Infectivity and Transmissibility of Avian H9N2 Influenza Virusesin Pigs

Jia Wang,a,b Maocai Wu,a Wenshan Hong,a Xiaohui Fan,c Rirong Chen,a Zuoyi Zheng,a Yu Zeng,a Ren Huang,d Yu Zhang,d

Tommy Tsan-Yuk Lam,b David K. Smith,b Huachen Zhu,a,b Yi Guana,b,c

Joint Influenza Research Center, Shantou University Medical College, Shantou, Chinaa; State Key Laboratory of Emerging Infectious Diseases and Centre of InfluenzaResearch, School of Public Health, The University of Hong Kong, Hong Kong, Chinab; Department of Microbiology, Guangxi Medical University, Nanning, Chinac;Guangdong Laboratory Animals Monitoring Institute, Guangzhou, Chinad

ABSTRACT

The H9N2 influenza viruses that are enzootic in terrestrial poultry in China pose a persistent pandemic threat to humans. Toinvestigate whether the continuous circulation and adaptation of these viruses in terrestrial poultry increased their infectivity topigs, we conducted a serological survey in pig herds with H9N2 viruses selected from the aquatic avian gene pool (Y439 lineage)and the enzootic terrestrial poultry viruses (G1 and Y280 lineages). We also compared the infectivity and transmissibility ofthese viruses in pigs. It was found that more than 15% of the pigs sampled from 2010 to 2012 in southern China were seroposi-tive to either G1 or Y280 lineage viruses, but none of the sera were positive to the H9 viruses from the Y439 lineage. Viruses ofthe G1 and Y280 lineages were able to infect experimental pigs, with detectable nasal shedding of the viruses and seroconversion,whereas viruses of the Y439 lineage did not cause a productive infection in pigs. Thus, adaptation and prevalence in terrestrialpoultry could lead to interspecies transmission of H9N2 viruses from birds to pigs. Although H9N2 viruses do not appear to becontinuously transmissible among pigs, repeated introductions of H9 viruses to pigs naturally increase the risk of generatingmammalian-adapted or reassorted variants that are potentially infectious to humans. This study highlights the importance ofmonitoring the activity of H9N2 viruses in terrestrial poultry and pigs.

IMPORTANCE

H9N2 subtype of influenza viruses has repeatedly been introduced into mammalian hosts, including humans and pigs, so aware-ness of their activity and evolution is important for influenza pandemic preparedness. However, since H9N2 viruses usuallycause mild or even asymptomatic infections in mammalian hosts, they may be overlooked in influenza surveillance. Here, wefound that the H9N2 viruses established in terrestrial poultry had higher infectivity in pigs than those from aquatic birds, whichsuggests that adaptation of the H9N2 viruses in terrestrial poultry might have increased the infectivity of the virus to mammals.Therefore, monitoring the prevalence and evolution of H9 viruses prevalent in terrestrial birds and conducting risk assessmentof their threat to mammals are critical for evaluating the pandemic potential of this virus.

Transmission of avian influenza viruses to mammals is regardedas a potential pandemic threat to humans. To date, several

subtypes (H5, H6, H7, H9, and H10) of avian influenza viruseshave occasionally been introduced to humans and swine (1–17).These interspecies transmissions mostly reflect the activity orprevalence of the viruses in birds in the field.

H9N2 influenza viruses have been enzootic in terrestrial poul-try in many Asian countries since the mid-1990s and have formedthree established lineages: the G1-like viruses that are enzootic inSoutheast and South Asia and the Middle East, the Y280-like (orCk/Bei-like) viruses mainly prevalent in China, and a subgroup ofY439-like viruses that circulate in Korea (18–24). Except for theKorean subclade, most of the terrestrial poultry H9N2 viruses arepart of the G1 or Y280 lineages, whereas Asian aquatic bird H9N2viruses are mainly from the Y439-like viruses (18–22, 24). Thebroad prevalence of H9N2 influenza viruses in poultry naturallyincreases their contact with, and risk of transmission, to mam-mals, especially humans and swine.

Sporadic human cases of H9N2 influenza infection were firstidentified in Guangdong in 1999 (2) and then in Hong Kong andother parts of China in the last two decades (6–8, 16). Althoughonly a small number of H9N2 viruses have been isolated fromhumans thus far, retrospective serosurveys revealed positive rates

for H9N2 antibodies of 1.3 to 1.4% in the general population andmore than 15% in retail poultry workers (25–27), indicating thatthe introduction of H9N2 viruses to humans is not rare.

