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Published in final edited form as: Virus Res. 2012 Jun 16;168(1-2):41–47. doi: 10.1016/j.virusres.2012.06.010

Isolation and characterization of an H9N2 influenza virus isolated in Argentina

Kemin Xu a, Lucas Ferreri b,1, Agustina Rimondi b,1, Valeria Olivera b, Marcelo Romano c,d, Hebe Ferreyra d, Virgina Rago d, Marcela Uhart d, Hongjun Chen a, Troy Sutton a, Ariel Pereda b,*, Daniel R Perez a,**
PMCID: PMC5003612  NIHMSID: NIHMS810074  PMID: 22709552

Abstract

As part of our ongoing efforts on animal influenza surveillance in Argentina, an H9N2 virus was isolated from a wild aquatic bird (Netta peposaca), A/rosy-billed pochard/Argentina/CIP051-559/2007 (H9N2) – herein referred to as 559/H9N2. Due to the important role that H9N2 viruses play in the ecology of influenza in nature, the 559/H9N2 isolate was characterized molecularly and biologically. Phylogenetic analysis of the HA gene revealed that the 559/H9N2 virus 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 clustered with viruses from South America. Experimental inoculation of the 559/H9N2 in chickens and quail revealed efficient replication and transmission only in quail. Our results add to the notion of the unique evolutionary trend of avian influenza viruses in South America. Our study increases our understanding of H9N2 viruses in nature and emphasizes the importance of expanding animal influenza surveillance efforts to better define the ecology of influenza viruses at a global scale.

Keywords: Low pathogenic avian influenza, Ecology, H9N2, transmission, South America, Argentina, Chicken, Quail, Wild bird, Rosy-billed pochard

1. Introduction

Influenza A viruses (IAVs) belong to the family Orthomyxoviridae. IAVs have in common 8 segments of genomic negative sense RNA. Wild aquatic birds are considered the natural reservoir of these viruses. Avian influenza viruses responsible for outbreaks in avian and mammalian species, including humans, are thought to evolve from wild bird strains. Two major lineages for avian influenza viruses have been defined, the Eurasian and North American lineages, respectively. Surveillance studies in Australia and South America suggest the presence of sub-lineages that evolve independently from other influenza genes in other regions. Avian influenza viruses from Australia appear to form a monophyletic lineage, whereas those from South American show independent evolution but share a common ancestor with North American viruses. Antigenic differences on the two viral surface proteins, hemagglutinin (HA) and neuraminidase (NA) have led to the classification of IAVs into subtypes; 16 HA and 9 NA subtype viruses have been described so far in wild aquatic birds (Bulach et al., 2010; Gonzalez-Reiche et al., 2012; Hansbro et al., 2010; Rimondi et al., 2011; Webster et al., 1992).

The H9N2 IAV subtype was first detected in the USA in 1966 (Homme and Easterday, 1970). In the late 1960s to early 1970s, H9N2 viruses were associated with a number of outbreaks in free-range turkeys in Minnesota, with viruses originating in wild ducks. To date, H9N2 influenza viruses are isolated sporadically from wild birds in North America (Halvorson et al., 1997; Kawaoka et al., 1988; Sharp et al., 1993, 1997). In Asia, H9N2 viruses were found in domestic ducks until the late 1980s before they crossed to domestic land-based poultry (Shortridge, 1992). Since then, H9N2s have caused disease outbreaks in poultry in a vast number of regions from South East Asia to the Middle East (Dong et al., 2011; Guo et al., 2000). China, Pakistan, Israel, Iran, and South Korea, among others, have reported H9N2 viruses in local live poultry markets where these viruses remain endemic (Fusaro et al., 2011; Lee et al., 2007; Naeem et al., 2007; Xu et al., 2007). More importantly, H9N2 viruses have been frequently transmitted to pigs, considered an intermediate host in the generation of influenza strains with pandemic potential (Peiris et al., 2001; Yu et al., 2011). Human infections with avian H9N2 viruses have also been reported and associated with mild influenza-like symptoms (Butt et al., 2005; Cheng et al., 2011; Lin et al., 2000). Serological studies suggest significant human exposure to these viruses in regions where they remain endemic (Khuntirat et al., 2011; Wang et al., 2009). Although no evidence of human-to-human transmission of H9N2 viruses has been observed, some H9N2 viruses circulating in poultry have evolved human-like receptor specificity and thus recognize sialic acids bound to the adjacent galactose in an α2,6 conformation (SAα2,6) (Matrosovich et al., 2001). Thus, the World Health Organization (WHO) considers H9N2 viruses as potentially pandemic strains (Capua and Alexander, 2002).

