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.

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.

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.

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.

Natural and Experimental Persistence of Highly Pathogenic H5 Influenza Viruses in Slurry of Domestic Ducks, with or without Lime Treatment.

Infections by A/H5 and A/H7 avian influenza viruses (AIVs) can cause acute disease and are therefore notifiable in poultry and wild birds. During winter 2015-2016, several cases of infection caused by highly pathogenic (HP) AIVs belonging to the A/H5N1, A/H5N2, and A/H5N9 subtypes were detected in southwestern France. Throughout winter 2016-2017, several cases of infections caused mainly by A/H5N8 HP AIV (A/goose/GD/1/1996, clade 2.3.4.4) were detected across Europe. On both occasions, the viruses were widely detected on palmiped farms in France. This study was designed to evaluate the persistence of A/H5 HP AIV in slurry from various duck productions. This was achieved (i) in the laboratory setting by artificially spiking four AIV-free slurry samples with known amounts of A/H5N9 HP AIV and monitoring virus infectivity, with or without lime treatment to achieve pH 10 or pH 12, and (ii) by sampling slurry tanks on five naturally A/H5N8 HP-contaminated farms. Experimental results in artificially spiked slurry suggested virus survival for 4 weeks in slurry from Muscovy or Pekin duck breeders and for 2 weeks in slurry from ducks for foie gras production during the assisted-feeding period, without lime treatment. Persistence of infectious A/H5N9 HP AIV in all slurry samples after lime treatment at pH 10 or pH 12 was less than 1 week. The A/H5N8 HP AIV persisted in naturally contaminated untreated slurry for 7 weeks. The results obtained provide experimental support for the 60-day storage period without treatment or the 7-day interval after lime treatment defined in French regulations for slurry sanitization.IMPORTANCE From November 2015 to July 2017, two successive episodes of H5 highly pathogenic avian influenza viruses (HP AIVs) infections occurred on poultry farms in France, mostly in domestic ducks raised for foie gras production in southwestern France. During the two epizootics, epidemiological investigations were carried out on infected farms and control and biosafety measures were implemented in association with surveillance in order to stop the spread of the viruses. Effluents are known to be an important factor in environmental dissemination of viruses, and suitable effluent management is needed to help prevent the spread of epizootics to other farms or pathogen persistence at the farm level. The present study was therefore designed to assess how long infectious A/H5 HP AIVs can persist in naturally or experimentally contaminated fecal slurry samples from ducks, with or without sanitization by lime treatment.

Highly Pathogenic Avian Influenza H5N1 A/Chicken/France/150169a/2015 Presents In Vitro Characteristics Consistent with Its Predicted Tropism for Avian Species.

Avian influenza A viruses are a major threat to animal and public health. Since 1997, several highly pathogenic H5N1 avian viruses have been directly transmitted from poultry to humans, caused numerous human deaths, and had considerable economic impact on poultry markets. During 2015-2016, a highly pathogenic avian influenza outbreak occurred in southwestern France. Different subtypes circulated, including the A/chicken/France/150169a/2015 H5N1 highly pathogenic virus, which did not possess the full set of genomic determinants known to promote transmission to humans. In order to evaluate the predicted absence of zoonotic potential, a quick method based on in vitro tests was developed to analyze some genetic and phenotypic host restriction determinants. A receptor-binding assay showed that the virus preferentially recognizes avian cell receptors. Temperature sensitivity revealed a cold-sensitive phenotype of the virus at 33 C as virus replication was reduced in contrast with what is expected for human influenza viruses, according to their primary infection sites. Altogether, our quick evaluation method suggests that the A/chicken/France/150169a/2015 H5N1 highly pathogenic virus has an avian phenotype in vitro, in accordance with in silico predictions based on genomic markers.