Infection of pigs with H9N2 influenza viruses has been ob-served since the late 1990s (9), and disease outbreaks were re-ported in several provinces in Mainland China in the 2000s (10,11, 14). Viruses isolated from diseased pigs were genetically closelyrelated to local enzootic poultry H9N2 viruses (9–15), suggestingthat poultry were the etiological source. Since pigs may facilitatethe introduction of avian viruses or viral genes to humans, trans-mission of avian H9N2 viruses to pigs raises concerns over thepossible generation of human pandemic influenza strains (9).

Received 11 October 2015 Accepted 10 January 2016

Accepted manuscript posted online 13 January 2016

Citation Wang J, Wu M, Hong W, Fan X, Chen R, Zheng Z, Zeng Y, Huang R, ZhangY, Lam TT-Y, Smith DK, Zhu H, Guan Y. 2016. Infectivity and transmissibility of avianH9N2 influenza viruses in pigs. J Virol 90:3506 –3514. doi:10.1128/JVI.02605-15.

Editor: S. Schultz-Cherry

Address correspondence to Huachen Zhu, zhuhch@hku.hk, orYi Guan, yguan@hku.hk.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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This has been heightened since the emergence of the swine-origin2009 H1N1 pandemic influenza virus (28, 29) and the subsequentrapid expansion of the genetic diversity of swine influenza viruses(30, 31).

One of the lessons learned from the 2009 pandemic is that apandemic strain could arise independently in pigs (28, 29), soignoring influenza virus activity in pigs may have serious conse-quences. Since limited investigation on the prevalence of H9N2viruses in pig herds has been conducted, the infectivity and trans-missibility of the enzootic poultry H9N2 viruses in pigs are stillunknown. We conducted here a large-scale serosurvey of healthypigs to investigate their levels of infection with avian H9N2 vi-ruses, and we also examined the infectivity and transmissibility ofthe different lineages of avian H9N2 viruses in a pig model. Thisinformation will help in assessing the risk of H9N2 viruses tomammalian hosts and assist in pandemic preparedness.

MATERIALS AND METHODSViruses. H9 viruses of the most prevalent genotype of each of the Y439,G1, and Y280 lineages (Table 1) (22, 32) were selected and passaged twicein 10-day-old embryonated chicken eggs. Swine/Guangdong/1361/2010

(SW1361, H1N1) which transmitted efficiently among pigs (33) was usedas a positive control in the transmission study.

Serological survey of H9 infection in domestic pigs. 2,500 swine se-rum samples, collected at abattoirs in the Guangxi Zhuang AutonomousRegion (n � 1,250, from March 2010 to July 2012) and Guangdong Prov-ince (n � 1,250, from January 2010 to July 2012) of China, were used forhemagglutination inhibition (HI) assays, and subsequent virus neutral-ization (VN) assays of HI-positive samples, according to standard proto-cols provided by the World Health Organization (http://www.who.int/csr/resources/publications/influenza/en/whocdscsrncs20025rev.pdf) using0.55% turkey red blood cells for the tests. All sera were treated withreceptor-destroying enzyme (Denka Seiken Co., Ltd.) and adsorbed byfresh turkey red blood cells to remove nonspecific inhibitors andhemagglutination factors, respectively. Eight H9 virus strains were used totest for the presence of H9 antibodies in the serum samples (Table 1).

Animals and animal experiments. This study was approved by theCommittee on the Use of Live Animals in Teaching and Research of TheUniversity of Hong Kong (reference number CULATR 2626-12) and the An-imal Ethics Committee of Shantou University Medical College (referencenumber SUMC 2012-127). Animal experiments were conducted in bio-safety level 2� containment facilities in strict compliance with the guide-lines of both institutes for the care and use of laboratory animals.

