Veterinary Microbiology 173 (2014) 132–135
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Lowly pathogenic avian influenza (H9N2) infection in Plateau pika (Ochotona curzoniae), Qinghai Lake, China Zhijun Yu a,b,1, Kaihui Cheng b,d,1, Weiyang Sun b, Yue Xin b, Jinshan Cai c, Ruilin Ma c, Quanbang Zhao c, Lin Li b, Jing Huang b,e, Xiaoyu Sang b, Xue Li b,f, Kun Zhang a, Tiecheng Wang b, Chuan Qin a, Jun Qian b, Yuwei Gao b,*, Xianzhu Xia a,b,* a
Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China b Military Veterinary Research Institute of Academy of Military Medical Sciences, Changchun, People’s Republic of China c Animal Disease Control and Prevention Center in Qinghai Province, Xining, People’s Republic of China d Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Jinan, People’s Republic of China e College of Animal Science and Technology, Jilin Agricultural University, Changchun, People’s Republic of China f Changchun Institute of Biological Products, Changchun, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 April 2014 Received in revised form 2 July 2014 Accepted 3 July 2014
Avian influenza viruses (AIVs) are globally important contagions. Several domestic mammals can be infected with AIVs and may play important roles in the adaptation and transmission of these viruses in mammals, although the roles of wild mammals in the natural ecology of AIVs are not yet clear. Here, we performed a serological survey of apparently healthy Plateau pikas at Qinghai Lake in China to assess the prevalence of exposure to AIVs. Ninety-two of 293 (31%) of wild Plateau pikas possessed serum antibodies against a lowly pathogenic avian influenza (LPAI) H9N2 virus. Experimental inoculation of Plateau pikas with a LPAI H9N2 virus resulted in productive viral replication in respiratory tissues without prior adaptation. Our findings suggest that Plateau pikas represent a natural mammalian host to H9N2 AIVs and may play a role in the ongoing circulation of H9N2 viruses at Qinghai Lake in China. Surveillance for AIV infection in Plateau pika populations and other mammals that have close contact with the Plateau pikas should be considered. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Qinghai Lake Plateau pika Avian influenza virus H9N2 Serological survey
1. Introduction
* Corresponding authors at: Research Center of Wildlife Disease, Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute, Academy of Military Medical Sciences, 666 Liuyingxi Rd, Changchun 130122, People’s Republic of China. Tel.: +86043186985511. E-mail addresses: [email protected] (Y. Gao), [email protected] (X. Xia). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.vetmic.2014.07.002 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
AIVs primarily circulate in wild waterfowl, although these viruses have also been isolated from various wild mammalian species (Song et al., 2009; Songserm et al., 2006; Sun et al., 2013). Infection of wild mammals with AIVs may contribute to the ongoing circulation of AIVs, lead to viral transmission to other wild and domestic animals, and present opportunities for avian viruses to adapt to mammalian hosts (Horimoto et al., 2011). Variant AIVs that have acquired the capacity for efficient replication in mammalian hosts could have pandemic potential if they
Z. Yu et al. / Veterinary Microbiology 173 (2014) 132–135
were able to infect and transmit between humans, thus giving rise to a serious public health concern. Therefore, the continuous monitoring of the exposure of wild mammals to avian influenza viruses is essential. The Plateau pika (Ochotona curzoniae) is a small nonhibernating mammal that resides throughout the Tibetan Plateau of China. In 2005, a severe outbreak of HPAI occurred among migratory wild birds in Qinghai Lake in the northeastern part of the Tibetan Plateau (Chen et al., 2005; Liu et al., 2005; Zhou et al., 2006). A previous report suggested that Plateau pikas can be asymptomatically infected with highly pathogenic avian influenza (HPAI) H5N1 (Zhou et al., 2009). These findings raise the possibility that wild Plateau pikas may play a role in the transmission of AIVs at Qinghai Lake and provide opportunities for the adaptation of AIV to mammalian hosts. Here, we conducted a serological survey to assess the prevalence of AIV exposure in wild Plateau pikas in the regions around Qinghai Lake. 2. Materials and methods 2.1. Serum samples and viruses During May through November of 2012, 293 serum samples were collected from Plateau pikas in the regions around Qinghai Lake, China. Sera samples were analyzed to assess exposure of wild Plateau pikas to AIVs. Influenza viruses used in this study included A/Duck/ China/66-70/2011(H3N6; D6670), A/Duck/China/46/ 2011(H4N6; D46), A/Tiger/Harbin/01/2003(H5N1; T01), A/ Mallard/SanJiang/275/2007(H6N1; SJ/275), and A/Chicken/ Jinan/Chai-1/2011 (H9N2; JN1). Viruses were propagated in eggs, and used to assess hemagglutination inhibition (HI) and virus neutralization (VN) serum antibody titers. 