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Serological reports of human infections of H7 and H9 avian influenza viruses in northern China
Journal of Clinical Virology 44 (2009) 225–229
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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Serological reports of human infections of H7 and H9 avian influenza viruses in northern China Na Jia a , Sake J. de Vlas c , Yun-Xi Liu d , Jiu-Song Zhang a , Lin Zhan a , Rong-Li Dang e , Yong-Hong Ma e , Xian-Jun Wang f , Ti Liu f , Guo-Ping Yang g , Qing-Li Wen g , Jan H. Richardus c , Shan Lu b,h,∗∗ , Wu-Chun Cao a,∗ a
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 20 Dong-Da Street, Fengtai District, Beijing 100071, PR China Jiangsu Key Laboratory of Infectious Diseases and China–US Vaccine Research Center, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China c Department of Public Health, Erasmus MC, University Medical Center, Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands d Chinese PLA General Hospital, 28 Fu-Xing Road, Hai-Dian District, Beijing 10853, PR China e Center for Disease Control and Prevention in Xinjiang Military Command, Xinjiang Uygur Autonomous Region, 83000, PR China f Center for Disease Control and Prevention in Shandong, Jinan 250014, PR China g Center for Disease Control and Prevention in Liaoning Military Command, Shenyang 110034, PR China h Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA b
a r t i c l e
i n f o
Article history: Received 3 September 2008 Received in revised form 4 December 2008 Accepted 17 December 2008 Keywords: Avian influenza Sero-prevalence H7 subtype H9 subtype
a b s t r a c t Background: H7 and H9 subtype avian influenza viruses pose a similar threat to humans as H5 virus. Objectives: This study aims to identify the potential existence of H7 and H9 avian influenza infections in farmers and in poultry workers in northern China regions with highly pathogenic avian influenza (HPAI) H5N1 outbreaks. Study design: Sera were collected from farmers in Xinjiang Uygur autonomous region and Liaoning province and poultry workers in Shandong province. Sera from healthy residents in Shanxi province were used as the controls. H7 and H9 virus infections were examined by hemagglutination inhibition (HI) assay using horse erythrocytes. The titer equal to or greater than 1:160 was considered positive. Results: A total of 583 sera collected from farmers in Xinjiang were tested, and 10 (1.7%) were positive for H9 virus infection. Out of 200 sera collected from Liaoning, two (1.0%) were infected by H9 virus. No H7 virus infection was detected in the above serum samples. Neither H7 nor H9 virus infection was identified in 277 poultry workers of Shandong and in 407 residents of Shanxi. Conclusions: Although H9 virus infection was limited in farmers from Xinjiang and Liaoning, a public health alert is needed as novel pandemic influenza strains may develop unnoticed given the presence of subclinical infections, and the possibility of re-assortment with prevailing H5N1 virus in these regions. Published by Elsevier B.V.
1. Introduction Influenza A viruses are classified on the basis of two surface proteins, i.e. the hemagglutinin (HA) and neuraminidase (NA).1 Among the 16 HA subtype viruses detected in wild birds and poultry throughout the world,2 H7 and H9 subtypes have generated significant concern for potential of human infection3 besides the H5N1
Abbreviations: HPAI, highly pathogenic avian influenza; HI, hemagglutination inhibition; Xinjiang, Xinjiang Uygur Autonomous Region of China. ∗ Corresponding author. Tel.: +86 10 63896082; fax: +86 10 63896082. ∗∗ Corresponding author. Tel.: +1 508 856 6791; fax: +1 508 856 6751. E-mail addresses: [email protected] (S. Lu), [email protected] (W.-C. Cao). 1386-6532/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jcv.2008.12.014
viruses which have caused worldwide threat to humans.4 Different from the severe illness and high case fatality of highly pathogenic avian influenza (HPAI) H5N1 virus,5 conjunctivitis and/or mild respiratory disease were common when humans are infected with H7 and H9 subtype viruses.6,7 However, H7 and H9 avian influenza viruses also pose a similar threat as H5 to human population because the nature of mild infections may allow the virus a greater opportunity to become more virulent through surreptitious spread and mutation or reassortment with influenza viruses of other serotypes.8,9 As only mild manifestations presented, few cases with H7 or H9 virus infection were reported.6 The knowledge is insufficient for understanding these viruses’ ability to transmit to humans. We hypothesize that human infections with H7 and H9 influenza may occur in areas with frequently reported outbreaks of HPAI H5N1 infections in poultry, as the special territorial and social
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Fig. 1. Survey sites in northern China where the sero-epidemiological study was conducted.
