Review Article

Low Pathogenicity Avian Influenza H9N2 Virus in Africa

Abdel Satar Arafa

Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center, Dokki, Giza, 12618, Egypt.

Received | May 23, 2022; Accepted | June 20, 2022; Published | July 19, 2022

*Correspondence | Abdel Satar Arafa, Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center, Dokki, Giza, 12618, Egypt; Email: abd.arafa@hotmail.com

Citation | Arafa, A.S., 2022. Low pathogenicity avian influenza H9N2 virus in Africa. Journal of Virological Sciences, 10(2): 16-32.

DOI | https://dx.doi.org/10.17582/journal.jvs/2022/10.2.16.23

Keywords | Low pathogenic avian influenza, H9N2, Africa, Surveillance, Detection

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Avian influenza virus (AIV) is a type of influenza A virus in the Orthomyxoviridae family. It contains eight negative-sense, single-strand RNA segments encoding 10 core proteins and other accessory proteins. Influenza A viruses are generally typed by their combinations of hemagglutinin (HA) and neuraminidase (NA) genes, lead to multiple different subtypes. AIVs are also classified into high pathogenicity avian influenza viruses (HPAIVs) and low pathogenicity avian influenza viruses (LPAIVs) according to their intravenous pathogenicity index (IVPI) in chickens.

Avian influenza virus (AIV) of the subtype H9N2 was isolated for the first time from turkey flocks in Wisconsin, United States in 1966 (Homme and Easterday, 1970). Since then, the virus has been detected worldwide in wild and domestic birds as well as in few occasions in mammals (Kawaoka et al., 1988). H9N2 AIVs can be classified into 2 major lineages: Eurasian and American. American lineage of H9N2 AIVS are mainly recorded in wild birds, while Eurasian lineage of H9N2 AIVs are subgrouped into several sublineages: The G1 lineage (A/quail/Hong Kong/G1/1997), the Y280 lineage (A/duck/Hong Kong/Y280/1997), and the Y439 lineage (A/duck/Hong Kong/Y439/1997) (Guo et al., 2000). The G1 and Y280 lineages have zoonotic potential with many cases have been reported in Asian countries and Egypt (Zhang et al., 2012; Pu et al., 2014; Carnaccini and Perez, 2020).

 

History and phylogeography of H9N2 virus in poultry

Avian influenza of subtype H9N2 has been recorded in several African countries, Egypt has an endemic status in poultry and the virus has been frequently reported in Libya and Tunisia (Alexander, 2007; Monne et al., 2013; Tombari et al., 2011; Kammon et al., 2015). Morocco, Burkina Faso, Ghana Algeria and Uganda have been isolated the virus since 2016 for the first time (El-Houadfi et al., 2016; Zecchin et al., 2017; Rubrum et al., 2018; Awuni et al., 2019). From those countries, Morocco has applied a mass vaccination programme for commercial poultry (El-Houadfi et al., 2016). The isolated viruses from poultry in Africa have been classified as G1 ‘Western’ Viruses and related to the circulating viruses in the Middle East in Israel, Jordan, Lebanon, Saudi Arabia and the United Arab Emirates.

Nigerian poultry and agricultural workers showed high seropositivity against H9N2 without virus isolation (Oluwayelu et al., 2016; Okoye et al., 2013). Although surveillance for HPAIVs is ongoing in Nigeria, serological investigations revealed presence of H9N2 antibodies in poultry without virus isolation. The presence of H9N2 virus in poultry across different regions of Africa suggests that other regions or countries may have infection. However, active surveillance is lacking or not found, and not being reported due to LPAIs such as H9N2 infections not being diseases that are notifiable to the World Organisation for Animal Health (OIE).

Phylogeographical analysis of the H9 subtype of AIV separated viruses into two basic lineages: American and Eurasian and subdivided into four clades based on HA gene segments (Hu et al., 2017; Carnaccini and Perez, 2020). The H9.1 clade is derived from the A/turkey/Wisconsin/1/1966 virus found in bird populations in North America. The H9.2 clade of the American lineage is related to A/quail/Arkansas/29209-1/93 strain. While the Eurasian lineage is composed of two primary subgroups: Y439-H9.3 (A/duck/HongKong/Y439/1997) and G1-H9.4 (A/Quail/Hong Kong/G1/1997). The Y439 lineage has spread in wild bird species across Asia, Africa, and Europe. The G1 lineage has been frequently reported in commercial poultry and live poultry markets in the Middle East region and Africa (Peacock et al., 2019). The H9.4 clade has been divided into two subclades: H9.4.1 and H9.4.2. Since 2013, the H9.4.2 subclade of the H9N2 AI virus has been discovered in China (Gu et al., 2017).

In this review, the evolutionary history of H9N2 viruses from Africa was inferred by using the maximum likelihood method and Tamura-Nei model (Tamura and Nei, 1993) (Figure 1). The tree with the highest log likelihood is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. This analysis involved 69 nucleotide sequences available in NCBI GenBank database for Africa. Codon positions included were 1st+2nd+3rd+Noncodingusing Mega11 (Tamura et al., 2021).

 

North Africa

LPAI H9N2 is currently enzootic in countries of North Africa. H9N2 viruses is circulating in North Africa causing economic losses even in the vaccinated flocks. Genetic analysis showed markers of adaptation of mammals, including humans (Moghoofei et al., 2018; Kaboudi, 2019).

Egypt recorded a genetic report of AIV H9N2 in 2006 from live poultry markets without virus isolation, while from February 2009 to April 2012, antibodies against H9 viruses were widespread in poultry in Egypt (Afifi et al., 2013). Virus isolation of H9N2 was recorded in Egypt from December 2010 and it was different from the first introduction recorded in 2006. Since then, AIV H9N2 infected a wide range of birds in Egypt including chickens, quails, ducks, turkeys and pigeons in commercial and backyard sectors (Kandeil et al., 2014; Arafa et al., 2012a, b; Abdel-Moneim et al., 2012; Monne et al., 2013; El-Zoghby et al., 2012; Awad et al., 2013; Li et al., 2020). Most of infected chickens and turkeys exhibited respiratory distress and/or decrease in egg production, but some quails and broilers flocks showed no overt symptoms (Afifi et al., 2013; Arafa et al., 2012b; Monne et al., 2013; El-Zoghby et al., 2012). The majority of outbreaks are reported during the winter months but outbreaks are observed year-round particularly in the Nile Delta (Arafa et al., 2012b; Abdelwhab and Abdel-Moneim, 2015). Co-infections with other viruses (like NDV, IBV, IBD) or mycoplasma (MG, MS) are common. Interestingly, out of 86 broiler flocks 42% were co-infected with H9N2 and IBV and in 41% a mixed triple infections with IBV-H5-H9-NDV were observed in 2012–2014 (Hassan et al., 2016; Naguib et al., 2017). There is evidence of reassortment between the co-circulating HPAI-H5N8 and LPAI-H9N2 viruses has been reported in 2018 leading to a new virus HPAI-H5N2 (Hagag et al., 2019; Hassan et al., 2020). And experimentally through coinfection of embryonated eggs with HPAI-H5N1 and LPAI-H9N2 (Naguib et al., 2017). Inactivated vaccines using local and non-local field strains of H9N2 are frequently used in Egypt and the emergence of antigenic drift variants has been reported. It is worth mentioning that the Egyptian viruses reacted poorly against serum samples from the ME including vaccination derived sera (Kandeil et al., 2014; Adel et al., 2017; Kandeil et al., 2017). In 2015, three children with a history of exposure to poultry were found positive for AIV H9N2 RNA (Abdelwhab and Abdel-Moneim, 2015) and up to 7·5% seroprevalence in exposed humans was reported (Gomaa et al., 2015). Human infections showed transient influenza like illness but subsided without sequelae.

H9N2 viruses was first recorded in Libya in commercial poultry in 2005–2006 (Alexander, 2007). Then further recorded in 2013 from layers, broilers chicken flocks and peacock (Kammon et al., 2015). The infection spread rapidly all over the country and the flocks were concurrently co-infected with NDV. Birds in the affected flocks showed respiratory signs and high mortality (Kammon et al., 2015). There are no reports of application of H9N2 vaccines in the field.

H9N2 viruses have emerged in Tunisia in 2009 causing several outbreaks in poultry flocks (Tombari et al., 2011). AIV H9N2 virus was also isolated from wild birds (Tombari, 2016). Moreover, a nationwide serosurvey of 800 flocks in 2010–2011 indicated widespread AIV infection including H9N2. A total of 223 flocks had anti-NP antibodies (28·7%) particularly in the coastal areas during the autumn and winter. The infection was mainly reported in layer and breeders. Low biosecurity measures and contact to wild birds were claimed to be the source of infection (Tombari, 2013). In 2012, H9N2 was isolated from a broiler flock (Aouini et al., 2016). Antivirals zanamivir and amantadine decreased virus replication in experimentally inoculated chickens (Umar, 2016).

There are no data available on the prevalence of H9N2 in Algerian poultry and without H9 antibodies were detected from broilers, turkeys or layers flocks in 2012-2013. It was also no vaccination against AIV is implemented in Algeria (Sid et al., 2015).

The first record of H9N2 in Morocco was in 2016 from broilers and breeders (El-Houadfi et al., 2016). Widespread of virus to several locations in the country was observed in layer and breeder chickens. The symptoms were mainly inducing respiratory signs and decrease in feed consumption, drop in egg production with mortalities from 2 to 15%. Similar signs were also reported in turkey flocks with mortality rates about 10%. Emergency vaccination was implemented for poultry in all production sectors. The control of the virus was announced the by April 2016 (El-Houadfi et al., 2016).

