Characterization of a New Toti-like Virus in Sea Bass, Dicentrarchus labrax.

The European sea bass Dicentrarchus labrax is the main species reared in Mediterranean aquaculture. Its larval stage, which is very sensitive and highly affected by sanitary and environmental conditions, is particularly scrutinized in hatcheries. Recently, a Mediterranean sea bass farm had to deal with an abnormal increase in mortality, especially between 20 and 35 days post-hatching (dph). Biological investigations led to the observation of cytopathic effects on three different fish cell lines after almost 3 weeks of culture at 14 °C in contact with homogenized affected larvae, suggesting the presence of a viral agent. High-throughput sequencing revealed a 6818-nucleotide-long RNA genome with six putative ORFs, corresponding to the organization of viruses belonging to the Totiviridae family. This genome clustered with the newly described and suggested Pistolvirus genus, sharing 45.5% to 37.2% nucleotide identity with other piscine toti-like viruses such as Cyclopterus lumpus toti-like virus (CLuTLV) or piscine myocarditis virus (PMCV), respectively. Therefore, we propose to name this new viral agent sea bass toti-like virus (SBTLV). Specific real-time RT-PCR confirmed the presence of the viral genome in the affected larval homogenate from different production batches and the corresponding cell culture supernatant. Experimental infections performed on sea bass fingerlings did not induce mortality, although the virus could be detected in various organs and a specific immune response was developed. Additional studies are needed to understand the exact involvement of this virus in the mortality observed in hatcheries and the potential associated cofactors.

Investigations into SARS-CoV-2 and other coronaviruses on mink farms in France late in the first year of the COVID-19 pandemic.

Soon after the beginning of the COVID-19 pandemic in early 2020, the Betacoronavirus SARS-CoV-2 infection of several mink farms breeding American minks (Neovison vison) for fur was detected in various European countries. The risk of a new reservoir being formed and of a reverse zoonosis from minks quickly became a major concern. The aim of this study was to investigate the four French mink farms to see whether SARS-CoV-2 was circulating there in late 2020. The investigations took place during the slaughtering period, thus facilitating different types of sampling (swabs and blood). On one of the four mink farms, 96.6% of serum samples were positive when tested with a SARS-CoV-2 ELISA coated with purified N protein recombinant antigen, and 54 out of 162 (33%) pharyngo-tracheal swabs were positive by RT-qPCR. The genetic variability among 12 SARS-CoV-2 genomes sequenced from this farm indicated the co-circulation of several lineages at the time of sampling. All the SARS-CoV-2 genomes detected were nested within the 20A clade (Nextclade), together with SARS-CoV-2 genomes from humans sampled during the same period. The percentage of SARS-CoV-2 seropositivity by ELISA varied between 0.3 and 1.1% on the other three farms. Interestingly, among these three farms, 11 pharyngo-tracheal swabs and 3 fecal pools from two farms were positive by end-point RT-PCR for an Alphacoronavirus very similar to a mink coronavirus sequence observed on Danish farms in 2015. In addition, a mink Caliciviridae was identified on one of the two farms positive for Alphacoronavirus. The clinical impact of these inapparent viral infections is not known. The co-infection of SARS-CoV-2 with other viruses on mink farms could help explain the diversity of clinical symptoms noted on different infected farms in Europe. In addition, the co-circulation of an Alphacoronavirus and SARS-CoV-2 on a mink farm would potentially increase the risk of viral recombination between alpha and betacoronaviruses as already suggested in wild and domestic animals, as well as in humans.

Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Domestic Cat, France, 2022.

We detected highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in a domestic cat that lived near a duck farm infected by a closely related virus in France during December 2022. Enhanced surveillance of symptomatic domestic carnivores in contact with infected birds is recommended to prevent further spread to mammals and humans.

Direct Xylella fastidiosa whole genome sequencing from various plant species using targeted enrichment.

