Phylogenetic and epidemiological characteristics of H9N2 Avian Influenza Viruses from 2020 to 2022 in Egypt

Phylogenetic and epidemiological characteristics of H9N2 Avian Influenza

 Viruses from 2020 to 2022 in Egypt

Zienab Mosaad^*, Naglaa M. Hagag^*, Moataz Mohamed^*, Wesam H. Mady^*, Zeinab A. El-Badiea^*, Osama Mahana^*, Neveen Rabie^*, Mohamed Samy^*, Ola abdel aziz^*, Abdel-Satar Arafa^*, Abdelhafiz Samir^*, Abdullah Selim^*, Samah Eid^*, Momtaz A. Shahein, Amany Adel^*

^*Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center (ARC), Giza, Egypt.

ABSTRACT

The H9 low-pathogenic avian influenza (LPAI) viruses cause enormous economic harm despite their low pathogenicity. It became common in Egypt in 2011 and has undergone ongoing genetic evolution since then. To limit the virus's transmission, regular monitoring of its evolution is essential. The current study concentrated on the frequency and molecular characteristics of LPAI H9N2 viruses spreading throughout different Egyptian areas between 2020 and 2022. Using real-time PCR, 503 positive LPAI H9 cases were detected out of 29,319 cases, for a total prevalence rate of 1.7%. However, live bird market (LBM) had the highest LPAI H9N2 prevalence rate (10.6%), followed by household sector and farm (2 % and 1.3% respectively). The 33 samples were isolated in 11-day-old embryonated chicken eggs (ECEs) before being sequenced for partial hemagglutinin (HA). The H9 isolates were phylogenetically related to the Egy-2 G1-B branch (pigeon-like), which has been the prevalent circulating H9N2 genotype in Egypt since 2016. The findings of the sequence analysis revealed a clear genetic evolution compared to the original virus (A/quail/Egypt/113413v/2011), which shared 93.2–95.4% and 94.7-97.1% homology at the nucleotide and amino acid levels, respectively. In comparison to the reference Egyptian strain, the molecular analysis found 12 alterations in amino acid residues with genetic stability in the major locations. The majority of examined strains had five glycosylation sites in HA. However, some strains had an extra sites at position 105, 145, 258. Comparable to A/quail/Hong Kong/G1/97, and all strains had the substitutions H191and L234 in the HA gene, indicating a predilection for binding to human-like receptors. Because of continues genetic development of H9 viruses reported in this work, frequent viral surveillance is required for better management.

INTRODUCTION

H9N2 low pathogenic avian influenza is a subtype of influenza viruses type A belonging to family Orthomyxoviridae. Since the first isolation of H9N2 prototype strain A/turkey/Wisconsin/1966 (Tu/WS/66) in 1966 in USA from turkey (Homme and Easterday, 1970), it was widely distributed among domestic and wild bird worldwide. The widespread and continuous circulation of H9N2 LPAI among different bird species cause persistent problems for the poultry industry (Alexander, 2000; Nili and Asasi, 2002), besides its ability to infect human beings causing threat to public health (Butt et al. 2005 Li et al. 2003).

Based on phylogenetic analysis, H9N2 was classified into two major genetic lineages; the North American and Eurasian lineages, the Eurasian lineage was further divided into 3 sub lineages; Korean like (A/chicken/Korea/38349), Y280-like (A/ duck/Hong Kong/Y280/9), and G1-like (A/quail/ Hong Kong/G1/97) (Guan et al. 2000), the LPAIV H9N2 from 1998 to 2010 in Central Asia and the Middle East, were clustered into four distinct groups (A, B, C, and D) (Fusaro et al. 2011). The H9N2 LPAI genome is unstable and continuously mutates through antigenic drift in the HA gene arising H9N2 variants (Peacock et al. 2018) as well as the antigenic shift as The H9N2 viruses frequently donate their internal genes to other AIVs during the co-infection (Hagag et al. 2019 Kandeil et al. 2017 Peacock et al. 2019).

In Egypt, Since the first detection of H9N2 LPAI from quail in 2011 (El-Zoghby et al. 2012), Egypt has been endemic with H9N2 LPAIV causing severe economic losses in poultry production, the circulated virus belonged to Asian G1-like and closely related to Israel strains (Monne et al. 2013). Genetic variability was detected in amino acid levels in the surface genes indicated continuous evolu­tion of H9N2 AIVs with complicated genetic reassortment in Egypt (Elsayed et al. 2021). In 2014, new antigenically distinct variant of H9N2 LPAIV was detected in quail (quail/2014 variant) (Adel et al. 2017). As well as a novel reassortament variant was reported in pigeon containing five internal gene segments (PB2, PB1, PA, NP, and NS) from wild bird like AIVs (Eurasian AIV) subtypes and (HA, NA, M) from Egyptian H9N2/2011 virus (Kandeil et al. 2017). The same genotype was found in backyard chickens in three Egyptian governorates in 2015 (Samir et al. 2019). Another reassortant virus has been evolved in 2014-2015 from the pigeon H9N2 virus with an Egyptian virus in late 2014, sharing PB2, PB1, PA, and NS genes (Hassan et al. 2020).

