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.

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.

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.

First genetic characterization of sturgeon mimiviruses in Ukraine.

A group of pathogenic nucleocytoplasmic large DNA viruses (NCLDVs) related to the Mimiviridae family infect farmed sturgeons across Europe, causing mild-to-severe losses. One of these viruses, Acipenser iridovirus-European (AcIV-E), was identified in six sturgeon species. During the 2018-2019 period, nine sick Siberian (A. baerii) and Russian (A. gueldenstaedtii) sturgeons were sampled in Ukrainian farms and tested for the presence of AcIV-E using real-time PCR. The presence of AcIV-E was confirmed in some samples. High-resolution melting (HRM) assay and Sanger sequencing demonstrated the presence in three farms of two alleles of the major capsid protein (MCP) gene, called var1 and var2. Five samples carried both var1 and var2 at varying ratios, and the sixth sample was infected with only var1. These results constitute the first detection of AcIV-E in Ukraine and the first detection of a sample carrying only var1. The full-length sequences of the MCP genes confirmed the existence of two genetic lineages of AcIV-E, tentatively named V1 and V2, each displaying multiple substitutions in the MCP gene. Some of the MCP sequences showed a genetic relationship to both V1 and V2 lineages, depending on the fragment examined. Most likely, these sequences resulted from recombination events.

Acipenser iridovirus-European encodes a replication factor C (RFC) sub-unit.

New genomic sequence data were acquired for the Acipenser iridovirus-European (AcIV-E), a virus whose complete genome and classification still remain to be elucidated. Here, we obtained the first full-length Major capsid protein (MCP) gene sequence for AcIV-E, as well as two additional open reading frames (ORFs) adjacent to the MCP gene. BLAST searches of the first ORF (α) resulted in no match to any gene or protein in the public databases. The other ORF (ß) was identified as a subunit of a replication factor C (RFC), known to function as a clamp loader in eukaryotes, archae and some viruses. The presence of similar RFC genes was confirmed in two distinct, yet related, viruses, the white sturgeon iridovirus and a European variant of Namao virus. The existence of an RFC gene in AcIV-E suggests a genome size larger than that of other classifiable members of the family Iridoviridae along with a mode of replication involving an interaction between a clamp loader and a proliferating nuclear cell antigen. Sequencing and comparison of the full-length RFC gene from various sturgeon samples infected with AcIV-E revealed two distinct clusters of sequences within one particular sample in which the coexistence of two lineages had previously been predicted based on analysis of the partial MCP gene sequence. These genetic data provide further evidence of the circulation of at least two concurrent AcIV-E lineages, sometimes co-infecting cultured European sturgeon.

Identification of a European interserotypic reassortant strain of infectious bursal disease virus.

Infectious bursal disease virus (IBDV, family Birnaviridae) is a bi-segmented double-stranded RNA virus for which two serotypes are described. Serotype 1 replicates in the bursa of Fabricius and causes an immunosuppressive and potentially fatal disease in young chickens. Serotype 2 is apathogenic in poultry species. Up to now, only one natural event of interserotypic reassortment has been described after the introduction of very virulent IBDV (vvIBDV) in the USA in 2009, resulting in an IBDV strain with its segment A related to vvIBDV and its segment B related to US serotype 2 strain OH. Here, we present the first European isolate illustrative of interserotypic reassortment. The reassorting isolate, named 100056, exhibits a genomic segment A typical of current European vvIBDV but a segment B close to European serotype 2 viruses, supporting an origin distinct from US strains. When inoculated into SPF chickens, isolate 100056 induced mild clinical signs in the absence of mortality but caused a severe bursal atrophy, which strongly suggests an immunosuppressive potential. These results illustrate that interserotypic reassortment is another mechanism that can create IBDV strains with a modified acute pathogenicity. As a consequence, and for a more precise inference of the possible phenotype, care should be taken that the molecular identification of IBDV strains is targeted to both genome segments.

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.

Taxonomy of the order Mononegavirales: update 2016.

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

Complete genome sequence of a novel avian paramyxovirus.

We report here the complete genome of a new avian paramyxovirus (APMV-11) isolated from common snipes. Sequence data from this virus showed that it has the largest genome of APMV and unusual P gene mRNA editing.

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.

High antigenic diversity of serotype 1 infectious bursal disease virus revealed by antigenic cartography.

