Introduction

Avian Influenza viruses belong to Type A Influenza viruses and have a genome composed of RNA comprised of eight segments (Killian 2008). Each segment is responsible to encode one or two specific proteins either structural or non-structural (Fiala et al. 2018). Segment 4 is responsible to encode for Hemagglutinin (HA) protein and segment 6 is responsible to encode for Neuraminidase (NA) protein (Bouvier and Palese 2008). Up till now, 18 HA and 11 NA subtypes of Influenza A types have been discovered (Tong et al. 2013). Both of these proteins are the surface glycoproteins of influenza viruses and thus responsible for virus entry and exit from the host cell (Jin et al. 2014). NA mainly breaks the mucin in the respiratory tract which is a protective secretion of respiratory tract and clears most of the bacterial and viral entry in the respiratory tract. Thus NA assists the virus to approach the sialic acid receptors on the host cell surface (Butt et al. 2010). Secondly, NA helps the virus to fuse with the host cell membrane and thirdly, its enzymatic activity cleaves the sialic acid from the sialo glycoconjugates at the time of viral exit from the cell. So it also helps in spread of viral infection to the new cells (Gubareva et al. 2000). HA protein mainly interacts with the terminal sialic acid on the host cell surface glycoconjugate and helps in viral adsorption (Tønnessen et al. 2013). Host immune responses are also generated against these proteins of the virus to control the infection (Kosik and Yewdell 2019). Low Pathogenic Avian Influenza (LPAI) viruses cause a significant level of morbidity but occasionally high mortality leading to high economic losses worldwide in the poultry (Foster 2018). Among these viruses, H9N2 subtype is of special concern regarding Pakistan (Ahad et al. 2013). This subtype was first discovered in 1966 from turkeys in Wisconsin (Dong et al. 2011). In Pakistan, it is endemic and was first reported in 1998 (Naeem et al. 1999). Since then, it has been continuously circulating and evolving among the poultry population (Cameron et al. 2000). A recent comprehensive study on broiler birds showed 92% prevalence of H9N2 in Pakistan (Kausar et al. 2018). The poultry sector plays an important role to meet the protein requirement of the nation (Hussain et al. 2015). Even after good management practices and vaccination strategies, every year losses due to various diseases are beard by poultry sector (Hafez and Attia 2020). Avian influenza also plays its role in progress inhibition of this sector (Gado et al. 2017). Various commercial farmers use imported as well as local vaccines to minimize this threat, yet this problem is faced by them each year (Irshad et al. 2018). The current study was designed to isolate recent field variants of H9N2 and their comparison was done with previous isolates as well as some common vaccines being used in the field against H9N2 in Pakistan.

Materials and Methods

Sampling and transportation

Total 500 oral swabs and organ samples (trachea) samples were collected from commercial and backyard farms of different localities of Faisalabad, Toba Tek Singh, Multan, and Bahawalpur districts. The samples were then transferred to the lab dipped in BHI (Brain Heart Infusion broth) as a transport medium added with antibiotic (Gentamycin at 0.5 mg/mL of BHI) in icebox.

Virus cultivation and harvesting

The samples were then cultivated on 9‒11 days old chicken embryonated eggs through chorio-allantoic sac (CAS) route (Senne 1989) and allantoic fluid (AF) was harvested after 48 h of incubation (Khalili et al. 2013).

Haemagglutination test (HA Test) and haemagglutination inhibition (HI) assay

The AF was checked for hemagglutination activity through HA test (Maff 1984). The positive samples were then checked for HI test using specific antiserum against subtype H9N2 of avian influenza (Villegas and Purchase 1998).

Molecular characterization and sequencing

Out of 21 positive samples, four representative samples of four different districts were then processed for RNA extraction using QIAmp viral RNA extraction mini kit using the manufacturer’s instructions. From this RNA, complementary DNA was synthesized using Revert Aid (Thermo Scientific^®) First Strand cDNA Synthesis Kit using the manufacturer’s instructions. Complete HA and NA genes were amplified using specific reported primers for H9 and N2 genes following (Ali et al. 2017). Sequencing was performed using specific primers which were used for PCR by Sangers’ sequencing method.

Phylogenetic and sequence analysis

The phylogenetic tree was generated neighbor-joining method using 1000 Bootstrap values in MEGA-X by using CLUSTAL W algorithm (Kumar et al. 2018). Sequences analysis of recent and previous isolates of Pakistan and vaccinal strains currently being used in the field was performed for percentage identity and divergence (Gado et al. 2017).

Results

Prevalence

A total of 500 samples were collected from various localities of District Faisalabad (n=105), Toba Tek Singh (n=113), Multan (n=122) and Bahawalpur (n=160). Out of 500 samples, 65 were found positive for HA test and 39 were found positive for H9 by using the specific anti-serum. The overall prevalence of H9N2 was found to be 7.8% in these areas (Table 1). Prevalence of each district was 8.57 (13/105), 8.84 (21/113), 6.55 (16/122) and 7.5% (15/107).

Sequence analysis

The HA and NA nucleotide sequences of four new isolates (one representative of each district) were submitted to GenBank with accession numbers mentioned in Table 2.