In contrast, no information regarding the presence of H9N2 viruses in South America is available. In this report, a H9N2 virus from an aquatic bird (559/H9N2) was isolated and characterized. Like other IAVs isolated in Argentina, the 559/H9N2 possesses a gene constellation consistent with a unique evolutionary group of IAVs from South America. Animal studies revealed limited replication with lack of transmission of 559/H9N2 in chickens, whereas in Japanese quail, efficient replication and transmission was observed. These results are consistent with previous studies showing that quail are more susceptible than chickens to H9N2 viruses isolated from ducks (Hossain et al., 2008; Makarova et al., 2003; Perez et al., 2003).

2. Materials and methods

2.1. Sample collection

Sampling activities were performed by trained biologists and veterinarians in the Lower Paraná River Valley. This valley is composed of a mosaic of rice fields, natural wetlands and marshes, native forests, and patches of land within the floodplain of the Parana River (30°41′S, 60°02′W) (Rimondi et al., 2011). Cloacal swabs were collected from carcasses of hunter-killed ducks donated by licensed hunters during the hunting seasons (April 15th to August 15th) of 2007 through 2008. Cloacal swabs were collected using single-use polyester sterile swabs and then stored separately in single plastic cryo-vials, containing 2 ml of Phosphate Buffer Solution (PBS) with 50% glycerol and Penicillin 10,000 IU/ml, Streptomycin 5 mg/ml, Gentamicin Sulfate 1 mg/ml, Kanamycin sulfate 700 µg/ml and Anphotericin B 10 µg/ml (Sigma Chemical Co, St. Louis, MO, USA). Samples were frozen in liquid nitrogen and transported on dry ice. Once in the laboratory all samples were stored at −80 °C until processed for molecular diagnosis and virus isolation.

2.2. Virus detection

Viral RNA was extracted from 140 µl of suspension from cloacal swabs using a QIAamp Viral RNA Mini Kit (Qiagen Inc., Valencia, CA, USA) in accordance with the manufacturer’s instructions. RNA was eluted in a final volume of 60 µl and stored at −80 °C. Viral cDNA was prepared using 30 µl of viral RNA and random hexamers in a final volume of 60 µl using a High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA). The cDNA was tested for avian influenza virus (AIV) by real-time reverse transcriptase PCR (RRT-PCR) using TaqMan Universal PCR Master Mix (Applied Biosystems) directed to the matrix (M) gene. This system detects all type A influenza viruses (Spackman et al., 2002). The PCR was performed on an ABI Prism 7500 SDS (Applied Biosystems).

2.3. Virus isolation

Swab samples positive by RRT-PCR were inoculated into 9–11 day old specific pathogen free (SPF) embryonated chicken eggs. Briefly, 200 µl of PBS suspension from the cloacal swabs was injected into the egg’s allantoic cavity and the eggs were incubated for 72 h and harvested in accordance with standard protocols described in the WHO Manual on Animal Influenza Diagnosis and Surveillance (Webster et al., 2005). Virus titration was performed by egg infectious dose 50 (EID50) following a previously described method (Reed and Muench, 1938) and using the hemagglutination assay as the final readout (Webster et al., 2005).