environments in these regions may have become favorable to the transmission of avian influenza viruses. Since 2004, HPAI H5N1 outbreaks have been repeatedly reported in Xinjiang Uygur Autonomous Region of China10 and in Liaoning province.11 Human cases of HPAI H5N1 infection were diagnosed in both regions after poultry outbreaks.11,12 In the two regions, agriculture plays a critical role in economic development and many families raise free-range domestic poultry. In addition, since 1996, H9N2 viruses were frequently isolated from a wide range of hosts, such as chicken and swine through routine surveillance in another northern China province, Shandong.13–15 It is thought that people directly exposing to live poultry as a work-duty run high risk for infection with H7 or H9 subtype avian influenza viruses. In this study, we conducted an epidemiological survey to detect H7 and H9 infections in farmers in Xinjiang and Liaoning where HPAI H5N1 were reported in both poultry and humans, and in professional poultry workers in Shandong. The same assay was performed in healthy residents from Shanxi, another province of northern China where no avian H5N1 outbreak occurred for comparison. 2. Methods
Heishan City (Fig. 1). HPAI H5N1 outbreaks had been reported in the above five villages in December 2005.10,11 These villages consist of a typical rural Chinese setting where the villagers usually raise various animals such as poultry, pigs, dogs and cows around their houses. We invited all residents of age 5 and above to participate in the study. All participants were interviewed with a standard questionnaire on demographic characteristics and history of poultry contact. Peripheral blood samples (5 ml each) were obtained once by trained healthcare workers. Volunteer sera were stored at −20 ◦ C until use. In April 2006, serological survey was also conducted in two poultry farms (Fig. 1) where about ten thousands of poultry were raised in cages, and in one poultry product processing factory where live poultry was slaughtered. These factories are located in Shandong province where H9N2 viruses were frequently isolated from a range of hosts.13–15 We did a cluster sampling. Sera samples were collected from the poultry workers. As a control, serum samples were collected from residents in Shanxi province (Fig. 1), a region where no avian influenza had been reported. This study protocol was reviewed and approved by the Institutional Review Board at the China Center for Disease Control and Prevention. An informed consent was provided to and signed by each participant.
2.1. Study protocol and sample collection 2.2. Hemagglutination inhibition (HI) assays The study was conducted in April 2006 in 2 villages around Ulumuqi, the capital city of Xinjiang, and in a village nearby a satellite city, Miquan. In February 2008, a similar survey was carried out in two villages in Liaoning, one was in Fuxin City and another was in
The influenza virus used for the HI assay was A/African Starling/England-Q/938/79(H7N1) and A/Chicken/Shanghai/10/01 (H9N2), provided by the Ha’erbin Veterinarian Institute, Chinese
N. Jia et al. / Journal of Clinical Virology 44 (2009) 225–229
227
Table 1 Characteristics of participants in the serological survey in northern China. Locationsa
Groups
XJ
Farmers living in H5N1 poultry outbreak areas
583 40 (5–87)
299 (51)
LN
As above
200 42 (15–72)
99 (49.5)
SD
Poultry workers
277 24 (17–60)
158 (57.0)
SX
Residents in an area without H5N1 poultry outbreak
407 32 (9–80)
161 (39.6)
No.
Demographic description Median of age (range)
a
No. of female (%)
Occupation (%) Farmer: 401(70) Else: 172 (30) Farmer: 164 (82) Else: 36 (18) Poultry farmer: 75 (27.1) Slaughterer: 10 (3.6) Processor: 192 (69.3) Farmer: 120 (30) Else: 287 (70)
Poultry contact (%) 374 (64.2) 134 (67) 277 (100) –
XJ: Xinjiang Uygur autonomous regions; LN: Liaoning province; SD: Shandong province; SX: Shanxi province.
