Open Access

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

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

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

International audienceThe 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

Modulation of Chromatin Remodelling Induced by the Freshwater Cyanotoxin Cylindrospermopsin in Human Intestinal Caco-2 Cells

International audienceCylindrospermopsin (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 mM 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

Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador

International audienceBluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV; however, no data are available about the presence of EHDV. In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT-qPCRs revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV1) and BTV serotypes 9 (BTV9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador

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

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

Complete genome sequence of bluetongue virus serotype 4 that emerged on the French island of Corsica in December 2016

International audienceIn November 2016, sheep located in the south of Corsica island exhibited clinical signs suggestive of bluetongue virus (BTV) infection. Laboratory analyses allowed to isolate and identify a BTV strain of serotype 4. The analysis of the full viral genome showed that all the 10 genomic segments were closely related to those of the BTV-4 present in Hungary in 2014 and involved in a large BT outbreak in the Balkan Peninsula. These results together with epidemiological data suggest that BTV-4 has been introduced to Corsica from Italy (Sardinia) where BTV-4 outbreaks have been reported in autumn 2016. This is the first report of the introduction in Corsica of a BTV strain previously spreading in eastern Europe