2.4. Phylogenetic and molecular analysis

Viral RNA was extracted from infected allantoic fluid using an RNEasy Mini kit (Qiagen). Reverse transcription followed by PCR was performed using specific primers for each gene segment as described previously (Hoffmann et al., 2001). PCR products were purified with a QIAQuick PCR purification kit (Qiagen). Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI PRISM 3700 DNA Analyzer (Applied Biosystems) following the manufacturer’s instructions. Phylogenetic analyses were performed using additional influenza virus sequence data available in GenBank. Sequences were assembled and edited with Lasergene 8.1 (DNASTAR); BioEdit 7 was used for alignment and residue analysis. Neighbor-joining (NJ) trees were constructed by using PAUP* 4.0. Estimates of the phylogenies were calculated by performing 1000 NJ bootstrap replicates. The TREEVIEW program, Version 1.6.6 was used for visualization and printing of phylogenetic trees.

2.5. Animal studies

2-Week old SPF White Leghorn chickens (Charles River Laboratories, Wilmington, MA) and 4-week old Japanese quail (Coturnix coturnix, B & D Game Farm) were used throughout the studies. Chickens (n = 4) or quail (n = 4) were inoculated intraocularly, intranasally, and intratracheally with 5 × 106 EID50 of virus. Transmission was monitored by bringing the directly inoculated birds in direct contact with naïve birds (n = 4 birds/group) at 1 day post-inoculation (dpi). Tracheal and cloacal swabs were collected at 1, 3, 5, and 7 dpi in 1 ml freezing media (50% glycerol in PBS containing 1% antibiotics) and stored at −80 °C until use for virus titration. Birds were observed daily for 14 days for signs of disease. Birds were monitored for appetite, activity, fecal output, and signs of distress including cyanosis of the tongue or legs, ruffled feathers and respiratory distress. Experiments were carried out under ABSL2+ conditions with investigators wearing appropriate protective equipment and compliant with protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland and adhered strictly to the Animal Welfare Act (AWA) regulations.

2.6. Nucleotide sequence accession numbers

The nucleotide sequences obtained in this study are available from GenBank under accession numbers CY111587 to CY111594.

3. Results and discussion

As part of ongoing animal influenza surveillance efforts an H9N2 IAV strain was isolated. Samples were collected between May 2007 and February 2008 at different hunting lodges in the Lower Paraná River Valley (30°41′S, 60°02′W) in Argentina. Details on species and samples have been previously described (Rimondi et al., 2011). Briefly, 1395 cloacal swabs representing 23 different bird species were collected and tested for the presence IAVs. Of these, 738 samples were obtained from rosy-billed pochards (Netta peposaca) and one of these samples resulted in the isolation of a H9N2 strain: A/rosy-billed pochard/Argentina/CIP051-559/2007 (H9N2) (559/H9N2). Given the ecological and epidemiological importance of H9N2 viruses elsewhere, the 559/H9N2 virus was subjected to molecular and biological characterization.

3.1. Genetic and phylogenetic analysis

Sequences corresponding to the entire ORF of the 8 gene segments of 559/H9N2 were compared to other H9N2 virus sequences deposited in GenBank. Blast search results suggested that the internal gene segments of 559/H9N2 were closely related to avian influenza viruses from South America (Table 1), with sequence homologies ranging from 95% (PB2 and PB1 gene segments) to 99% (M gene segment). With respect to the HA gene, the closest homology identified was 91% with A/Quail/Arkansas/29209-1/93 (H9N2), reflecting the lack of H9N2 reference viruses from South America in the database. The NA gene had 98% nucleotide identity with A/rosy-billed pochard/Argentina/CIP051-557/2007 (H6N2), which has been recently characterized (Rimondi et al., 2011).

Table 1.

