Emergence of a novel cluster of influenza A ( H 5 N 1 ) virus clade 2 . 2 . 1 . 2 with putative human health impact in Egypt , 2014 / 15

A S Arafa1,2, M M Naguib1,2,3, C Luttermann3, A A Selim1, W H Kilany1, N Hagag1, A Samy1, A Abdelhalim1, M K Hassan1, E M Abdelwhab1, Y Makonnen4, G Dauphin5, J Lubroth5, T C Mettenleiter3, M Beer3, C Grund3, T C Harder (timm.harder@fli.bund.de)3 1. National Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Dokki, Giza, Egypt 2. These authors have contributed equally 3. Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald Insel-Riems, Germany 4. Food and Agriculture Organization of the United Nations (FAO), Emergency Centre for Transboundary animal Diseases (ECTAD), Cairo, Egypt 5. Food and Agriculture Organisation of the United Nations (FAO), Animal Production and Health division, Viale delle Terme di Caracalla, Rome, Italy

A distinct cluster of highly pathogenic avian influenza viruses of subtype A(H5N1) has been found to emerge within clade 2.2.1.2 in poultry in Egypt since summer 2014 and appears to have quickly become predominant.Viruses of this cluster may be associated with increased incidence of human influenza A(H5N1) infections in Egypt over the last months.
In Egypt, highly pathogenic avian influenza (HPAI) influenza A(H5N1) viruses of clade 2.2.1 and their descendants have been circulating in poultry populations since 2006, causing sporadic human infections [1].Human influenza A(H5N1) infections in Egypt have been reported since the introduction of the virus in 2006 with 204 cases occurring until end of 2014 and a fatality rate of 35,8% in laboratory-confirmed cases reported to the World Health Organization (WHO).However, since January 2015, the incidence of human H5N1 cases in Egypt has increased dramatically: as of 21 March 2015, 116 human cases including 36 deaths have been reported to WHO [2].This study was initiated to analyse molecular properties of H5N1 viruses that have caused outbreaks in poultry in Egypt since summer 2014 and to compare them with published sequences from H5N1 viruses obtained from recent human cases.

Sample origin
Between October 2014 and February 2015, a new wave of 435 outbreaks of H5N1 infections in poultry in Egypt was reported to the National Laboratory for Quality Control on Poultry Production (NLQP) by Egyptian veterinary authorities (Figure 1).Affected poultry species included chickens, ducks, turkeys and quails on commercial farms as well as in backyard holdings.In this study, 29 H5N1-positive samples, mostly obtained by passive surveillance and submitted to NLQP for routine analysis, were selected so as to represent different poultry species, sectors of poultry holdings (commercial farms, backyards and live bird markets) and locations (Table 1).

Phylogenetic analyses
Nucleotide sequence data for the haemagglutinin (HA) gene of all 29 viruses and of the neuraminidase (NA) gene of 15 viruses were generated by Sanger sequencing; whole genome sequencing was carried out for four virus isolates selected to represent different locations, moments in time and sectors of poultry holdings.Phylogenetic analysis of the HA and NA gene sequences was done with the maximum likelihood methodology using the IQTree software [3,4].The authors gratefully acknowledge the originating and submitting laboratories who contributed sequences used in the phylogenetic analysis to the Global Initiative on Sharing All Influenza Data (GISAID) EpiFlu database, and recognise in particular Alice Fusaro and colleagues (Istituto Zooprofilattico Sperimentale Delle Venezie, Padova, Italy) as well as Mee Poh (Centers for Disease Control and Prevention, WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Influenza Division, Atlanta, United States).Phylogenetic analysis placed the H5N1 viruses sequenced in this study into a separate cluster within the previously defined clade 2.2.1.2(Figure 2A and 2B).While the H5N1 isolate A/ duck/Egypt/14VIR784-4-133AD/2013 (KP035030) harboured the closest related ancestral sequences of this cluster, two further poultry viruses sampled in June and July 2014 were placed directly at the root of this cluster: A/quail/Egypt/BSU5514-AR2219/2014 (EPI557138) was at the basis of the HA phylogeny while A/duck/ Egypt/14154-FAOS/2014 (EPI573331) marked the basis of the NA tree (Figure 2A and 2B, green colour).The cluster has expanded since October 2014; the fact that no more sequences of the older range of 2.2.1.2viruses were detected thereafter indicates that this cluster had become predominant over previously circulating phylotypes.GenBank sequences of two recent H5N1 HPAI viruses obtained from infected humans in Egypt in November 2014 (AJM70734 and AJM70746), fell into the same expanding cluster (Figure 2A and 2B, blue colour).Calculation of the time to the most recent common ancestor (TMRCA) of the emerging phylotype by BEAST analysis (Figure 2C) [5] suggested that ancestors of this phylotype emerged around February 2014.Similar phylogenetic relationships were observed for the internal gene segments of these viruses (data not shown, available from authors upon request).The phylogenetic information of the internal genes supports the results for the HA and NA gene sequences, indicating that the new viruses represent a distinct cluster that originated from previously circulating viruses of clade 2.2.1.2.So far, no reassortment events were found to be involved in generating this newly emerging phylotype.