2.2. HI assay HI tests were performed as previously described (WHO, 2002). Briefly, serum samples were treated with receptordestroying enzyme (RDE; Sigma-Aldrich, St. Louis, MO, USA; 1 part serum:4 parts RDE) overnight at 37 8C to remove nonspecific inhibitors prior to analysis. Five parts of 1.5% sodium citrate were added and the samples heatinactivated at 56 8C for 30 min. Serum samples were then serially diluted two-fold in phosphate buffered saline (PBS). Twenty-five microliters of the undiluted and serially diluted serum was mixed with 25 ml of test virus containing four hemagglutination units in each well of a microplate and incubated at room temperature for 30 min. Then 50 ml of 0.5% chicken erythrocytes was added to each well. Results were read after incubation at room temperature for 30 min. HI antibody titers were defined as the reciprocal of the highest serum dilution that prevented virus-mediated hemagglutination of the chicken erythrocytes. 2.3. VN assay VN assays were performed as previously described (WHO, 2002). Briefly, treated sera were serially diluted two-fold in duplicate and incubated with the indicated test virus at final concentration of 100 50% tissue culture
133
infection dose (TCID50)/100 ml for 1 h at 37 8C. The serumvirus mixture was transferred onto a MDCK monolayer maintained in minimum essential medium supplemented with TPCK trypsin (2 mg/ml; Worthington Biochemical Corp., Lakewood, NJ. USA) and incubated for 72 h. The virus neutralizing antibody titer was calculated using the method of Reed–Muench (Reed and Muench, 1938). 2.4. Animal experiment To assess the replication capability of an H9N2 avian influenza A virus in Plateau pikas, three Plateau pikas were intranasally (i.n.) inoculated with 107 50% egg infectious doses (EID50) of the LPAI virus A/Chicken/Shandong/Li-1/ 2009 (H9N2). All Plateau pikas were healthy and showed no serological evidence of prior H9N2 infection before inoculation. Clinical signs of infection (sneezing, nasal discharge, and coughing) of all Plateau pikas were monitored for 3 days post-inoculation (dpi). At 3 dpi, all Plateau pikas were humanely euthanized, and organs including the brain, spleen, kidneys, liver, colon, nasal turbinates, trachea, and lungs were collected for virus titration in eggs. Infectious virus titers were reported as log10 EID50/ml, and were calculated from 3 replicates by the method of Reed–Muench (Reed and Muench, 1938). All animal studies were conducted in a biosafety level 2+ laboratory approved by the Institute of Military Veterinary, Changchun, China.
3. Results and discussion Serum samples collected from 293 Plateau pikas residing in the regions near Qinghai Lake, China were analyzed for serological evidence of AIV exposure. Ninetytwo samples (31.4%) had detectable HI antibody titers against the JN1 H9N2 virus (Table 1). H9N2 HI antibody titers ranged from 10 to 2560. Forty-six samples had titers of 10–80 and 46 had titers >160 (Table 1 and Fig. 1). Serum specimens did not react to viruses of other HA subtypes, including H3, H4, H5, and H6. All serum samples with detectable HI antibody titers were further evaluated using a virus neutralization (VN) assay with the JN1 H9N2 virus according to standard protocols (WHO, 2002). Of the 92 HI-positive serum samples, 75 (25.6% of the original 293 serum samples) also had detectable neutralizing antibodies to the JN1 H9N2 virus (Table 1). H9N2 VN antibody titers ranged from 10 to 1280. Thirty-six samples had VN antibody titers of 10–80 and 39 had VN antibody titers >160 (Table 1 and Fig. 2).
Table 1 Plateau pika serum antibodies against subtype H9N2 virus A/Chicken/ Jinan/Chai-1/2011, Tibetan Plateau, People’s Republic of China*. Titer
10–80 160 Total
No. (%), n=293 HI
VN
46 46 92(31.4)
36 39 75(25.6)
* HI, hemagglutination inhibition; VN, virus neutralization.