West and central Africa

Since 2017, H9N2 viruses have been detected in several Sub-Saharan African countries like Ghana, Nigeria, Burkina Faso, Uganda, Kenya, Benin, Togo and Senegal, where a human case was recently reported (Jallow et al., 2020; Fusade-Boyer et al., 2021).

H9N2 subtype virus was identified in West Africa, where highly pathogenic H5 strains of the A/goose/Guangdong/1/1996 lineage (Gs/GD) have been widely distributed since 2015. The H9N2 has a concern because of animal health implications and its negative effect on local economies as well as possible emergence of reassortant viruses with zoonotic risk. 

The H9N2 subtype virus identified in Burkina Faso in 2017 and it seems to be introduced from Morocco (Zecchin et al., 2017). Then between November 2017 and February 2018 it was identified as GI-19 lineage in Ghana (Awuni et al., 2019).

The H9N2 viruses from Benin and Togo were clustered with previously isolated in Western Africa, however, viruses from Uganda were genetically distant and clustered with viruses from the Middle East. Antigenic drifting viruses were suggested in Benin that showed decreased cross-reactivity with those from Togo and Uganda. The viruses exhibited mammalian adaptation markers similar to those of the human strain A/Senegal/0243/2019 (H9N2) (Jallow et al., 2020; Fusade-Boyer et al., 2021). The H9N2 Nigerian strains were also belonging to the G1 lineage, which has zoonotic potential, and are clustered with H9N2 viruses identified in Africa between 2016 and 2020. There are two distinct clusters of H9N2 viruses in Nigeria, suggesting different introductions into the country (Sulaiman et al., 2021).

In Kenya, previous AIV surveillance detected AIV in 0.8% of chickens sampled in live bird markets (LBMs), but no virus was successfully isolated and the detected sample was not further subtyped (Kariithi et al., 2020).

South Africa

South Africa isolated H9N2 viruses from farmed ostriches and they were similar to wild bird viruses of the Y439 lineage, so these viruses are representing dead-end spill over actions from wild migratory birds from 2017-2018 (Abolnik et al., 2006; Wang et al., 2018; Peacock et al., 2019).

H9N2 virus pathogenesis

H9N2 viruses are of low pathogenicity in experimental infections when tested by IVPI (Guo et al., 2000; Alexander, 2003; Iqbal et al., 2009), however, they frequently display moderate-to-high morbidity and

 

Table 1: African countries with laboratory confirmed H9N2 infections.

Country	Years of poultry isolates	Lineages	Species	Status	Recorded human cases/ Serology
North Africa
Egypt	2006, 2011–2021	G1-W	Chicken, Quail, Duck, Turkey, Pigeon, Bat, pig	Endemic	4 cases /and serology
Libya	2006 & 2013	G1-W	Chicken	Sporadic	No
Tunisia	2010–2012, 2014-2020	G1-W	Chicken Turkey, Wild birds	Potentially endemic	No
Morocco	2016-2019	G1-W	Chicken	Potentially endemic	No
Algeria	2017	G1-W	Chicken	Sporadic	No
Central Africa
Kenyia	2017	G1-W	Chicken	Sporadic	No
West Africa
Nigeria	2013 & 2019	n/a	Chicken, Wild birds	Potentially endemic	Serology only
Burkina Faso	2017	G1-W	Chicken	Sporadic	No
Ghana	2017–2018	G1-W	Chicken	Sporadic	No
Uganda	2017-2019	G1-W	Chicken, Wild birds	Potentially endemic	No
Benin	2017-2020	G1-W	Chicken, Duck	Potentially endemic	No
Senegal	2017-2019	G1-W	Chicken	Potentially endemic	1 case
Togo	2018-19	G1-W	Chicken	Sporadic	No
South Africa
South Africa	1995, 2008–2009 2018-2019	Y439 G1-W	Ostrich, Chicken, Wild birds	H9N2-free	No

 

Table 2: Available HA gene sequence data on 2 public databases for African H9N2 viruses.

Country	Year	Host	No.	Database
South Africa	1995	Avian	2	IVD
	2008	Avian	1	IVD
	2018	Bat	1	IVD
	2009	Ostrich	1	GISAID
Egypt	2004	Avian	1	IVD
	2011	Avian	17	IVD
	2012	Avian	52	IVD
	2013	Avian	28	IVD
	2014	Avian	41	IVD
	2015	Avian /1 Swine	80	IVD
	2016	Avian	38	IVD
	2017	Avian/1 Bat	26	IVD
	2018	Avian	42	IVD
	2019	Avian	94	IVD
	2020	Avian	108	IVD
	2021	Avian	22	IVD
	2011-2021	Avian	441	GISAID
Libya	2006	Avian	1	IVD
	2013	Avian	3	IVD
	2006	Avian	1	GISAID
Tunisia	2010	Avian	4	IVD
	2011	Avian	3	IVD
	2012	Avian	2	IVD
	2013	Avian	1	IVD
	2014	Avian	3	IVD
	2015	Avian	2	IVD
	2016	Avian	1	IVD
	2018	Avian/ 1 Env	8	IVD
	2020	Avian	2	IVD
	2010-2018	Avian/ 1 Env	15	GISAID
Morocco	2016	Avian	17	IVD
	2017	Avian	3	IVD
	2018	Avian	3	IVD
	2019	Avian	4	IVD
	2016-2018	Avian	22	GISAID
Burkina Faso	2017	Avian	1	IVD
	2017	Avian	1	GISAID
Uganda	2017	Avian	59	IVD
	2018	Avian	76	IVD
	2019	Avian	92	IVD
	2017-2019	Avian	191	GISAID
Algeria	2017	Avian	37	IVD
	2017	Avian	37	GISAID
Senegal	2017	Avian	3	IVD
	2019	Human	1	GISAID
	Table continued on next column........
Country	Year	Host	No.	Database
Ghana	2017	Avian	3	IVD
	2018	Avian	4	IVD
	2017-2018	Avian	7	GISAID
Benin	2018	Avian	2	IVD
	2019	Avian	31	IVD
	2020	Avian	2	IVD
	2018-2020	Avian	35	GISAID
Togo	2019	Avian	3	IVD
	2019	Avian	3	GISAID
Nigeria	2019	Avian	19	IVD
	2019	Avian	19	GISAID
Kenya	2017	Avian	5	IVD
	2017	Avian	5	GISAID

 

GISAID: Global Initiative on sharing all influenza data; IVD: Influenza virus database.

 

 

mortality in the field. There are many reports of mortality rates more commonly associated with HPAIV outbreaks (Banet-Noach et al., 2007; Awuni et al., 2019). They also reported with co-infection with bacterial or viral pathogens, and many other factors such as poor biosecurity, mal-nutrition and housing problems (Kishida et al., 2004; Seifi et al., 2010; Wang et al., 2016). H9N2 viruses are considered as a source of gene doner to produce a highly pathogenic or zoonotic viruses, therefore it is important to control H9N2 viruses to prevent the emergence of new zoonotic viruses (Liu et al., 2014).

HPAIV phenotype was not observed in H9N2 virus when HPAIV-like polybasic cleavage site was engineered into H9 virus background. However, when the polybasic H9 HA was combined with the remaining genes from an HPAIV strain the reassortant virus did develop an HPAIV phenotype (Gohrbandt et al., 2011). This implies H9N2 virus internal genes may not be compatible with an HPAIV phenotype in some cases.

H9N2 virus transmission and host tropism in poultry

The main routes of influenza virus transmission are droplet, aerosol, faecal-oral and direct contact (Killingley et al., 2013). Droplet transmission occurred through the upper respiratory tract inhalation of infectious particles (more than 10 nm). While, aerosol droplets are typically less than 5 nm and can reach to the lower respiratory tract (Killingley et al., 2013). Contact transmission occurred through mucous membranes directly, or through an intermediate contact fomite. H9N2 viruses can be transmitted is determined via several viral, host, and environmental aspects, including viral replication and viral titres shedding; distance and frequency between contacts and virus stability in different environmental conditions. In wild aquatic birds such as ducks, the gastrointestinal tropism of AIVs exhibit through the oral-faecal route. LPAIVs H9N2 in chickens tend to show more respiratory tropism and some gastrointestinal tropism (Wang et al., 2016; Killingley et al., 2013; Sorrell et al., 2010). There is good evidence to suggest that many LPAIV strains transmit by the airborne route, the oral-faecal route and the waterborne route (Killingley et al., 2013; Lv et al., 2015). However, the main route of transmission among individuals varies by host species and viral strain.

Direct contact is the important transmission route for H9N2 viruses in chickens, then aerosol and faecal-oral have been also shown to be significant. The respiratory tropism is favored by many strains, however, some other H9N2 strains have been shown to have an extended tropism for the kidneys or oviducts (Bonfante et al., 2018; Perez et al., 2003; Yao et al., 2014). Both in the field and experimentally poultry adapted H9N2 viruses are mostly detected from oral/tracheal rather than cloacal swabs (Peacock et al., 2017). Additionally, inoculation of some H9N2 viruses into the respiratory tract is 40 times more effective than gastrointestinal inoculation at initiating infection (Yao et al., 2014). LBMs have been considered as the major player for transmission of endemic H5N1 and H9N2 viruses as they enhance direct contact transmission among birds. So, respiratory and contact transmission are the primary routes of transmission of H9N2 and has implications for zoonotic transmission.

Vaccination and control

H9N2 causes marked economic damage in many countries including African countries, so vaccination has been adopted at a national level or in a local area to control disease in poultry (El-Houadfi et al., 2016; Kilany et al., 2016). Inactivated vaccines are the most common vaccines used. H9N2 viruses have a wide antigenic variability in different lineages (Peacock et al., 2018). Efficacy trials are generally fewer for H9N2 vaccines than other HPAI H5 vaccines specially against antigenically drifted viruses.