A targeted enrichment method was developed to sequence Xylella fastidiosa genomic DNA directly from plant samples. The method was evaluated on various plant species infected with different strains at different levels of contamination. After enrichment, X. fastidiosa genome coverage was above 99.9% for all tested samples.

Investigations on SARS-CoV-2 and other coronaviruses in mink farms in France at the end of the first year of COVID-19 pandemic.

Soon after the beginning of the COVID-19 pandemic in early 2020, the Betacoronavirus SARS-CoV-2 infection of several mink farms breeding American minks ( Neovison vison ) for fur was detected in several countries of Europe. The risk of a new reservoir formation and of a reverse zoonosis from minks was then a major concern. The aim of this study was to investigate the four French mink farms for the circulation of SARS-CoV-2 at the end of 2020. The investigations took place during the slaughtering period thus facilitating different types of sampling (swabs and blood). In one of the four mink farms, 96.6% of serum samples were positive in SARS-CoV-2 ELISA coated with purified N protein recombinant antigen and 54 out of 162 (33%) pharyngo-tracheal swabs were positive by RT-qPCR. The genetic variability among 12 SARS-CoV-2 genomes sequenced in this farm indicated the co-circulation of several lineages at the time of sampling. All SARS-CoV-2 genomes detected were nested within the 20A clade (Nextclade), together with SARS-CoV-2 genomes from humans sampled at the same period. The percentage of SARS-CoV-2 seropositivity by ELISA varied between 0.5 and 1.2% in the three other farms. Interestingly, among these three farms, 11 pharyngo-tracheal swabs and 3 fecal pools from two farms were positive by end-point RT-PCR for an Alphacoronavirus highly similar to a mink coronavirus sequence observed in Danish farms in 2015. In addition, a mink Caliciviridae was identified in one of the two positive farms for Alphacoronavirus . The clinical impact of these unapparent viral infections is not known. The co-infection of SARS-CoV-2 with other viruses in mink farms could contribute to explain the diversity of clinical symptoms noted in different infected farms in Europe. In addition, the co-circulation of an Alphacoronavirus and SARS-CoV-2 within a mink farm would increase potentially the risk of viral recombination between alpha and betacoronaviruses already suggested in wild and domestic animals, as well as in humans. Author summary: France is not a country of major mink fur production. Following the SARS-CoV-2 contamination of mink farms in Denmark and the Netherlands, the question arose for the four French farms.The investigation conducted at the same time in the four farms revealed the contamination of one of them by a variant different from the one circulating at the same time in Denmark and the Netherlands mink farms. Investigation of three other farms free of SARS-CoV-2 contamination revealed the circulation of other viruses including a mink Alphacoronavirus and Caliciviridae , which could modify the symptomatology of SARS-CoV-2 infection in minks.

Research Note: Analysis of immune responses in broilers after vaccination against Campylobacter jejuni.

Campylobacter infections traced mainly to poultry products are major bacterial foodborne zoonoses. Among the many control strategies evaluated at primary poultry level to reduce these infections, vaccination could be a solution, but no effective vaccines are available to date. A better understanding of the immune mechanisms involved in protection against Campylobacter would be helpful for designing novel vaccine strategies. The present study was designed to analyze in more depth the immune responses developed in broilers in order to potentially identify which immune parameters may be important for establishing protection against Campylobacter by comparing the immune responses obtained here with those obtained in a previous study performed on vaccinated specific-pathogen-free Leghorn chickens that presented a partial reduction of Campylobacter after experimental challenge. The protection against Campylobacter colonization was evaluated at different time points over 40 d of rearing, by measuring specific IgY levels in serum and IgA antibodies in bile reflecting the systemic and mucosal humoral responses respectively and the relative expressions of 9 cecal immune marker genes (cytokines and antimicrobial peptides), which reflect the innate and cellular immune responses. Despite no reduction of Campylobacter in the cecum, a systemic immune response over time characterized by the production of specific anti-flagellin IgY was observed, in addition to upregulation of the antimicrobial peptide avian ß-defensin (AvBD) 12 gene expression in the cecum of vaccinated broilers compared with the placebo group. However, the levels of specific anti-flagellin mucosal IgA antibodies in the bile as well as the relative expression of other cecal cytokines studied was underexpressed in the vaccinated group or similar in both groups.

