China

In mainland China, LPAI H9N2 subtype was first isolated from chickens in Guangdong province in 1994, and then the viruses spread to several other southern provinces and resulted in severe economic losses for poultry industries. Recently H9N2 has been the most prevalent subtype of influenza virus in Chinese poultry populations (Cong et al., Reference CONG, PU, LIU, WANG, ZHANG, ZHANG, FAN, BROWN and LIU2007; Yu et al., Reference YU, HUA, WEI, ZHOU, TIAN, LI, LIU and TONG2008; Zhang et al., Reference ZHANG, YIN, BI, WANGA, XU, WANGA, ZHOU, SUN and YOON2012). Genetic studies showed that three distinct lineages of LPAI H9N2 have been responsible for AI outbreaks in China: represented by Ck/Beijing/1/94 (BJ/94-like), Qa/HK/G1/97 (G1-like) and Dk/HK/Y439/97 (Korea-like), respectively. For controlling H9N2 infections, an oil-emulsion, formalin-inactivated vaccine was derived from an early isolates A/ck/Shandong/6/96 (A/CK/SD/6/96) and has been used in layers and breeders on chicken farms since 1998. However, viral infection still occurred, sometimes even in the vaccinated flocks. (Chengjun et al., Reference CHENGJUN, TIAN, YANBING, DANDAN, KANGZHEN and CHEN2004; Zhang, et al., 2012; Yu et al., Reference YU, HUA, WEI, ZHOU, TIAN, LI, LIU and TONG2008). Chengjun et al. (Reference CHENGJUN, TIAN, YANBING, DANDAN, KANGZHEN and CHEN2004) conducted the antigenic analysis of 20 H9N2 viruses isolated from domestic poultry in China, and showed that most viruses isolated after 1996 could not be inhibited efficiently by the antisera induced by A/CK/SD/6/96, which suggested that the vaccine could not induce enough protection to the prevalent strains. The antisera induced by A/CK/SJZ/2/98 exhibited much broader reactions to the virus isolated after 1998. Therefore, it was proposed that A/CK/SJZ/2/98 could be chosen as a vaccine strain to replace the current one. Moreover, they analysed 27 LPAI H9N2 viruses isolated from chickens and ducks from 1996 to 2002, and demonstrated that these were antigenically different from the vaccine strain currently used in China. Phylogenetic analysis revealed that all of the viruses originated from CK/BJ/1/94 had formed multiple genotypes through complicated reassortment with Qa/HK/G1/97-, Ck/HK/G9/97-, Ck/SH/F/98- and TY/WI/66-like viruses.

Sun et al. (Reference SUN, PU, JIANG, GUAN, XIA, XU, LIU, MA, TIAN, BROWN and LIU2010) analysed the evolution of H9N2 influenza viruses in China from 1994 to 2008 and demonstrated that the H9N2 viruses in China underwent extensive reassortment, and novel genotypes continued to emerge. Among 330 viruses, 54 genotypes were observed including 19 novel genotypes that have not been recognised before.

Ji et al. (Reference JI, JIANG, LIU, CHEN, CHEN, HOU, LI and HUANG2010) conducted three surveys of LPAI H9 subtypes, and the HA gene of 55 H9 subtypes isolated in the China in 2007-2009 were sequenced and analysed. Their results indicated that LPAI H9 isolated between 2007 and 2009 were a little different from previous strains. This indicated that the strains used for production of the LPAI H9 vaccines used in poultry in China needed to be updated. Additionally, mutations in the viral HA gene and surveys of the viruses suggested that they have become of a greater risk for public health in the recent years.

To help understand the problems associated with vaccine failure, Zhang et al. (Reference ZHANG, YIN, BI, WANGA, XU, WANGA, ZHOU, SUN and YOON2012) analyzed 41 LPAI H9N2 subtypes isolated from chicken flocks in various regions of China between 1998 and 2007. The results showed that LPAI H9N2 subtypes had continued to evolve over time, forming distinct antigenic groups among the viruses over time. Two earlier H9N2 strains, that previously had been commonly used in commercial vaccines, were not able to provide sufficient protection to all of the antigenic groups. From this work it was strongly recommended that a national or regional surveillance program was required in order to maintain better prevention and control of H9N2 in China.