So, vaccination failure has been noticed in many areas applying H9N2 vaccination with high virus shedding and infection to vaccinated birds due to sub-optimal vaccination that may produce antigenic drift and increase zoonotic potential and pathogenicity (Park et al., 2011; Zhang et al., 2008; Peacock et al., 2017; Sealy et al., 2019).

H9N2 vaccination can provide protection for birds by reducing clinical signs and virus shedding. Inactivated whole-virus vaccines may fail to provide sufficient protection when there are differences in antigenicity between the H9N2 vaccine strain and the circulating viruses (Pu et al., 2014; Sun et al., 2012). The H9N2 vaccine needs to be optimized to fit the current status of multiple antigenic groups. The inactivated vaccines induce mainly humoral immunity, which mask virus infection and increase shedding of virus from upper respiratory tract. Besides, field H9N2 viruses can induce efficient chicken-to-chicken aerosol transmission in vaccinated chickens (Zhong et al., 2014) that will increase the difficulty of virus prevention and control. Therefore, it is important to select vaccines that capable to reduce virus shedding to help in controlling H9N2 infections.

Inactivated whole-virus vaccines

Inactivated whole-virus vaccines has many advantages including ease of production and its lack of ability to revert to a virulent state. The immunogenicity of the vaccine depends on vaccine candidate strains used and its protective antigenic epitopes as well as the compatibility between HA and NA (Wang et al., 2019). There are multiple H9N2 antigenic groups were prevalent worldwide that makes antigen-matched vaccine challenging and difficult to predict antigenic variation for the control of H9N2. Antigenicity prediction models based on HA sequences show good potential (Du et al., 2012). Strains with broad-spectrum cross-protection may exist among the naturally prevalent strains (Radvak et al., 2021). An inactivated vaccine modified for the H9 gene was successfully prepared, but its broad-spectrum protection still needs to be further invetigated (Li et al., 2021). A graph-based vaccine design algorithm has been applied and demonstrated its broad protection against H3 subtypes (Bullard et al., 2021). Furthermore, H9/H5N2 recombinant vaccine, that expressed the HA1 of H9N2 and the HA2 of H5N8 viruses, protected chickens against lethal challenge by HPAI H5N8 viruses and significantly reduced virus shedding post infection with both H9N2 and HPAI H5N8 viruses (Kim et al., 2017).

The inactivated whole-virus vaccines are mainly prepared from the formaldehyde inactivation or beta-propiolactone (BPL) or the application of gamma radiation of virus-containing allantoic fluids from infected chicken embryos. A study showed that antibody-mediated immune responses were increased in chickens that received BPL and gamma inactivated whole-virus vaccines compared to formaldehyde prepared vaccines against H9N2 AIV (Astill et al., 2018). Inactivated vaccines mainly induce humoral immunity. Adjuvants could improve the cellular immunity or mucosal immunity induced by inactivated vaccines. CpG oligodeoxynucleotides assist the whole inactivated H9N2 influenza virus in crossing the intestinal epithelial barriers via transepithelial uptake of dendritic cell dendrites (Qin et al., 2015; Yin et al., 2015).

Vector vaccines are developed and licensed for use in the control of avian influenza (Aida et al., 2021). Vector vaccines induce cellular immunity and mucosal immunity to provide extended immunity. There are a variety of vectors have been used for H9 vaccine development, including fowlpox virus (FPV), fowl adenoviruses (FAdVs), Marek’s disease virus (MDV) and NDV. In addition to other non-viral vectors, like Lactobacillus and Eimeria acervuline (Shi et al., 2014, 2015; Bo et al., 2019; Li et al., 2020; Zhang et al., 2021a) are also used as vectors to prepare vaccines to control H9N2 avian influenza. However, overcoming maternal antibody interference is a major challenge. Attenuated fowlpox virus (FPV) strains have been used as vaccines against wild-type virus infection. Recombinant fowlpox virus (rFPV-HA) expressing the HA gene of H9N2 AIV- could prevent virus shedding and replication in internal organs in response to H9N2 (Chen et al., 2011). However, the efficacy of recombinant FPV-based vaccines can be affected by preexisting immunity (Swayne et al., 2000). Fowl adenoviruses (FAdVs), with a linear, 26–45 kb, double-stranded DNA molecule, can also be used as virus vectors. By inserting the nucleotide sequence encoding the VP2 protein of IBDV into rFAdV, rFAdV-VP2 was generated (Pan et al., 2021). The protection induced by rFAdV-VP2 vaccination in SPF chickens induced good immune protection, suggesting its potential in the vector vaccine development of H9N2-subtype avian influenza virus. Currently, there is no recombinant FAdVs are being produced as vectored vaccines. Preexisting immunities hamper the application of FAdV vectors in vaccine development.

Marek’s disease virus (MDV) which are divided into three species, gallid herpesvirus 2, gallid herpesvirus 3 and meleagrid herpesvirus 1, formally named MDV serotype 1 (MDV-1), MDV serotype 2 (MDV-2) and MDV serotype 3 (MDV-3), respectively.

Turkey herpesvirus (HVT) (of meleagrid herpesvirus 1) has been extensively used as a vaccine against Marek’s disease for over the past decades. Attenuated MDV-1 strains and HVT have many advantages that make them appropriate for the development of recombinant vector-based vaccines for poultry diseases. The attenuated CVI988/Rispens MDV-1 strain has been used to express the S1 glycoprotein of IBV (Zhang et al., 2012) and the VP2 of IBDV (Zhou et al., 2010).

Chickens vaccinated with MDV-H9 induced less than 50% protection (Ma et al., 2014). The vector vaccine candidate HVT-H9 could induce humoral and cellular immunity in chickens. In a challenge study, no chicken shed H9N2 virus from the oropharynx and cloaca, and no H9N2 virus was found in the viscera in the vaccination groups when challenged with homologous virus, suggesting that HVT-H9 provides effective protection against H9N2 AIV in chickens (Liu et al., 2019). NDV-based viral vectors expressing the influenza NA and HA glycoproteins (Nagy et al., 2016; Park et al., 2006) have been obtained and evaluated as immunogens for chickens. Recombinant NDV expressing H9 HA protects SPF chickens against heterologous avian influenza H9N2 virus challenge (Nagy et al., 2016; Liu et al., 2018; Xu et al., 2019; Zhang et al., 2021b).

Conclusions and Recommendations

Avian influenza virus (AIV) of the subtype H9N2 virus has been detected in several African countries with endemic status in Egypt and frequently reported in North Africa (Morocco, Tunisia, Libya, Algeria), West and Central Africa (Nigeria, Burkina Faso, Ghana, Uganda, Benin, Senegal, Togo and Kenyia) since 2016 and South Africa.

The isolated viruses from poultry in Africa have been classified as G1 ‘Western’ Viruses and related to the circulating viruses in the Middle East. In addition to Y439 lineage recorded in South Africa farmed ostriches. The infection with H9N2 is displaying moderate-to-high morbidity and mortality in the field especially when associated with bacterial or viral infections or with other factors related to biosecurity, nutrition and management. LPAIVs H9N2 in chickens showed mainly respiratory tropism with some strains also show gastrointestinal tropism.

The respiratory and contact transmission are the primary routes of transmission and may arise as an adaptation to poultry which clearly has implication for zoonotic transmission.

Inactivated vaccines are the most common vaccines used to control H9N2 viruses, however they have a wide antigenic variability in different lineages. Egypt and Morocco have applied a mass vaccination programme for commercial poultry to control the infection.

Africa now is under threat from zoonotic H9N2 infections. Several H9N2 viruses have mammalian receptor binding properties that could allow efficient transmission to humans with internal gene cassettes that allow efficient replication in humans. There is a clear risk of H9N2 reassortant with the human influenza virus emergence as well as efficient mammalian polymerase reassortant emergence that pose a high zoonotic and pandemic threat.

There is a real need for better understanding of H9 antigenicity based on the molecular determinants and identification of antigenic drifting viruses and their implications on zoonotic potential. Vaccination can protect against current circulating strains in countries applying vaccination like Egypt.

Further surveillance efforts are highly needed particularly in non-officially declared countries for H9N2 as well as in endemic countries applying vaccination. Continual phenotypic characterisation of H9N2 viruses circulating in poultry farms need to assessed over time to examine their properties such as viral pathogenicity, antigenicity and zoonotic potential.

Novelty Statement

This review discuss the findings of the previous studies published and specifically from Africa for H9N2 virus infection and summarizes the updated siltation and highlights on important findings.

Conflict of interest

The author has declared no conflict of interest.

References

Abdel-Moneim, A.S., Afifi, M.A. and El-Kady, M.F., 2012. Isolation and mutation trend analysis of influenza A virus subtype H9N2 in Egypt. Virol. J., 9: 173. https://doi.org/10.1186/1743-422X-9-173

Abdelwhab, E.M. and Abdel-Moneim, A.S., 2015. Epidemiology, ecology and gene pool of influenza A virus in Egypt: Will Egypt be the epicentre of the next influenza pandemic? Virulence, 6(1): 6-18. https://doi.org/10.4161/21505594.2014.992662

Abolnik, C., Bisschop, S., Gerdes, T., Olivier, A. and Horner, R., 2007. Outbreaks of avian influenza H6N2 viruses in chickens arose by a reassortment of H6N8 and H9N2 ostrich viruses. Virus Genes, 34(1): 37-45. https://doi.org/10.1007/s11262-006-0007-6

Abolnik, C., Cornelius, E., Bisschop, S.P., Romito, M. and Verwoerd, D., 2006. Phylogenetic analyses of genes from South African LPAI viruses isolated in 2004 from wild aquatic birds suggests introduction by Eurasian migrants. Dev. Biol. (Basel). 124: 189-99.