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.

Impact of DNA Prime/Protein Boost Vaccination against Campylobacter jejuni on Immune Responses and Gut Microbiota in Chickens.

Campylobacteriosis is reported to be the leading zoonosis in Europe, and poultry is the main reservoir of Campylobacter. Despite all the efforts made, there is still no efficient vaccine to fight this bacterium directly in poultry. Recent studies have reported interactions between the chicken immune system and gut microbiota in response to Campylobacter colonisation. The present study was designed to analyse in more depth the immune responses and caecal microbiota following vaccination with a DNA prime/protein boost flagellin-based vaccine that induces some protection in specific-pathogen-free White Leghorn chickens, as shown previously. These data may help to improve future vaccination protocols against Campylobacter in poultry. Here a vaccinated and a placebo group were challenged by C. jejuni at the age of 19 days. A partial reduction in Campylobacter loads was observed in the vaccinated group. This was accompanied by the production of specific systemic and mucosal antibodies. Transient relatively higher levels of Interleukin-10 and antimicrobial peptide avian ß-defensin 10 gene expressions were observed in the vaccinated and placebo groups respectively. The analysis of caecal microbiota revealed the vaccination's impact on its structure and composition. Specifically, levels of operational taxonomic units classified as Ruminococcaceae and Bacillaceae increased on day 40.

Description of the first isolates of guinea fowl corona and picornaviruses obtained from a case of guinea fowl fulminating enteritis.

Guinea fowl fulminating enteritis has been reported in France since the 1970s. In 2014, a coronavirus was identified and appeared as a possible viral pathogen involved in the disease. In the present study, intestinal content from a guinea fowl involved in a new case of the disease in 2017 was analysed by deep sequencing, revealing the presence of a guinea fowl coronavirus (GfCoV) and a picornavirus (GfPic). Serial passage assays into the intra-amniotic cavity of 13-day-old specific pathogen-free chicken eggs and 20-day-old conventional guinea fowl eggs were attempted. In chicken eggs, isolation assays failed, but in guinea fowl eggs, both viruses were successfully obtained. Furthermore, two GfCoV and two GfPic isolates were obtained from the same bird but from different sections of its intestines. This shows that using eggs of the same species, in which the virus has been detected, can be the key for successful isolation. The consensus sequence of the full-length genomes of both GfCoV isolates was highly similar, and correlated to those previously described in terms of genome organization, ORF length and phylogenetic clustering. According to full-length genome analysis and the structure of the Internal Ribosome Entry Site, both GfPic isolates belong to the Anativirus genus and specifically the species Anativirus B. The availability of the first isolates of GfCoV and GfPic will now provide a means of assessing their pathogenicity in guinea fowl in controlled experimental conditions and to assess whether they are primary viral pathogens of the disease "guinea fowl fulminating enteritis".RESEARCH HIGHLIGHTSFirst isolation of guinea fowl coronaviruses and picornaviruses.Eggs homologous to the infected species are key for isolation.Isolates available to precisely evaluate the virus roles in fulminating enteritis.First full-length genome sequences of guinea fowl picornaviruses.

Phenotypic and Genetic Evolutions of a Porcine Reproductive and Respiratory Syndrome Modified Live Vaccine after Limited Passages in Pigs.