Korea

In Korea, the first outbreak of H9N2 influenza viruses occurred in 1996, when five of these viruses were isolated from broiler breeder flocks showing clinical signs. Phylogenic analysis revealed that the 1996 Korean isolates were different from two previously established lineages, the G1-like and Y280-like groups, represented by A/Qa/HK/G1/97 and A/ck/HK/Y280/97 respectively. Thus, they were assigned to the Korean-like or Y439-like group represented by A/Dk/HK/Y439/97 (Kim et al., Reference KIM, CHO, KIM and SEO2006; Song et al., Reference SONG, LEE, JEONG, KANG, KIM, KWON, KIM, SEONG and KIM2008).

Korean animal health authorities initiated stamping-out and compensation control policies with regards to H9N2 influenza virus when they occurred between 1996 and 1999. At that time, vaccines were prohibited in Korea. However, LPAI H9N2 became endemic and the policy was not reliable enough to cover each outbreak. According to the 2004 AI standard operating procedures, the Korean Veterinary Authority permitted the use of an inactivated oil adjuvant LPAI H9N2 vaccine, and the Committee on the National AI Vaccine Campaign determined that using a single vaccine strain was the most effective strategy with which to simplify the LPAI H9N2 situation in Korea. (Lee et al., Reference LEE, PARK, LEE, SONG, KANG, LEE, PARK, CHOI and SONG2011).

Seo and Kim (Reference SEO and KIM2004) conducted surveillance of LPAI at live poultry markets, and isolated H9N2, H3N2, and H6N1 influenza viruses from poultry. Serological surveillance in chickens showed that over 50% of chickens were infected with H9N2 viruses. Kim et al. (Reference KIM, CHO, KIM and SEO2006) determined that LPAI H9N2 isolated from poultry in Korean live bird markets were genetically different from viruses isolated from China and Hong Kong. When they infected layer hens with H9N2 isolates, they had around 30% mortality and reduced egg production. Since no vaccine was available for controlling LPAI H9N2 in Korea in that time, they recommended developing an effective vaccine for LPAI H9N2 to prevent the economic losses in Korean chicken industry from such infections and to prevent the possible human casualties.

Lee et al. (Reference LEE, SHIN, SONG, LEE, CHOI, LEE, JEONG, SUNG, KIM, KWON, KWON, KIM, WEBBY, WEBSTER and CHOI2007) have analysed the evolution of LPAI H9 isolated from Korean chicken farms from 2002 to 2004. Korean LPAI H9 formed two antigenically distinct groups: those isolated from 1996 to mid 2003 and those from late 2003 through 2004. Phylogenetic analysis of 12 viruses studied revealed three genotypes of LPAI H9N2 and showed that reassortment had occurred. One isolate, Ck/Kor/164/04, belonged to the H9N8 subtype, whereby its HA and PB1 genes were similar to those of the LPAI H9N2, but other genes were more closely related to H3N8 viruses. They reported that the pathogenicity of the early isolates altered due to antigenic drift and reassortment, leading to LPAI H9 in Korea that potentially could expand their host range to mammals.

Woo and Park (Reference WOO and PARK2008) studied the sero-prevalence of LPAI H9N2 in poultry flocks in the Gyeonggi-do region of Korea (GPK) during 2005-2006. They indicated that LPAI H9N2 occurred in portions of layers and broilers in the GPK, but it had remained largely undetected or undiagnosed. In older birds were over twice as likely to be at risk compared to younger birds when comparing sero-positivity by age. This may have been due to increased susceptibility with age due to decreased immunity and a better opportunity for viral exposure via personnel and transportation, which were the main source of spread of LPAI.