Nota de Investigación - El virus de la influenza aviar altamente patógeno H5N1 A/Pollo/Francia/150169a/2015 presenta características in vitro consistentes con el tropismo que ha sido predicho para especies aviares. Los virus de la influenza aviar A son una amenaza importante para la salud animal y pública. Desde el año 1997, varios virus aviares H5N1 altamente patógenos que se han transmitido directamente de la avicultura comercial a los humanos, han causado numerosas muertes humanas y han tenido un impacto económico considerable en los mercados avícolas. Durante los años 2015-2016, se produjo un brote de influenza aviar altamente patógena en el suroeste de Francia. Diferentes subtipos circularon, incluido el virus H5N1 A/pollo/Francia/150169a/2015, altamente patógeno, que no poseía completamente el conjunto de determinantes genómicos conocidos por promover la transmisión a los humanos. Para evaluar la ausencia prevista de potencial zoonótico, se desarrolló un método rápido basado en pruebas in vitro para analizar algunos determinantes genéticos y fenotípicos de restricción para el hospedero. Un ensayo de unión al receptor mostró que el virus reconoce preferentemente los receptores de células aviares. La sensibilidad a la temperatura reveló un fenotipo del virus sensible al frío a 33 C ya que la replicación del virus se redujo en contraste con lo esperado para los virus de la influenza humana, según sus sitios de infección primaria. En conjunto, el presente método de evaluación rápida sugiere que el virus altamente patógeno A/pollo/Francia/150169a/2015 H5N1 tiene un fenotipo aviar in vitro, que está de acuerdo con las predicciones in silico basadas en marcadores genómicos.

Exploring First Interactions Between Ostreid Herpesvirus 1 (OsHV-1) and Its Host, Crassostrea gigas: Effects of Specific Antiviral Antibodies and Dextran Sulfate.

Viral entry mechanisms of herpesviruses constitute a highly complex process which implicates several viral glycoproteins and different receptors on the host cell surfaces. This initial infection stage was currently undescribed for Ostreid herpes virus 1 (OsHV-1), a herpesvirus infecting bivalves including the Pacific oyster, Crassostrea gigas. To identify OsHV-1 glyproteins implicated in the attachment of the virus to oyster cells, three viral putative membrane proteins, encoded by ORF 25, 41, and 72, were selected and polyclonal antibodies against these targets were used to explore first interactions between the virus and host cells. In addition, effects of dextran sulfate, a negative charged sulfated polysaccharide, were investigated on OsHV-1 infection. Effects of antiviral antibodies and dextran sulfate were evaluated by combining viral DNA and RNA detection in spat (in vivo trials) and in oyster hemolymph (in vitro trials). Results showed that viral protein encoded by ORF 25 appeared to be involved in interaction between OsHV-1 and host cells even if other proteins are likely implicated, such as proteins encoded by ORF 72 and ORF 41. Dextran sulfate at 30 µg/mL significantly reduced the spat mortality rate in the experimental conditions. Taken together, these results contribute to better understanding the pathogenesis of the viral infection, especially during the first stage of OsHV-1 infection, and open the way toward new approaches to control OsHV-1 infection in confined facilities.

Haemocytes collected from experimentally infected Pacific oysters, Crassostrea gigas: Detection of ostreid herpesvirus 1 DNA, RNA, and proteins in relation with inhibition of apoptosis.

Recent transcriptomic approaches focused on anti-viral immunity in molluscs lead to the assumption that the innate immune system, such as apoptosis, plays a crucial role against ostreid herpesvirus type 1 (OsHV-1), infecting Pacific cupped oyster, Crassostrea gigas. Apoptosis constitutes a major mechanism of anti-viral response by limiting viral spread and eliminating infected cells. In this way, an OsHV-1 challenge was performed and oysters were monitored at three times post injection to investigate viral infection and host response: 2h (early after viral injection in the adductor muscle), 24h (intermediate time), and 48h (just before first oyster mortality record). Virus infection, associated with high cumulative mortality rates (74% and 100%), was demonstrated in haemocytes by combining several detection techniques such as real-time PCR, real-time RT PCR, immunofluorescence assay, and transmission electron microscopy examination. High viral DNA amounts ranged from 5.46×104 to 3.68×105 DNA copies ng-1 of total DNA, were detected in dead oysters and an increase of viral transcripts was observed from 2, 24, and 48hpi for the five targeted OsHV-1 genes encoding three putative membrane proteins (ORFs 25, 41, and 72), a putative dUTPase (ORF 75), and a putative apoptosis inhibitor (ORF 87). Apoptosis was studied at molecular and cellular levels with an early marker (phosphatidyl-serine externalisation measured by flow cytometry and epifluorescence microscopy) and a later parameter (DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (TUNEL)). The down-regulation of genes encoding proteins involved in the activation of the apoptotic pathway (TNF and caspase 3) and the up-regulation of genes encoding anti-apoptotic proteins (IAP-2, and Bcl-2) suggested an important anti-apoptosis phenomenon in haemocytes from OsHV-1 infected oysters at 24 and 48hpi. Additionally, more phosphatidyl-serines were externalized and more cells with DNA fragmentation were observed in haemocytes collected from artificial seawater injected oysters than in haemocytes collected from OsHV-1 infected oysters at 24 and 48hpi, suggesting an inhibition of the apoptotic process in presence of the virus. In conclusion, this study is the first to focus on C. gigas haemocytes, cells involved in the host immune defense, during an OsHV-1 challenge in controlled conditions by combining various and original approaches to investigate apoptosis at molecular and cellular levels.