Type A influenza virus isolates with the highest nucleotide identity to A/rosy-billed pochard/Argentina/CIP051-559/2007 (H9N2) by gene segment.

Gene
segment		 Highest sequence identity with % nt identity
PB2 A/chicken/Chile/176822/2002 (H7N3) 95%
PB1 A/cinnamon teal/Bolivia/4537/2001 (H7N3) 95%
PA A/rosy-billed pochard/Argentina/CIP051-557/2007
(H6N2)		 97%
HA A/quail/Arkansas/29209-1/1993 (H9N2) 91%
NP A/rosy-billed pochard/Argentina/CIP051-575/2007
(H6N8)		 97%
NA A/rosy-billed pochard/Argentina/CIP051-557/2007
(H6N2)		 98%
M A/rosy-billed pochard/Argentina/CIP051-557/2007
(H6N2)		 99%
NS A/rosy-billed pochard/Argentina/CIP051-925/2008
(H6N2)		 96%
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Phylogenetic analysis revealed that the 559/H9N2 virus genes clustered with avian influenza viruses from South America (Fig. 1). Four clades (H9.1, H9.2, H9.3, H9.4) can be identified for the H9 HA gene based on the nomenclature proposed recently (Liu et al., 2009). Clade H9.1 includes the old H9N2 viruses from North America and one unusual H9N2 virus from China, A/Chicken/HLJ/35/2000. Clade H9.3 contains viruses from both Eurasia and North America while clade H9.4 contains H9N2 viruses that have become established in poultry in Asia. The HA gene of 559/H9N2 virus belongs to H9.2 clade, together with 3 H9N2 viruses from North America, suggesting a recent common ancestor, although the Argentine H9N2 virus maintains an out-group relationship with the other viruses, indicating it has diverged and may have formed a unique sub-clade during evolution. If more H9 subtype viruses are isolated from South America, this trend is likely to become more evident.

Fig. 1.

Fig. 1

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Phylogenetic trees of HA and NA gene segments. Trees were generated by the neighbor-joining method in the PAUP* program. Numbers above branches indicate neighbor-joining bootstrap values. Not all supports are shown because of space constraints. Analysis was based on nucleotides: H9, 129–1042; and N2, 249–1303. The H9 and N2 trees were rooted to A/duck/Alberta/60/1976 (H12N5) and A/equine/Prague/1/56 (H7N7), respectively. The 559/H9N2 strain is highlighted in red. Scale bar at the bottom of each tree indicates substitutions per site.

Phylogenetic analysis of the NA gene showed that 559/H9N2 is closely related to the Argentine H6N2 viruses characterized recently (Fig. 1). Likewise, phylogenetic analysis of the internal gene segments (PB2, PB1, PA, NP, M and NS) revealed that 559/H9N2 belongs to the South American clade (Figs. 2 and 3) (Alvarez et al., 2010; Pereda et al., 2008; Rimondi et al., 2011). It is interesting to note that 559/H9N2, along with viruses from South America, shows a sister-group relationship with equine viruses in the PA and NP phylogenetic trees, suggesting a recent common ancestor. The NS gene of 559/H9N2 belongs to Allele A in the South American clade.

Fig. 2.

Fig. 2

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Phylogenetic trees of PB2, PB1, and PA gene segments, performed as described in materials and methods and in legend of Fig. 1. Analysis was based on nucleotides: PB2, 1079–2138; PB1, 42–1217; and PA, 1429–2127. Phylogenetic trees were rooted to A/equine/Prague/1/56 (H7N7) for the PB2 gene and A/Brevig Mission/1/1918 (H1N1) for the PB1 and PA genes. Scale bar at the bottom of each tree indicates substitutions per site.

Fig. 3.

Fig. 3

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Phylogenetic trees of NP, M and NS gene segments, performed as described in materials and methods and in legend of Fig. 1. Analysis was based on nucleotides: NP, 31–917; M, 49–864; and NS, 88–815. The trees were rooted to A/equine/Prague/1/56 (H7N7). Scale bar at the bottom of each tree indicates substitutions per site.