Hong Kong

Since the mid 1990s, three strains of LPAI H9N2 have become established in domestic poultry in Hong Kong and southern China: Dk/HK/Y280/97, Qa/HK/G1/97 and Ck/Kor/006/96. Among these, H9N2 viruses of Qa / HK / G1/97 lineages have been sporadically introduced into humans causing flu-like illnesses. The Qa/HK/G1/97 (H9N2) virus has a fast evolving rate, especially in their PB2, HA; NP and NA genes, which indicated that this virus was in the process of adapting to a new host. Another lineage co-circulated in the poultry markets in Hong Kong and south-eastern China, is represented by Dk/HK/Y280/97 (Y280-like). H9N2 viruses belonging to this Y280-like lineage are closely related genetically to Dk/HK/G9/97 (H9N2). It was found that the majority of the H9N2 influenza virus isolated in the same region was antigenically related to Dk/HK/G9/97 (H9N2) and were mainly found in chicken, duck, geese, pigeon and silky chickens. In the meantime, H9N2 influenza Y280-like viruses were isolated from domestic pigs in Hong Kong in 1998. These findings indicate that interspecies transmissions of H9N2 influenza virus from avian to mammalian hosts have continued to occur (Butt et al., Reference BUTT, SMITH, CHEN, ZHANG, LEUNG, XU, LIM, WEBSTER, YUEN, PEIRIS and GUAN2005).

In 2003, a total of 294 influenza viruses were isolated from the poultry markets of Hong Kong. At this time the LPAI H9N2 subtype was concurrently circulating with Newcastle disease virus (NDV). In 2004, two other subtypes H5 and H6 were found to be co-circulating with H9N2 viruses. The majority of the samples tested were H9 viruses and these were mainly found in chicken. In 2005, only H9 influenza viruses and NDV were isolated with similar isolation rates. Although influenza viruses, mainly of the H9N2 subtype, and NDV have been isolated from poultry, no symptoms of disease were observed over the entire sampling period (Butt et al., Reference BUTT, SMITH, CHEN, ZHANG, LEUNG, XU, LIM, WEBSTER, YUEN, PEIRIS and GUAN2005).

Japan

In 2001, the Japanese Government decided to stop the import of duck meat and to survey the presence of the viruses in imported chicken products from China at the Animal Quarantine Service (AQS). During this process, LPAI H9N2 and NDV were isolated from chicken products such as meat and bone marrow (Mase et al., Reference MASE, ETO, IMAI, TSUKAMOTO and YAMAGUCHI2007). In addition, LPAI H9N2 was isolated from the meat and bone marrow of chickens imported from China to Japan in 2001. These isolates were experimentally inoculated intranasally into specific pathogen free (SPF) chickens, and viruses were recovered from the meat and bone marrow of the birds without any clinical syndromes. On the other hand, chickens co-infected with the H9N2 virus and with either Staphylococcus aureus or Haemophilus paragallinarum showed clinical signs which were more severe than those shown by chickens infected only with S. aureus or H. paragallinarum. Higher titres of H9N2 viruses recovered from the chickens co-infected with S. aureus or H. paragallinarum compared with those from birds infected only by the virus, which indicated that co-infection with S. aureus, or H. paragallinarum enhanced the replication of the LPAI H9N2, resulting in exacerbation of the disease (Kishida et al., Reference KISHIDA, ETO, SUNAGA, SUNAGA and KIDA2004).

A study by Mase et al. (Reference MASE, ETO, IMAI, TSUKAMOTO and YAMAGUCHI2007) characterized 11 LPAI H9N2 viruses isolated from chicken products at AQS in Japan. Genetically, they were classified into six distinct genotypes, including five that were already known and one novel genotype. This suggested that multiple genotypes of the LPAI H9N2 had already become widespread and endemic in China. The pathogenicity index indicated that the LPAI H9N2 isolated from chicken products were a low pathogenicity threat to chickens.

Vietnam

The first reported genetic characterisation of LPAI H9N2 from domestic ducks in northern Vietnam was completed in 2009 (Hotta et al., Reference HOTTA, TAKAKUWA, LE, PHUONG, MURASE, ONO, ITO, OTSUKI and YAMASHIRO2012). They conducted the phylogenetic analysis of all the eight gene segments of H9N2 and found that all isolates were closely related to those isolated from the coastal regions of Far East Asia. They concluded that there was no evidence that the H9N2 subtypes shared gene segments with H5N1 subtypes.