Modified live vaccines (MLVs) against the porcine reproductive and respiratory syndrome virus (PRRSV) have been regularly associated with safety issues, such as reversion to virulence. In order to characterize the phenotypic and genetic evolution of the PRRSV-1 DV strain from the Porcilis® PRRS MLV after limited passages in pigs, three in vivo experiments were performed. Trial#1 aimed (i) at studying transmission of the vaccine strain from vaccinated to unvaccinated contact pigs. Trial#2 and Trial#3 were designed (ii) to assess the reproducibility of Trial#1, using another vaccine batch, and (iii) to compare the virulence levels of two DV strains isolated from vaccinated (passage one) and diseased contact pigs (passage two) from Trial#1. DV strain isolates from vaccinated and contact pigs from Trial#1 and Trial#2 were submitted to Next-Generation Sequencing (NGS) full-genome sequencing. All contact animals from Trial#1 were infected and showed significantly increased viremia compared to vaccinated pigs, whereas no such change was observed during Trial#2. In Trial#3, viremia and transmission were higher for inoculated pigs with passage two of the DV strain, compared with passage one. In this study, we showed that the re-adaptation of the DV strain to pigs is associated with faster replication and increased transmission of the vaccine strain. Punctually, a decrease of attenuation of the DV vaccine strain associated with clinical signs and increased viremia may occur after limited passages in pigs. Furthermore, we identified three mutations linked to pig re-adaptation and five other mutations as potential virulence determinants.

Highly Pathogenic Avian Influenza A(H5N8) Virus Spread by Short- and Long-Range Transmission, France, 2016-17.

We detected 3 genotypes of highly pathogenic avian influenza A(H5N8) virus in France during winter 2016-17. Genotype A viruses caused dramatic economic losses in the domestic duck farm industry in southwestern France. Our phylogenetic analysis suggests that genotype A viruses formed 5 distinct geographic clusters in southwestern France. In some clusters, local secondary transmission might have been started by a single introduction. The intensity of the viral spread seems to correspond to the density of duck holdings in each production area. To avoid the introduction of disease into an unaffected area, it is crucial that authorities limit the movements of potentially infected birds.

Genetic and Antigenic Evolution of European Swine Influenza A Viruses of HA-1C (Avian-Like) and HA-1B (Human-Like) Lineages in France from 2000 to 2018.

This study evaluated the genetic and antigenic evolution of swine influenza A viruses (swIAV) of the two main enzootic H1 lineages, i.e., HA-1C (H1av) and -1B (H1hu), circulating in France between 2000 and 2018. SwIAV RNAs extracted from 1220 swine nasal swabs were hemagglutinin/neuraminidase (HA/NA) subtyped by RT-qPCRs, and 293 virus isolates were sequenced. In addition, 146 H1avNy and 105 H1huNy strains were submitted to hemagglutination inhibition tests. H1avN1 (66.5%) and H1huN2 (25.4%) subtypes were predominant. Most H1 strains belonged to HA-1C.2.1 or -1B.1.2.3 clades, but HA-1C.2, -1C.2.2, -1C.2.3, -1B.1.1, and -1B.1.2.1 clades were also detected sporadically. Within HA-1B.1.2.3 clade, a group of strains named "Δ146-147" harbored several amino acid mutations and a double deletion in HA, that led to a marked antigenic drift. Phylogenetic analyses revealed that internal segments belonged mainly to the "Eurasian avian-like lineage", with two distinct genogroups for the M segment. In total, 17 distinct genotypes were identified within the study period. Reassortments of H1av/H1hu strains with H1N1pdm virus were rarely evidenced until 2018. Analysis of amino acid sequences predicted a variability in length of PB1-F2 and PA-X proteins and identified the appearance of several mutations in PB1, PB1-F2, PA, NP and NS1 proteins that could be linked to virulence, while markers for antiviral resistance were identified in N1 and N2. Altogether, diversity and evolution of swIAV recall the importance of disrupting the spreading of swIAV within and between pig herds, as well as IAV inter-species transmissions.

Intranasal inoculations of naked or PLGA-PEI nanovectored DNA vaccine induce systemic and mucosal antibodies in pigs: A feasibility study.