The antigenic characterization of IBDV, a virus that causes an immunosuppressive disease in young chickens, has been historically addressed using cross virus neutralization (VN) assay and antigen-capture enzyme-linked immunosorbent (AC-ELISA). However, VN assay has been usually carried out either in specific antibody negative embryonated eggs, for non-cell culture adapted strains, which is tedious, or on chicken embryo fibroblasts (CEF), which requires virus adaptation to cell culture. AC-ELISA has provided crucial information about IBDV antigenicity, but this information is limited to the epitopes included in the tested panel with a lack of information of overall antigenic view. The present work aimed at overcoming those technical limitations and providing an extensive antigenic landscape based on original cross VN assays employing primary chicken B cells, where no previous IBDV adaptation is required. Sixteen serotype 1 IBDV viruses, comprising both reference strains and documented antigenic variants were tested against eleven chicken post-infectious sera. The VN data were analysed by antigenic cartography, a method which enables reliable high-resolution quantitative and visual interpretation of large binding assay datasets. The resulting antigenic cartography revealed i) the existence of several antigenic clusters of IBDV, ii) high antigenic relatedness between some genetically unrelated viruses, iii) a highly variable contribution to global antigenicity of previously identified individual epitopes and iv) broad reactivity of chicken sera raised against antigenic variants. This study provides an overall view of IBDV antigenic diversity. Implementing this approach will be instrumental to follow the evolution of IBDV antigenicity and control the disease.

Evaluation of three hemagglutinin-based vaccines for the experimental control of a panzootic clade 2.3.4.4b A(H5N8) high pathogenicity avian influenza virus in mule ducks.

In France during winter 2016-2017, 487 outbreaks of clade 2.3.4.4b H5N8 subtype high pathogenicity (HP) avian influenza A virus (AIV) infections were detected in poultry and captive birds. During this epizootic, HPAIV A/decoy duck/France/161105a/2016 (H5N8) was isolated and characterized in an experimental infection transmission model in conventional mule ducks. To investigate options to possibly protect such ducks against this HPAIV, three vaccines were evaluated in controlled conditions. The first experimental vaccine was derived from the hemagglutinin gene of another clade 2.3.4.4b A(H5N8) HPAIV. It was injected at three weeks of age, either alone (Vac1) or after a primer injection at day-old (Vac1 + boost). The second vaccine (Vac2) was a commercial bivalent adjuvanted vaccine containing an expressed hemagglutinin modified from a clade 2.3.2 A(H5N1) HPAIV. Vac2 was administered as a single injection at two weeks of age. The third experimental vaccine (Vac3) also incorporated a homologous 2.3.4.4b H5 HA gene and was administered as a single injection at three weeks of age. Ducks were challenged with HPAIV A/decoy duck/France/161105a/2016 (H5N8) at six weeks of age. Post-challenge virus excretion was monitored in vaccinated and control birds every 2-3 days for two weeks using real-time reverse-transcription polymerase chain reaction and serological analyses (haemagglutination inhibition test against H5N8, H5 ELISA and AIV ELISA) were performed. Vac1 abolished oropharyngeal and cloacal shedding to almost undetectable levels, whereas Vac3 abolished cloacal shedding only (while partially reducing respiratory shedding) and Vac2 only partly reduced the respiratory and intestinal excretion of the challenge virus. These results provided relevant insights in the immunogenicity of recombinant H5 vaccines in mule ducks, a rarely investigated hybrid between Pekin and Muscovy duck species that has played a critical role in the recent H5 HPAI epizootics in France.

Full-Length Genome Sequence of a Novel European Antigenic Variant Strain of Infectious Bursal Disease Virus.

We report the full-length genome sequence (compared to reference sequences) of a novel European variant strain of infectious bursal disease virus (IBDV), designated 19P009381 (AxB1). This should help to further identify such viruses in Europe.

Phylodynamic analysis of the highly pathogenic avian influenza H5N8 epidemic in France, 2016-2017.

In 2016-2017, France experienced a devastating epidemic of highly pathogenic avian influenza (HPAI) H5N8, with more than 400 outbreaks reported in poultry farms. We analyzed the spatiotemporal dynamics of the epidemic using a structured-coalescent-based phylodynamic approach that combined viral genomic data (n = 196; one viral genome per farm) and epidemiological data. In the process, we estimated viral migration rates between départements (French administrative regions) and the temporal dynamics of the effective viral population size (Ne) in each département. Viral migration rates quantify viral spread between départements and Ne is a population genetic measure of the epidemic size and, in turn, is indicative of the within-département transmission intensity. We extended the phylodynamic analysis with a generalized linear model to assess the impact of multiple factors-including large-scale preventive culling and live-duck movement bans-on viral migration rates and Ne. We showed that the large-scale culling of ducks that was initiated on 4 January 2017 significantly reduced the viral spread between départements. No relationship was found between the viral spread and duck movements between départements. The within-département transmission intensity was found to be weakly associated with the intensity of duck movements within départements. Together, these results indicated that the virus spread in short distances, either between adjacent départements or within départements. Results also suggested that the restrictions on duck transport within départements might not have stopped the viral spread completely. Overall, we demonstrated the usefulness of phylodynamics in characterizing the dynamics of a HPAI epidemic and assessing control measures. This method can be adapted to investigate other epidemics of fast-evolving livestock pathogens.

