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.

Improvement of NASH and liver fibrosis through modulation of the gut-liver axis by a novel intestinal FXR agonist.

Farnesoid X receptor (FXR) plays a pivotal role in the regulation of bile acid homeostasis and is involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). Although FXR agonists effectively alleviate pathological features of NASH, adverse effects such as disturbance of cholesterol homeostasis and occurrence of pruritus remain to be addressed. Here, we identified a novel FXR agonist, ID119031166 (ID166), and explored the pharmacological benefits of ID166 in the treatment of NASH. ID166, a potent and selective non-bile acid FXR agonist, exhibits preferential distribution in the intestine and shows no agonist activity against potential itch receptors including Mas-related G protein-coupled receptor X4 (MRGPRX4). Interestingly, ID166 significantly attenuated total nonalcoholic fatty liver disease (NAFLD) activity and liver fibrosis in a free choice diet-induced NASH hamster model. In addition, ID166 drastically modulated the relative abundance of five gut microbes and reduced the increase in plasma total bile acid levels to normal levels in NASH hamsters. Moreover, long-term treatment with ID166 significantly improved key histological features of NASH and liver fibrosis in a diet-induced NASH mouse model. In the NASH mouse livers, RNA-seq analysis revealed that ID166 reduced the gene expression changes associated with both NASH and liver fibrosis. Notably, ID166 exhibited no substantial effects on scratching behavior and serum IL-31 levels in mice. Our findings suggest that ID166, a novel FXR agonist with improved pharmacological properties, provides a preclinical basis to optimize clinical benefits for NASH drug development.

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.

Molecular imaging of liver inflammation using an anti-VCAM-1 nanobody.

To date, a biopsy is mandatory to evaluate parenchymal inflammation in the liver. Here, we evaluated whether molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) could be used as an alternative non-invasive tool to detect liver inflammation in the setting of chronic liver disease. To do so, we radiolabeled anti-VCAM-1 nanobody (99mTc-cAbVCAM1-5) and used single-photon emission computed tomography (SPECT) to quantify liver uptake in preclinical models of non-alcoholic fatty liver disease (NAFLD) with various degree of liver inflammation: wild-type mice fed a normal or high-fat diet (HFD), FOZ fed a HFD and C57BL6/J fed a choline-deficient or -supplemented HFD. 99mTc-cAbVCAM1-5 uptake strongly correlates with liver histological inflammatory score and with molecular inflammatory markers. The diagnostic power to detect any degree of liver inflammation is excellent (AUROC 0.85-0.99). These data build the rationale to investigate 99mTc-cAbVCAM1-5 imaging to detect liver inflammation in patients with NAFLD, a largely unmet medical need.

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.

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.

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.

Diet-Induced Obesity and NASH Impair Disease Recovery in SARS-CoV-2-Infected Golden Hamsters.

Obese patients with non-alcoholic steatohepatitis (NASH) are prone to severe forms of COVID-19. There is an urgent need for new treatments that lower the severity of COVID-19 in this vulnerable population. To better replicate the human context, we set up a diet-induced model of obesity associated with dyslipidemia and NASH in the golden hamster (known to be a relevant preclinical model of COVID-19). A 20-week, free-choice diet induces obesity, dyslipidemia, and NASH (liver inflammation and fibrosis) in golden hamsters. Obese NASH hamsters have higher blood and pulmonary levels of inflammatory cytokines. In the early stages of a SARS-CoV-2 infection, the lung viral load and inflammation levels were similar in lean hamsters and obese NASH hamsters. However, obese NASH hamsters showed worse recovery (i.e., less resolution of lung inflammation 10 days post-infection (dpi) and lower body weight recovery on dpi 25). Obese NASH hamsters also exhibited higher levels of pulmonary fibrosis on dpi 25. Unlike lean animals, obese NASH hamsters infected with SARS-CoV-2 presented long-lasting dyslipidemia and systemic inflammation. Relative to lean controls, obese NASH hamsters had lower serum levels of angiotensin-converting enzyme 2 activity and higher serum levels of angiotensin II-a component known to favor inflammation and fibrosis. Even though the SARS-CoV-2 infection resulted in early weight loss and incomplete body weight recovery, obese NASH hamsters showed sustained liver steatosis, inflammation, hepatocyte ballooning, and marked liver fibrosis on dpi 25. We conclude that diet-induced obesity and NASH impair disease recovery in SARS-CoV-2-infected hamsters. This model might be of value for characterizing the pathophysiologic mechanisms of COVID-19 and evaluating the efficacy of treatments for the severe forms of COVID-19 observed in obese patients with NASH.

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.

Alteration of the gut microbiota's composition and metabolic output correlates with COVID-19-like severity in obese NASH hamsters.