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Z. Yu et al. / Veterinary Microbiology 173 (2014) 132–135
Fig. 1. HI titers of positive serum samples against avian H9N2 virus in wild Plateau pikas. A total of 293 serum samples were tested by HI assay of which 92 samples had detectable HI antibody titers. The geometric mean and 95% confidence intervals are indicated by long and short horizontal lines, respectively.
Collectively, these data suggest that wild Plateau pikas generate H9N2-specific antibody responses due to natural exposure to H9N2 avian influenza viruses. To explore the replication capability of H9N2 avian influenza A viruses in Plateau pikas, three Plateau pikas were intranasally (i.n.) inoculated with 107 EID50 A/Chicken/ Shandong/Li-1/2009 (H9N2). On day 3 post-infection, virus was recovered from the nasal turbinates of all three Plateau pikas (2.75–4.75 log10 EID50/ml) and in the trachea and lungs of one pika (Fig. 3). These results demonstrate that intranasal inoculation of Plateau pikas with a LPAI H9N2 virus results in productive infection and viral replication. The potential spillover of AIVs into humans has been a major public health concern following the unprecedented report of an H5N1 outbreak in migratory wild birds at Qinghai Lake, China in 2005 (Liu et al., 2005). There have been numerous reports documenting the persistent circulation of HPAI virus at Qinghai Lake (Hu et al., 2011; Li et al., 2010). In this study, we confirmed H9N2 subtype lowly pathogenic avian influenza virus (LPAI) infection in mammalian species at Qinghai Lake of China. Additionally, an outbreak of human infection with LPAI (H7N9) was reported in China in 2013, further underscoring the potential risk of AIVs to human health (Gao et al., 2013).
Fig. 3. Virus replication in Plateau pikas inoculated with LPAI H9N2. Three Plateau pikas were intranasally inoculated with 107 50% EID50 of the LPAI H9N2 virus (A/Chicken/Shandong/Li-1/2009). Three days postinoculation, the indicated tissues were collected and analyzed for the presence of replicating virus by titration in eggs. Viral loads in each tissue are expressed as log10 EID50/ml. The dashed black lines indicate the lower limit of detection (0.5 log10 EID50/ml).
Although the sporadic infection of humans with H5N1 and H7N9 AIVs has not yet led to sustained human-tohuman transmission, a major question concerns the potential of these AIVs to acquire adaptive mutations that enable more efficient replication in mammals and respiratory droplet-mediated transmission between mammals, as these characteristics are widely thought to be prerequisites for the emergence of an avian-origin pandemic influenza virus. Like H5N1 and H7N9 AIVs, H9N2 influenza viruses also may pose a pandemic threat due to their widespread distribution and documented ability to infect humans and pigs (Malik Peiris, 2009). In this study, we provide serological and experimental evidence that strongly suggests wild Plateau pikas are susceptible to LPAI H9N2 AIV infection. Specifically, we found that LPAI H9N2 could infect Plateau pikas and replicate in their respiratory organs, and that nearly a third of the surveyed wild Plateau pikas had serological evidence of natural exposure to H9N2 viruses. Plateau pikas are the primary wild mammals of Qinghai Lake, and serve as the main prey for most of the predatory animals in this region, including raptorial birds and carnivorous mammals. Therefore, we speculate that Plateau pikas may serve as an LPAI reservoir and play a role in LPAI transmission at Qinghai Lake, although the role of these animals in the natural history of avian influenza viruses is not clear. Infection of Plateau pikas with H9N2 AIVs may provide opportunities for the acquisition of mammalian adaptive mutations which may potentially lessen the barriers to interspecies transmission of H9N2 viruses to humans. Therefore, surveillance for LPAI H9N2 and other AIVs among Plateau pika populations and other animals that have close contact with Plateau pikas should be considered.
Conflict of interest statement There are no potential conflicts of interest. Fig. 2. VN titers of positive serum samples against avian H9N2 virus in wild Plateau pikas. A total of 92 HI-positive serum samples were tested by VN assay of which 75 had detectable VN antibody titers. The geometric mean and 95% confidence intervals are indicated by long and short horizontal lines, respectively.
