Novel real-time PCR-based patho- and phylotyping of potentially zoonotic avian influenza A subtype H5 viruses at risk of incursion into Europe in 2017

摘要

Since November 2016, Europe witnesses another wave of incursion of highly pathogenic avian influenza (HPAI) A(H5) viruses of the Asian origin goose/Guangdong (gs/GD) lineage. Infections with H5 viruses of clade 2.3.4.4b affect wild bird and poultry populations. H5 viruses of clades 2.2, 2.3.1.2c and 2.3.4.4a were detected previously in Europe in 2006, 2010 and 2014. Clades 2.2.1.2 and 2.3.2.1.c are endemic in Egypt and Western Africa, respectively and have caused human fatalities. Evidence exists of their co-circulation in the Middle East. Subtype H5 viruses of low pathogenicity (LPAI) are endemic in migratory wild bird populations. They potentially mutate into highly pathogenic phenotypes following transmission into poultry holdings. However, to date only the gs/GD H5 lineage had an impact on human health. Rapid and specific diagnosis marks the cornerstone for control and eradication of HPAI virus incursions. We present the development and validation of five real-time RT-PCR assays (RT-qPCR) that allow sequencing-independent pathotype and clade-specific distinction of major gs/GD HPAI H5 virus clades and of Eurasian LPAI viruses currently circulating. Together with an influenza A virus-generic RT-qPCR, the assays significantly speed up time-to-diagnosis and reduce reaction times in a OneHealth approach of curbing the spread of gs/GD HPAI viruses. Keywords: Zoonotic avian influenza, phylotyping, pathotyping, real-time RT-qPCR, diagnosisIntroductionInfluenza A viruses constitute a virus species in the family Orthomyxoviridae . They harbour single-stranded negative-sense RNA arranged into eight genomic segments. Members of this species which infect avian hosts (avian influenza viruses, AIV) are grouped into 16 (H1 to H16) and 9 (N1 to N9) subtypes, respectively, based on phylogenetic and antigenic properties of their haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins [1]. Different species of aquatic wild birds are the natural reservoirs for all AIV subtypes. Novel subtypes and gene constellations continue to evolve in aquatic wild birds or in infected poultry populations by genetic reassortment during infection of a single host cell with two or more distinct AIV genotypes. In addition to reassortment, the intrinsically error-prone influenza virus genome replication machinery promotes the generation of quasi-species that can be shaped by directional selection pressures, e.g. following host species switches or by specific herd immunity. In the latter case, antigenic drift variants are selected that may escape immunity by very few amino acid substitutions in the HA [2].Based on their virulence in galliform poultry (e.g. chicken, turkey), AIV are distinguished into groups of highly pathogenic (HP) and low pathogenic (LP) phenotypes [3]. Correct AI diagnosis includes determining the HA subtype and, in case of subtypes H5 or H7, also the pathotype. So far, HPAI phenotypes detected in the field (i.e. ‘free’ natural environment), were only described among AIV of subtypes H5 and H7 [4]. Some of these viruses including those of the HPAI H5 goose/Guangdong (gs/GD) lineage that emerged in southern China in 1996, have zoonotic potential and are sporadically transmitted from infected birds to humans [5,6]. HPAI viruses of the gs/GD lineage have continued to circulate and evolved into numerous clades. Viruses of three major phylogenetic clades (2.2.1.2, 2.3.2.1 and 2.3.4.4) as well as of three further minor clades (1.1.2, 2.1.3.2 and 7.2) have become endemic in poultry populations in several countries in Asia, Africa and the Middle East [7]. Occasionally, spillover transmission from infected poultry may cause infection and viral spread in wild birds with increased mortality in some species. Infected migratory wild birds may spread such viruses across wider distances and act as the source of transmission back to poultry [7,8].Europe has experienced several incursions by viruses of the gs/GD lineage over the past decade; both wild birds and poultry were affected but no human cases were reported [9]. This is in sharp contrast to Egypt and Asian countries where the endemicity of HPAI H5 viruses in poultry is associated with repeated spillover transmission to and infection of humans. In fact, the majority of human HPAI H5 cases worldwide were registered in Egypt [10,11]. Moreover, a new major clade, designated 2.2.1.2, evolved along with transient spread of an escape mutant-based lineage, 2.2.1.1, in this country [12].Further potentially zoonotic gs/GD viruses of clade 2.3.2.1c are widespread in Central and Southern Asia and they were sporadically detected along the European Black Sea coast as well as in the Middle East [13-15]. In addition, viruses of this clade have caused major outbreaks among poultry in several Western African countries with ongoing virus circulation to date [16]. Interestingly, 2.3.2.1c viruses have not (yet) been reported from Egypt. Since 2010, another gs/GD cluster, termed 2.3.4.4, has e