Mucosa are the routes of entry of most pathogens into animals' organisms. Reducing the important global burden of mucosal infectious diseases in livestock animals is required in the field of veterinary public health. For veterinary respiratory pathogens, one possible strategy is the development of intranasal (IN) DNA vaccination. The aim of this study was to assess the feasibility of IN DNA vaccination in pigs, an important species in livestock production industry, and a source of zoonotic diseases. To achieve this goal, we used a DNA vaccine against pseudorabies virus (PrV) encoding the immunogenic glycoprotein B (pcDNA3-gB plasmid). When pigs were inoculated with the naked DNA vaccine through the IN route, PrV-specific IgG and IgA type antibodies were detected in porcine sera. Interestingly, mucosal salivary IgA antibodies against PrV were also detected, at similar levels to those measured following intramuscular injection (positive controls). Furthermore, the IN delivery of pcDNA3-gB combined with PLGA-PEI nanoparticles resulted in similar levels of antibodies but was associated with an increase in the duration of detection of mucosal IgA for 2 out of 3 pigs. Our results suggest that there is room to improve the efficacy of IN DNA vaccination in pigs through optimization of IN inoculations, for example by using nanoparticles such as PLGA-PEI. Further studies will be dedicated to optimizing and testing the protective potential of IN DNA vaccination procedures against PrV.

Virus persistence in pig herds led to successive reassortment events between swine and human influenza A viruses, resulting in the emergence of a novel triple-reassortant swine influenza virus.

This report describes the detection of a triple reassortant swine influenza A virus of H1avN2 subtype. It evolved from an avian-like swine H1avN1 that first acquired the N2 segment from a seasonal H3N2, then the M segment from a 2009 pandemic H1N1, in two reassortments estimated to have occurred 10 years apart. This study illustrates how recurrent influenza infections increase the co-infection risk and facilitate evolutionary jumps by successive gene exchanges. It recalls the importance of appropriate biosecurity measures inside holdings to limit virus persistence and interspecies transmissions, which both contribute to the emergence of new potentially zoonotic viruses.

Bidirectional Human-Swine Transmission of Seasonal Influenza A(H1N1)pdm09 Virus in Pig Herd, France, 2018.

In 2018, a veterinarian became sick shortly after swabbing sows exhibiting respiratory syndrome on a farm in France. Epidemiologic data and genetic analyses revealed consecutive human-to-swine and swine-to-human influenza A(H1N1)pdm09 virus transmission, which occurred despite some biosecurity measures. Providing pig industry workers the annual influenza vaccine might reduce transmission risk.

Lleida Bat Lyssavirus isolation in Miniopterus schreibersii in France.

Bat rabies cases are attributed in Europe to five different Lyssavirus species of 16 recognized Lyssavirus species causing rabies. One of the most genetically divergent Lyssavirus spp. has been detected in a dead Miniopterus schreibersii bat in France. Brain samples were found positive for the presence of antigen, infectious virus and viral RNA by classical virological methods and molecular methods respectively. The complete genome sequence was determined by next-generation sequencing. The analysis of the complete genome sequence confirmed the presence of Lleida bat lyssavirus (LLEBV) in bats in France with 99.7% of nucleotide identity with the Spanish LLEBV strain (KY006983).

Identification of a Divergent Avian Influenza H3N2 Virus from Domestic Ducks in France.

An avian influenza H3N2 virus was isolated from domestic ducks in France in 2016. Although this French H3N2 virus possesses traits of an avian virus, the genetic distances observed for hemagglutinin (HA) and neuraminidase (NA) show that these two genes most likely evolved independently from other avian influenza sequences.

Spatiotemporal Distribution and Evolution of the A/H1N1 2009 Pandemic Influenza Virus in Pigs in France from 2009 to 2017: Identification of a Potential Swine-Specific Lineage.