Acknowledgments This work was supported by the National Key Technologies R&D Program (no. 2013BAD12B04, KJYJ-2013-01-01,
Z. Yu et al. / Veterinary Microbiology 173 (2014) 132–135
and 2012AA022006), and by the Department of Wildlife Conservation and Nature Reserve Management of the State Forestry Administration (SFA), People’s Republic of China. References Chen, H., Smith, G.J., Zhang, S.Y., Qin, K., Wang, J., Li, K.S., Webster, R.G., Peiris, J.S., Guan, Y., 2005. Avian flu: H5N1 virus outbreak in migratory waterfowl. Nature 436, 191–192. Gao, R., Cao, B., Hu, Y., Feng, Z., Wang, D., Hu, W., Chen, J., Jie, Z., Qiu, H., Xu, K., Xu, X., Lu, H., Zhu, W., Gao, Z., Xiang, N., Shen, Y., He, Z., Gu, Y., Zhang, Z., Yang, Y., Zhao, X., Zhou, L., Li, X., Zou, S., Zhang, Y., Li, X., Yang, L., Guo, J., Dong, J., Li, Q., Dong, L., Zhu, Y., Bai, T., Wang, S., Hao, P., Yang, W., Zhang, Y., Han, J., Yu, H., Li, D., Gao, G.F., Wu, G., Wang, Y., Yuan, Z., Shu, Y., 2013. Human infection with a novel avian-origin influenza A (H7N9) virus. N. Engl. J. Med. 368, 1888–1897. Horimoto, T., Maeda, K., Murakami, S., Kiso, M., Iwatsuki-Horimoto, K., Sashika, M., Ito, T., Suzuki, K., Yokoyama, M., Kawaoka, Y., 2011. Highly pathogenic avian influenza virus infection in feral raccoons, Japan. Emerg. Infect. Dis. 17, 714–717. Hu, X., Liu, D., Wang, M., Yang, L., Wang, M., Zhu, Q., Li, L., Gao, G.F., 2011. Clade 2.3.2 avian influenza virus (H5N1), Qinghai Lake region China, 2009-2010. Emerg. Infect. Dis. 17, 560–562. Li, Y., Li, P., Lei, F., Guo, S., Ding, C., Xin, Z., He, Y., Yan, B., Kou, Z., Tang, S., Zhang, Z., Hu, Z., Li, T., 2010. Persistent circulation of highly pathogenic influenza H5N1 virus in Lake Qinghai area of China. Avian Dis. 54, 821–829. Liu, J., Xiao, H., Lei, F., Zhu, Q., Qin, K., Zhang, X.W., Zhang, X.L., Zhao, D., Wang, G., Feng, Y., Ma, J., Liu, W., Wang, J., Gao, G.F., 2005. Highly
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pathogenic H5N1 influenza virus infection in migratory birds. Science 309, 1206. Malik Peiris, J.S., 2009. Avian influenza viruses in humans. Rev. Sci. Tech. 28, 161–173. Reed, L.J., Muench, H., 1938. A simple method for estimating fifty percent endpoints. Am. J. Hyg. (Lond.) 27, 493–497. Song, D., Lee, C., Kang, B., Jung, K., Oh, T., Kim, H., Park, B., Oh, J., 2009. Experimental infection of dogs with avian-origin canine influenza A virus (H3N2). Emerg. Infect. Dis. 15, 56–58. Songserm, T., Amonsin, A., Jam-on, R., Sae-Heng, N., Pariyothorn, N., Payungporn, S., Theamboonlers, A., Chutinimitkul, S., Thanawongnuwech, R., Poovorawan, Y., 2006. Fatal avian influenza A H5N1 in a dog. Emerg. Infect. Dis. 12, 1744–1747. Sun, X., Xu, X., Liu, Q., Liang, D., Li, C., He, Q., Jiang, J., Cui, Y., Li, J., Zheng, L., Guo, J., Xiong, Y., Yan, J., 2013. Evidence of avian-like H9N2 influenza A virus among dogs in Guangxi, China. Infect. Genet. Evol. 20, 471–475, Journal of molecular epidemiology and evolutionary genetics in infectious diseases. World Health Organization, 2002. WHO Manual on Animal Influenza Diagnosis and Surveillance. World Health Organization Available: hhttp://www.who.int/csr/resources/publications/influenza/en/ whocdscsrncs20025rev.pdfi Zhou, J., Sun, W., Wang, J., Guo, J., Yin, W., Wu, N., Li, L., Yan, Y., Liao, M., Huang, Y., Luo, K., Jiang, X., Chen, H., 2009. Characterization of the H5N1 highly pathogenic avian influenza virus derived from wild pikas in China. J. Virol. 83, 8957–8964. Zhou, J.Y., Shen, H.G., Chen, H.X., Tong, G.Z., Liao, M., Yang, H.C., Liu, J.X., 2006. Characterization of a highly pathogenic H5N1 influenza virus derived from bar-headed geese in China. J. Gen. Virol. 87, 1823–1833.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Lowly pathogenic avian influenza (H9N2) infection in Plateau pika (Ochotona curzoniae), Qinghai Lake, China Zhijun Yu a,b,1, Kaihui Cheng b,d,1, Weiyang Sun b, Yue Xin b, Jinshan Cai c, Ruilin Ma c, Quanbang Zhao c, Lin Li b, Jing Huang b,e, Xiaoyu Sang b, Xue Li b,f, Kun Zhang a, Tiecheng Wang b, Chuan Qin a, Jun Qian b, Yuwei Gao b,*, Xianzhu Xia a,b,* a
Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China b Military Veterinary Research Institute of Academy of Military Medical Sciences, Changchun, People’s Republic of China c Animal Disease Control and Prevention Center in Qinghai Province, Xining, People’s Republic of China d Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Jinan, People’s Republic of China e College of Animal Science and Technology, Jilin Agricultural University, Changchun, People’s Republic of China f Changchun Institute of Biological Products, Changchun, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 April 2014 Received in revised form 2 July 2014 Accepted 3 July 2014
Avian influenza viruses (AIVs) are globally important contagions. Several domestic mammals can be infected with AIVs and may play important roles in the adaptation and transmission of these viruses in mammals, although the roles of wild mammals in the natural ecology of AIVs are not yet clear. Here, we performed a serological survey of apparently healthy Plateau pikas at Qinghai Lake in China to assess the prevalence of exposure to AIVs. Ninety-two of 293 (31%) of wild Plateau pikas possessed serum antibodies against a lowly pathogenic avian influenza (LPAI) H9N2 virus. Experimental inoculation of Plateau pikas with a LPAI H9N2 virus resulted in productive viral replication in respiratory tissues without prior adaptation. Our findings suggest that Plateau pikas represent a natural mammalian host to H9N2 AIVs and may play a role in the ongoing circulation of H9N2 viruses at Qinghai Lake in China. Surveillance for AIV infection in Plateau pika populations and other mammals that have close contact with the Plateau pikas should be considered. ß 2014 Elsevier B.V. All rights reserved.
Keywords: Qinghai Lake Plateau pika Avian influenza virus H9N2 Serological survey
1. Introduction
* Corresponding authors at: Research Center of Wildlife Disease, Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute, Academy of Military Medical Sciences, 666 Liuyingxi Rd, Changchun 130122, People’s Republic of China. Tel.: +86043186985511. E-mail addresses: [email protected] (Y. Gao), [email protected] (X. Xia). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.vetmic.2014.07.002 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
AIVs primarily circulate in wild waterfowl, although these viruses have also been isolated from various wild mammalian species (Song et al., 2009; Songserm et al., 2006; Sun et al., 2013). Infection of wild mammals with AIVs may contribute to the ongoing circulation of AIVs, lead to viral transmission to other wild and domestic animals, and present opportunities for avian viruses to adapt to mammalian hosts (Horimoto et al., 2011). Variant AIVs that have acquired the capacity for efficient replication in mammalian hosts could have pandemic potential if they
Z. Yu et al. / Veterinary Microbiology 173 (2014) 132–135
were able to infect and transmit between humans, thus giving rise to a serious public health concern. Therefore, the continuous monitoring of the exposure of wild mammals to avian influenza viruses is essential. The Plateau pika (Ochotona curzoniae) is a small nonhibernating mammal that resides throughout the Tibetan Plateau of China. In 2005, a severe outbreak of HPAI occurred among migratory wild birds in Qinghai Lake in the northeastern part of the Tibetan Plateau (Chen et al., 2005; Liu et al., 2005; Zhou et al., 2006). A previous report suggested that Plateau pikas can be asymptomatically infected with highly pathogenic avian influenza (HPAI) H5N1 (Zhou et al., 2009). These findings raise the possibility that wild Plateau pikas may play a role in the transmission of AIVs at Qinghai Lake and provide opportunities for the adaptation of AIV to mammalian hosts. Here, we conducted a serological survey to assess the prevalence of AIV exposure in wild Plateau pikas in the regions around Qinghai Lake. 2. Materials and methods 2.1. Serum samples and viruses During May through November of 2012, 293 serum samples were collected from Plateau pikas in the regions around Qinghai Lake, China. Sera samples were analyzed to assess exposure of wild Plateau pikas to AIVs. Influenza viruses used in this study included A/Duck/ China/66-70/2011(H3N6; D6670), A/Duck/China/46/ 2011(H4N6; D46), A/Tiger/Harbin/01/2003(H5N1; T01), A/ Mallard/SanJiang/275/2007(H6N1; SJ/275), and A/Chicken/ Jinan/Chai-1/2011 (H9N2; JN1). Viruses were propagated in eggs, and used to assess hemagglutination inhibition (HI) and virus neutralization (VN) serum antibody titers. 