3.2. Molecular analysis

Within the receptor binding site, the HA protein of 559/H9N2 contains amino acid residues consistent with binding to avian-like SAα2,3 receptors (Q226 and G228). The HA also contains the typical low pathogenic A-S-D-R/G motif at the cleavage site. No mutations associated with drug resistance were found in the M2 and NA proteins and no mutations associated with increased virulence in mammals were found in PB2, PB1, and NS proteins (data not shown).

3.3. Replication and transmission of 559/H9N2 in chickens and quail

H9N2 viruses have established stable lineages in different types of poultry in Asia and the Middle East (Dong et al., 2011; Lee et al., 2007; Naeem et al., 2007; Xu et al., 2007). To determine the potential risk of 559/H9N2 virus to local poultry, the replication and transmission of this virus in chickens and quail was evaluated. In chickens, 559/H9N2 showed limited replication in inoculated chickens (2 out of 4). Only one chicken shed significant amounts of virus from the respiratory tract up to 3 dpi with titers of 4.5 log10EID50/ml and 2.5 log10EID50/ml at 1 and 3 dpi, respectively (Table 2). No virus shedding was detected by 5 dpi (or thereafter) in chickens. No evidence of virus shedding in cloaca and no evidence of transmission was observed in contact chickens either by virus isolation or serology (not shown). Inoculated chickens showed no signs of disease during the course of the experiment.

Table 2.

Replication and transmission of A/rosy-billed pochard/Argentina/CIP051-559/2007 in chickens and quail.

Species Group 1 dpi 3 dpi 5 dpi 7 dpi
Trachea Cloaca Trachea Cloaca Trachea Cloaca Trachea Cloaca
Chicken Inoculated 2/4 (4.5)a 0/4 1/4 (2.5) 0/4 0/4 0/4 0/4 0/4
Contact 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4
Quail Inoculated 4/4 (3.8) 0/4 2/4 (2.5) 1/4 (4.5) 0/4 0/4 0/4 1/4 (0.7)
Contact 0/4 0/4 4/4 (3.8) 0/4 4/4 (2.8) 0/4 3/4 (2.5) 0/4
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a

Number of birds that shed virus vs. number of birds in group. Peak virus titer shown in parentheses (log10EID50/ml).

In quail, 559/H9N2 virus was detected from the respiratory tract in 4 out 4 inoculated quail at 1 dpi, which represents also the time when peak virus titers were observed (4.5 log10EID50/ml, Table 2). By 3 dpi, 2 out 4 inoculated quail were positive for virus in the trachea and 1 quail was shedding virus in the cloaca. The cloaca positive bird was again weakly positive by 7 dpi. More importantly, direct contact quail became positive for virus in the trachea (4/4) by 3 dpi with peak virus titers of 3.8 log10EID50/ml and 3/4 remained positive by 7 dpi. Direct contact quail remained negative for virus isolation in the cloaca for the duration of the study. Neither directly inoculated quail nor direct contact quail showed clinical signs of disease, which is consistent with previous studies (Hossain et al., 2008; Makarova et al., 2003; Perez et al., 2003). Although further studies would be needed to determine whether transmission occurred by either respiratory droplets or fomites or drinking water or a combination of those routes, our studies are also consistent with a previous study highlighting the relative higher susceptibility of Japanese quail to H9N2 viruses compared to chickens (Perez et al., 2003). Interestingly, the 559/H9N2, which has not been adapted to domestic poultry, shows a pattern of transmission equal or better than H9N2 strains isolated from ducks in the 1970s in Asia (Perez et al., 2003). It was also interesting to observe that direct contact quail had a tendency to show higher levels of virus shedding than the directly inoculated quail; however, the limited number of animals in these studies precludes any major conclusions.