A total of 54 piglets, aged 6 to 8 weeks, were obtained through a labo-

TABLE 1 Influenza virus strains used in this study

Virus Subtype Lineage Abbreviation Expta

A/duck/Shantou/2030/2000 H9N1 Y439 ST2030 S, IA/duck/Jiangxi/7554/2007 H9N2 Y439 JX7554 S, IA/quail/Hong Kong/G1/1997 H9N2 G1 HKG1 SA/chicken/Hong Kong/NT449/2007 H9N2 G1 HK449 S, I, TA/Hong Kong/33982/2009 H9N2 G1 HK33982 SA/chicken/Hong Kong/NT10/2011 H9N2 G1 HK10 IA/duck/Hong Kong/Y280/1997 H9N2 Y280 HKY280 SA/chicken/Hong Kong/SSP177W/2009 H9N2 Y280-1 HK177W S, I, TA/chicken/Hong Kong/YU341/2008 H9N2 Y280-2 HK341 S, IA/swine/Guangdong/1361/2010 H1N1 Swine SW1361 Ta S, serological test; I, test for infectivity; T, test for transmissibility.

TABLE 2 Seroprevalence of anti-H9 influenza virus antibodies in pig herdsa

Lineage Virus

No. of positives (HI/VN)

Guangxi Guangdong

Subtotal(n � 2,500)

2010(n � 500)

2011(n � 500)

2012(n � 250)

2010(n � 500)

2011(n � 500)

2012(n � 250)

Y439 ST2030 0/0 0/0 0/0 0/0 0/0 0/0 0/0JX7554 0/0 0/0 0/0 0/0 0/0 0/0 0/0Any Y439* 0/0 0/0 0/0 0/0 0/0 0/0 0/0

G1 HKG1 0/0 1/0 1/0 3/2 2/1 0/0 7/3HK449 1/0 0/0 1/0 1/1 1/1 0/0 4/2HK33982 3/0 0/0 1/0 1/0 2/1 0/0 7/1Any G1* 4/0 1/0 1/0 4/3 4/2 0/0 14/5

Y280 HKY280 26/1 45/2 17/0 51/4 51/8 34/1 224/16HK177W 39/1 29/3 22/1 35/2 32/4 16/1 173/12HK341 33/1 39/8 17/0 39/2 59/11 29/2 216/24Any Y280* 67/3 72/8 36/1 78/5 85/16 43/2 379/35

Total (any H9)* 68/3 72/8 37/1 82/8 87/18 43/2 389/40a See Table 1 for the virus names. HI, number of sera positive (HI titer �1:40) in the HI assay; VN, number of sera positive (VN titer �1:16) in the VN assay. *, Samples thatreacted with more than one strain of a lineage were counted only once in the lineage total.

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ratory pig-breeding program at the Guangdong Laboratory AnimalsMonitoring Institute. They were confirmed to be free of influenza virusesby inoculation of nasal swabs in Madin-Darby canine kidney cells and tobe seronegative for influenza antibodies by HI assays. Baselines of body

weight and temperature were established over 3 days prior to the com-mencement of experiments.

To investigate the infectivity of avian H9 influenza viruses in pigs, 26piglets were allocated, via a random number generator, into six groups

FIG 1 Prevalence of H9 influenza virus antibody in pigs. Pig sera were collected from Guangxi (n � 1,250, from March 2010 to July 2012) and Guangdong (n �1,250, from January 2010 to July 2012). H9 antibodies were detected with a panel of H9 viruses (Table 1) in the hemagglutination inhibition (HI) assay. HI titersof 1:40 or above against any of the H9 viruses tested were considered positive.

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FIG 2 Virus shed by pigs inoculated with avian H9 influenza viruses. Nasal swabs were collected from pigs inoculated with H9 influenza viruses at 1 to 14 dpiand titrated by plaque-forming assays. Dotted lines indicate a detection limit of 1.1 log PFU/ml (or 12.5 PFU/ml). No virus was detected after 8 dpi.

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(n � 4), each inoculated with one virus, and one mock-infected group(n � 2). The viruses used in this study are summarized in Table 1. Each pigwas intranasally inoculated with one H9 virus strain at a dose of 107 PFUin 2 ml of phosphate-buffered saline (PBS) or with 2 ml of PBS. All pigswere allowed to have free access to feeding materials and water. Bodyweight, body temperature and disease signs were recorded daily duringthe 14-day course of the experiment. Nasal and rectal swabs were collectedfrom the pigs at 1 to 14 days postinoculation (dpi) and titrated by plaque-forming assays. Sera were collected at 14 dpi for the determination ofseroconversion using HI and VN tests.