India

In India, the first reported isolation and identification of LPAI H9N2 chickens was made between 2003 and 2004 (Nagarajan et al., Reference NAGARAJAN, RAJUKUMAR, TOSH, RAMASWAMY, PUROHIT, SAXENA, BEHERA, PATTNAIK, PRADHAN and DUBEY2009). This confirmed that all the isolates were low pathogenic strains. Nucleotide sequence analysis of the HA gene showed that the Indian isolates were closely related to H9N2 isolates from German and Asian regions rather than from mainland China. Phylogenetic analysis revealed they shared a common ancestor - the Qa/HK/G1/97 isolate - which contributed to the internal genes of H5N1 virus circulating in Vietnam at the time. Amino acid sequencing showed that the Indian H9N2 isolates might have the potential to infect humans.

To understand the genetic relationships between Indian viruses, Tosh et al. (Reference TOSH, NAGARAJAN, BEHERA, RAJUKUMAR, PUROHIT, KAMAL, MURUGKAR, GOUNALAN, PATTNAIK, VANAMAYYA, PRADHAN and DUBEY2008) determined the nucleotide sequence of five H9N2 viruses. The amino acid sequences revealed that four isolates shared an R–S–S–R/G motif at the cleavage site of HA, while one virus harboured an R–S–N–R/G motif at the same position. Phylogenetic analysis showed that 50% of the genes (HA, NA, NP and M) were similar to G1-like viruses. These finding provided evidence of a novel reassorted H9N2 genotype of G1-like viruses circulating in India.

Vegad (Reference VEGAD2014) published a review on the emergence of drift variants of LPAI viruses, with particular focus on their occurrence in India. Influenza viruses are dynamic and continuously evolving, and variants emerge from the instability of these viruses due to their constant mutations through antigenic drift. The drift variants emerging from the LPAI viruses gradually acquire higher virulence.

Bangladesh

Paritosh et al. (Reference TOSH, NAGARAJAN, BEHERA, RAJUKUMAR, PUROHIT, KAMAL, MURUGKAR, GOUNALAN, PATTNAIK, VANAMAYYA, PRADHAN and DUBEY2008) detected the influenza virus subtype H9N2 in chickens on three farms in Bangladesh, whilst Isabella et al. (Reference ISABELLA, MAT, GWENAELLE, FILIP, GARBA, MOHAMMED, ANNALISA, SILVIA, FRANCESCO, ALESSIA and GIOVANNI2013) identified a natural reassorted HPAI (H5N1) virus containing a H9N2-PB1 gene. Their findings highlighted the risks for prolonged co-circulation of avian influenza viruses and the need to monitor their evolution.

Karthik et al. (Reference KARTHIK, MOHAMMED, LISA, DAVID, SMRABIUL, MKAMRUL, PAMELA, SCOTT, RICHARD and ROBERT2014) characterised the genetic sequences of 37 Bangladeshi H9N2 viruses isolated between 2011-2013 and investigated their inter- and intra-subtypic genetic diversities by tracing their genesis in relationship to other H9N2 viruses isolated from neighbouring countries. These strains were homogenous with several mammalian host-specific markers and provided a new H9N2 sublineage, wherein the haemagglutinin (HA) gene was derived from an Iranian H9N2 lineage (Mideast_B Iran), the neuraminidase (NA) and polymerase basic 2 (PB2) genes were from Dubai H9N2 (Mideast_C Dubai), and the non-structural protein (NS), nucleoprotein (NP), matrix protein (MP), polymerase acidic (PA) and polymerase basic 1 (PB1) genes were from HP H7N3 originated from Pakistan. Phylogenetic and molecular analyses suggested that the current genotype was originally descended from the prototypical H9N2 lineage (G1).

