Abstract
Recently, three novel avian-origin swine influenza viruses (SIVs) were first isolated from pigs in Guangdong Province, southern China, yet little is known about the seroprevalence of avian influenza viruses among pigs in southern China. Here, we report for the first time the seroprevalence of avian H3, H4, and H6 influenza viruses in swine populations and the lack of seroepidemiological evidence of avian H5 influenza virus transmission to pigs in China.
TEXT
Influenza A viruses are known to infect a wide variety of animals, including humans, pigs, horses, birds, and sea mammals. The primary reservoir of the influenza A virus is aquatic waterfowl, and birds are the source of all influenza viruses in other species (1). Pigs are susceptible to both human and avian influenza viruses (AIVs) and have been proposed to be intermediate hosts, or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host (2). Experimentally, the pig is able to be infected by all of the avian H1 to H13 subtypes studied (3). In nature, however, interspecies transmission of avian influenza viruses to pigs is not often documented (4). Recently, three novel avian-origin swine influenza viruses (SIVs) were first isolated from pigs in Guangdong Province, southern China (5–7). The appearance of AIVs among pigs poses concerns for both veterinary and human health, and yet little is known about the seroprevalence of avian influenza viruses among pigs in southern China.
To investigate the prevalence of AIV infections in pigs in southern China, a total of 1,080 blood samples were collected from April 2010 to June 2012 from 20 swine farms distributed throughout Guangdong, Guangxi, Fujian, and Jiangxi Provinces, southern China (Fig. 1). The 1,080 serum samples from 21- to 25-week-old growing-finishing pigs were chosen using a stratified random sampling method for farms and for pigs within farms. In addition, 550 serum samples were retrospectively analyzed from apparently healthy pigs in Guangdong Province in 2001. All animal researches were conducted under the guidance of CDC's Institutional Animal Care and Use Committee and in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility. Our animal research in this study has been approved by the Guangdong Province Animal Disease Control Center. These serum samples were separated by centrifugation at 3,000 rpm for 15 min. The serum samples were transferred to new Eppendorf tubes and stored at −20°C until tested for antibodies against influenza A virus. All serum samples were treated with a receptor-destroying enzyme and absorbed with erythrocytes to remove nonspecific inhibitors before the assays. All samples were tested by hemagglutination inhibition (HI) and virus neutralization (VN) assays according to standard protocols (8). According to previous reports, the HI assay does not reliably detect antibodies to AIVs in mammalian sera because nonspecific hemagglutination inhibitors in the mammalian sera, even inactivated, can cause false-positive results for AIVs (9). Therefore, in this study, the VN test was carried out in parallel with the HI test by four viruses, A/Swine/Guangdong/L21/2011(H3N2), A/Swine/Guangdong/K4/2011(H4N8), A/Swine/Guangdong/K6/2010(H6N6), and A/Chicken/Guangdong/178/04(H5N1). The following antigens were also used for HI testing: H1N1 influenza virus [A/Swine/Guangdong/L6/2009(H1N1)] for classical H1N1 SIV and H3N2 influenza virus [A/Swine/Guangdong/01/2005(H3N2)] for human-like H3N2 SIV. These influenza viruses were provided by the College of Veterinary Medicine, South China Agricultural University. An HI titer of ≥40 or a VN titer of ≥40 is considered seropositive and indicates previous infection.
Fig 1.
Survey sites in southern China where the seroepidemiological study was conducted. GX, GD, FJ, and JX, provinces of Guangxi, Guangdong, Fujian, and Jiangxi, respectively.
A total of 1,630 serum samples (including 550 serum samples collected in 2001) were examined by VN and HI for AIV antibodies. Results of the VN and HI tests are reported in Table 1. The serum samples collected in 2001 had no HI or VN antibodies against any of these AIVs. In addition, 35.2% (380/1,080) of the serum samples were HI positive for A/Swine/Guangdong/L6/2009(H1N1), and 19.7% (213/1,080) of the serum samples were HI positive for A/Swine/Guangdong/01/2005(H3N2).
Table 1.
