Phylogeny and genotyping of recent avian low-pathogenic H5 subtype influenza viruses from French ducks F.-X. Briand, G. Le Gall-Recule ´ , C. Guillou-Cloarec, K. Ogor and V. Jestin Correspondence V. Jestin v.jestin@afssa.fr AFSSA, French Reference Laboratory for Avian Influenza and Newcastle Disease, Avian and Rabbit Virology, Immunology and Parasitology Unit, BP 53, 22440 Ploufragan, France Received 25 September 2009 Accepted 11 December 2009 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. INTRODUCTION Aquatic birds are the primary reservoir of type A influenza viruses. These avian influenza viruses (AIV) are classified into subtypes based on antigenic differences in their surface glycoproteins (haemagglutinin, HA; and neuraminidase, NA). So far, sixteen haemagglutinin (H1–H16) and nine neuraminidase (N1–N9) subtypes have been reported (Alexander, 2000; Fouchier et al., 2007). The AIV genome is segmented into eight negative strands, permitting the occurrence of reassortment events. Each segment contains either one gene (PB2, PA, HA, NP, NA) or two genes (PB1/ PB1-F2, M1/M2, NS1/NS2) (Chen et al., 2001). Two main lineages (American and Eurasian) have evolved indepen- dently, due to limited contacts between birds from the old and new continents (Ito et al., 1995; Kawaoka et al., 1988; Lin et al., 1994; Okazaki et al., 2000; Webster et al., 1992). Exchanges of AIV genes between these two lineages are, in consequence, very rare (Krauss et al., 2007). Avian influenza viruses can evolve genetically via mutations (substitutions, insertions, deletions) and reassortments (Domingo et al., 2001; Webster et al., 1992). Mutations are due to mistakes generated by the polymerase complex during replication. Some insertions, such as the addition of polybasic amino acids at the cleavage site of H5 or H7 low- pathogenic (LP) AIV, allow the virus to become highly pathogenic (De Marco et al., 2005; Donatelli et al., 2001). This mechanism was reported for Italian H5N2 in 1997, for Italian H7N1 in 1999, for Dutch H7N7 in 2003, for South African H5N2 and for the current ‘Asian’ H5N1. Deletions in the neuraminidase have been observed as an adaptation of AIV to terrestrial poultry (Matrosovich et al., 1999). A further evolutionary phenomenon of AIV is its capacity to undergo reassortment. Co-infection of the same cell of a given host by at least two different viruses can result in mixed segments from both parental viruses. This phenom- enon has given rise to pandemic viruses (Chen et al., 2009; Kawaoka et al., 1989). Poultry infection with H5 or H7 LPAIV can be asymptomatic or produce a wide range of signs, varying from mild respiratory disorders to more severe disease in the case of aggravating circumstances such as concomitant infection with other pathogens (Alexander, 2000). Because these viruses can become highly pathogenic, the presence of H5 or H7 LPAIV in poultry is notifiable and therefore regulated (European Commission, 2006). In order to further our understanding of AIV evolution and the consequences of new mutations, LPAIV H5 and LPAIV H7 from both wild and domestic birds need to be monitored and characterized. Although the characteristics The GenBank/EMBL/DDBJ accession numbers for the new virus sequences determined in this work are CY046117–CY046186. A supplementary figure with details of the phylogenetic analysis of the genes is available with the online version of this paper. Journal of General Virology (2010), 91, 960–970 DOI 10.1099/vir.0.016733-0 960 016733 G 2010 SGM Printed in Great Britain