The H1N1 influenza virus responsible for the most recent pandemic in 2009 (H1N1pdm) has spread to swine populations worldwide while it replaced the previous seasonal H1N1 virus in humans. In France, surveillance of swine influenza A viruses in pig herds with respiratory outbreaks led to the detection of 44 H1N1pdm strains between 2009 and 2017, regardless of the season, and findings were not correlated with pig density. From these isolates, 17 whole-genome sequences were obtained, as were 6 additional hemagglutinin (HA)/neuraminidase (NA) sequences, in order to perform spatial and temporal analyses of genetic diversity and to compare evolutionary patterns of H1N1pdm in pigs to patterns for human strains. Following mutation accumulation and fixation over time, phylogenetic analyses revealed for the first time the divergence of a swine-specific genogroup within the H1N1pdm lineage. The divergence is thought to have occurred around 2011, although this was demonstrated only through strains isolated in 2015 to 2016 in the southern half of France. To date, these H1N1pdm swine strains have not been related to any increased virulence in swine herds and have not exhibited any antigenic drift compared to seasonal human strains. However, further monitoring is encouraged, as diverging evolutionary patterns in these two species, i.e., swine and humans, may lead to the emergence of viruses with a potentially higher risk to both animal and human health.IMPORTANCE Pigs are a "mixing vessel" for influenza A viruses (IAVs) because of their ability to be infected by avian and human IAVs and their propensity to facilitate viral genomic reassortment events. Also, as IAVs may evolve differently in swine and humans, pigs can become a reservoir for old human strains against which the human population has become immunologically naive. Thus, viruses from the novel swine-specific H1N1pdm genogroup may continue to diverge from seasonal H1N1pdm strains and/or from other H1N1pdm viruses infecting pigs and lead to the emergence of viruses that would not be covered by human vaccines and/or swine vaccines based on antigens closely related to the original H1N1pdm virus. This discovery confirms the importance of encouraging swine IAV monitoring because H1N1pdm swine viruses could carry an increased risk to both human and swine health in the future as a whole H1N1pdm virus or gene provider in subsequent reassortant viruses.

Ruminant and chicken: important sources of campylobacteriosis in France despite a variation of source attribution in 2009 and 2015.

Pathogen source attribution studies are a useful tool for identifying reservoirs of human infection. Based on Multilocus Sequence Typing (MLST) data, such studies have identified chicken as a major source of C. jejuni human infection. The use of whole genome sequence-based typing methods offers potential to improve the precision of attribution beyond that which is possible from 7 MLST loci. Using published data and 156 novel C. jejuni genomes sequenced in this study, we performed probabilistic host source attribution of clinical C. jejuni isolates from France using three types of genotype data: comparative genomic fingerprints; MLST genes; 15 host segregating genes previously identified by whole genome sequencing. Consistent with previous studies, chicken was an important source of campylobacteriosis in France (31-63% of clinical isolates assigned). There was also evidence that ruminants are a source (22-55% of clinical isolates assigned), suggesting that further investigation of potential transmission routes from ruminants to human would be useful. Additionally, we found evidence of environmental and pet sources. However, the relative importance as sources varied according to the year of isolation and the genotyping technique used. Annual variations in attribution emphasize the dynamic nature of zoonotic transmission and the need to perform source attribution regularly.

A DNA prime/protein boost vaccine protocol developed against Campylobacter jejuni for poultry.

Vaccination of broilers is one of the potential ways to decrease Campylobacter intestinal loads and therefore may reduce human disease incidence. Despite many studies, no efficient vaccine is available yet. Using the reverse vaccinology strategy, we recently identified new vaccine candidates whose immune and protective capacities need to be evaluated in vivo. Therefore, the goal of the present study was to develop and evaluate an avian subunit vaccine protocol for poultry against Campylobacter jejuni. For this, flagellin was used as vaccine antigen candidate. A DNA prime/protein boost regimen was effective in inducing a massive protective immune response against C. jejuni in specific pathogen free Leghorn chickens. Contrastingly, the same vaccine regimen stimulated the production of antibodies against Campylobacter in conventional Ross broiler chickens harbouring maternally derived antibodies against Campylobacter, but not the control of C. jejuni colonization. These results highlight the strength of the vaccine protocol in inducing protective immunity and the significance of the avian strain and/or immune status in the induction of this response. Nevertheless, as such the vaccine protocol is not efficient in broilers to induce protection and has to be adapted; this has been done in one of our recent published work.