Obese patientss with nonalcoholic steatohepatitis (NASH) are particularly prone to developing severe forms of coronavirus disease 19 (COVID-19). The gut-to-lung axis is critical during viral infections of the respiratory tract, and a change in the gut microbiota's composition might have a critical role in disease severity. Here, we investigated the consequences of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the gut microbiota in the context of obesity and NASH. To this end, we set up a nutritional model of obesity with dyslipidemia and NASH in the golden hamster, a relevant preclinical model of COVID-19. Relative to lean non-NASH controls, obese NASH hamsters develop severe inflammation of the lungs and liver. 16S rRNA gene profiling showed that depending on the diet, SARS-CoV-2 infection induced various changes in the gut microbiota's composition. Changes were more prominent and transient at day 4 post-infection in lean animals, alterations still persisted at day 10 in obese NASH animals. A targeted, quantitative metabolomic analysis revealed changes in the gut microbiota's metabolic output, some of which were diet-specific and regulated over time. Our results showed that specifically diet-associated taxa are correlated with disease parameters. Correlations between infection variables and diet-associated taxa highlighted a number of potentially protective or harmful bacteria in SARS-CoV-2-infected hamsters. In particular, some taxa in obese NASH hamsters (e.g. Blautia and Peptococcus) were associated with pro-inflammatory parameters in both the lungs and the liver. These taxon profiles and their association with specific disease markers suggest that microbial patterns might influence COVID-19 outcomes.

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.

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.

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.

Alteration of the gut microbiota following SARS-CoV-2 infection correlates with disease severity in hamsters.

Mounting evidence suggests that the gut-to-lung axis is critical during respiratory viral infections. We herein hypothesized that disruption of gut homeostasis during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may associate with early disease outcomes. To address this question, we took advantage of the Syrian hamster model. Our data confirmed that this model recapitulates some hallmark features of the human disease in the lungs. We further showed that SARS-CoV-2 infection associated with mild intestinal inflammation, relative alteration in intestinal barrier property and liver inflammation and altered lipid metabolism. These changes occurred concomitantly with an alteration of the gut microbiota composition over the course of infection, notably characterized by a higher relative abundance of deleterious bacterial taxa such as Enterobacteriaceae and Desulfovibrionaceae. Conversely, several members of the Ruminococcaceae and Lachnospiraceae families, including bacteria known to produce the fermentative products short-chain fatty acids (SCFAs), had a reduced relative proportion compared to non-infected controls. Accordingly, infection led to a transient decrease in systemic SCFA amounts. SCFA supplementation during infection had no effect on clinical and inflammatory parameters. Lastly, a strong correlation between some gut microbiota taxa and clinical and inflammation indices of SARS-CoV-2 infection severity was evidenced. Collectively, alteration of the gut microbiota correlates with disease severity in hamsters making this experimental model valuable for the design of interventional, gut microbiota-targeted, approaches for the control of COVID-19.Abbreviations: SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; COVID-19, coronavirus disease 2019; SCFAs, short-chain fatty acids; dpi, day post-infection; RT-PCR, reverse transcription polymerase chain reaction; IL, interleukin. ACE2, angiotensin converting enzyme 2; TMPRSS2, transmembrane serine protease 2.

Effects of excessive sodium chloride loading in the spontaneously diabetic torii (SDT) fatty rats, a preclinical model of type 2 diabetes mellitus.

Type 2 diabetes mellitus represents an international health concern with its growing number of patients worldwide. At the same time, excessive salt consumption is also seen as a major cause of diseases such as hypertension and may expedite renal complications in diabetic patients. In this study, we investigated the effects of excessive sodium chloride supplementation on the kidney of the Spontaneously Diabetic Torii-Leprfa (SDT fatty) rat, an obese type 2 diabetes model. Male and female SDT fatty rats and normal Sprague-Dawley (SD) rats at 5 weeks of age were loaded with 0.3% sodium chloride (NaCl) in drinking water for 13 weeks. Blood serum and urinary parameters were observed throughout the experiment and kidney samples were examined in histopathological and genetical analyses. Significant changes on the body weight, blood pressure, urine volume, creatinine clearance, blood urea nitrogen (BUN), relative gene expressions of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-ß (TGF-ß) were observed in the salt-loaded male SDT fatty rats. Urinary L-type fatty acid-binding protein (L-FABP) and albumin levels were higher observed in the salt-loaded male SDT fatty rats throughout the period, but urinary albumin levels in the female SDT fatty rats remain unchanged. In the kidney, slight Armani-Ebstein changes, tubular degeneration, hyaline cast, and inflammatory cell infiltration were observed in female SDT fatty rats while the levels of some changes were higher in the salt-loaded group. The kidney of the salt-loaded male SDT fatty rats demonstrated a higher degree of lesions compared to the female group and the male unloaded group. Histopathological changes in salt-loaded SDT fatty rats show that excessive salt consumption may act as a diabetic pathology exacerbation factor, but the pathology may be influenced by gender difference. Urinary L-FABP levels may act as a useful biomarker to detect slight tubular damages in the kidney. Excessive salt loading was shown to exacerbate the renal injury in SDT fatty rats.

The hepatic compensatory response to elevated systemic sulfide promotes diabetes.

Impaired hepatic glucose and lipid metabolism are hallmarks of type 2 diabetes. Increased sulfide production or sulfide donor compounds may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver- enriched mitochondrial SOP enzyme thiosulfate sulfurtransferase (Tst-/- mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia, and fatty liver. Unexpectedly, hepatic sulfide levels are normal in Tst-/- mice because of exaggerated induction of sulfide disposal, with associated suppression of global protein persulfidation and nuclear respiratory factor 2 target protein levels. Hepatic proteomic and persulfidomic profiles converge on gluconeogenesis and lipid metabolism, revealing a selective deficit in medium-chain fatty acid oxidation in Tst-/- mice. We reveal a critical role of TST in hepatic metabolism that has implications for sulfide donor strategies in the context of metabolic disease.

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.

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.

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.