phosphatases (PP) inhibition potency that would explain these differences in toxicity, others mechanisms of action are thought to be involved. Therefore, we investigated and compared which mechanisms are involved in the toxicity of these three analogues. As the intestine is one of the target organs, we studied the transcriptomic profiles of human intestinal epithelial Caco-2 cells exposed to OA, DTX-1, and DTX-2. The pathways specifically affected by each toxin treatment were further confirmed through the expression of key genes and markers of toxicity. Our results did not identify any distinct biological mechanism for OA and DTX-2. However, only DTX-1 induced up-regulation of the MAPK transduction signalling pathway, and down-regulation of gene products involved in the regulation of DNA repair. As a consequence, based on transcriptomic results, we demonstrated that the higher toxicity of DTX-1 compared to OA and DTX-2 was consistent with certain specific pathways involved in intestinal cell response.

Identification of key pathways involved in the toxic response of the cyanobacterial toxin cylindrospermopsin in human hepatic HepaRG cells.

The hepatotoxin cylindrospermopsin (CYN) has been involved in cases of poisoning in humans following ingestion. As its liver toxicity process is complex, we studied the transcriptomic profile of HepaRG cells exposed to CYN. The affected pathways were confirmed through the expression of key genes and the investigation of toxicity markers. In addition, CYP450 activities and cell redox homeostasis were investigated following acute and repeated exposure. CYN induced the down-regulation of genes involved in xenobiotic metabolism and cell cycle progression. There was cell cycle disturbance characterised by an accumulation of G1/S and G2/M cells and an increase in phospho-H3-positive cells. This was linked to the induction of DNA damage demonstrated by an increase in γH2AX-positive cells as well as an accumulation of sub-G1 cells indicating apoptosis but not involving caspase-3. While glutathione (GSH) content sharply decreased following acute exposure to CYN, it increased following repeated exposure, reflecting an adaptive response of cell redox homeostasis. However, our data also suggested that CYN induced the down-regulation of phase I and II metabolism gene products, and CYP450 activities were affected following both acute and repeated exposure to CYN. Our study indicated that repeated exposure of liver cells to low concentrations of CYN may affect their detoxification capacities.

A single amino acid change in the non-structural NV protein impacts the virulence phenotype of Viral hemorrhagic septicemia virus in trout.

Novirhabdoviruses like the Viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses infecting fish. In the current study, RNA genomes of different VHSV field isolates classified as high, medium or low virulent phenotypes have been sequenced by next-generation sequencing and compared. Various amino acid changes, depending on the VHSV phenotype, have been identified in all the VHSV proteins. As a starting point, we focused our study on the non-virion (NV) non-structural protein in which an arginine residue (R116) is present in all the virulent isolates and replaced by a serine/asparagine residue S/N116 in the attenuated isolates. A recombinant virus derived from a virulent VHSV strain in which the NV R116 residue has been replaced by a serine, rVHSVNVR116S, was generated by reverse genetics and used to infect juvenile trout. We showed that rVHSVNVR116S was highly attenuated and that surviving fish were almost completely protected from a challenge with the wild-type VHSV.

Complete Genome Sequence of a Recombinant Porcine Reproductive and Respiratory Syndrome Virus Strain from Two Genotype 1 Modified Live Virus Vaccine Strains.

This paper provides information on the complete genome sequence of a porcine reproductive and respiratory syndrome virus (PRRSV) strain isolated on a French pig farm which was identified as a recombinant strain from two commercial modified live virus vaccine strains of genotype 1 (VP-046BIS and DV strains).

Emerging highly pathogenic H5 avian influenza viruses in France during 2015/16: phylogenetic analyses and markers for zoonotic potential.

Several new highly pathogenic (HP) H5 avian influenza virus (AIV) have been detected in poultry farms from south-western France since November 2015, among which an HP H5N1. The zoonotic potential and origin of these AIVs immediately became matters of concern. One virus of each subtype H5N1 (150169a), H5N2 (150233) and H5N9 (150236) was characterised. All proved highly pathogenic for poultry as demonstrated molecularly by the presence of a polybasic cleavage site in their HA protein - with a sequence (HQRRKR/GLF) previously unknown among avian H5 HPAI viruses - or experimentally by the in vivo demonstration of an intravenous pathogenicity index of 2.9 for the H5N1 HP isolate. Phylogenetic analyses based on the full genomes obtained by NGS confirmed that the eight viral segments of the three isolates were all part of avian Eurasian phylogenetic lineage but differed from the Gs/Gd/1/96-like lineage. The study of the genetic characteristics at specific amino acid positions relevant for modulating the adaptation to and the virulence for mammals showed that presently, these viruses possess most molecular features characteristic of AIV and lack some major characteristics required for efficient respiratory transmission to or between humans. The three isolates are therefore predicted to have no significant pandemic potential.

Complete Genome Sequence of Bluetongue Virus Serotype 8, Which Reemerged in France in August 2015.