Academy of Agriculture Science. Sera were treated with receptor destroying enzyme (RDE) and absorbed with the erythrocytes to remove non-specific hemagglutination. Our HI assay used horse erythrocytes instead of turkey red blood cells, in order to increase the assay sensitivity.16 This assay was carried out following the WHO influenza laboratory procedures, with a modification as reported by Meijer et al.16 The sensitivity and specificity of the horse erythrocytes HI assay detecting avian influenza antibodies was validated using a panel of immunized rabbit sera in our previous study.17
3. Results The total numbers of volunteers participating the current study were 583, 200, and 407 from Xinjiang (XJ), Liaoning (LN) and Shanxi (SX), respectively (Table 1). The age distribution in the three regions was comparable. About half of participants were female among participants in XJ and LN, and about 40% were female in SX province. The majority of volunteers from XJ and LN provinces were farmers, representing 70% and 82% of the total volunteers, respectively. On the other hand, only 30% from SX control group were farmers and the remaining 70% worked in schools, small factories, restaurants and hotels. Most villagers from XJ and LN were directly exposed to poultry around their household, and chicken represented the most frequently contacted species, followed by waterfowl (ducks or geese) and other birds. A total of 75 workers from two poultry farms and 202 from a poultry product processing factory, all located in Shandong province (SD), took part in the study. The age in this group was relatively young (median was 24) due to the work-related duties and 57% were female. In the poultry factory, most of the workers were involved in poultry product and a small number of them were responsible for slaughtering live poultry (Table 1). The sanitation in the poultry farms and factory was good in general and the workers were required to wear respirator, glove and protective clothing when they were exposed to poultry.
In the current study, an HI titer of 1:160 or greater was considered positive, which was also the standard used in other serological identifications of human infection of avian influenza A (H5N1) viruses.18 We found that 10 (1.7%) and 2 (1.0%) of the samples had a positive response to the H9 virus in XJ and in LN groups, respectively. While no positive sample in SD and in SX groups was identified. In addition, none of the samples was positive for H7 virus infection (Table 2). Based on the questionnaire, age, gender and history of poultry contact were all not statistically associated with H9 virus infection. Although no significant association between H9 positive antibodies response and exposure to dead or ill poultry, 3 of 4 subjects who had the highest HI titers against H9 virus (greater than 1:320) from XJ and LN provinces reported exposure to dead or ill poultry at their homes (Table 2). 4. Discussion In the current study, human infection with H9 virus was identified in farmers from Xinjiang and Liaoning where HPAI H5N1 infection in poultry and humans have been reported. Our study indicated that H9 and H5 avian influenza viruses may co-circulate in the two regions, where both serotypes infected the human. On the other hand, there was no detectable serological evidence to suggest H7 virus infection. The limitation of the study is that only HI assay was applied for serological test. As there is no established HI titer standard to detect a mild or asymptomatic H7 or H9 influenza infection, we used WHO HI criteria for detecting human H5N1 infection by a single serum sample collection.18 The lower titers might result from cross-reactivity with previous human influenza virus infections,19 or could be the result of asymptomatic influenza infections because mild human infections may not result in marked antibody responses.20 Furthermore, these results could reflect the decay of antibody titers over time.21 The primary objective of this study is not to investigate the precise prevalence of H7 or H9 human infection, rather to investigate if there is any possible H7 or H9 infec-
Table 2 Antibody titers of H9 and H7 viruses infections using hemagglutination inhibition (HI) assays. Viral strains
Study sitesa
No.
H9
XJ LN SD SX XJ LN SD SX
583 200 277 407 583 200 277 407
H7
a
Sum of titer ≥1:160 (%)
HI titer (%) <1:20
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
501 (85.9) 195 (97.5) 272 (98.2) 384 (94.3) 531 (91.1) 188 (94) 266 (96.0) 406 (99.8)
36 (6.2) 3 (1.5) 3 (1.1) 21 (5.2) 36 (6.2) 10 (5) 11 (4) 1 (0.2)
20 (3.4) 0 (0) 1 (0.4) 0 (0) 12 (2.1) 2 (1) 0 (0) 0 (0)
16 (2.7) 0 (0) 1 (0.4) 2 (0.5) 4 (0.7) 0 (0) 0 (0) 0 (0)
8 (1.4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
1 (0.2) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
1 (0.2) 1 (0.5) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
0(0) 1 (0.5) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
XJ: Xinjiang Uygur autonomous regions; LN: Liaoning province; SD: Shandong province; SX: Shanxi province.