Avian influenza outbreaks: evaluating the efficacy of cleaning and disinfection of vehicles and transport crates.

In 2021, France faced large avian influenza outbreaks, like in 2016 and 2017. Controlling these outbreaks required the preventive depopulation of a large number of duck farms. A previous study in 2017 showed that the quality of decontamination of trucks and transport crates used for depopulation was often insufficient. A new study was then set up to evaluate cleaning and disinfection (C&D) of trucks and crates used for duck depopulation and whether practices had changed since 2017. Three methods were used to assess decontamination: 1) detection of avian influenza virus (AIV) genome, 2) visual inspection of cleanliness, and 3) microbial counts, considering that 2 and 3 are commonly used in abattoirs. Another objective of the study was to evaluate the correlation between results obtained with the 3 methods. In 5 abattoirs, 8 trucks and their crates were sampled by swabbing to detect AIV genome by rRT-PCR before and after decontamination. Visual cleanliness scores and coliform counts were also determined on crates after C&D. Trucks and crates were decontaminated according to the abattoirs' protocols. Before C&D, 3 quarters of crates (59/79) and 7 of 8 trucks were positive for AIV genome. C&D procedures were reinforced in 2021 compared to 2017; use of detergent solution and warm water were more common. Nevertheless, 28% of the crates were positive for AIV genome after C&D, despite the fact that cleaning scores and microbiological counts were satisfactory for 84% and 91% of the crates, respectively. No correlation was observed between results for AIV genome detection and results from visual control or from coliform counts. Abattoirs are encouraged to use environmental sampling coupled with AIV genome detection to monitor the quality of cleaning and disinfection of trucks and crates during AI outbreaks. Reinforcement of biosecurity measures at abattoirs is still needed to avoid residual contamination of the equipment and cross-contamination during the decontamination process.

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.

Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021.

Phylogenetic evidence from the recent resurgence of high-pathogenicity avian influenza (HPAI) virus subtype H5N1, clade 2.3.4.4b, observed in European wild birds and poultry since October 2021, suggests at least two different and distinct reservoirs. We propose contrasting hypotheses for this emergence: (i) resident viruses have been maintained, presumably in wild birds, in northern Europe throughout the summer of 2021 to cause some of the outbreaks that are part of the most recent autumn/winter 2021 epizootic, or (ii) further virus variants were reintroduced by migratory birds, and these two sources of reintroduction have driven the HPAI resurgence. Viruses from these two principal sources can be distinguished by their hemagglutinin genes, which segregate into two distinct sublineages (termed B1 and B2) within clade 2.3.4.4b, as well as their different internal gene compositions. The evidence of enzootic HPAI virus circulation during the summer of 2021 indicates a possible paradigm shift in the epidemiology of HPAI in Europe.

Serological Evidence of Backyard Pig Exposure to Highly Pathogenic Avian Influenza H5N8 Virus during 2016-2017 Epizootic in France.

In autumn/winter 2016-2017, HPAI-H5N8 viruses belonging to the A/goose/Guandong/1/1996 (Gs/Gd) lineage, clade 2.3.4.4b, were responsible for outbreaks in domestic poultry in Europe, and veterinarians were requested to reinforce surveillance of pigs bred in HPAI-H5Nx confirmed mixed herds. In this context, ten pig herds were visited in southwestern France from December 2016 to May 2017 and serological analyses for influenza A virus (IAV) infections were carried out by ELISA and hemagglutination inhibition assays. In one herd, one backyard pig was shown to have produced antibodies directed against a virus bearing a H5 from clade 2.3.4.4b, suggesting it would have been infected naturally after close contact with HPAI-H5N8 contaminated domestic ducks. Whereas pigs and other mammals, including humans, may have limited sensitivity to HPAI-H5 clade 2.3.4.4b, this information recalls the importance of implementing appropriate biosecurity measures in pig and poultry farms to avoid IAV interspecies transmission, a prerequisite for co-infections and subsequent emergence of new viral genotypes whose impact on both animal and human health cannot be predicted.