Pakistan

The first outbreak of LPAI H9N2 subtype reported in poultry in Pakistan was in 1998 and resulted in 10-20% mortality with a decrease in egg production from 10% up to 75% in severe cases. It was named A/ck/ Pakistan/3/99 (H9N2), and since then, the disease has been reported in various poultry rearing areas at different locations throughout the country. A new outbreak of LPAI involving serotype H7N3 and H9N2 was reported in Pakistan from November of 2003 to May 2004, and affected commercial layer and breeder flocks. The disease caused a high mortality rate (Khawaja et al., Reference KHAWAJA, NAEEM, AHMED and AHMAD2005). These authors monitored the prevalence of LPAI in wild birds in certain areas in Pakistan during the outbreak of 2003. Seven wild bird species were selected, and blood, cloacal and tissue samples were collected for serological evaluation and virus isolation. Data indicated that antibodies to LPAI H9N2 were present in 10% of wild birds, whereas the virus was isolated only from 6.72% of the samples. They concluded that the wild birds should be considered as one of the major carriers of LPAI infection.

Iqbal et al. (Reference IQBAL, YAQUB, REDDY and MCCAULEY2009) investigated the molecular features and genetic relationships of H9N2 viruses prevalent in Pakistan. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The HA sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007. The HA, NA, NP and M genes showed close identity with LPAI H9N2 isolated during 1999 in Pakistan and were part of the same cluster as the A/Qa/HK/G1/97 lineage. In contrast, the polymerase genes clustered with LPAI H9N2 from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity to NS genes from either H5 or H7 subtypes rather than to established H9N2 Eurasian lineages. The authors indicated that, during recent years, the H9N2 viruses have undergone extensive genetic reassortment, which has led to the generation of H9N2 viruses of novel genotypes on the Indian sub-continent.

Egypt

In Egypt the first isolation and genetic characterisation of H9N2 viruses were made from commercial bobwhite quail in May 2011. Virus isolation and rRT-PCR confirmed the presence of the H9N2 virus in Egypt, and HA and NA genes showed a close relationship to Middle Eastern isolates. Phylogenetic analysis of the Egyptian H9N2 virus demonstrated its close relationship to G1-like lineages isolated from neighbouring countries (El-Zoghby et al., Reference EL-ZOGHBY, ARAFA, HASSAN, ALY, SELIM, KILANY, SELIM, NASEF, AGGOR, ABDELWHAB and HAFEZ2012).

Arafa et al. (Reference ARAFA, HAGAG, ERFAN, MADY, EL-HUSSEINY, ADEL and NASEF2012) described the genetic characterisation of Qa/Egypt/11; H9N2 previously isolated from the infected quail found in Giza in May 2011. Phylogenetic analysis indicated that the Qa/Egypt/11 isolate belonged to the A/Qa/HK/G1/1997-like lineage with new mutations identified in all viral proteins. The phylogenetic analysis for the eight genes indicated placement of the Egyptian virus within the same lineage of H9N2 viruses that have been circulating in the region since 2006.

In Alexandria, Abdel-Moneim et al. (Reference ABDEL-MONEIM, AFIFI and EL-KADY2012) isolated H9N2 virus from chicken flocks suffering from respiratory symptom with high morbidity rates (up to 70%) and mortality rate reaching 15%. Blast analysis of the nucleotide sequences from the eight viral genes showed that the recently isolated Egyptian H9N2 strain, A/chicken/Egypt/BSU-CU/2011 was closely related to the other Middle East H9N2 strains. The virus shared the common ancestor - the A/Qa/HK/G1/97 isolate - which contributed to the internal genes of the H5N1 virus circulating in Asia.

Libya

Serious economic losses occurred during March-May 2005 as a result of the high mortality rates in broiler chickens and declines in egg production for both breeder and layer flocks, with LPAI H9N2 being identified as one of the main causes of such losses (Al-Garib et al., Reference AL-GARIB, KAMMON, ASHEG, HAMID, FATHALLA and LAWALA2007). The high mortality seen between 37 and 48 days of age correlated with the detection of specific antibodies directed against LPAI H9N2. Fares et al. (Reference FARES, ELHAFI, ALZALETNY and GERISH2010) performed seroprevalence work on LPAI H9N2 in commercial broiler and layer flocks northwest of Libya and found that all positive samples had antibodies against only LPAI H9 subtype. The positive birds had no history of clinical signs to suggest the existence of LPAI in the region, although recently, according to RT-PCR results, nucleic acid of avian influenza type A viruses has been detected. The H9 subtype was detected using rRT-PCR assay.