Phylogenetic analysis

Phylogenetic analysis of HA and NA genes (Figs. 1, 2 and 3) of new isolates in comparison with isolates of 2015‒2016 and previous Pakistani isolates showed that the recent isolates have more resemblance with the isolates of 2015‒2016. The study isolates form a distinct clade with 2015‒2016 isolates in G1 lineage from those which were isolated before 2010. Moreover, the local vaccine virus (A/chicken/Pakistan/vac/2018) falls in the same clade and as those of recent isolates. While the imported vaccine viruses are being currently used in the field in Pakistan A/chicken/Guangdong/SS/94, Table 1: Study isolates with their accession numbers for HA and NA genes on GenBank

S. No.	Isolates of H9N2	District	Type of Poultry	HA accession No.	NA accession No.
1	A/Chicken/Pakistan/041CP/2020	Faisalabad	Commercial	MW767044	MW786686
2	A/Chicken/Pakistan/061CP/2020	Multan	Commercial	MW774320	MW786666
3	A/Chicken/Pakistan/062BYP/2020	Bahawalpur	Backyard	MW769799	MW786665
4	A/Chicken/Pakistan/046CP/2019	Toba Tek Sing	Commercial	MW767039	MW786664

Nucleotide sequences analysis for percentage identity and nucleotide diversity among the study isolates, previous recent isolates and vaccine viruses showed a maximum identity of field isolates with the local vaccine virus and minimum identity with imported vaccine viruses. This shows that study isolates have 98.5 % homology with local vaccine virus as compared to imported vaccine viruses (89.95%) based on HA gene (Table 2) while based on NA gene, 99.59% homology with local vaccine virus as compared to imported vaccine viruses (90.58%) as mentioned in Table 3. Moreover, the nucleotide divergence showed that NA gene is more conserved among the field isolates as compared to HA gene

Table 2: Nucleotide diversity & percentage identity between field isolates and vaccine viruses based on HA gene

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Fig. 1: Prevalence of H9N2 in various districts of Punjab, Pakistan

A/chicken/Egypt/114940v/NLQP/2011, A/chicken/Iran/Av1221/1998 fall in different clade than those of recent isolates.

Discussion

H9N2 is most prevalent influenza virus among terrestrial poultry throughout Eurasia (Dong et al. 2011). The virus is endemic in Pakistan and outbreaks are continuously being reported among the poultry every year in Punjab province of Pakistan (Shaukat et al. 2016). In current study the overall prevalence in various districts of Punjab was found to be 7.8%. Another recent study showed a comparable prevalence of 6.7% in commercial and 2.7% in backyard poultry of Pakistan (Ali et al. 2017). A study in Quetta district of Balochistan province showed the seroprevalence of 14.03% H9N2 in broiler birds (Arif et al. 2015). Another country wide study showed 92% prevalence of H9N2 in broiler birds (Kausar et al. 2018) while a study of Faisalabad district showed a prevalence of 60% in commercial layers (Akhter et al. 2017). These all show that H9N2 has a higher prevalence in commercial poultry as compared to backyard poultry. On the basis of phylogenetic studies, there are mainly three lineages H9N2 viruses including Y280 like (A/Duck/Hong Kong/Y280/97), G1 like (A/Quail/Hong Kong/G1/97) and Korean like (A/Chicken/Korea/38349-p96323/96) (Hosseini et al. 2017). The Pakistani isolates of H9N2 are found to be linked with G1 like lineage (Ali et al. 2019). Study isolates in this research, also fall in G1 lineage and form a distinct clade as compared to previous isolates before 2010 which shows that the virus has evolved much during the recent decade from 2011‒2020. Sequence analysis for percentage identity and nucleotide diversity on the basis of HA and NA genes showed that study isolates from commercial and backyard poultry are not much variant and have a maximum diversity of 7.3 and 7.4% on the basis of HA and NA genes respectively. The local vaccine isolate is much more identical to the study isolates and previous isolates of Pakistan. A study from Sharkia Governorate of Egypt also showed similar results (Gado et al. 2017) and indicated that due to wide host range, H9N2 viruses (Dong et al. 2011) rapidly evolved with time, and there is a need for continuous monitoring to upgrade the vaccinal seed for immunization of birds.

Conclusion

Current study shows that H9N2 is continuously circulating and evolving in Pakistan. So there is a need to upgrade the vaccine virus on regular basis to combat this problem.

Table 3: Nucleotide diversity & percentage identity between field isolates and vaccine viruses based on NA gene

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Fig. 2: Phylogenetic tree (based on HA gene) showing recent isolates of H9N2 viruses in Pakistan compared with the previous isolates and the vaccine viruses

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Fig. 3: Phylogenetic tree (based on NA gene) showing recent isolates of H9N2 viruses in Pakistan compared with the previous isolates and the vaccine viruses

Acknowledgements

Author is thankful to University of Veterinary and Animal Sciences Lahore, Pakistan for providing work area to conduct the research work.

Author Contribution

This article is a part of Ph. D research work of KK of this manuscript while the KK, TY and MZS have contributed in making the research plan and conducting the research work. All the authors contributed equally in write up of this work and approval of final copy.

Conflict of Intrests

Authors declear no confilict of intrest

Data Availibilty

Data will be availbal on demand

Ethics Approval

During this study, oral samples were taken from the field from suspected birds and virus isolates were further studied through sequencing and bioinformatics tools. No trial on birds was done such as disease induction or immune protection analysis. So, there is no need of such approval.

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