Seroprevalence of avian-origin H3N2 SIV, avian-origin H4N8 SIV, avian-origin H6N6 SIV, and H5N1 AIV in swine farms in different provinces of southern China
| Province or sample group | H3N2 |
H4N8 |
H5N1 |
H6N6 |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. examined | % seroprevalence (no. positive/total no.)a |
No. examined | % seroprevalence (no. positive/no. total)a |
No. examined | % seroprevalence (no. positive/total no.)a |
No. examined | % seroprevalence (no. positive/total no.)a |
|||||
| VN | HI | VN | HI | VN | HI | VN | HI | |||||
| Guangdong | 300 | 2 (6/300) | 1.0 (3/300) | 300 | 2.3 (7/300) | 0.7 (2/300) | 300 | 0 (0/300) | 0 (0/300) | 300 | 2.7 (8/300) | 0 (0/300) |
| Guangxi | 280 | 0 (0/280) | 0 (0/280)c | 280 | 2.9 (8/280) | 0.4 (1/280) | 280 | 0 (0/280) | 0 (0/280) | 280 | 1.4 (4/280) | 0 (0/280) |
| Fujian | 200 | 2 (4/200) | 0 (0/200) | 200 | 0 (0/200) | 0 (0/200) | 200 | 0 (0/200) | 0 (0/200) | 200 | 2.5 (5/200) | 0 (0/200) |
| Jiangxi | 300 | 0 (0/300) | 0 (0/300) | 300 | 0.7 (2/300) | 0.7 (2/300) | 300 | 0 (0/300) | 0 (0/300) | 300 | 0.7 (2/300) | 0 (0/300) |
| GD2001b | 550 | 0 (0/550) | 0 (0/550) | 550 | 0 (0/550) | 0 (0/550) | 550 | 0 (0/550) | 0 (0/550) | 550 | 0 (0/550) | 0 (0/550) |
| Totalc | 1,080 | 0.93 (10/1,080) | 0.27 (3/1,080) | 1,080 | 1.6 (17/1,080) | 0.5 (5/1,080) | 1,080 | 0 (0/1,080) | 0 (0/1,080) | 1,080 | 1.8 (19/1,080) | 0 (0/1,080) |
HI titers of ≥40 or VN titers of ≥40 are considered seropositive results and indicate previous infection.
Five hundred fifty serum samples were collected from apparently healthy pigs in Guangdong Province in 2001.
The total does not include the 550 serum samples that were collected in 2001.
In the present study, antibodies against H3, H4, and H6 AIVs were detected in the serum samples collected from pigs in southern China. None of the samples tested showed seropositivity against the avian H5 virus according to the HI and VN tests, suggesting that they have been sporadically infected with H3, H4, and H6 AIVs. H3N2, H4N8, and H6N6 viruses were recently isolated from pigs in China, and the viral genes were derived wholly from AIVs of the Asia lineage (5–7). Therefore, we report for the first time the seroprevalence of avian H3, H4, and H6 influenza viruses in swine populations and the lack of seroepidemiological evidence of avian H5 influenza virus transmission to pigs in China. All the four provinces of southern China participating in the investigation have now been confirmed with seropositivity for avian-origin SIVs in pigs, indicating that the avian-origin SIVs have already been prevailing among the swine population in southern China, not only in Guangdong Province. The number of commercial pig farms is certainly increasing in southern China, and most of these are large-scale swine farms. Thus, humans and pigs are in close proximity in farming villages, providing the opportunity for the interspecies transmission of influenza viruses. Given the evidence that pigs can sustain reassortment of human and avian influenza viruses, it is important to be cautious and to enhance surveillance for atypical influenza viruses in pigs as part of our overall pandemic preparedness plans and to consider the potential for avian virus-like SIVs, or the novel reassortant viruses, to enter the human population.
ACKNOWLEDGMENTS
This study was funded by the National Key Basic Research Program (project 973) of China (grant no. 2011CB504700-G). This work was supported by the International Sci & Tech Cooperation Program (2010DFB33920) and the Modern Agricultural Industry Technology System (CARS-36).
We give our heartfelt thanks to Fu Gao, of the Chinese Academy of Sciences, and Guangzhi Tong, of the Shanghai Veterinary Research Institute, CAAS, who gave us guidance and help with scientific research ideas during the years of the study. We also give many thanks to Xiufeng Wan, of the University of Mississippi, and Chuanling Qiao, of the Harbin Veterinary Research Institute, CAAS, who gave us technical support for our epidemiological survey.
The funding organizations had no role in the study design, data collection and analysis, ownership of the materials, or preparation of the manuscript.
There are no competing interests to declare.
Footnotes
Published ahead of print 21 November 2012
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