Tunisia

In Tunisia, the first emergence of LPAI H9N2 was seen in poultry at the end of 2009. The virus has caused many outbreaks in Tunisian flocks and resulted in major economic losses in the poultry industry since then. A second wave of LPAI H9N2 was reported during July to October 2010 (Tombari et al., Reference TOMBARI, NSIRI, LARBI, GUERIN and GHRAM2011). These authors studied the phylogenetic analysis and demonstrated that A/Ck/TUN/12/10 and A/Migratory Bird/TUN/51/10 was closely related to the Middle Eastern isolates belonging to the G1- like lineage in the H9N2 subtype. H9N2 isolates did not harbour multiple basic amino acids in HA cleavage site motif. This indicated that Tunisian H9N2 strains were LPAI, although the motif for these viruses is similar to the sequence required for the highly pathogenic H5 and H7 subtypes. Such genetic findings suggested that Tunisian H9N2 viruses could have the potential to acquire basic amino changes in order to become highly pathogenic strains.

Lebanon

Barbour et al. (Reference BARBOUR, SAGHERIAN, SAGHERIAN, DANKER, JABER, USAYRAN and FARRAN2006) described the first occurrence of an outbreak of LPAI H9N2 in poultry farms in Lebanon in 2005. Both broiler breeder and commercial layers showed a significant drop in egg production, equivalent to 46% and 47.3% respectively. Mortality rates among breeder, commercial layers, free-range layers and broilers averaged 2, 2, 1 and 35% respectively. Serum samples were examined using ELISA technique, and the positive samples for AI from meat breeders and commercial layers were confirmed by the HI test to contain H9 specific antibodies. In addition, the direct immunofluorescent antibody test indicated the presence of an H9 antigen in congested brains and in tracheal lesions of broilers. The virus isolated from the brains of broilers was subtyped H9N2. In addition, the study demonstrated 100% H9 specific HI antibodies in pigs fed with carcasses of affected chicken. Furthermore, they found antibodies in the sera of farmers (32.3%) who managed the affected chickens, which has worrying human health implications.

Jordan

Al-Natour et al. (2005) studied the seroprevalence of AI in commercial Jordanian broiler breeder flocks in 2001. Random blood samples collected were examined using the ELISA test for specific antibodies to avian influenza. The sero-positive birds had no history of clinical signs and flock mortality rates were low, indicating that all AI isolates from Jordan were classified as LPAI and subtyped as H9N2. All infected flocks were near migratory waterfowl routes and large bodies of surface water, which could be a major contributor for exposure.

Roussan et al. (Reference ROUSSAN, KHAWALDEH, AL RIFAI, TOTANJI and SHAHEEN2009) investigated the prevalence of LPAI H9 subtypes in commercial chicken flocks in Jordan by serological and molecular methods. Serum samples from 180 chicken flocks (120 broilers and 60 layers) which were free from respiratory symptoms were examined by HI test for specific antibodies against LPAI subtype H9. Eighty-three chicken flocks (60 broilers and 23 layers) with respiratory symptoms with mortality rates of 15-25%, and a drop in egg production (15-18%) were examined by RT-PCR, using universal primers for influenza A viruses. Specific primers targeting LPAI H9 gene were used for the flocks that were determined to be positive by universal primers. Overall, 65 out of 120 broiler flocks (54.2%), and 47 out of 60 layer flocks (78.3%) were positive for LPAI H9 subtype antibodies. Nucleic acid sequences from influenza A viruses were detected in 31 out of 60 broiler flocks (51.7%), and 15 out of 23 layer flocks (65.2%). The study confirmed the endemic nature of LPAI H9 subtype in broiler and layer flocks in Jordan.