Abolnik, C., Gerdes, G.H., Sinclair, M., Ganzevoort, B.W., Kitching, J.P., Burger, C.E., Romito, M., Dreyer, M., Swanepoel, S., Cumming, G.S. and Olivier, A.J., 2010. Phylogenetic analysis of influenza A viruses (H6N8, H1N8, H4N2, H9N2, H10N7) isolated from wild birds, ducks, and ostriches in South Africa from 2007 to 2009. Avian Dis., 54(1 Suppl): 313-322. https://doi.org/10.1637/8781-040109-Reg.1

Adel, A., Arafa, A., Hussein, H.A. and El-Sanousi, A.A., 2017. Molecular and antigenic traits on hemagglutinin gene of avian influenza H9N2 viruses: Evidence of a new escape mutant in Egypt adapted in quails. Res. Vet. Sci., 112: 132-140. https://doi.org/10.1016/j.rvsc.2017.02.003

Adel, A., Mosaad, Z., Shalaby, A.G., Selim, K., Samy, M., Abdelmagid, M.A., Hagag, N.M., Arafa, A.S., Hassan, W.M., and Shahien, M.A., 2021. Molecular evolution of the hemagglutinin gene and epidemiological insight into low-pathogenic avian influenza H9N2 viruses in Egypt. Res. Vet. Sci., 136: 540-549. https://doi.org/10.1016/j.rvsc.2021.04.006

Afifi, M.A., El-Kady, M.F., Zoelfakar, S.A. and Abdel-Moneim, A.S. 2013. Serological surveillance reveals widespread influenza A H7 and H9 subtypes among chicken flocks in Egypt. Trop. Anim. Health Prod., 45: 687–690. https://doi.org/10.1007/s11250-012-0243-9

Aida, V., Pliasas, V.C., Neasham, P.J., North, J.F., McWhorter, K.L., Glover, S.R. and Kyriakis, C.S., 2021. Novel vaccine technologies in veterinary medicine: A herald to human medicine vaccines. Front. Vet. Sci., 8: 654289. https://doi.org/10.3389/fvets.2021.654289

Alexander, D.J., 2003. Report on avian influenza in the eastern hemisphere during 1997–2002. Avian Dis., 47: 792–797. https://doi.org/10.1637/0005-2086-47.s3.792

Alexander, D.J., 2007. Summary of avian influenza Activity in Europe, Asia, Africa, and Australasia, 2002–2006. Avian Dis., 51: 161–166. https://doi.org/10.1637/7602-041306R.1

Amal, E.B., Saâdi, N., Asma, F., Moncef, B., and Ouafae, F.F., 2020. Characterization and phylogenetic analysis of the hemagglutinin gene in H9 influenza viruses from chickens in Morocco from 2017 to 2019. Avian Dis., 64(3): 310-314. https://doi.org/10.1637/aviandiseases-D-20-00009

Arafa, A.S., Hagag, N., Erfan, A., Mady, W., El-Husseiny, M., Adel, A., and Nasef, S., 2012a. Complete genome characterization of avian influenza virus subtype H9N2 from a commercial quail flock in Egypt. Virus Genes, 45(2): 283-294. https://doi.org/10.1007/s11262-012-0775-0

Arafa, A.S., Hagag, N.M., Yehia, N., Zanaty, A.M., Naguib, M.M., and Nasef, S.A., 2012b. Effect of cocirculation of highly pathogenic avian influenza H5N1 subtype with low pathogenic H9N2 subtype on the spread of infections. Avian Dis., 56(4 Suppl): 849-857. https://doi.org/10.1637/10152-040812-Reg.1

Arbi, M., Souiai, O., Rego, N., Larbi, I., Naya, H., Ghram, A., and Houimel, M., 2020. Historical origins and zoonotic potential of avian influenza virus H9N2 in Tunisia revealed by Bayesian analysis and molecular characterization. Arch. Virol., 165(7): 1527-1540. https://doi.org/10.1007/s00705-020-04624-4

Astill, J., Alkie, T., Yitbarek, A., Taha-Abdelaziz, K., and Bavananthasivam, J., Nagy, E., Petrik, J.J., and Sharif, S., 2018. Examination of the effects of virus inactivation methods on the induction of antibody- and cell-mediated immune responses against whole inactivated H9N2 avian influenza virus vaccines in chickens. Vaccine, 36: 3908–3916. https://doi.org/10.1016/j.vaccine.2018.05.093

Aouini, R., Laamiri, N., Ghram, A. 2016. Novel gene mutations in Tunisian isolate of avian H9N2 influenza virus. J. Vet. Sci. Technol., 8: 405.

Awad, A.A., Arafa, A., and Hagag, S.Y., 2013. Incidence of avian influenza among commercial and native breeds in west Delta region. Alex. J. Vet. Sci., 39: 31–39.

Awuni, J.A., Bianco, A., Dogbey, O.J., Fusaro, A., Yingar, D.T., Salviato, A., Ababio, P.T., Milani, A., Bonfante, F., and Monne, I., 2019. Avian influenza H9N2 subtype in Ghana: virus characterization and evidence of co-infection. Avian Pathol., 48(5): 470-476. https://doi.org/10.1080/03079457.2019.1624687

Banet-Noach, C., Perk, S., Simanov, L., Grebenyuk, N., Rozenblut, E., Pokamunski, S., Pirak, M., Tendler, Y., Barberis, A., Boudaoud, A., Gorrill, A., Loupias, J., Ghram, A., Lachheb, J., Alloui, N., and Ducatez, M.F., 2020. Full-length genome sequences of the first H9N2 avian influenza viruses isolated in the Northeast of Algeria. Virol. J., 17(1): 108. https://doi.org/10.1186/s12985-020-01377-z

Belkasmi, S.F.Z., Fellahi, S., Touzani, C.D., Faraji, F.Z., Maaroufi, I., Delverdier, M., Guérin, J.L., Fihri, O.F., El-Houadfi, M., and Ducatez, M.F., 2020. Co-infections of chickens with avian influenza virus H9N2 and Moroccan Italy 02 infectious bronchitis virus: Effect on pathogenesis and protection conferred by different vaccination programmes. Avian Pathol., 49(1): 21-28. https://doi.org/10.1080/03079457.2019.1656328

Bo, F., Yang, W.T., Shonyela, S.M., Jin, Y.B., Huang, K.Y., Shao, L.N., Wang, C., Zhou, Y., Li, Q.Y., Jiang, Y.L., Huang, H.B., Shi, C.W., Wang, J.Z., Wang, G., Kang, Y.H., Yang, G.L., Wang, C.F. 2019. Immune responses of mice inoculated with recombinant Lactobacillus plantarum NC8 expressing the fusion gene HA2 and 3M2e of the influenza virus and protection against different subtypes of influenza virus. Virus Res., 263: 64–72. https://doi.org/10.1016/j.virusres.2019.01.001

Bonfante, F., Mazzetto, E., Zanardello, C., Fortin, A., Gobbo, F., Maniero, S., Bigolaro, M., Davidson, I., Haddas, R., Cattoli, G. and Terregino, C., 2018. A G1-lineage H9N2 virus with oviduct tropism causes chronic pathological changes in the infundibulum and a long-lasting drop in egg production. Vet. Res., 49: 83. https://doi.org/10.1186/s13567-018-0575-1

Boumart, Z., Bamouh, Z., Jazouli, M., Zecchin, B., Fusaro, A., Salviato, A., Monne, I., Tadlaoui, K.O., and Harrak, M.E., 2019. Pathogenicity and full genome sequencing of the avian influenza H9N2 moroccan isolate 2016. Avian Dis., 63(1): 24-30. https://doi.org/10.1637/11941-080418-Reg.1

Brown, I.H., 2010. Summary of avian influenza activity in Europe, Asia, and Africa, 2006–2009. Avian Dis., 54: 187–193. https://doi.org/10.1637/8949-053109-Reg.1

Bullard, B.L., Corder, B.N., DeBeauchamp, J., Rubrum, A., Korber, B., Webby, R.J., and Weaver, E.A., 2021. Epigraph hemagglutinin vaccine induces broad cross-reactive immunity against swine H3 influenza virus. Nat. Commun., 12: 1203. https://doi.org/10.1038/s41467-021-21508-6

Carnaccini, S., and Perez, D.R., 2020. H9 influenza viruses: An emerging challenge. Cold Spring Harb. Perspect. Med., 10(6): a038588. https://doi.org/10.1101/cshperspect.a038588

Chen, H.Y., Shang, Y.H., Yao, H.X., Cui, B.A., Zhang, H.Y., Wang, Z.X., Wang, Y.D., Chao, A.J., and Duan, T.Y., 2011. Immune responses of chickens inoculated with a recombinant fowlpox vaccine coexpressing HA of H9N2 avain influenza virus and chicken IL-18. Antivir. Res., 91: 50–56. https://doi.org/10.1016/j.antiviral.2011.04.007

Clements, A.L., Sealy, J.E., Peacock, T.P., Sadeyen, J.R., Hussain, S., Lycett, S.J., Shelton, H., Digard, P., and Iqbal, M., 2020. Contribution of segment 3 to the acquisition of virulence in contemporary H9N2 avian influenza viruses. J. Virol., 94(20): e01173-20. https://doi.org/10.1128/JVI.01173-20