From 2006, a long-term systematic influenza surveillance network was established in Argentina between the National Institute of Agricultural Technology (INTA), the Argentine National Animal Health Service (SENASA), the Wildlife Conservation Society (WCS), and the University of Maryland, College Park. This surveillance network has helped us define the presence of a unique clade in the natural gene pool of IAVs in South America (Alvarez et al., 2010; Pereda et al., 2008; Rimondi et al., 2011). This present study further supports this notion. The Argentine H9N2 virus belongs to clade H9.2, sharing a sister-group relationship with North American H9N2 viruses. Unlike the Asian H9N2 viruses which appear to have gone through a rather lengthy process of adaptation from wild birds to domestic ducks and from there to domestic land-based birds (Perez et al., 2003; Shortridge, 1992), the 559/H9N2 virus appears very effective in terms of replication and transmission in Japanese quail. In this regard, the 559/H9N2 resembles its North American counterparts that have been associated with disease outbreaks in domestic turkeys after exposure to H9N2 viruses present in wild ducks. Due to improvements in biosecurity, H9N2 viruses have not become established in domestic poultry in the Americas. Our study suggests that these viruses have the makings of viruses that could easily jump to other bird species and thus, highlights the potential threat posed to local poultry. In summary, our study increases our understanding of H9N2 viruses in nature and emphasizes the importance of expanding animal influenza surveillance efforts to better define the ecology of influenza viruses at a global scale.

Acknowledgments

The authors would like to thank Andrea Ferrero, Qiong Chen, and Johanna Lavigne for their technical support. This work was partially supported by the USDA and by the NIAID, Center for Research on Influenza Pathogenesis (CRIP) through University of Maryland College Park contract No. HHSN266200700010C. This work was also supported by P.E. INTA Exoticas y Emergentes and by the European Community (Proyecto Integrado Cadena Carne Aviar - BiotecSur) and partially supported by the Global Avian Influenza Network for Surveillance (GAINS) program, funded in part by USAID Grant No. LAG-A-00-99-00047-00. The opinions expressed herein are those of the author(s) and do not necessarily reflect the views of the US Agency for International Development.