2.2. HI assay HI tests were performed as previously described (WHO, 2002). Briefly, serum samples were treated with receptordestroying enzyme (RDE; Sigma-Aldrich, St. Louis, MO, USA; 1 part serum:4 parts RDE) overnight at 37 8C to remove nonspecific inhibitors prior to analysis. Five parts of 1.5% sodium citrate were added and the samples heatinactivated at 56 8C for 30 min. Serum samples were then serially diluted two-fold in phosphate buffered saline (PBS). Twenty-five microliters of the undiluted and serially diluted serum was mixed with 25 ml of test virus containing four hemagglutination units in each well of a microplate and incubated at room temperature for 30 min. Then 50 ml of 0.5% chicken erythrocytes was added to each well. Results were read after incubation at room temperature for 30 min. HI antibody titers were defined as the reciprocal of the highest serum dilution that prevented virus-mediated hemagglutination of the chicken erythrocytes. 2.3. VN assay VN assays were performed as previously described (WHO, 2002). Briefly, treated sera were serially diluted two-fold in duplicate and incubated with the indicated test virus at final concentration of 100 50% tissue culture
133
infection dose (TCID50)/100 ml for 1 h at 37 8C. The serumvirus mixture was transferred onto a MDCK monolayer maintained in minimum essential medium supplemented with TPCK trypsin (2 mg/ml; Worthington Biochemical Corp., Lakewood, NJ. USA) and incubated for 72 h. The virus neutralizing antibody titer was calculated using the method of Reed–Muench (Reed and Muench, 1938). 2.4. Animal experiment To assess the replication capability of an H9N2 avian influenza A virus in Plateau pikas, three Plateau pikas were intranasally (i.n.) inoculated with 107 50% egg infectious doses (EID50) of the LPAI virus A/Chicken/Shandong/Li-1/ 2009 (H9N2). All Plateau pikas were healthy and showed no serological evidence of prior H9N2 infection before inoculation. Clinical signs of infection (sneezing, nasal discharge, and coughing) of all Plateau pikas were monitored for 3 days post-inoculation (dpi). At 3 dpi, all Plateau pikas were humanely euthanized, and organs including the brain, spleen, kidneys, liver, colon, nasal turbinates, trachea, and lungs were collected for virus titration in eggs. Infectious virus titers were reported as log10 EID50/ml, and were calculated from 3 replicates by the method of Reed–Muench (Reed and Muench, 1938). All animal studies were conducted in a biosafety level 2+ laboratory approved by the Institute of Military Veterinary, Changchun, China.
3. Results and discussion Serum samples collected from 293 Plateau pikas residing in the regions near Qinghai Lake, China were analyzed for serological evidence of AIV exposure. Ninetytwo samples (31.4%) had detectable HI antibody titers against the JN1 H9N2 virus (Table 1). H9N2 HI antibody titers ranged from 10 to 2560. Forty-six samples had titers of 10–80 and 46 had titers >160 (Table 1 and Fig. 1). Serum specimens did not react to viruses of other HA subtypes, including H3, H4, H5, and H6. All serum samples with detectable HI antibody titers were further evaluated using a virus neutralization (VN) assay with the JN1 H9N2 virus according to standard protocols (WHO, 2002). Of the 92 HI-positive serum samples, 75 (25.6% of the original 293 serum samples) also had detectable neutralizing antibodies to the JN1 H9N2 virus (Table 1). H9N2 VN antibody titers ranged from 10 to 1280. Thirty-six samples had VN antibody titers of 10–80 and 39 had VN antibody titers >160 (Table 1 and Fig. 2).
Table 1 Plateau pika serum antibodies against subtype H9N2 virus A/Chicken/ Jinan/Chai-1/2011, Tibetan Plateau, People’s Republic of China*. Titer
10–80 160 Total
No. (%), n=293 HI
VN
46 46 92(31.4)
36 39 75(25.6)
* HI, hemagglutination inhibition; VN, virus neutralization.