Couacy-Hymann, E, Kouakou, A.V., Kouamé, C.K., Kouassi, A.L., Koffi, Y.M., Godji, P., Nana, P., Tarnagda, Z., and Akoua-Koffi, C., 2012. Surveillance for avian influenza and Newcastle disease in backyard poultry flocks in Côte d’Ivoire, 2007-2009. Rev. Sci. Tech., 31(3): 821-828. https://doi.org/10.20506/rst.31.3.2158

Du, X., Dong, L., Lan, Y., Peng, Y., Wu, A., Zhang, Y., Huang, W., Wang, D., Wang, M., Guo, Y., Shu, Y. and Jiang, T. 2012. Mapping of H3N2 influenza antigenic evolution in China reveals a strategy for vaccine strain recommendation. Nat. Commun., 3: 709. https://doi.org/10.1038/ncomms1710

El-Houadfi, M., Fellahi, S., Nassik, S., Guérin, J.L., and Ducatez, M.F., 2016. First outbreaks and phylogenetic analyses of avian influenza H9N2 viruses isolated from poultry flocks in Morocco. Virol. J., 13(1): 140. https://doi.org/10.1186/s12985-016-0596-1

Eladl, A.H., Alzayat, A.A., Ali, H.S., Fahmy, H.A., and Ellakany, H.F., 2019. Comparative molecular characterization, pathogenicity and seroprevalence of avian influenza virus H9N2 in commercial and backyard poultry flocks. Comp. Immunol. Microbiol. Infect. Dis., 64: 81-89. https://doi.org/10.1016/j.cimid.2019.02.011

El-Zoghby, E.F., Arafa, A.S., Hassan, M.K., Aly, M.M., Selim, A., Kilany, W.H., Selim, U., Nasef, S., Aggor, M.G., Abdelwhab, E.M., and Hafez, H.M., 2012. Isolation of H9N2 avian influenza virus from bobwhite quail (Colinus virginianus) in Egypt. Arch. Virol., 157(6): 1167-1172. https://doi.org/10.1007/s00705-012-1269-z

Erfan, A.M., Selim, A.A., Helmy, S.A., Eriksson, P., and Naguib, M.M., 2019. Chicken anaemia virus enhances and prolongs subsequent avian influenza (H9N2) and infectious bronchitis viral infections. Vet. Microbiol., 230: 123–129. https://doi.org/10.1016/j.vetmic.2019.01.024

European Food Safety Authority, 2020. European centre for disease prevention and control and European union reference laboratory for avian influenza; Adlhoch, C., Fusaro, A., Kuiken, T., Niqueux, E., Staubach, C., Terregino, C., Guajardo, I.M., Baldinelli, F., Avian influenza overview November 2019- February2020. EFSA J., 18(3): e06096. https://doi.org/10.2903/j.efsa.2020.6096

Fellahi, S., Nassik, S., Maaroufi, I., Tligui, N.S., Touzani, C.D., Rawi, T., Delvecchio, A., Ducatez, M.F., and Houadfi, M.E., 2021. Pathogenesis of avian influenza virus subtype H9N2 in Turkeys and evaluation of inactivated vaccine efficacy. Avian Dis., 65(1): 46-51. https://doi.org/10.1637/aviandiseases-D-20-00067

Fusade-Boyer, M., Djegui, F., Batawui, K., Byuragaba, D.K., Jones, J.C., Wabwire-Mangeni, F., Erima, B., Atim, G., Ukuli, Q.A., Tugume, T., Dogno, K., Adjabli, K., Nzuzi, M., Adjin, R., Jeevan, T., Rubrum, A., Go-Maro, W., Kayali, G., McKenzie, P., Webby, R.J., and Ducatez, M.F., 2021. Antigenic and molecular characterization of low pathogenic avian influenza A(H9N2) viruses in sub-Saharan Africa from 2017 through 2019. Emerg. Microbes Infect., 10(1): 753-761. https://doi.org/10.1080/22221751.2021.1908097

Fusaro, A., Monne, I., Salviato, A., Valastro, V., Schivo, A., Amarin, N.M., Gonzalez, C., Ismail, M.M., Al-Ankari, A.R., Al-Blowi, M.H., Khan, O., Ali, A.S.M., Hedayati, A., García, J.G., Ziay, G., Shoushtari, A., Qahtani, K.N.A., Capua, I., Holmes, E.C. and Cattoli, G., 2011. Phylogeography and evolutionary history of reassortant H9N2 viruses with potential human health implications. J. Virol., 85: 8413–8421. https://doi.org/10.1128/JVI.00219-11

Gohrbandt, S., Veits, J., Breithaupt, A., Hundt, J., Teifke, J.P., Stech, O., Mettenleiter, T.C., and Stech, J., 2011. H9 avian influenza reassortant with engineered polybasic cleavage site displays a highly pathogenic phenotype in chicken. J. Gen. Virol., 92: 1843–1853. https://doi.org/10.1099/vir.0.031591-0

Gomaa, M.R., Kayed, A.S., Elabd, M.A., Zeid, D.A., Zaki, S.A., El-Rifay, A.S., Sherif, L.S., McKenzie, P.P., Webster, R.G., Webby, R.J., Ali, M.A., and Kayali, G., 2015. Avian influenza A(H5N1) and A(H9N2) seroprevalence and risk factors for infection among Egyptians: a prospective, controlled seroepidemiological study. J. Infect. Dis., 211(9): 1399-1407. https://doi.org/10.1093/infdis/jiu529

Guo, Y.J., Krauss, S., Senne, D.A., Mo, I.P., Lo, K.S., Xiong, X.P., Norwood, M., Shortridge, K.F., Webster, R.G., and Guan, Y., 2000. Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia. Virology, 267: 279–288. https://doi.org/10.1006/viro.1999.0115

Gu, M., Xu, L., Wang, X., 2017. Current situation of H9N2 subtype avian influenza in China. Vet Res 48: 49. https://doi.org/10.1186/s13567-017-0453-2

Hagag, N.M., Erfan, A.M., El-Husseiny, M., Shalaby, A.G., Saif, M.A., Tawakol, M.M., Nour, A.A., Selim, A.A., Arafa, A.S., Hassan, M.K., Hassan, W.M.M., Fahmy, H.A., Ibraheem, E., Attia, M., Abdelhakim, A.M.M., Shahein, M.A., and Naguib, M.M., 2019. Isolation of a novel reassortant highly pathogenic avian influenza (H5N2) virus in Egypt. Viruses, 11(6): 565. https://doi.org/10.3390/v11060565

Hassan, K.E., Saad, N., Abozeid, H.H., Shany, S., El-Kady, M.F., Arafa, A., El-Sawah, A.A.A., Pfaff, F., Hafez, H.M., Beer, M., and Harder, T., 2020. Genotyping and reassortment analysis of highly pathogenic avian influenza viruses H5N8 and H5N2 from Egypt reveals successive annual replacement of genotypes. Infect. Genet. Evol., 84: 104375. https://doi.org/10.1016/j.meegid.2020.104375

Hassan, K.E., Shany, S.A., Ali, A., Dahshan, A.H., El-Sawah, A.A., and El-Kady, M.F., 2016. Prevalence of avian respiratory viruses in broiler flocks in Egypt. Poult. Sci., 95(6): 1271-1280. https://doi.org/10.3382/ps/pew068

Henry, M.K., Catharine, N.W., Helena, L.F., Elizabeth, A.P., Leonard, O.A., Yatinder, S.B., Auleria, A.A., Thomas, D.D., Claudio, L.A., and David, L.S., 2020. Genetic characterization and pathogenesis of the first H9N2 low pathogenic avian influenza viruses isolated from chickens in Kenyan live bird markets. Infect. Genet. Evol., 78: 104074. https://doi.org/10.1016/j.meegid.2019.104074

Homme, P.J., and Easterday, B.C., 1970. Avian influenza virus infections. I: characteristics of influenza A/turkey/Wisconsin/1966 virus. Avian Dis., 14: 66–74. https://doi.org/10.2307/1588557

Hu, M., Jin, Y., Zhou, J., Huang, Z., Li, B., Zhou, W., Ren, H., Yue, J., and Liang, L., 2017. Genetic characteristic and global transmission of influenza A H9N2 virus. Front. Microbiol., 8: 2611. https://doi.org/10.3389/fmicb.2017.02611

Iqbal, M., Yaqub, T., Reddy, K., and McCauley, J.W., 2009. Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing ns genes similar to highly pathogenic H7N3 and H5N1 viruses. PLoS One, 4: e5788. https://doi.org/10.1371/journal.pone.0005788

Jallow, M.M., Fall, A., Barry, M.A., Diop, B., Sy, S., Goudiaby, D., Fall, M., Enouf, V., Niang, M.N., and Dia, N., 2020. Genetic characterization of the first detected human case of low pathogenic avian influenza A/H9N2 in sub-Saharan Africa, Senegal. Emerg. Microb. Infect., 9(1): 1092-1095. https://doi.org/10.1080/22221751.2020.1763858

Jeevan, T., Darnell, D., Gradi, E.A., Benali, Y., Kara, R., Guetarni, D., Rubrum, A., Seiler, P.J., Crumpton, J.C., Webby, R.J., and Derrar, F.A., 2019. H9N2 influenza viruses associated with chicken mortality in outbreaks in Algeria 2017. Influenza Other Respir Viruses. 13(6): 622-626. https://doi.org/10.1111/irv.12675

Kaboudi, K., 2019. Low pathogenic avian influenza virus subtype H9N2 in poultry in north Africa: Current status. Vet. Sci. Res. Rev., 5(2): 73-79. https://doi.org/10.17582/journal.vsrr/2019/5.2.73.79