In situ localization and tissue distribution of ostreid herpesvirus 1 proteins in infected Pacific oyster, Crassostrea gigas.

Immunohistochemistry (IHC) assays were conducted on paraffin sections from experimentally infected spat and unchallenged spat produced in hatchery to determine the tissue distribution of three viral proteins within the Pacific oyster, Crassostrea gigas. Polyclonal antibodies were produced from recombinant proteins corresponding to two putative membrane proteins and one putative apoptosis inhibitor encoded by ORF 25, 72, and 87, respectively. Results were then compared to those obtained by in situ hybridization performed on the same individuals, and showed a substantial agreement according to Landis and Koch numeric scale. Positive signals were mainly observed in connective tissue of gills, mantle, adductor muscle, heart, digestive gland, labial palps, and gonads of infected spat. Positive signals were also reported in digestive epithelia. However, few positive signals were also observed in healthy appearing oysters (unchallenged spat) and could be due to virus persistence after a primary infection. Cellular localization of staining seemed to be linked to the function of the viral protein targeted. A nucleus staining was preferentially observed with antibodies targeting the putative apoptosis inhibitor protein whereas a cytoplasmic localization was obtained using antibodies recognizing putative membrane proteins. The detection of viral proteins was often associated with histopathological changes previously reported during OsHV-1 infection by histology and transmission electron microscopy. Within the 6h after viral suspension injection, positive signals were almost at the maximal level with the three antibodies and all studied organs appeared infected at 28h post viral injection. Connective tissue appeared to be a privileged site for OsHV-1 replication even if positive signals were observed in the epithelium cells of different organs which may be interpreted as a hypothetical portal of entry or release for the virus. IHC constitutes a suited method for analyzing the early infection stages of OsHV-1 infection and a useful tool to investigate interactions between OsHV-1 and its host at a protein level.

Detection of undescribed ostreid herpesvirus 1 (OsHV-1) specimens from Pacific oyster, Crassostrea gigas.

The ostreid herpesvirus 1 (OsHV-1) and variants were implicated in mass mortality affecting the young Pacific cupped oysters, Crassostrea gigas, in European countries and those around the world. From 2008 onwards, oyster mortality had greatly increased on the French coast and was associated with the detection of a new OsHV-1 variant, entitled OsHV-1 µVar. The OsHV-1 µVar is predominant in oysters; however, other OsHV-1 variants have been detected in samples collected during mortality periods or collected out of mortality periods in France, Ireland, Spain, Portugal, Italy, Mexico, United States, South Korea, Australia, and New Zealand. A retrospective study conducted on 1047 OsHV-1 specimens sampled mainly in France between 2009 and 2012, revealed 17 undescribed OsHV-1 variants found in 65 oyster samples. These specimens presented point mutations situated downstream and upstream from the microsatellite area in the C region (ORF 4/5) which were different from the OsHV-1 reference and the OsHV-1 µVar. In the present work, investigation was performed to further characterize these OsHV-1 specimens by sequencing two habitually targeted regions to study genetic polymorphism of the virus: ORF 41/42 and ORF 35-38. An OsHV-1 variant detected in six oyster samples, contained a nucleotide substitution in the C region which impacted the amino acid sequence and might modify the function of the unknown protein encoding by ORF 4. For the ORF 41/42 region, only two specimens presented a synonymous mutation in comparison with the OsHV-1 µVar. All specimens contained the same deletion with the OsHV-1 µVar in ORF 35-38. Then, a phylogenetic analysis based on the C region was performed to investigate the distribution of undescribed specimens among 21 OsHV-1 DNA sequences notified in GenBank and collected from different countries (France, Japan, New Zealand, China, Ireland, and United States) between 1995 and 2012. All analyzed samples and the OsHV-1 µVar were placed in the same group, excepted for a Japan specimen. Our results contribute to improve the description of the genetic diversity of the OsHV-1 and the C region (ORF 4/5) appears to be a better target than ORF 42/42 and 35-38 to distinguish variants between themselves.

Genome exploration of six variants of the Ostreid Herpesvirus 1 and characterization of large deletion in OsHV-1µVar specimens.