To study the transmissibility of H9N2 viruses, 28 pigs were randomlyallocated into three groups (n � 8 each) for the HK449 (G1 lineage),HK177W (Y280 lineage), and SW1361 (positive control) viruses and onegroup (n � 4) for a mock infection. In each of the virus-inoculatedgroups, sets of four pigs were held separately and inoculated with107 PFUof virus in 2 ml of PBS. At 1 dpi, these inoculated pigs were transferred toa room holding two pigs (designated “physical-contact” pigs) and twoairborne-virus-exposed pigs. In the mock control group, two pigs wereinoculated with 2 ml of PBS; one was used as a physical-contact animaland one as an airborne-virus-exposed animal.

Airborne-virus-exposed animals were separated from inoculated andphysically exposed (contact) pigs by two stainless steel mesh partitionsseparated by 10 cm (33). Body weights, temperatures, and clinical signs ofinfection were recorded daily. Nasal and rectal swabs were collected dailyand titrated as described above. Sera were collected at 14 dpi for HI andVN assays. At 4 and 6 dpi, two pigs from each inoculated group weresacrificed. Turbinate, trachea, lobar bronchus, segmental bronchus, andvarious lung tissues were collected for the pathology study.

Pathological examinations. Tissues were immediately fixed in 4%(vol/vol) formaldehyde in PBS at room temperature for 24 h, embeddedin paraffin, and serial sections (3 �m) were prepared for hematoxylin andeosin staining and immunohistochemical (IHC) staining as previouslydescribed (34). The primary antibody used in the IHC staining was amouse monoclonal antibody against influenza virus nucleoprotein (NP),which was kindly provided by N. S. Xia, Xiamen University, at a dilutionof 1:2,500, and the secondary antibody was a goat anti-mouse IgG-specificbiotin conjugate (Calbiochem) at a 1:50 dilution.

RESULTSPrevalence of avian H9 influenza viruses in domestic pig herds.Pig serum samples were tested against a panel of H9 viruses se-lected from the antigenically distinct lineages (Table 1). A total of389 samples (15.6%) reacted against at least one of the tested H9viruses with an HI titer of �1:40 (Table 2). H9 antibodies weredetected in samples collected from each surveillance area and inalmost all survey months, except for January 2010 in Guangdongand January 2012 in Guangxi (Fig. 1). Positive rates of �20% werefrequent, and occasionally rates of �30% were observed (Fig. 1).Regional variation was not significant (Pearson’s �2, P � 0.05),with overall HI-positive rates of 14.2% for Guangxi and 17.0% forGuangdong (Table 2).

Most of the positive samples (n � 379, 97.4%) reacted againstone or more of the HKY280, HK177W, or HK341 viruses from theY280 lineage. In contrast, only 14 (3.6%) of the positive samplesreacted against viruses of the G1 lineage (HKG1, HK33982, orHK449), and no sera were positive against the ST2030 or JX7554viruses from the Y439 lineage (Table 2). Thus, the introduction ofH9 viruses into pigs varies by lineage, which is consistent withtheir prevalence in the field (21, 23).

To confirm the presence of H9 neutralizing antibodies, the 389samples with an HI titer of �1:40 against any of the H9 viruseswere subjected to a VN test. Only 40 of these 389 samples con-tained H9 neutralizing antibodies with a titer of �1:16. Samples

with higher HI titers did not necessarily have higher or detectablelevels of neutralizing antibodies, which could reflect the presenceof cross-reactions with other antibodies in the HI tests. The find-ing of anti-H9 neutralizing antibodies in pigs suggests that naturalinfections with avian H9 influenza viruses can induce the produc-tion of protective antibodies, but at much lower levels than HIantibodies.

Infectivity of H9 viruses in pigs. Six viruses (two from eachlineage) were used to inoculate groups of four pigs. At the inocu-lation dose of 107 PFU, none of the viruses caused a loss of bodyweight or other signs of disease in the animals.

Shedding of virus was detected from the nasal swabs of pigsinoculated with either the G1 or Y280, but not the Y439, lineagesof viruses (Fig. 2), nor from the rectal swabs of any pig. Virusshedding started as early as 1 to 2 dpi, lasted for 5 to 6 days, andmostly peaked at 3 to 5 dpi. An HK10 (G1 lineage)-inoculated pigshowed the highest peak titer of 105.2 PFU/ml and shed virusat �104 PFU/ml for 5 days.