Iran

An avian influenza outbreak occurred during the summer in July 1998 and included broiler, layer and breeder farms. All viruses isolated were subtyped as H9N2, and the LPAI strains isolated from layer, breeder and broiler flocks were identified respectively as: A / ck / Iran / ZMT-101(101) / 1998 (H9N2), A/ck/Iran/ZMT- 102 (102) /1998 (H9N2) and A/ck/Iran/ZMT-103(103)/1998 (H9N2). The viruses were further classified as H9N2, subtyped by haemagglutination inhibition (HI) and neuraminidase inhibition (NI) test. These H9N2 strains were used to infect 4-6 weeks old chicken, and the birds did not die although performance was temporary depressed (Marandi and Fard, 2002). Mixed infections of the influenza virus with other respiratory pathogens, particularly IBV and Mycoplasma gallisepticum, were thought to be responsible for such high mortality seen on farms, which resulted in great economic losses (Nili and Asasi Reference NILI and ASASI2002).

Kianizadeh et al. (Reference KIANIZADEH, POURBAKHSH, TOROGHI and MOMAYEZ2006) studied pathogenicity, HA gene variation and phylogenetic analysis of 16 LPAI H9N2 isolated from outbreaks in Iran from 1998 to 2001. The results in both pathogenicity and HA gene cleavage site sequence detection represented a low pathogenic feature for all Iranian AI isolates studied. The cleavage site motif of all influenza viral isolates indicated that they had the capability of becoming highly pathogenic viruses following nucleotide substitutions in this region.

Pazani et al. (Reference PAZANI, VASFI MARANDI, ASHRAFIHELAN, HOSSEIN MARJANMEHR and GHODS2008) carried out pathological studies in chickens experimentally inoculated with A/Ck/Tehran/ZMT-173/99 (H9N2) influenza virus. Two groups of 30-day-old, containing 40 Mycoplasma gallisepticum positive and 40 negative commercial broiler chickens were used. One experimental subgroup was inoculated intravenously (IV) through the brachial vein and the other via both IV and oculonasal routes with a volume of 0.2 ml of a 1:10 dilution of infectious amino allantoic fluid with an infectivity titre of 107.5 ELD50 (50% embryo lethal doses). They found that renal lesions and tracheal casts were the principal causes of mortality and simultaneous inoculation played an important role in the formation of tracheal casts. In addition, immunosuppression due to depletion of lymphoid organs as well as concurrent infections such as M. gallisepticum might have increased the pathogenic potential of LPAI H9N2 subtype.

United Arab Emirates (UAE)

Aamir et al. (Reference AAMIR, WERNERY, ILYUSHINA and WEBSTER2007) characterised seven viruses isolated from poultry in the UAE between 2000 and 2003. The quail isolates caused a decrease of up to 70% in egg production, whereas the chicken isolates resulted in rapid mortality due to tracheitis and respiratory congestion. Phylogenetic analysis indicated that all the genes, with the exception of the NS and PB2 genes, were highly similar to the genes of isolates from Iran and Pakistan which also belonged to the A/Qa/HK/G1/97-like lineage. Genetic analysis of UAE H9N2 viruses revealed that the amino acid sequences at the HA cleavage sites had an RSSR motif identical to that of G1 and Y280 viruses. Although the motif in these viruses was similar to the RX-RYK-R required for the highly pathogenic H5 and H7 subtypes, the absence of multi-basic amino acids at the cleavage sites in these isolates was associated with the low pathogenicity of these H9 viruses. However, the presence of this particular motif emphasised that these viruses had the potential to become pathogenic, should they acquire any further nucleotide substitutions in the HA-connecting peptide region.

Argentina

In Argentina the isolation of LPAI H9N2 from a wild aquatic bird (Netta peposaca) was first reported between 2007 and 2008 (Xu et al., Reference XU, FERRERI, RIMONDI, OLIVERA, ROMANO, FERREYRA, RAGOD, UHARTD, CHENA, SUTTONA, PEREDAB and PEREZ2012). The isolated H9N2 strain was assigned as A/rosy-billed pochard/ Argentina/ CIP051-559/ 2007 (H9N2). Phylogenetic analysis of the HA gene revealed that the 559/H9N2 virus had maintained an independent evolutionary pathway and shared a sister-group relationship with North American viruses, suggesting a common ancestor. The rest of the genome segments were part of the cluster from South America. Experimental inoculation of the 559/H9N2 in chickens and quail revealed efficient replication and transmission only in quail.