10 (1.7) 2 (1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
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tion among people of risk, so a conservative cut-off value of HI titer was used. Historically, low pathogenic avian influenza (LPAI) viruses have not been the priority, however, LPAI H9N2 virus infections in poultry, mainly in chicken, have occurred in many countries since the mid 1990s.22 H9N2 viruses were isolated from 5 influenza like illness patients in southeastern China in 1998,23 from 2 children in Hong Kong in 1999,24 and one more child in 2003.25 It should be noted that H9N2 viruses isolated from poultry in Asia have human influenza virus-like receptor specificity, indicating an enhanced propensity for introduction into humans.26,27 If H9N2 viruses can succeed in transmitting between humans, these viruses will have more chance as the source of new influenza pandemic.6 Human infections with the H7 serotype have been reported. The earliest report of infection from avian source was in the United Kingdom in 1995.28 In 2003, 89 H7 human infections were reported in the Netherlands, of which one veterinarian died from respiratory distress.29 At the same time, the H7 virus was circulating in Italy, and 3.8% (7 out of 185) poultry workers had serological evidence of infection.30 One year later, two human cases of H7 infection were reported in Canada.8 Recently, a LPAI influenza A/H7N2 virus infection was reported in 4 exposed individuals during the poultry outbreak in UK.31 There has been only limited H7 virus isolated in China, and the first report was in 2002 when a routine surveillance was carried out in poultry in northern China32 . A previous seroepidemiological study on H7 infection was carried out in southern China, with a positive rate of 25%.33 In another study, HI assay was used to test antibodies against H9N2 avian influenza virus. The rate of positive HI titer ≥ 20 was about 13% in poultry workers in Shenzhen, a city in southern China.34 Until now, there was very limited information on H7 and H9 infections from the northern provinces. The current report identified possible H9 infections in humans in northern China. In Liaoning province, H9N2 virus was isolated from chicken in 1999 and 2000.14,15 Although there was no direct report of H9 virus isolated in Xinjiang, its adjacent regions, such as Pakistan and Central Asia countries, H9N2 viruses have been responsible for several outbreaks in poultry.35,36 These countries are part of an important bird migratory flyway, called the “East Africa–West Asia” flyway,37 making these birds a potential source for the rapid spread of influenza virus strains to other areas. In the study H5N1 and H9N2 influenza viruses were found cocirculating in the poultry markets in Hong Kong in 1997, and the six genes encoding the internal components of these H9N2 viruses were similar to those of the H5N1 genes isolated from both human and avian sources. This feature highlighted the potential mechanism for the emergence of avian influenza viruses that may infect humans.38,39 It is not clear whether the positive H9-specific HI antibodies had any relationship to the co-circulating of both H5 and H9 viruses in the study sites. The risk of human infections with avian influenza was thought greater through contact with outdoor-reared domestic poultry than with indoor commercial or industrial poultry where biosecurity and worker protection are generally higher.40 Our report confirmed that no serological evidence of human infection with H7 or H9 subtype avian influenza was detected in poultry workers. It is likely that reasonable protection measures and good working environment have efficiently prevented them from the infection. However, the use of gloves and respirators was inconsistent and infrequent, making it unlikely that we would be able to detect any effect of personal protective equipment using statistical methods. In conclusion, we have demonstrated H9 sero-positivity in northern China. Although no documented clinical cases of H9 infection were reported, it does not necessarily indicate a lower pandemic potential considering the possibility of mild or subclinical infections. The co-circulation of two influenza subtypes, H5 and H9, in these regions implies that there is a risk for virus