References

  1. Alvarez P, Mattiello R, Rivailler P, Pereda A, Davis CT, Boado L, et al. First isolation of an H1N1 avian influenza virus from wild terrestrial non-migratory birds in Argentina. Virology. 2010;396(1):76–84. doi: 10.1016/j.virol.2009.10.009. [DOI] [PubMed] [Google Scholar]
  2. Bulach D, Halpin R, Spiro D, Pomeroy L, Janies D, Boyle DB. Molecular analysis of H7 avian influenza viruses from Australia and New Zealand: genetic diversity and relationships from 1976 to 2007. J Virol. 2010;84(19):9957–9966. doi: 10.1128/JVI.00930-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Butt KM, Smith GJ, Chen H, Zhang LJ, Leung YH, Xu KM, et al. Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003. J Clin Microbiol. 2005;43(11):5760–5767. doi: 10.1128/JCM.43.11.5760-5767.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Capua I, Alexander DJ. Avian influenza and human health. Acta Trop. 2002;83(1):1–6. doi: 10.1016/s0001-706x(02)00050-5. [DOI] [PubMed] [Google Scholar]
  5. Cheng VC, Chan JF, Wen X, Wu WL, Que TL, Chen H, et al. Infection of immunocompromised patients by avian H9N2 influenza A virus. J Infect. 2011;62(5):394–399. doi: 10.1016/j.jinf.2011.02.007. [DOI] [PubMed] [Google Scholar]
  6. Dong G, Luo J, Zhang H, Wang C, Duan M, Deliberto TJ, et al. Phylogenetic diversity and genotypical complexity of H9N2 influenza A viruses revealed by genomic sequence analysis. PLoS One. 2011;6(2):e17212. doi: 10.1371/journal.pone.0017212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fusaro A, Monne I, Salviato A, Valastro V, Schivo A, Amarin NM, et al. Phylogeography and evolutionary history of reassortant H9N2 viruses with potential human health implications. J Virol. 2011;85(16):8413–8421. doi: 10.1128/JVI.00219-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gonzalez-Reiche AS, Morales-Betoulle ME, Alvarez D, Betoulle JL, Muller ML, Sosa SM, et al. Influenza a viruses from wild birds in guatemala belong to the north american lineage. PLoS One. 2012;7(3):e32873. doi: 10.1371/journal.pone.0032873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guo YJ, Krauss S, Senne DA, Mo IP, Lo KS, Xiong XP, et al. Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia. Virology. 2000;267(2):279–288. doi: 10.1006/viro.1999.0115. [DOI] [PubMed] [Google Scholar]
  10. Halvorson DA, Frame DD, Friendshuh AJ, Shaw DP. Outbreaks of low pathogenecity avian influenza in USA. In: Swayne D, Slemons R, editors. Proceedings of the Fourth International Symposium on Avian Influenza. Tallahassee, FL: United States Animal Health Association, Rose Printing Company; 1997. pp. 36–46. [Google Scholar]
  11. Hansbro PM, Warner S, Tracey JP, Arzey KE, Selleck P, O’Riley K, et al. Surveillance and analysis of avian influenza viruses, Australia. Emerg Infect Dis. 2010;16(12):1896–1904. doi: 10.3201/eid1612.100776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol. 2001;146(12):2275–2289. doi: 10.1007/s007050170002. [DOI] [PubMed] [Google Scholar]
  13. Homme PJ, Easterday BC. Avian influenza virus infections I. Characteristics of influenza A-Turkey-Wisconsin-1966 virus. Avian Dis. 1970;14(1):66–74. [PubMed] [Google Scholar]
  14. Hossain MJ, Hickman D, Perez DR. Evidence of expanded host range and mammalian-associated genetic changes in a duck H9N2 influenza virus following adaptation in quail and chickens. PLoS One. 2008;3(9):e3170. doi: 10.1371/journal.pone.0003170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kawaoka Y, Chambers TM, Sladen WL, Webster RG. Is the gene pool of influenza viruses in shorebirds and gulls different from that in wild ducks? Virology. 1988;163(1):247–250. doi: 10.1016/0042-6822(88)90260-7. [DOI] [PubMed] [Google Scholar]
  16. Khuntirat BP, Yoon IK, Blair PJ, Krueger WS, Chittaganpitch M, Putnam SD, et al. Evidence for subclinical avian influenza virus infections among rural Thai villagers. Clin Infect Dis. 2011;53(8):e107–e116. doi: 10.1093/cid/cir525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lee YJ, Shin JY, Song MS, Lee YM, Choi JG, Lee EK, et al. Continuing evolution of H9 influenza viruses in Korean poultry. Virology. 2007;359(2):313–323. doi: 10.1016/j.virol.2006.09.025. [DOI] [PubMed] [Google Scholar]
