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.

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.