Emergence and multiple reassortments of French 2015-2016 highly pathogenic H5 avian influenza viruses.

From November 2015 to August 2016, 81 outbreaks of highly pathogenic (HP) H5 avian influenza virus were detected in poultry farms from South-Western France. These viruses were mainly detected in farms raising waterfowl, but also in chicken or guinea fowl flocks, and did not induce severe signs in waterfowl although they did meet the HP criteria. Three different types of neuraminidases (N1, N2 and N9) were associated with the HP H5 gene. Full genomes sequences of 24 H5HP and 6 LP viruses that circulated in the same period were obtained by next generation sequencing, from direct field samples or after virus isolation in SPF embryonated eggs. Phylogenetic analyses of the eight viral segments confirmed that they were all related to the avian Eurasian lineage. In addition, analyses of the "Time of the Most Recent Common Ancestor" showed that the common ancestor of the H5HP sequences from South-Western France could date back to early 2014 (±1 year). This pre-dated the first detection of H5 HP in poultry farms and was consistent with a silent circulation of these viruses for several months. Finally, the phylogenetic study of the different segments showed that several phylogenetic groups could be established. Twelve genotypes of H5HP were detected implying that at least eleven reassortment events did occur after the H5HP cleavage site emerged. This indicates that a large number of co-infections with both highly pathogenic H5 and other avian influenza viruses must have occurred, a finding that lends further support to prolonged silent circulation.

Phylogeny and genotyping of recent avian low-pathogenic H5 subtype influenza viruses from French ducks.

H5 low-pathogenic avian influenza virus (LPAIV) has the potential to become highly pathogenic and to cause serious problems in animal and public health. AIV surveillance and characterization in both wild and domestic species is therefore necessary. In order to acquire molecular information and to identify possible reassortments in French viruses, we analysed the entire genome of five H5N3, three H5N2 and two H5N1 LPAIV, isolated in France between 2002 and 2008 mostly from captive ducks (free-range commercial poultry or decoy ducks). Some of the genome sequences showed atypical characteristics, such as an insertion of 1 aa in the PB1 protein of one H5N3, a highly truncated PB1-F2 protein (11 aa in length instead of 90 aa) in one H5N2, and an insertion of 8 aa in the NS1 protein of H5N1. These two last molecular characteristics have not been described previously. Phylogenetic analysis demonstrated that all genes of French LPAIV, except the closely related matrix protein genes, clustered within the Eurasian avian influenzavirus lineage and fell into at least two phylogenetic subgroups. In addition, the French H5 LPAIV were segregated into eight genotypes, suggesting that many reassortment events have occurred in H5 LPAIV in Europe. However, it is not known whether the reassortment events have occurred in wild waterfowl and/or in captive birds in direct or indirect contact with wild birds.

Virologic findings in selected free-range mule duck farms at high risk for avian influenza infection.

Prevalence of avian influenza infection in free-range mule ducks (a cross between Muscovy [Cairina moschata domesticus] and Pekin ducks [Anas platyrhychos domesticus]) is a matter of concern and deserves particular attention. Thus, cloacal swabs were collected blindly from 30 targeted mule flocks at 4, 8, and 12 wk of age between October 2004 and January 2005. They were stored until selection. On the basis of a positive H5 antibody detection at 12 wk of age with the use of four H5 antigens, the samples from eight flocks were selectively analyzed. Positive samples were first screened with a matrix gene-based real-time reverse transcriptase-polymerase chain reaction assay before virus isolation. Eight avian influenza subtypes (H5N1, H5N2, H5N3, H6N2, H6N8, and H11N9) and three avian paramyxovirus type 1 viruses were isolated. All 11 are characterized as low pathogenicity (LP) and avirulent, respectively, by in vivo tests and, when relevant, nucleotide sequencing of the hemagglutinin (or fusion [F]) protein cleavage site. Regarding H5 isolates, all of their eight genes belong to the avian lineage and some particular genetic traits were determined. H5 genes were fully sequenced and phylogenetically analyzed; they all belong to the Eurasian lineage, lack additional glycosylation sites, and do not cluster, suggesting separate introductions from the wild reservoir. None were grouped with the Asian isolates. The N1 gene (H5N1 isolate) was very close genetically to an Italian LP-H7N1 gene. Antigenic relationships between these H5 isolates and others were assessed comparatively by crossed hemagglutination inhibition tests. All these data are very useful to control the evolution of H5 viruses at the genetic and antigenic level to better understand the source of new outbreaks (new introductions from wild birds or the result of spread among poultry) and to assess the immunity afforded by available vaccines. These data are useful also to update antigens for avian influenza survey and to choose the most suitable vaccine in the case of preventive vaccination of ducks.