At 14 dpi, sera were collected and tested for specific antibodiesby HI and VN assays. Although pigs inoculated with the Y439lineage of viruses did not shed virus, three of the four ST2030-inoculated pigs seroconverted (two with HI titers of 40 and 160and three with VN titers of 16 and 32; Table 3). In the G1 lineage-inoculated pigs, all of the HK449-inoculated pigs and two of theHK10-inoculated pigs seroconverted, with HI titers ranging from

TABLE 3 Antibody titers of pigs inoculated with H9 influenza viruses at14 dpia

Lineage Virus Pig

Antibody titerb

HI VN

Y439 ST2030 1 �10 82 �10 163 160 324 40 32

JX7554 1 �10 �82 �10 �83 10 84 �10 �8

G1 HK449 1 80 �82 80 83 20 324 40 32

HK10 1 �10 82 �10 �83 80 324 160 256

Y280 HK177W 1 160 82 1280 1283 �1,280 644 �1,280 64

HK341 1 320 322 1,280 323 320 5124 1,280 512

a Pigs were screened 3 days before inoculation and confirmed to have HI titers � 1:10to H1, H3, and H9 influenza viruses. At 14 dpi, sera were tested by HI and VN assays.An HI titer �1:40 and a VN titer �1:16 were considered seroconversion. See Table 1for the virus names.b That is, against the homologous virus.

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40 to 160 and VN titers ranging from 32 to 256 (Table 3). All pigsinoculated with viruses from the Y280 lineage (HK177W andHK341) seroconverted with HI titers of �160, although theHK1777W-inoculated pig with the lowest HI titer of 160 had a VNtiter of only 8 (Table 3). Y280 lineage viruses generally inducedmuch higher antibody levels than did the G1 or Y439 lineage vi-ruses.

Transmissibility of H9N2 viruses in pigs. HK449 (G1 lineage)and HK177W (Y280 lineage) viruses were inoculated into pigs,with the swine H1N1 SW1361 virus acting as a positive control.Shedding of virus by the H9-inoculated pigs was similar to thatobserved in the infectivity test, with a peak titer at around 103.6

PFU/ml (Fig. 3). SW1361-inoculated pigs shed virus at up to 107

PFU/ml. None of the physical-contact or airborne-virus-exposedpigs of the HK449 and HK177W groups shed virus or serocon-verted. In the SW1361 group, efficient transmission was observedto both the physical-contact and airborne-virus-exposed pigs,with virus shedding (Fig. 3) and convalescent antibody titers (datanot shown) as previously seen (33). Although the number of con-

tact animals in these experiments was small, this difference be-tween the avian origin H9N2 viruses and the swine H1N1 SW1361virus might reflect limits on the ability of H9N2 viruses to transmitamong pigs.

Pathological changes in the respiratory tract of pigs infectedwith avian H9 viruses. Two inoculated pigs from each of thetransmission study groups were sacrificed at 4 dpi and two weresacrificed at 6 dpi. No obvious gross lesions were observed in pigsinoculated with HK449 (G1 lineage) at 4 dpi, whereas at 6 dpi anarea of 2 by 1.5 cm2 was found to have slight congestion in a leftcaudal lung lobe (data not shown). For pigs inoculated withHK177W (Y280 lineage), a large area of pleural hemorrhagic fi-brin exudates was observed in the right lung lobes of one pig at 4dpi, but no change was observed in the lungs of the other pigs(data not shown). Whether this was due to a bacterial infectionwas not tested. In SW1361-inoculated pigs, local congestion wascommonly observed in the lungs at both time points (data notshown). Microscopic observations of pulmonary sections showedlesions of various extents with red blood cell exudation, infiltra-

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dpi

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Inoculated Airborne exposedPhysical contact

Inoculated Airborne exposedPhysical contact dpc dpc

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FIG 3 Transmission and virus shedding of H9N2 influenza viruses in pigs. Nasal swabs were collected from four inoculated pigs, two physical-contact pigs andtwo airborne-virus-exposed pigs, and titrated by plaque-forming assays. Dotted lines indicate a detection limit of 1.1 log PFU/ml (or 12.5 PFU/ml). No sheddingof virus was detected after 10 dpi. Asterisks (**) indicate that two inoculated pigs were sacrificed at 4 dpi and another two were sacrificed at 6 dpi. dpc, dayspostcontact.