reassortment. Thus, extensive public health surveillance needs to be established and maintained in areas where low-level avian influenza infections may exist. Competing interests The authors declare that they have no competing interests. Acknowledgements We thank Dr. Jill M. Grimes-Serrano (UMass Medical School) for editing the manuscript. This study was supported by the grants of Natural Science Fund for Distinguished Young Scholars (30725032), Natural Science for Major International Joint Research Project (30810103903), China’s National High-tech 863 Program (2006AA02Z457), China’s National Scientific and Technical Supporting Program (2006BAD06A02), the Foundation for State Key Laboratory of Pathogen and Biosecurity (200701), a Jiangsu Province Natural Science Foundation Major Research Grant (BK2006728), and Jiangsu Province ID Key Laboratory Program. References 1. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiol Rev 1992;56:152–79. 2. Fouchier RA, Munster V, Wallensten A, Bestebroer TM, Herfst S, Smith D, et al. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 2005;79:2814–22. 3. Claas EC, Osterhaus AD. New clues to the emergence of flu pandemics. Nat Med 1998;4:1122–3. 4. Monto AS. The threat of an avian influenza pandemic. N Engl J Med 2005;352:323–5. 5. Abdel-Ghafar AN, Chotpitayasunondh T, Gao Z, Hayden FG, Nguyen DH, de Jong MD, et al. Update on avian influenza A (H5N1) virus infection in humans. N Engl J Med 2008;358:261–73. 6. Lipatov AS, Govorkova EA, Webby RJ, Ozaki H, Peiris M, Guan Y, et al. Influenza: emergence and control. J Virol 2004;78:8951–9. 7. Wong SS, Yuen KY. Avian influenza virus infections in humans. Chest 2006;129:156–68. 8. Tweed SA, Skowronski DM, David ST, Larder A, Petric M, Lees W, et al. Human illness from avian influenza H7N3, British Columbia. Emerg Infect Dis 2004;10:2196–9. 9. Li KS, Xu KM, Peiris JS, Poon LL, Yu KZ, Yuen KY, et al. Characterization of H9 subtype influenza viruses from the ducks of southern China: a candidate for the next influenza pandemic in humans? J Virol 2003;77:6988–94. 10. World Health Organisation. Avian influenza A (H5N1) – update 15: additional confirmed human case in Thailand; China announces suspected spread of infection in poultry; investigation of possible human-to-human transmission; 2004 February 2. http://www.who.int/csr/don/2004 02 02/en/index.html [accessed April 2006]. 11. World Health Organisation. Avian influenza – situation in China, Thailand – update 47. http://www.who.int/csr/don/2005 12 09/en/index.html [accessed April 2006]. 12. World Health Organisation. Avian influenza – situation in China – update 14. http://www.who.int/csr/don/2006 08 14/en/index.html [accessed October 2007]. 13. Cong YL, Pu J, Liu QF, Wang S, Zhang GZ, Zhang XL, et al. Antigenic and genetic characterization of H9N2 swine influenza viruses in China. J Gen Virol 2007;88:2035–41. 14. Liu H, Liu X, Cheng J, Peng D, Jia L, Huang Y. Phylogenetic analysis of the hemagglutinin genes of twenty-six avian influenza viruses of subtype H9N2 isolated from chickens in China during 1996–2001. Avian Dis 2003;47:116–27. 15. Liu JH, Okazaki K, Mweene A, Shi WM, Wu QM, Su JL, et al. Genetic conservation of hemagglutinin gene of H9 influenza virus in chicken population in Mainland China. Virus Genes 2004;29:329–34. 16. Meijer A, Bosman A, van de Kamp EE, Wilbrink B, van Beest Holle Mdu R, Koopmans M. Measurement of antibodies to avian influenza virus A(H7N7) in humans by hemagglutination inhibition test. J Virol Methods 2006;132:113– 20. 17. Jia N, Wang SX, Liu YX, Zhang PH, Zuo SQ, Lin Z, et al. Increased sensitivity for detecting avian influenza-specific antibodies by a modified hemagglutination inhibition assay using horse erythrocytes. J Virol Methods 2008;153:43– 8. 18. World Health Organization. Recommendations and laboratory procedures for detection of avian influenza A(H5N1) virus in specimens from suspected human cases. http://www.who.int/csr/disease/avian influenza/guidelines/labtests/ en/index.html [accessed December 2007]. 19. Couch RB. An overview of serum antibody responses to influenza virus antigens. Dev Biol (Basel) 2003;115:25–30.