Genetic characterisation
Compared with viruses sampled before October 2014 in Egypt, the emerging cluster contained distinct fixed mutations in several genome segments (PB2, PB1-F2, HA, NA, M1).
In the HA gene, differences in the nucleotide composition of the coding sequence of up to 1.3% were found, which included up to 14 specific fixed nucleotide substitutions distinguishing these viruses from earlier Egyptian isolates of 2014.A total of 12 of these mutations were synonymous (silent) and only two resulted in amino acid substitutions (K373R and F537S).Of these, only K373R, located in the stalk domain at the oligomerisation interface of the HA, is characteristic of the emerging cluster; this mutation has sporadically been reported in very few older isolates from Egypt [6].
The mutation F537S had already been detected in several older isolates.In addition, the HA protein of the viruses in the new cluster contained mutations D94N, T156A, K189R and P235S, which are associated with improved binding to SAα2,6-Gal, the human type of influenza virus receptors [7].However, these mutations were present also in earlier clade 2.2.1 H5N1 viruses  The phylogenetic analysis was done by maximum likelihood method using the IQTree algorithm [3,4].BEAST [5] analysis was carried out to determine time to the most recent common ancestor (TMRCA; indicated by horizontal blue bars overlaying nodes).TMRCA calculations were based on an uncorrelated log-normal relaxed clock model.The maximum clade credibility tree was scaled to time using the collection dates (day/month/year) of all samples.Red labels indicate poultry viruses sampled after August 2014 and sequenced in this study.Blue labels denote sequences of viruses retrieved from human influenza A(H5N1) infections (AJM70734; AJM70746).Green labels highlight viruses close to the basis of the emerging cluster of selected H5N1 HPAI viruses circulating in Egypt.The authors gratefully acknowledge the originating and submitting laboratories who contributed sequences used in the phylogenetic analysis to the Global Initiative on Sharing All Influenza Data (GISAID) EpiFlu database.
in Egypt.Since no substituting mutations were found in HA epitopes, we do not expect marked differences in the antigenic properties of the emerging phylotype compared with the previously circulating clade.This was partially confirmed by haemagglutination inhibition assays using sera against different clades of H5 viruses (Table 2).
The NA gene of the emerging cluster differed by seven nucleotide substitutions from recent H5N1 HPAI viruses of clade 2.2.1.2.Four of the seven mutations encoded amino acid substitutions not previously reported in 2.2.1.2viruses: V34I, I74V, V244I and V284I (Table 3).Mutation V34I has been reported in H5N1 strains from Cambodia from 2013 where an increase in human H5N1 infections was observed [8].No biological function has been associated with the V34I substitution, while positions 74, 244 and 284 are located in B-or T-cell antigenic regions of the NA protein [9].For four viruses, whole genome sequences were generated and further signature mutations of the emerging viruses were found in the internal gene segments as well: Non-silent cluster-specific mutations were confined to the PB2 (M66I, T106A), PB1-F2 (G22E), and M1 (I15V) proteins (Table 3).

Discussion
Influenza pandemics remain one of the major threats posed by communicable diseases to the human population.The avian reservoir of influenza viruses contributed by reassortment to the emergence of most previous pandemic human influenza viruses [10].Since their emergence in Asia in 2003, HPAI viruses of subtype H5N1 and their recent descendants continue to cause significant economic losses to commercial poultry not only in Asia, but also in Egypt where high mortality in poultry has continuously been observed since 2006 [11,12].They also exhibit strain-specific zoonotic potential resulting in sporadic avian-to-human spillover transmissions which lead to human infections associated with a high case fatality rate [13].However, apart from sporadic cases (e.g., family clusters) sustained human-to-human transmission of any of these viruses has not ensued so far.
Our data confirm the emergence of an additional virus cluster within the Egyptian 2. Given the endemic status of influenza H5N1 in poultry and the limitations of the reporting system of H5N1 HPAI virus outbreaks in poultry in Egypt, it is difficult to assess whether the altered epidemiological pattern of the emerging phylotype is due to altered biological properties in poultry or whether the increased incidence of infections in poultry merely reflects an increased viral burden across all poultry sectors in Egypt.In any case, the observed recent rise in outbreaks in poultry probably resulted in increased exposure risks for humans in contact with poultry, which may have caused an increased incidence in human cases.However, it can at this point not be excluded with certainty that the emerging phylotype of viruses may have increased zoonotic potential and may be transmitted more efficiently to humans, although this assumption cannot be drawn from the molecular evidence described here.Further studies of the pathogenicity and transmissibility of these viruses in humans, e.g. in the ferret model, are required.Concerted efforts of both veterinary and public health authorities are urgently needed to interrupt virus circulation in poultry in Egypt efficiently.This will help decrease the risk of human exposure to the virus.

Figure 2
Figure 2 Phylogenetic analysis of the HA (A) and NA (B) genes (coding regions) and maximum clade credibility tree (C) based on the HA open reading frame of selected highly pathogenic avian influenza A(H5N1) viruses, Egypt, June 2014-January 2015 (n=29)
2.1.2clade of H5N1 HPAI viruses.Since November 2014, viruses of this new cluster appear to have become dominant over the previously described clade 2.2.1.2phylotypes circulating a

Table 3
Amino acid residues distinguishing recently emerging highly pathogenic avian influenza A(H5N1) viruses from virus lineages circulating before November 2014, Egypt, March 2015 Data on the new cluster are based on sequences established in this study: HA (29 sequences), NA (15 sequences), internal gene segments (4 sequences).Data for older clades were retrieved from public databases.a Some of the listed mutations have been infrequently observed among single isolates from previous years.