Furthermore, a novel reassortant H5N2 HPAI virus emerged in late 2017–2018, with 7 genes of the Egyptian LPAI H9N2 virus and only the HA gene from the Egyptian HPAI H5N8 virus (Hagag et al. 2019; Hassan et al. 2020).

 Based on the phylogenetic analysis, the EgyptianH9N2 LPAIVs have been further diversified into three groups clustered within G1-B lineage based on their HA gene segment (Kandeil et al. 2014; Naguib et al. 2017). So, this study aimed to molecular detection, isolation and Genetic characterization of H9N2 LPAI currently circulating in Egypt. The current study was aimed to monitor the LPAI H9N2 virus. The collected LPAIH9N2viruses samples during 2020-2022 in different governorate in Egypt.

MATERIALS and METHODS

Samples collection

Total of 496,166 Cloaca and oropharyngeal swabs were collected from 29319 cases from different poultry sectors (307 Household, 27762 Farm and 1250 LBM, for regular screening at the Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP, Egypt) in 2020-2022. The samples have been collected during active and passive surveillance from 27 Egyptian governorates. Several poultry species were involved in the surveillance, including chicken (no. of cases: 25947), ducks (no. of cases: 1466), mixed flocks (no. of cases: 1150 turkey (no. of cases: 698), wild birds (no. of cases:5) , other species including geese, pigeon , quail, ostrich and environmental samples (no. of cases: 53)

Virus detection and isolation

For each collected sample, RNA was extracted from the supernatant liquid using QIAamp viral RNA Mini kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions. Using specific primers and probes (Adel et al. 2017; Adel et al. 2019; Shabat et al. 2010), the RNA was examined against the Matrix (M) gene of influenza A viruses. Positive sample was further tested against AIV subtypes using real-time reverse transcriptase quantitative polymerase chain reaction RT-qPCR. Further, all samples were screened against Newcastle disease virus (NDV), infectious bronchitis virus (IBV) to explore the possibility for co-infection with other viruses. Reaction mixes were prepared using RT-qPCR Verso 1-step™ Real Time PCR kit (Catalog no.AB4101A) and performed using Stratagene MX3005real-time PCR machine.

For each positive sample 0.1ml of the supernatant fluid was injected into three separate specific pathogen free embryonated chicken eggs (SPF-ECE) of 9-11 days of age. The inoculated eggs were then incubated at 37 °C and monitored daily for 3–5 days. Allantoic fluid was retrieved from the collected or dead eggs and tested for virus haemagglutination activity by HA assay (Manual, 2015).

Nucleotide sequencing and phylogenetic analysis

The HA gene of selected positive samples were amplified using specific primers. The PCR was carried out using applied biosystem thermal cycler (ProFlexTM PCR System) using an Easyscript one-step RT-PCR kit (Trans Gen Biotech)). Size-specific PCR products for each gene were separated by gel electrophoresis and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden,  Germany).

Further, purified products were using for nucleotide sequencing using Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA) and purified using Centrisep spin column, (Thermo Fisher,Waltham, MA,  USA). Sequencing was performed using ABI PRISM® 3100 Genetic Analyzer (Life Technologies, USA). Further, the obtained nucleotide sequences were assembled and edited using Bio-edit programme version 7.2.5 (Hall et al. 2011). Generated sequences in this study were deposited at the GenBank database under accession numbers provided in (Table 1). A Blast search was performed using (http://www.ncbi.nlm.nih.gov/blast/) on the NCBI website

The nucleotide sequences were aligned using BioEdit version 7.0 (Hall, 2004) with other AIV strains representing different clades as well as vaccine strains against H5N1 used in Egypt, obtainedfrom the National Center for Biotechnology Information (NCBI). The Phylogenetic analyses were conducted out using MEGA 6 (Tamura et al. 2013). best models were the General Time Reversible (GTR) substitution with Gamma distribution (G) and estimate of proportion of invariable sites (I), a moderate strength neighbor-joining approach, and 1000 bootstrap repeats (Kumar et al. 2016). The pairwise nucleotide percent identity was calculated using BioEdit version 7.0 (Hall, 2004). Further, the N-linked glycosylation pattern on HA gene H9N2 AIVs were analyzed via by NetNGlyc 1.0Server http://www.cbs.dtu.dk/services/NetNGlyc/.