We announce here the complete genome sequence (coding and noncoding) of the bluetongue virus (BTV) serotype 8, isolated from a ram in Allier department, France, 2015. Sequence analysis confirms the reemergence of the BTV-8 strain that previously circulated in France until 2009 and other European countries until 2010.

Complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france in december 2014.

We report the first and only case of a porcine epidemic diarrhea (PED) outbreak occurring in December 2014 in northern France, and we show using the full-length genome sequence of the French PED virus (PEDV) isolate that it was a PEDV indel strain close to German PEDV strains recently isolated.

Modulation of chromatin remodelling induced by the freshwater cyanotoxin cylindrospermopsin in human intestinal caco-2 cells.

Cylindrospermopsin (CYN) is a cyanotoxin that has been recognised as an emerging potential public health risk. Although CYN toxicity has been demonstrated, the mechanisms involved have not been fully characterised. To identify some key pathways related to this toxicity, we studied the transcriptomic profile of human intestinal Caco-2 cells exposed to a sub-toxic concentration of CYN (1.6 µM for 24hrs) using a non-targeted approach. CYN was shown to modulate different biological functions which were related to growth arrest (with down-regulation of cdkn1a and uhrf1 genes), and DNA recombination and repair (with up-regulation of aptx and pms2 genes). Our main results reported an increased expression of some histone-modifying enzymes (histone acetyl and methyltransferases MYST1, KAT5 and EHMT2) involved in chromatin remodelling, which is essential for initiating transcription. We also detected greater levels of acetylated histone H2A (Lys5) and dimethylated histone H3 (Lys4), two products of these enzymes. In conclusion, CYN overexpressed proteins involved in DNA damage repair and transcription, including modifications of nucleosomal histones. Our results highlighted some new cell processes induced by CYN.

Differential cellular gene expression in duck trachea infected with a highly or low pathogenic H5N1 avian influenza virus.

BACKGROUND: Avian influenza A (AI) viruses of subtypes H5 can cause serious disease outbreaks in poultry including panzootic due to H5N1 highly pathogenic (HP) viruses. These viruses are a threat not only for animal health but also public health due to their zoonotic potential. The domestic duck plays a major role in the epidemiological cycle of influenza virus subtypes H5 but little is known concerning host/pathogen interactions during influenza infection in duck species. In this study, a subtracted library from duck trachea (a primary site of influenza virus infection) was constructed to analyse and compare the host response after a highly or low pathogenic (LP) H5N1-infection. RESULTS: Here, we show that more than 200 different genes were differentially expressed in infected duck trachea to a significant degree. In addition, significant differentially expressed genes between LPAI- and HPAI-infected tracheas were observed. Gene ontology annotation was used and specific signalling pathways were identified. These pathways were different for LPAI and HPAI-infected tracheas, except for the CXCR4 signalling pathway which is implicated in immune response. A different modulation of genes in the CXCR4 signalling pathway and TRIM33 was induced in duck tracheas infected with a HPAI- or a LPAI-H5N1. CONCLUSION: First, this study indicates that Suppressive Subtractive Hybridization (SSH) is an alternative approach to gain insights into the pathogenesis of influenza infection in ducks. Secondly, the results indicate that cellular gene expression in the duck trachea was differently modulated after infection with a LPAI-H5N1 or after infection with a HPAI-H5N1 virus. Such difference found in infected trachea, a primary infection site, could precede continuation of infection and could explain appearance of respiratory symptoms or not.

Transcriptomic comparison of cyanotoxin variants in a human intestinal model revealed major differences in oxidative stress response: effects of MC-RR and MC-LR on Caco-2 cells.

Microcystins (MCs) are cyclic hepatotoxins produced by various species of cyanobacteria. Their structure includes two variable amino acids (AA) giving rise to more than 90 MC variants, however most of the studies to date have focused on the most toxic variant: microcystin LR (MC-LR). Ingestion is the major route of human exposure to MCs and several in vivo studies have demonstrated macroscopic effects on the gastro-intestinal tract. However, little information exists concerning the pathways affected by MC variants on intestinal cells. In the current study, we have investigated the effects of MC-RR and MC-LR on the human intestinal cell line Caco-2 using a non-selective method and compared their response at the pangenomic scale. The cells were incubated for 4h or 24h with a range of non-toxic concentrations of MC-RR or MC-LR. Minimal effects were observed after short term exposures (4h) to either MC variant. In contrast, dose dependent modulations of gene transcription levels were observed with MC-RR and MC-LR after 24h. The transcriptomic profiles induced by MC-RR were quite similar to those induced by MC-LR, suggestive of a largely common mechanism of toxicity. However, changes in total gene expression were more pronounced following exposure to MC-LR compared to MC-RR, as revealed by functional annotation. MC-LR affected two principal pathways, the oxidative stress response and cell cycle regulation, which did not elicit significant alteration following MC-RR exposure. This work is the first comparative description of the effects of MC-LR and MC-RR in a human intestinal cell model at the pangenomic scale. It has allowed us to propose differences in the mechanism of toxicity for MC-RR and MC-LR. These results illustrate that taking into account the toxicity of MC variants remains a key point for risk assessment.