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tion of inflammatory cells, widened alveolar walls, and interlobu-lar septa in all pigs inoculated with the H9N2 or the SW1361viruses at 4 dpi (Fig. 4).

Staining of NP-positive cells in the respiratory tracts of theinoculated pigs revealed that the H9 viruses affected fewer cellsthan the SW1361 virus (Table 4 and Fig. 4). At 4 dpi, viral NP waspresent in the trachea, segmental bronchi, and lung lobes of oneHK449 (G1 lineage)-inoculated pig and in the lung lobes of each

HK177W (Y280 lineage)-inoculated pig, whereas in the SW1361group, NP was detected in both the upper and lower respiratorytracts. Clearance of influenza viruses was observed in the H9-infected respiratory tissues at 6 dpi, with no NP-positive cellsfound in the HK177W (Y280 lineage)-inoculated pigs and onlysporadic positive cells in one HK449 (G1 lineage)-inoculated pig.The SW1361 virus was still widely distributed in the lungs of onepig at 6 dpi but had a limited presence in the other pig.

FIG 4 Pathological changes and virus replication in the lungs of inoculated pigs at 4 dpi. Hematoxylin and eosin staining (A to D) and immunohistochemicalstaining (E to H) of swine lungs. Pigs were inoculated with PBS (A and E), HK449 H9N2 virus (B and F), HK177W H9N2 virus (C and G), or SW1361 H1N1 virus(D and H). Influenza NP antigen staining was visible as a brown color (F to H). Scale bars, 100 �m.

TABLE 4 Virus distribution in the respiratory tracts of the pigs inoculated with the H9 influenza virusesa

Virus and pig Turbinate Trachea Lobe bronchus Segmental bronchus

Lung

A B C1 C2 D E1 E2 F G

HK4491 – – – – – – – – – – – – –2 – �� – � ��� �� – � ��� �� �� �� ���3 – – – – – – – – – – – � �4 – – – – – – – – – – – – –

HK177W1 – – – – � � � – � �� – – ��2 – – – – � � � � � � � � �3 – – – – – – – – – – – – –4 – – – – – – – – – – – – –

SW13611 �� � � � � �� ��� � � � � ��� ��2 �� � �� ��� � � �� � – � � � ���3 – – – – – – – – � – – – –4 – – � � – � � � � – – � �

a In each group, pigs 1 and 2 were sacrificed on 4 dpi, and pigs 3 and 4 were sacrificed at 6 dpi. The symbols �, �, ��, and ��� indicate that the numbers of cells with viral NPpositive signal were 0, 1 to 20, 20 to 100, and �100, respectively, in each section. Lung lobes: A, left cranial; B, left middle; C1, left caudal (upper); C2, left caudal (lower); D,accessory; E1, right caudal (lower); E2, right caudal (upper); F, right middle; G, right cranial.

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DISCUSSION

Serological survey conducted in this study revealed that more than15% of pigs were seropositive to H9N2 viruses from the Y280lineage that is established in terrestrial poultry in China. Trans-missions or introductions of viruses from avian sources to swineseem to occur more frequently than previously reported (9, 35,36). This was observed across our whole survey period and in tworegions of China (Fig. 1). No sera were positive for aquatic birdviruses and only 0.6% were positive for G1 viruses. This mightreflect the Y280 viruses being more infectious to pigs or indicatedifferences in virus prevalence in the field.

The serological results were consistent with the infectivitystudies using viruses from different lineages. Inoculation with ter-restrial poultry H9N2 viruses led to virus shedding and serocon-version in most pigs, with the Y280 lineage viruses being moreinfective. The H9 viruses from aquatic birds tested here causedonly limited seroconversion and no shedding of virus in pigs.Thus, terrestrial poultry H9N2 viruses may be generally more in-fective toward pigs, and their long-term establishment in land-based poultry might be a factor in this, although the mechanism oftransmission to mammals remains unclear.