  18. Lin YP, Shaw M, Gregory V, Cameron K, Lim W, Klimov A, et al. Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates. Proc Natl Acad Sci USA. 2000;97(17):9654–9658. doi: 10.1073/pnas.160270697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Liu S, Ji K, Chen J, Tai D, Jiang W, Hou G, et al. Panorama phylogenetic diversity and distribution of type A influenza virus. PLoS One. 2009;4(3):e5022. doi: 10.1371/journal.pone.0005022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Makarova NV, Ozaki H, Kida H, Webster RG, Perez DR. Replication and transmission of influenza viruses in Japanese quail. Virology. 2003;310(1):8–15. doi: 10.1016/s0042-6822(03)00094-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Matrosovich MN, Krauss S, Webster RG. H9N2 influenza A viruses from poultry in Asia have human virus-like receptor specificity. Virology. 2001;281(2):156–162. doi: 10.1006/viro.2000.0799. [DOI] [PubMed] [Google Scholar]
  22. Naeem K, Siddique N, Ayaz M, Jalalee MA. Avian influenza in Pakistan: outbreaks of low- and high-pathogenicity avian influenza in Pakistan during 2003–2006. Avian Dis. 2007;51(1 Suppl):189–193. doi: 10.1637/7617-042506R.1. [DOI] [PubMed] [Google Scholar]
  23. Peiris JS, Guan Y, Markwell D, Ghose P, Webster RG, Shortridge KF. Cocirculation of avian H9N2 and contemporary human H3N2 influenza A viruses in pigs in southeastern China: potential for genetic reassortment? J Virol. 2001;75(20):9679–9686. doi: 10.1128/JVI.75.20.9679-9686.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pereda AJ, Uhart M, Perez AA, Zaccagnini ME, La Sala L, Decarre J, et al. Avian influenza virus isolated in wild waterfowl in Argentina: evidence of a potentially unique phylogenetic lineage in South America. Virology. 2008;378(2):363–370. doi: 10.1016/j.virol.2008.06.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Perez DR, Lim W, Seiler JP, Yi G, Peiris M, Shortridge KF, et al. 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. 2003;77(5):3148–3156. doi: 10.1128/JVI.77.5.3148-3156.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Reed LJ, Muench H. A simple method for estimating fifty percent endpoints. Am J Hyg. 1938;27:493–497. [Google Scholar]
  27. Rimondi A, Xu K, Craig MI, Shao H, Ferreyra H, Rago MV, et al. Phylogenetic analysis of H6 influenza viruses isolated from rosy-billed pochards (Netta peposaca) in Argentina reveals the presence of different HA gene clusters. J Virol. 2011;85(24):13354–13362. doi: 10.1128/JVI.05946-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sharp GB, Kawaoka Y, Jones DJ, Bean WJ, Pryor SP, Hinshaw V, et al. Coinfection of wild ducks by influenza A viruses: distribution patterns and biological significance. J Virol. 1997;71(8):6128–6135. doi: 10.1128/jvi.71.8.6128-6135.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sharp GB, Kawaoka Y, Wright SM, Turner B, Hinshaw V, Webster RG. Wild ducks are the reservoir for only a limited number of influenza A subtypes. Epidemiol Infect. 1993;110(1):161–176. doi: 10.1017/s0950268800050780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shortridge KF. Pandemic influenza: a zoonosis? Semin Respir Infect. 1992;7(1):11–25. [PubMed] [Google Scholar]
  31. Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, et al. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol. 2002;40(9):3256–3260. doi: 10.1128/JCM.40.9.3256-3260.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang M, Fu CX, Zheng BJ. Antibodies against H5 and H9 avian influenza among poultry workers in China. N Engl J Med. 2009;360(24):2583–2584. doi: 10.1056/NEJMc0900358. [DOI] [PubMed] [Google Scholar]
  33. Webster R, Cox N, Stohr K. WHO manual on animal influenza diagnosis and surveillance. World Health Organization Department of Communicable Disease Surveillance and Response. 2005
  34. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiol Rev. 1992;56(1):152–179. doi: 10.1128/mr.56.1.152-179.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Xu KM, Smith GJ, Bahl J, Duan L, Tai H, Vijaykrishna D, et al. The genesis and evolution of H9N2 influenza viruses in poultry from southern China, 2000 to 2005. J Virol. 2007;81(19):10389–10401. doi: 10.1128/JVI.00979-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yu H, Zhou YJ, Li GX, Ma JH, Yan LP, Wang B, et al. Genetic diversity of H9N2 influenza viruses from pigs in China: a potential threat to human health? Vet Microbiol. 2011;149(1–2):254–261. doi: 10.1016/j.vetmic.2010.11.008. [DOI] [PubMed] [Google Scholar]

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