Kalonda, A., Saasa, N., Nkhoma, P., Kajihara, M., Sawa, H., Takada, A., and Simulundu, E., 2020. Avian influenza viruses detected in birds in Sub-Saharan Africa: A systematic review. Viruses, 12(9): 993. https://doi.org/10.3390/v12090993

Kammon, A., Heidari, A., Dayhum, A., Eldaghayes, I., Sharif, M., Monne, I., Cattoli, G., Asheg, A., Farhat, M., and Kraim, E., 2015. Characterization of avian influenza and Newcastle disease viruses from poultry in Libya. Avian Dis. 59: 422–430. https://doi.org/10.1637/11068-032215-ResNote.1

Kandeil, A., El-Shesheny, R., Maatouq, A., Moatasim, Y., Cai, Z., McKenzie, P., Webby, R., Kayali, G., and Ali, M.A., 2017. Novel reassortant H9N2 viruses in pigeons and evidence for antigenic diversity of H9N2 viruses isolated from quails in Egypt. J. Gen. Virol., 98(4): 548-562. https://doi.org/10.1099/jgv.0.000657

Kandeil, A., El-Shesheny, R., Maatouq, A.M., Moatasim, Y., Shehata, M.M., Bagato, O., Rubrum, A., Shanmuganatham, K., Webby, R.J., Ali, M.A., and Kayali, G., 2014. Genetic and antigenic evolution of H9N2 avian influenza viruses circulating in Egypt between 2011 and 2013. Arch. Virol., 159(11): 2861-2876. https://doi.org/10.1007/s00705-014-2118-z

Kandeil, A., Gomaa, M.R., Shehata, M.M., El-Taweel, A.N., Mahmoud, S.H., Bagato, O., Moatasim, Y., Kutkat, O., Kayed, A.S., Dawson, P., Qiu, X., Bahl, J., Webby, R.J., Karesh, W.B., Kayali, G., and Ali, M.A., 2019. Isolation and characterization of a distinct influenza a virus from Egyptian bats. J. Virol., 93(2): e01059-18. https://doi.org/10.1128/JVI.01059-18

Kandeil, A., Hicks, J.T., Young, S.G., El-Taweel, A.N., Kayed, A.S., Moatasim, Y., Kutkat, O., Bagato, O., McKenzie, P.P., Cai, Z., Badra, R., Kutkat, M., Bahl, J., Webby, R.J., Kayali, G., and Ali, M.A., 2019. Active surveillance and genetic evolution of avian influenza viruses in Egypt, 2016-2018. Emerg. Microb. Infect., 8(1): 1370-1382. https://doi.org/10.1080/22221751.2019.1663712

Kariithi, H.M., Welch, C.N., Ferreira, H.L., Pusch, E.A., Ateya, L.O., Binepal, Y.S., Apopo, A.A., Dulu, T.D., Afonso, C.L., and Suarez, D.L., 2020. Genetic characterization and pathogenesis of the first H9N2 low pathogenic avian influenza viruses isolated from chickens in Kenyan live bird markets. Infect. Genet. Evol., 78: 104074. https://doi.org/10.1016/j.meegid.2019.104074

Kawaoka, Y., Chambers, T.M., Sladen, W.L., and Webster, R.G., 1988. Is the gene pool of influenza viruses in shorebirds and gulls different from that in wild ducks? Virology, 163: 247–250. https://doi.org/10.1016/0042-6822(88)90260-7

Kayali, G., Kandeil, A., El-Shesheny, R., Kayed, A.S., Gomaa, M.M., Maatouq, A.M., Shehata, M.M., Moatasim, Y., Bagato, O., Cai, Z., Rubrum, A., Kutkat, M.A., McKenzie, P.P., Webster, R.G., Webby, R.J., and Ali, M.A., 2014. Active surveillance for avian influenza virus, Egypt, 2010-2012. Emerg Infect Dis., 20(4): 542-551. https://doi.org/10.3201/eid2004.131295

Kilany, W.H., Ali, A., Bazid, A.H., El-Deeb, A.H., El-Abideen, M.A., Sayed, M.E., and El-Kady, M.F., 2016. A dose-response study of inactivated low pathogenic avian influenza H9N2 virus in specific-pathogen-free and commercial broiler chickens. Avian Dis., 60: 256–261. https://doi.org/10.1637/11143-050815-Reg

Killingley, B. and Nguyen-Van-Tam, J., 2013. Routes of influenza transmission. Influenza Other Respir. Viruses, 7: 42–51. https://doi.org/10.1111/irv.12080

Kim, S.M., Kim, Y.I., Park, S.J., Kim, E.H., Kwon, H.I., Si, Y.J., Lee, I.W., Song, M.S., and Choi, Y.K., 2017. Vaccine efficacy of inactivated, chimeric hemagglutinin H9/H5N2 avian influenza virus and its suitability for the marker vaccine strategy. J. Virol., 91: e01693-16. https://doi.org/10.1128/JVI.01693-16

Kim, S.H., 2018. Challenge for one health: Co-circulation of zoonotic H5N1 and H9N2 avian influenza viruses in Egypt. Viruses, 10(3): 121. https://doi.org/10.3390/v10030121

Kishida, N., Sakoda, Y., Eto, M., Sunaga, Y., and Kida, H., 2004. Co-infection of Staphylococcus aureus or Haemophilus paragallinarum exacerbates H9N2 influenza A virus infection in chickens. Arch. Virol., 149: 2095–2104. https://doi.org/10.1007/s00705-004-0372-1

Li, L., Tang, G.Y., Feng, H.L., Xue, Y.H., Ren, Z., Wang, G.K., Jia, M.M., Shang, Y., Luo, Q.P., Shao, H.B. and Wen, G., 2021. Evaluation of immune efficacy of H9 subtype avian influenza virus inactivated vaccine based on Mosaic HA sequence. Acta Vet. Zoot. Sin., 52: 3569–3577.

Li, Q.Y., Xu, M.M., Dong, H., Zhao, J.H., Xing, J.H., Wang, G., Yao, J.Y., Huang, H.B., Shi, C.W., Jiang, Y.L., Wang, J., Kang, Y., Ullah, N., Yang, W., Yang, G. and Wang, C.. 2020. Lactobacillus plantarum surface-displayed influenza antigens (NP-M2) with FliC flagellin stimulate generally protective immune responses against H9N2 influenza subtypes in chickens. Vet. Microbiol., 249: 108834. https://doi.org/10.1016/j.vetmic.2020.108834

Li, R., Adel, A., Bohlin, J., Lundkvist, Å., Olsen, B., Pettersson, J.H.O. and Naguib, M.M., 2020. Phylogeographic dynamics of influenza A (H9N2) virus crossing Egypt. Front. Microbiol., 11: 392. https://doi.org/10.3389/fmicb.2020.00392

Liu, J., Xue, L., Hu, S., Cheng, H., Deng, Y., Hu, Z., Wang, X., and Liu, X., 2018. Chimeric Newcastle disease virus-vectored vaccine protects chickens against H9N2 avian influenza virus in the presence of pre-existing NDV immunity. Arch. Virol., 163: 3365–3371. https://doi.org/10.1007/s00705-018-4016-2

Liu, L., Wang, T., Wang, M., Tong, Q., Sun, Y., Pu, J., Sun, H., and Liu, J., 2019. Recombinant turkey herpesvirus expressing H9 hemagglutinin providing protection against H9N2 avian influenza. Virology, 529: 7–15. https://doi.org/10.1016/j.virol.2019.01.004

Liu, D., Shi, W., and Gao, G.F., 2014. Poultry carrying H9N2 act as incubators for novel human avian influenza viruses. Lancet, 383: 869–875. https://doi.org/10.1016/S0140-6736(14)60386-X

Lv, J., Wei, L., Yang, Y., Wang, B., Liang, W., Gao, Y., Xia, X., Gao, L., Cai, Y., Hou, P., Yang, H., Wang, A., Huang, R., Gao, J. and Chai, T., 2015. Amino acid substitutions in the neuraminidase protein of an H9N2 avian influenza virus a_ect its airborne transmission in chickens. Vet. Res., 46: 44. https://doi.org/10.1186/s13567-014-0142-3

Ma, C., Zhang, Z., Zhao, P., Duan, L., Zhang, Y., Zhang, F., Chen, W., and Cui, Z., 2014. Comparative transcriptional activity of five promoters in BAC-cloned MDV for the expression of the hemagglutinin gene of H9N2 avian influenza virus. J. Virol. Methods, 206: 119–127. https://doi.org/10.1016/j.jviromet.2014.05.023

Moghoofei, M., Monavari, S.H., Mostafaei, S., Hadifar, S., Ghasemi, A., Babaei, F., Kavosi, H., Tavakoli, A., Javanmard, D., Esghaei, M. and Khodabandehlou, N., 2018. Prevalence of influenza A infection in the Middle-East: A systematic review and meta-analysis. Clin. Respir. J., 12: 1787–1801. https://doi.org/10.1111/crj.12758

Monne, I., Hussein, H.A., Fusaro, A., Valastro, V., Hamoud, M.M., Khalefa, R.A., Dardir, S.N., Radwan, M.I., Capua, I., and Cattoli, G., 2013. H9N2 influenza A virus circulates in H5N1 endemically infected poultry population in Egypt. Influenza Other Respir. Viruses, 7(3): 240-243. https://doi.org/10.1111/j.1750-2659.2012.00399.x

Monne, I., Meseko, C., Joannis, T., Shittu, I., Ahmed, M., Tassoni, L., Fusaro, A., and Cattoli, G., 2015. Highly pathogenic avian influenza A(H5N1) virus in poultry, Nigeria, 2015. Emerg. Infect. Dis., 21: 1275–1277. https://doi.org/10.3201/eid2107.150421