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Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Serological reports of human infections of H7 and H9 avian influenza viruses in northern China Na Jia a , Sake J. de Vlas c , Yun-Xi Liu d , Jiu-Song Zhang a , Lin Zhan a , Rong-Li Dang e , Yong-Hong Ma e , Xian-Jun Wang f , Ti Liu f , Guo-Ping Yang g , Qing-Li Wen g , Jan H. Richardus c , Shan Lu b,h,∗∗ , Wu-Chun Cao a,∗ a
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 20 Dong-Da Street, Fengtai District, Beijing 100071, PR China Jiangsu Key Laboratory of Infectious Diseases and China–US Vaccine Research Center, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China c Department of Public Health, Erasmus MC, University Medical Center, Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands d Chinese PLA General Hospital, 28 Fu-Xing Road, Hai-Dian District, Beijing 10853, PR China e Center for Disease Control and Prevention in Xinjiang Military Command, Xinjiang Uygur Autonomous Region, 83000, PR China f Center for Disease Control and Prevention in Shandong, Jinan 250014, PR China g Center for Disease Control and Prevention in Liaoning Military Command, Shenyang 110034, PR China h Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA b
a r t i c l e
i n f o
Article history: Received 3 September 2008 Received in revised form 4 December 2008 Accepted 17 December 2008 Keywords: Avian influenza Sero-prevalence H7 subtype H9 subtype
a b s t r a c t Background: H7 and H9 subtype avian influenza viruses pose a similar threat to humans as H5 virus. Objectives: This study aims to identify the potential existence of H7 and H9 avian influenza infections in farmers and in poultry workers in northern China regions with highly pathogenic avian influenza (HPAI) H5N1 outbreaks. Study design: Sera were collected from farmers in Xinjiang Uygur autonomous region and Liaoning province and poultry workers in Shandong province. Sera from healthy residents in Shanxi province were used as the controls. H7 and H9 virus infections were examined by hemagglutination inhibition (HI) assay using horse erythrocytes. The titer equal to or greater than 1:160 was considered positive. Results: A total of 583 sera collected from farmers in Xinjiang were tested, and 10 (1.7%) were positive for H9 virus infection. Out of 200 sera collected from Liaoning, two (1.0%) were infected by H9 virus. No H7 virus infection was detected in the above serum samples. Neither H7 nor H9 virus infection was identified in 277 poultry workers of Shandong and in 407 residents of Shanxi. Conclusions: Although H9 virus infection was limited in farmers from Xinjiang and Liaoning, a public health alert is needed as novel pandemic influenza strains may develop unnoticed given the presence of subclinical infections, and the possibility of re-assortment with prevailing H5N1 virus in these regions. Published by Elsevier B.V.
1. Introduction Influenza A viruses are classified on the basis of two surface proteins, i.e. the hemagglutinin (HA) and neuraminidase (NA).1 Among the 16 HA subtype viruses detected in wild birds and poultry throughout the world,2 H7 and H9 subtypes have generated significant concern for potential of human infection3 besides the H5N1
Abbreviations: HPAI, highly pathogenic avian influenza; HI, hemagglutination inhibition; Xinjiang, Xinjiang Uygur Autonomous Region of China. ∗ Corresponding author. Tel.: +86 10 63896082; fax: +86 10 63896082. ∗∗ Corresponding author. Tel.: +1 508 856 6791; fax: +1 508 856 6751. E-mail addresses: [email protected] (S. Lu), [email protected] (W.-C. Cao). 1386-6532/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.jcv.2008.12.014
viruses which have caused worldwide threat to humans.4 Different from the severe illness and high case fatality of highly pathogenic avian influenza (HPAI) H5N1 virus,5 conjunctivitis and/or mild respiratory disease were common when humans are infected with H7 and H9 subtype viruses.6,7 However, H7 and H9 avian influenza viruses also pose a similar threat as H5 to human population because the nature of mild infections may allow the virus a greater opportunity to become more virulent through surreptitious spread and mutation or reassortment with influenza viruses of other serotypes.8,9 As only mild manifestations presented, few cases with H7 or H9 virus infection were reported.6 The knowledge is insufficient for understanding these viruses’ ability to transmit to humans. We hypothesize that human infections with H7 and H9 influenza may occur in areas with frequently reported outbreaks of HPAI H5N1 infections in poultry, as the special territorial and social
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N. Jia et al. / Journal of Clinical Virology 44 (2009) 225–229
Fig. 1. Survey sites in northern China where the sero-epidemiological study was conducted.