The genetic polymorphism of the Ostreid Herpesvirus 1 (OsHV-1) has generally been investigated in three areas: ORFs 4/5, ORFs 42/43, and ORFs 35 to 38. The present study, however, focuses on 40 ORFs, representing 30% of the OsHV-1 genome, encoding four categories of putative proteins: 4 ORFs encoding putative inhibitor of apoptosis proteins; 17 ORFs encoding membrane proteins; 10 ORFs encoding secreted proteins; and 9 ORFs encoding RING finger proteins. The potential role of these proteins in major steps of the life cycle of the OsHV-1 motivated their selection. Seven specimens have been selected in accordance with their nucleotide variations in the C region (area located between the end of the ORF4 and the beginning of ORF 5): 3 OsHV-1µVar specimens, 2 OsHV-1µVar Δ9, one specimen of OsHV-1µVar Δ15, and one OsHV-1 specimen (reference control) close to the reference genome to validate PCRs. The OsHV-1µVar is mainly characterized by a deletion of 12 consecutive nucleotides followed by a deletion of one adenine in a microsatellite area located in the C region. A representation of nucleotide modifications between the different specimens was performed by building evolutionary trees with respect to the category of ORFs. This phylogenetic analysis revealed two groups: the first one corresponded to the reference control and the reference genome AY509253, and the second one included the 6 OsHV-1 variants. These results suggest that the two main groups come from the same common ancestor, and that the divergence between the reference OsHV-1 and its variants occurred quite far back in time. Moreover, consequences of nucleotide variations in the amino acid sequences, especially the change of the N glycoslyation sites, were investigated. Herein is the first report of four important deletions in these OsHV-1µVar variants: a deletion of 1385bp in ORF 11; a deletion of 599bp in ORF 48; a deletion of 3549bp in ORFs 61 to 64; and a deletion of 712bp in ORF 114. The size of the deletions differed between OsHV-1µVar specimens, OsHV-1µVar Δ9 specimens, and the OsHV-1µVar Δ15 specimen. These zones seem to correspond to special points of gene rearrangements for producing new proteins. Further investigation necessary proves to link such nucleotide modifications with consequences of protein functions in the OsHV-1 life cycle.

Variants of herpetic virus ostreid herpesvirus-1 (OsHV-1) in young Pacific oyster Crassostrea gigas.

Since the 1990s, high mortality rates of young Pacific oysters Crassostrea gigas have been regularly recorded in France and affect mainly spat (oysters less than 1 year old). Pathogens infecting marine bivalve mollusks have been studied particularly due to their economic and ecologic impacts on the farmed and wild oysters. A herpes virus belonging to the family Malacoherpesviridae and called Ostreid Herpesvirus-1 (OsHV-1) has been detected in infected oysters. Since the summer 2008, the mortality of spat has greatly increased on the French coast (with a mortality rate ranging from 40 to 80%) and may be linked to a variant of the OsHV-1, named OsHV-1 µVar. Few variants of the OsHV-1 and genotypes closed to the OsHV-1 µVar have been described in several areas in the world. However, the OsHV-1 µVar is predominant in analyzed samples and the reference genotype of the OsHV-1 has not been detected since 2008. The causes of the emergence of the OsHV-1 µVar and its rapid spreading in the world are currently unknown.

Detection of different variants of Ostreid Herpesvirus 1 in the Pacific oyster, Crassostrea gigas between 2008 and 2010.

Since summer 2008, high mortality rates of young Pacific oysters Crassostrea gigas have been recorded in association with the detection of the Ostreid Herpesvirus 1 (OsHV-1). A new variant called µVar has been recently described, characterized mainly by 12 consecutive deletions followed by one deletion of an adenine in the C region. The purpose of this study is to characterize the genotype (variants or OsHV-1 reference) of 300 positive samples of C. gigas analyzed between July 2008 and July 2010 collected along the French, Jersey, and Irish coasts. Samples were quantified by TaqMan PCR, amplified with conventional PCR, targeting the area of the deletion, and then sequenced. Eighty-seven percent of the samples were characterized and the OsHV-1 µVar was detected in 257 oyster samples. The genotype OsHV-1 reference was never detected during the 25 months of the present survey. Thirty-eight samples could not be determined and the majority of them had a low viral load. A novel genotype containing only 9 consecutive deletions named OsHV-1 µVar Δ9 was found in 5 samples. These observations indicate the emergence of different OsHV-1 variants.