Mostafa, A., Blaurock, C., Scheibner, D., Müller, C., Blohm, U., Schäfer, A., Gischke, M., Salaheldin, A.H., Nooh, H.Z., Ali, M.A., Breithaupt, A., Mettenleiter, T.C., Pleschka, S., and Abdelwhab, E.M., 2020. Genetic incompatibilities and reduced transmission in chickens may limit the evolution of reassortants between H9N2 and panzootic H5N8 clade 2.3.4.4 avian influenza virus showing high virulence for mammals. Virus Evol. 6(2): veaa077. https://doi.org/10.1093/ve/veaa077

Mostafa, A., Mahmoud, S.H., Shehata, M., Müller, C., Kandeil, A., El-Shesheny, R., Nooh, H.Z., Kayali, G., Ali, M.A., and Pleschka, S., 2020. PA from a recent H9N2 (G1-Like) avian influenza a virus (AIV) strain carrying lysine 367 confers altered replication efficiency and pathogenicity to contemporaneous H5N1 in mammalian systems. Viruses, 12(9): 1046. https://doi.org/10.3390/v12091046

Naguib, M.M., Arafa, A.S., El-Kady, M.F., Selim, A.A., Gunalan, V., Maurer-Stroh, S., Goller, K.V., Hassan, M.K., Beer, M., Abdelwhab, E.M., and Harder, T.C., 2015. Evolutionary trajectories and diagnostic challenges of potentially zoonotic avian influenza viruses H5N1 and H9N2 co-circulating in Egypt. Infect. Genet. Evol., 34: 278-291. https://doi.org/10.1016/j.meegid.2015.06.004

Naguib, M.M., Arafa, A.S., Parvin, R., Beer, M., Vahlenkamp, T., and Harder, T.C., 2017. Insights into genetic diversity and biological propensities of potentially zoonotic avian influenza H9N2 viruses circulating in Egypt. Virology, 511: 165-174. https://doi.org/10.1016/j.virol.2017.08.028

Naguib, M.M., Grund, C., Arafa, A.S., Abdelwhab, E.M., Beer, M., and Harder, T.C., 2017. Heterologous post-infection immunity against Egyptian avian influenza virus (AIV) H9N2 modulates the course of subsequent infection by highly pathogenic AIV H5N1, but vaccination immunity does not. J. Gen. Virol., 98(6): 1169-1173. https://doi.org/10.1099/jgv.0.000767

Naguib, M.M., and Harder, T., 2018. Endemic situation of multiple avian influenza strains in poultry in Egypt: A continuing nightmare. Zoon. Publ. Hlth., 65(8): 908-910. https://doi.org/10.1111/zph.12486

Naguib, M.M., Verhagen, J.H., Samy, A., Eriksson, P., Fife, M., Lundkvist, Å., Ellström, P., and Järhult, J.D., 2019. Avian influenza viruses at the wild-domestic bird interface in Egypt. Infect. Ecol. Epidemiol., 9(1): 1575687. https://doi.org/10.1080/20008686.2019.1575687

Naguib, M.M., Ulrich, R., Kasbohm, E., Eng, C.L.P., Ho_mann, D., Grund, C., Beer, M., and Harder, T.C., 2017. Natural reassortants between potentially zoonotic avian influenza viruses H5N1 and H9N2 from Egypt display distinct pathogenic phenotypes in experimentally infected chickens and ferrets. J. Virol., 91: e01300-17. https://doi.org/10.1128/JVI.01300-17

Nagy, A., Lee, J., Mena, I., Henningson, J., Li, Y., Ma, J., Duff, M., Li, Y., Lang, Y., Yang, J., Abdallah, F., Richt, J.A., Ali, A., García-Sastre, A. and Ma, W., 2016. Recombinant Newcastle disease virus expressing H9 HA protects chickens against heterologous avian influenza H9N2 virus challenge. Vaccine, 34: 2537–2545. https://doi.org/10.1016/j.vaccine.2016.04.022

Nagy, A., Mettenleiter, T.C., and Abdelwhab, E.M., 2017. A brief summary of the epidemiology and genetic relatedness of avian influenza H9N2 virus in birds and mammals in the middle east and North Africa. Epidemiol. Infect., 145: 3320–3333. https://doi.org/10.1017/S0950268817002576

Okoye, J., Eze, D., Krueger, W.S., Heil, G.L., Friary, J.A., and Gray, G.C., 2013. Serologic evidence of avian influenza virus infections among Nigerian agricultural workers. J. Med. Virol., 85: 670–676. https://doi.org/10.1002/jmv.23520

Oluwayelu, D.O., Omolanwa, A., Adebiyi, A.I., and Aiki-Raji, C.O., 2016. Flock-based surveillance for low pathogenic avian influenza virus in commercial breeders and layers, southwest nigeria. Afr. J. Infect. Dis., 11; 44–49. https://doi.org/10.21010/ajid.v11i1.5

Pan, Q., Zhang, Y., Liu, A., Cui, H., Gao, Y., Qi, X., Liu, C., Zhang, Y., Li, K., and Gao, L., 2021. Development of a novel avian vaccine vector derived from the emerging fowl adenovirus 4. Front. Microbiol., 12: 780978. https://doi.org/10.3389/fmicb.2021.780978

Panshin, A., 2007. H9N2 influenza viruses from Israeli poultry: A five-year outbreak. Avian Dis., 51: 290–296. https://doi.org/10.1637/7590-040206R1.1

Park, M.S., Steel, J., García-Sastre, A., Swayne, D., and Palese, P., 2006. Engineered viral vaccine constructs with dual specificity: Avian influenza and Newcastle disease. Proc. Natl. Acad. Sci. USA. 103: 8203–8208. https://doi.org/10.1073/pnas.0602566103

Park, K.J., Kwon, H.I., Song, M.S., Pascua, P.N., Baek, Y.H., Lee, J.H., Jang, H.L., Lim, J.Y., Mo, I.P. and Moon, H.J., 2011. Rapid evolution of low-pathogenic H9N2 avian influenza viruses following poultry vaccination programmes. J. Gen. Virol., 92: 36–50. https://doi.org/10.1099/vir.0.024992-0

Peacock, T.H.P., James, J., Sealy, J.E., and Iqbal, M., 2019. A global perspective on H9N2 avian influenza virus. Viruses, 11(7): 620. https://doi.org/10.3390/v11070620

Peacock, T.P., Benton, D.J., James, J., Sadeyen, J.R., Chang, P., Sealy, J.E., Bryant, J.E., Martin, S.R., Shelton, H. and Barclay, W.S., 2017. Immune escape variants of H9N2 influenza viruses containing deletions at the haemagglutinin receptor binding site retain fitness in vivo and display enhanced zoonotic characteristics. J. Virol., 91: e00218-17. https://doi.org/10.1128/JVI.00218-17

Peacock, T.P., Harvey, W.T., Sadeyen, J.R., Reeve, R., and Iqbal, M., 2018. The molecular basis of antigenic variation among a(H9N2) avian influenza viruses. Emerg. Microbes Infect., 7: 176. https://doi.org/10.1101/312967

Perez, D.R., Lim, W., Seiler, J.P., Yi, G., Peiris, M., Shortridge, K.F., and Webster, R.G., 2003. Role of quail in the interspecies transmission of H9 influenza A viruses: Molecular changes on ha that correspond to adaptation from ducks to chickens. J. Virol., 77: 3148–3156. https://doi.org/10.1128/JVI.77.5.3148-3156.2003

Pu, J., Wang, S., Yin, Y., Zhang, G., Carter, R.A., Wang, J., Xu, G., Sun, H., Wang, M., Chu, W., Wei, Y., Wang, D., Zhu, B., Lemmon, G., Jiao, Y., Duan, S., Wang, Q., Du, Q., Sun, M., Baoa, J., Suna, Y., Zhaoa, J. , Zhang, H., Wu, G., Liua, J. and Webster, R.G., 2015. Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus. Proc. Natl. Acad. Sci. USA, 112: 548–553. https://doi.org/10.1073/pnas.1422456112

Qin, T., Yin, Y., Yu, Q., Huang, L., Wang, X., Lin, J., and Yang, Q., 2015. CpG oligodeoxynucleotides facilitate delivery of whole inactivated H9N2 influenza virus via transepithelial dendrites of dendritic cells in Nasal Mucosa. J. Virol., 89: 5904–5918. https://doi.org/10.1128/JVI.00296-15

Radvak, P., Kosikova, M., Kuo, Y.C., Li, X., Garner, R., Schmeisser, F., Kosik, I., Ye, Z., Weir, J.P., Yewdell, J.W. and Xie, H. 2021. Highly pathogenic avian influenza A/Guangdong/17SF003/2016 is immunogenic and induces cross-protection against antigenically divergent H7N9 viruses. NPJ Vaccines, 6: 30. https://doi.org/10.1038/s41541-021-00295-7

Rubrum, A., Jeevan, T., Darnell, D., Webby, R., Derrar, F., and Gradi, E.A., 2018. Hemagglutinin (influenza A virus). Accession no. Azf86190.1. GenBank. 2018. Available online: https://www.ncbi.nlm.nih.gov/protein/Azf86190.1

Samy, A., and Naguib, M., 2018. Avian respiratory coinfection and impact on avian influenza pathogenicity in domestic poultry: Field and experimental findings. Vet. Sci., 5: 23. https://doi.org/10.3390/vetsci5010023

Sealy, J.E., Yaqub, T., Peacock, T.P., Chang, P., Ermetal, B., Clements, A., Sadeyen, J.R., Mehboob, A., Shelton, H. and Bryant, J.E., 2019. Association of increased receptor-binding avidity of influenza A(H9N2) viruses with escape from antibody-based immunity and enhanced zoonotic potential. Emerg. Infect. Dis., 25: 63–72. https://doi.org/10.3201/eid2501.180616

Seifi, S., Asasi, K., and Mohammadi, A., 2010. Natural co-infection caused by avian influenza H9 subtype and infectious bronchitis viruses in broiler chicken farms. Vet. Arch., 80: 269–281.