environments in these regions may have become favorable to the transmission of avian influenza viruses. Since 2004, HPAI H5N1 outbreaks have been repeatedly reported in Xinjiang Uygur Autonomous Region of China10 and in Liaoning province.11 Human cases of HPAI H5N1 infection were diagnosed in both regions after poultry outbreaks.11,12 In the two regions, agriculture plays a critical role in economic development and many families raise free-range domestic poultry. In addition, since 1996, H9N2 viruses were frequently isolated from a wide range of hosts, such as chicken and swine through routine surveillance in another northern China province, Shandong.13–15 It is thought that people directly exposing to live poultry as a work-duty run high risk for infection with H7 or H9 subtype avian influenza viruses. In this study, we conducted an epidemiological survey to detect H7 and H9 infections in farmers in Xinjiang and Liaoning where HPAI H5N1 were reported in both poultry and humans, and in professional poultry workers in Shandong. The same assay was performed in healthy residents from Shanxi, another province of northern China where no avian H5N1 outbreak occurred for comparison. 2. Methods
Heishan City (Fig. 1). HPAI H5N1 outbreaks had been reported in the above five villages in December 2005.10,11 These villages consist of a typical rural Chinese setting where the villagers usually raise various animals such as poultry, pigs, dogs and cows around their houses. We invited all residents of age 5 and above to participate in the study. All participants were interviewed with a standard questionnaire on demographic characteristics and history of poultry contact. Peripheral blood samples (5 ml each) were obtained once by trained healthcare workers. Volunteer sera were stored at −20 ◦ C until use. In April 2006, serological survey was also conducted in two poultry farms (Fig. 1) where about ten thousands of poultry were raised in cages, and in one poultry product processing factory where live poultry was slaughtered. These factories are located in Shandong province where H9N2 viruses were frequently isolated from a range of hosts.13–15 We did a cluster sampling. Sera samples were collected from the poultry workers. As a control, serum samples were collected from residents in Shanxi province (Fig. 1), a region where no avian influenza had been reported. This study protocol was reviewed and approved by the Institutional Review Board at the China Center for Disease Control and Prevention. An informed consent was provided to and signed by each participant.
2.1. Study protocol and sample collection 2.2. Hemagglutination inhibition (HI) assays The study was conducted in April 2006 in 2 villages around Ulumuqi, the capital city of Xinjiang, and in a village nearby a satellite city, Miquan. In February 2008, a similar survey was carried out in two villages in Liaoning, one was in Fuxin City and another was in
The influenza virus used for the HI assay was A/African Starling/England-Q/938/79(H7N1) and A/Chicken/Shanghai/10/01 (H9N2), provided by the Ha’erbin Veterinarian Institute, Chinese
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227
Table 1 Characteristics of participants in the serological survey in northern China. Locationsa
Groups
XJ
Farmers living in H5N1 poultry outbreak areas
583 40 (5–87)
299 (51)
LN
As above
200 42 (15–72)
99 (49.5)
SD
Poultry workers
277 24 (17–60)
158 (57.0)
SX
Residents in an area without H5N1 poultry outbreak
407 32 (9–80)
161 (39.6)
No.
Demographic description Median of age (range)
a
No. of female (%)
Occupation (%) Farmer: 401(70) Else: 172 (30) Farmer: 164 (82) Else: 36 (18) Poultry farmer: 75 (27.1) Slaughterer: 10 (3.6) Processor: 192 (69.3) Farmer: 120 (30) Else: 287 (70)
Poultry contact (%) 374 (64.2) 134 (67) 277 (100) –
XJ: Xinjiang Uygur autonomous regions; LN: Liaoning province; SD: Shandong province; SX: Shanxi province.