In this study, all H9N1 and H9N2 strains tested had threonineat HA-155 position (H3 numbering), which has been associatedwith the binding of H9 viruses to -2,6-sialylglycopolymers (37).The HA-226 was leucine in the Y280 lineage viruses (HKY280,HK341, and HK177W) and the G1 prototype virus (HKG1) butglutamine in the later G1 viruses (HK449, HK33982, and HK10)and the Y439 lineage viruses (ST2030 and JX7554). Leucine, in-stead of glutamine, at this position has been found to be respon-sible for the transmission of H9N2 viruses among ferrets and forpreferential infection of nonciliated cells (38, 39). However, sinceall of the Y280 and G1 lineage viruses tested here (HK341,HK177W, HK449, and HK10) were infectious to pigs, although atdifferent levels, the HA-Q226L was probably not a determinativefactor for the H9N2 virus infectivity in pigs.

At the HA1-HA2 cleavage site, the G1 and Y280 strains, but notthe Y439 virus, had a second arginine in the R-S-[NST]-R motifcompatible with a minimum furin cleavage motif (40, 41),whereas ST2030 and JX7554 had A-S-D-R, indicating that possi-ble cleavage by furin-like proteases could heighten the activity ofthe G1 and Y280 viruses in pigs.

The amino acid sequences of the PB2, PB1, PA, NP, and Mproteins had sequence identities of �94.8% among the H9 strainsexamined here. All of these strains had 627E and 701D in the PB2gene, a characteristic of avian viruses. NS1 genes of the G1 andY280 viruses were from allele A, whereas those of the Y439 strainswere from allele B. Allele B is rarely observed in influenza virusesisolated from mammals and has been shown to attenuate influ-enza viruses in mammalian systems (42, 43). This could probablyexplain the limited ability of the Y439 strains to infect pigs.

There is currently no evidence suggesting that avian H9 viruseshave acquired efficient transmissibility among pigs, and this wasconfirmed in our study. Viruses of the G1 and Y280 lineages wereinfective in pigs but could not be transmitted through either phys-ical or airborne contact. In the field, outbreaks caused by avian H9viruses in pig herds are rare and have only been reported fromfarms in the Shandong and Henan provinces in China (10, 11, 14).As shown by previous (9, 35, 36) and this serological surveillancein pigs, H9 infections, which may be derived from direct introduc-

tions of viruses from avian sources, have occurred frequentlywithout causing obvious clinical signs or disease outbreaks. Apartfrom the viruses from Korea, all of the other available swine H9N2viruses are from China and belong to the Y280 lineage (10–15).Although this is the most prevalent lineage in the field, its persis-tent presence and adaptation in terrestrial poultry might have as-sisted its ability to infect pigs.

Human infections with H9N2 viruses, while rare, have comefrom the G1 and Y280 lineages (2, 6–8, 16, 17). H9N2 viruses ofthe Y280 lineage have recently contributed to human infections byproviding the internal genes to the H7N9 (and H10N8) virusesthat have caused severe outbreaks in humans (4, 44, 45). Whetherreassortment of H9N2 viruses containing internal genes similar tothose of the H7N9 viruses with the prevailing swine viruses mightlead to a more human infectious virus is not known. Since H9viruses usually cause asymptomatic infections, it is likely that in-fections in pigs are going undetected, and this could lead to moreadapted or transmissible H9N2 viruses developing in pigs orH9N2/H7N9-like internal genes appearing in established swinevirus lineages. Surveillance of pigs for the H9 and other subtypesof influenza viruses would be able to detect and avert the progres-sion of these scenarios and should be a major part of pandemicpreparedness and zoonotic disease control.

ACKNOWLEDGMENTS

We gratefully acknowledge our colleagues from the State Key Laboratoryof Emerging Infectious Diseases (Hong Kong) and the Joint InfluenzaResearch Center (Shantou) for their technical assistance.

FUNDING INFORMATIONGuangxi Science and Technology Development Plan provided funding toXiaohui Fan under grant number GKH 1347004-27.

This project was supported by the Research Fund for the Control of In-fectious Diseases (Hong Kong SAR Government; grant 12111252), theGuangdong Top-Tier University Development Scheme, the Li Ka ShingFoundation, and the Distinguished Expert Scheme of Guangxi. Thefunders had no role in study design, data collection and interpretation, orthe decision to submit the work for publication. We declare no competingfinancial interests.

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