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Fig. 1. HI titers of positive serum samples against avian H9N2 virus in wild Plateau pikas. A total of 293 serum samples were tested by HI assay of which 92 samples had detectable HI antibody titers. The geometric mean and 95% confidence intervals are indicated by long and short horizontal lines, respectively.
Collectively, these data suggest that wild Plateau pikas generate H9N2-specific antibody responses due to natural exposure to H9N2 avian influenza viruses. To explore the replication capability of H9N2 avian influenza A viruses in Plateau pikas, three Plateau pikas were intranasally (i.n.) inoculated with 107 EID50 A/Chicken/ Shandong/Li-1/2009 (H9N2). On day 3 post-infection, virus was recovered from the nasal turbinates of all three Plateau pikas (2.75–4.75 log10 EID50/ml) and in the trachea and lungs of one pika (Fig. 3). These results demonstrate that intranasal inoculation of Plateau pikas with a LPAI H9N2 virus results in productive infection and viral replication. The potential spillover of AIVs into humans has been a major public health concern following the unprecedented report of an H5N1 outbreak in migratory wild birds at Qinghai Lake, China in 2005 (Liu et al., 2005). There have been numerous reports documenting the persistent circulation of HPAI virus at Qinghai Lake (Hu et al., 2011; Li et al., 2010). In this study, we confirmed H9N2 subtype lowly pathogenic avian influenza virus (LPAI) infection in mammalian species at Qinghai Lake of China. Additionally, an outbreak of human infection with LPAI (H7N9) was reported in China in 2013, further underscoring the potential risk of AIVs to human health (Gao et al., 2013).
Fig. 3. Virus replication in Plateau pikas inoculated with LPAI H9N2. Three Plateau pikas were intranasally inoculated with 107 50% EID50 of the LPAI H9N2 virus (A/Chicken/Shandong/Li-1/2009). Three days postinoculation, the indicated tissues were collected and analyzed for the presence of replicating virus by titration in eggs. Viral loads in each tissue are expressed as log10 EID50/ml. The dashed black lines indicate the lower limit of detection (0.5 log10 EID50/ml).
Although the sporadic infection of humans with H5N1 and H7N9 AIVs has not yet led to sustained human-tohuman transmission, a major question concerns the potential of these AIVs to acquire adaptive mutations that enable more efficient replication in mammals and respiratory droplet-mediated transmission between mammals, as these characteristics are widely thought to be prerequisites for the emergence of an avian-origin pandemic influenza virus. Like H5N1 and H7N9 AIVs, H9N2 influenza viruses also may pose a pandemic threat due to their widespread distribution and documented ability to infect humans and pigs (Malik Peiris, 2009). In this study, we provide serological and experimental evidence that strongly suggests wild Plateau pikas are susceptible to LPAI H9N2 AIV infection. Specifically, we found that LPAI H9N2 could infect Plateau pikas and replicate in their respiratory organs, and that nearly a third of the surveyed wild Plateau pikas had serological evidence of natural exposure to H9N2 viruses. Plateau pikas are the primary wild mammals of Qinghai Lake, and serve as the main prey for most of the predatory animals in this region, including raptorial birds and carnivorous mammals. Therefore, we speculate that Plateau pikas may serve as an LPAI reservoir and play a role in LPAI transmission at Qinghai Lake, although the role of these animals in the natural history of avian influenza viruses is not clear. Infection of Plateau pikas with H9N2 AIVs may provide opportunities for the acquisition of mammalian adaptive mutations which may potentially lessen the barriers to interspecies transmission of H9N2 viruses to humans. Therefore, surveillance for LPAI H9N2 and other AIVs among Plateau pika populations and other animals that have close contact with Plateau pikas should be considered.
Conflict of interest statement There are no potential conflicts of interest. Fig. 2. VN titers of positive serum samples against avian H9N2 virus in wild Plateau pikas. A total of 92 HI-positive serum samples were tested by VN assay of which 75 had detectable VN antibody titers. The geometric mean and 95% confidence intervals are indicated by long and short horizontal lines, respectively.
Acknowledgments This work was supported by the National Key Technologies R&D Program (no. 2013BAD12B04, KJYJ-2013-01-01,
Z. Yu et al. / Veterinary Microbiology 173 (2014) 132–135
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