Shehata, A.A., Parvin, R., Sultan, H., Halami, M.Y., Talaat, S., Abd Elrazek, A., Ibrahim, M., Heenemann, K., and Vahlenkamp, T., 2015. Isolation and full genome characterization of avian influenza subtype H9N2 from poultry respiratory disease outbreak in Egypt. Virus Genes, 50(3): 389-400. https://doi.org/10.1007/s11262-015-1188-7

Shi, S.H., Yang, W.T., Yang, G.L., Cong, Y.L., Huang, H.B., Wang, Q., Cai, R.P., Ye, L.P., Hu, J.T. and Zhou, J.Y., 2014. Immunoprotection against influenza virus H9N2 by the oral administration of recombinant Lactobacillus plantarum NC8 expressing hemagglutinin in BALB/c mice. Virology, 464-465: 166–176. https://doi.org/10.1016/j.virol.2014.07.011

Shi, S.H., Yang, W.T., Yang, G.L., Zhang, X.K., Liu, Y.Y., Zhang, L.J., Ye, L.P., Hu, J.T., Xing, X., and Qi, C., 2016. Lactobacillus plantarum vaccine vector expressing hemagglutinin provides protection against H9N2 challenge infection. Virus Res., 211: 46–57. https://doi.org/10.1016/j.virusres.2015.09.005

Sid, H., Benachour, K., Rautenschlein, S. 2015. Co-infection with multiple respiratory pathogens contributes to increased mortality rates in Algerian poultry flocks. Avian Diseases, 59: 440–446.

Soo-Jeong, K., Min-Ji, P., Na-Young, K., Yu-Na, L., Gyeong-Beom, H., Yoon-Ki, B., Jae-In, S., Myoung-Heon, L., and Youn-Jeong, L., 2021. Pathogenicity of H9N2 low pathogenic avian influenza viruses of different lineages isolated from live bird markets tested in three animal models: SPF chickens, Korean native chickens, and ducks. Poult. Sci., 100(9): 101318. https://doi.org/10.1016/j.psj.2021.101318

Sorrell, E.M., Song, H., Pena, L., and Perez, D.R., 2010. A 27-amino-acid deletion in the neuraminidase stalk supports replication of an avian H2N2 influenza A virus in the respiratory tract of chickens. J. Virol., 84: 11831–11840. https://doi.org/10.1128/JVI.01460-10

Sulaiman, L., Shittu, I., Fusaro, A., Inuwa, B., Zecchin, B., Gado, D., Schivo, A., Bianco, A., Laleye, A., Gobbo, F., Vakuru, C., Joannis, T., Monne, I., and Meseko, C., 2021. Live bird markets in Nigeria: A potential reservoir for H9N2 avian influenza viruses. Viruses, 13(8): 1445. https://doi.org/10.3390/v13081445

Sun, Y., Pu, J., Fan, L., Sun, H., Wang, J., Zhang, Y., Liu, L., and Liu, J., 2012. Evaluation of the protective efficacy of a commercial vaccine against different antigenic groups of H9N2 influenza viruses in chickens. Vet. Microbiol., 156: 193–199. https://doi.org/10.1016/j.vetmic.2011.10.003

Swayne, D.E., Beck, J.R., and Kinney, N., 2000. Failure of a recombinant fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunized with a fowl pox vaccine. Avian Dis., 44: 132–137. https://doi.org/10.2307/1592516

Tamura, K. and Nei, M., 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol., 10: 512-526.

Tamura, K., Stecher, G., and Kumar, S., 2021. MEGA 11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol., https://doi.org/10.1093/molbev/msab120

Tombari ,W., 2013. Risk factors and characteristics of low pathogenic avian influenza virus isolated from commercial poultry in Tunisia. PLoS ONE. 8: e53524.

Tombari, W., Nsiri, J., Larbi, I., Guerin, J.L., and Ghram, A., 2011. Genetic evolution of low pathogenecity H9N2 avian influenza viruses in Tunisia: Acquisition of new mutations. Virol. J., 8: 467. https://doi.org/10.1186/1743-422X-8-467

Tombari, W., 2016. Variability of tropism and replicative capacity of two naturally occurring influenza A H9N2 viruses in cell cultures from different tissues. Avian Pathology, 45: 212–220.

Umar, S., 2016. Comparative antiviral efficacy of zanamivir and amantadine against Tunisian isolate of avian influenza virus (H9N2). Pakistan Journal of Zoology, 48: 1443–1447.

Wang, Z., Li, Z., Su, X., Qiao, Y., Fan, W., Li, H., Shi, B., Qin, T., Chen, S. and Peng, D., 2019.Enhanced cross-lineage protection induced by recombinant H9N2 avian influenza virus inactivated vaccine. Vaccine, 37: 1736–1742. https://doi.org/10.1016/j.vaccine.2019.02.012

Wang, D., Wang, J., Bi, Y., Fan, D., Liu, H., Luo, N., Yang, Z., Wang, S., Chen, W., Wang, J., Xu, S., Chen, J., Zhang, Y. and Yin, Y. 2018. Characterization of avian influenza H9N2 viruses isolated from ostriches (Struthio camelus). Sci. Rep., 8: 2273. https://doi.org/10.1038/s41598-018-20645-1

Wang, J., Cao, Z., Guo, X., Zhang, Y., Wang, D., Xu, S., and Yin, Y., 2016. Cytokine expression in 3 chicken host systems infected with H9N2 influenza viruses with different pathogenicities. Avian Pathol., 45: 1–26. https://doi.org/10.1080/03079457.2016.1193665

Xu, X., Xue, C., Liu, X., Li, J., Fei, Y., Liu, Z., Mu, J., Bi, Y., Qian, J. and Yin, R., 2019. A novel recombinant attenuated Newcastle disease virus expressing H9 subtype hemagglutinin protected chickens from challenge by genotype VII virulent Newcastle disease virus and H9N2 avian influenza virus. Vet. Microbiol., 228: 173–180. https://doi.org/10.1016/j.vetmic.2018.11.006

Yao, M., Lv, J., Huang, R., Yang, Y., and Chai, T., 2014. Determination of infective dose of H9N2 avian influenza virus in different routes: Aerosol, intranasal, and gastrointestinal. Intervirology, 57: 369–374. https://doi.org/10.1159/000365925

Yin, Y., Qin, T., Wang, X., Lin, J., Yu, Q., and Yang, Q., 2015. CpG DNA assists the whole inactivated H9N2 influenza virus in crossing the intestinal epithelial barriers via transepithelial uptake of dendritic cell dendrites. Mucosal Immunol., 8: 799–814. https://doi.org/10.1038/mi.2014.110

Young, S.G., Carrel, M., Malanson, G.P., Ali, M.A., and Kayali, G., 2016. Predicting avian influenza co-infection with H5N1 and H9N2 in Northern Egypt. Int. J. Environ. Res. Publ. Health, 13(9): 886. https://doi.org/10.3390/ijerph13090886

Zecchin, B., Minoungou, G., Fusaro, A., Moctar, S., Ouedraogo-Kaboré, A., Schivo, A., Salviato, A., Marciano, S., and Monne, I., 2017. Influenza A(H9N2) Virus, Burkina Faso. Emerg. Infect. Dis., 23(12): 2118-2119. https://doi.org/10.3201/eid2312.171294

Zhang, S., Tang, X., Wang, S., Shi, F., Duan, C., Bi, F., Suo, J., Hu, D., Liu, J. and Wang, C., 2021. Establishment of recombinant eimeria acervulina expressing multi-copies M2e derived from avian influenza virus H9N2. Vaccines, 9: 791. https://doi.org/10.3390/vaccines9070791

Zhang, X., Bo, Z., Meng, C., Chen, Y., Zhang, C., Cao, Y., and Wu, Y., 2021. Generation and evaluation of recombinant thermostable Newcastle disease virus expressing the HA of H9N2 avian influenza virus. Viruses, 13: 1606. https://doi.org/10.3390/v13081606

Zhang, X., Wu, Y., Huang, Y., and Liu, X., 2012. Protection conferred by a recombinant Marek’s disease virus that expresses the spike protein from infectious bronchitis virus in specific pathogen-free chicken. Virol. J., 9: 1–10. https://doi.org/10.1186/1743-422X-9-85

Zhang, P., Tang, Y., Liu, X., Peng, D., Liu, W., Liu, H., Lu, S., and Liu, X., 2008. Characterization of H9N2 influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation in eastern China during a 5 years period (1998–2002). J. Gen. Virol., 89: 3102–3112. https://doi.org/10.1099/vir.0.2008/005652-0

Zhong, L., Wang, X., Li, Q., Liu, D., Chen, H., Zhao, M., Gu, X., He, L., Liu, X., and Gu, M., 2014. Molecular mechanism of the airborne transmissibility of H9N2 avian influenza A viruses in chickens. J. Virol., 88: 9568–9578. https://doi.org/10.1128/JVI.00943-14

Zhou, X., Wang, D., Xiong, J., Zhang, P., Li, Y., and She, R., 2010. Protection of chickens, with or without maternal antibodies, against IBDV infection by a recombinant IBDV-VP2 protein. Vaccine, 28: 3990–3996. https://doi.org/10.1016/j.vaccine.2010.03.021