Academy of Agriculture Science. Sera were treated with receptor destroying enzyme (RDE) and absorbed with the erythrocytes to remove non-specific hemagglutination. Our HI assay used horse erythrocytes instead of turkey red blood cells, in order to increase the assay sensitivity.16 This assay was carried out following the WHO influenza laboratory procedures, with a modification as reported by Meijer et al.16 The sensitivity and specificity of the horse erythrocytes HI assay detecting avian influenza antibodies was validated using a panel of immunized rabbit sera in our previous study.17
3. Results The total numbers of volunteers participating the current study were 583, 200, and 407 from Xinjiang (XJ), Liaoning (LN) and Shanxi (SX), respectively (Table 1). The age distribution in the three regions was comparable. About half of participants were female among participants in XJ and LN, and about 40% were female in SX province. The majority of volunteers from XJ and LN provinces were farmers, representing 70% and 82% of the total volunteers, respectively. On the other hand, only 30% from SX control group were farmers and the remaining 70% worked in schools, small factories, restaurants and hotels. Most villagers from XJ and LN were directly exposed to poultry around their household, and chicken represented the most frequently contacted species, followed by waterfowl (ducks or geese) and other birds. A total of 75 workers from two poultry farms and 202 from a poultry product processing factory, all located in Shandong province (SD), took part in the study. The age in this group was relatively young (median was 24) due to the work-related duties and 57% were female. In the poultry factory, most of the workers were involved in poultry product and a small number of them were responsible for slaughtering live poultry (Table 1). The sanitation in the poultry farms and factory was good in general and the workers were required to wear respirator, glove and protective clothing when they were exposed to poultry.
In the current study, an HI titer of 1:160 or greater was considered positive, which was also the standard used in other serological identifications of human infection of avian influenza A (H5N1) viruses.18 We found that 10 (1.7%) and 2 (1.0%) of the samples had a positive response to the H9 virus in XJ and in LN groups, respectively. While no positive sample in SD and in SX groups was identified. In addition, none of the samples was positive for H7 virus infection (Table 2). Based on the questionnaire, age, gender and history of poultry contact were all not statistically associated with H9 virus infection. Although no significant association between H9 positive antibodies response and exposure to dead or ill poultry, 3 of 4 subjects who had the highest HI titers against H9 virus (greater than 1:320) from XJ and LN provinces reported exposure to dead or ill poultry at their homes (Table 2). 4. Discussion In the current study, human infection with H9 virus was identified in farmers from Xinjiang and Liaoning where HPAI H5N1 infection in poultry and humans have been reported. Our study indicated that H9 and H5 avian influenza viruses may co-circulate in the two regions, where both serotypes infected the human. On the other hand, there was no detectable serological evidence to suggest H7 virus infection. The limitation of the study is that only HI assay was applied for serological test. As there is no established HI titer standard to detect a mild or asymptomatic H7 or H9 influenza infection, we used WHO HI criteria for detecting human H5N1 infection by a single serum sample collection.18 The lower titers might result from cross-reactivity with previous human influenza virus infections,19 or could be the result of asymptomatic influenza infections because mild human infections may not result in marked antibody responses.20 Furthermore, these results could reflect the decay of antibody titers over time.21 The primary objective of this study is not to investigate the precise prevalence of H7 or H9 human infection, rather to investigate if there is any possible H7 or H9 infec-
Table 2 Antibody titers of H9 and H7 viruses infections using hemagglutination inhibition (HI) assays. Viral strains
Study sitesa
No.
H9
XJ LN SD SX XJ LN SD SX
583 200 277 407 583 200 277 407
H7
a
Sum of titer ≥1:160 (%)
HI titer (%) <1:20
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
501 (85.9) 195 (97.5) 272 (98.2) 384 (94.3) 531 (91.1) 188 (94) 266 (96.0) 406 (99.8)
36 (6.2) 3 (1.5) 3 (1.1) 21 (5.2) 36 (6.2) 10 (5) 11 (4) 1 (0.2)
20 (3.4) 0 (0) 1 (0.4) 0 (0) 12 (2.1) 2 (1) 0 (0) 0 (0)
16 (2.7) 0 (0) 1 (0.4) 2 (0.5) 4 (0.7) 0 (0) 0 (0) 0 (0)
8 (1.4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
1 (0.2) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
1 (0.2) 1 (0.5) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
0(0) 1 (0.5) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
XJ: Xinjiang Uygur autonomous regions; LN: Liaoning province; SD: Shandong province; SX: Shanxi province.
10 (1.7) 2 (1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
228
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tion among people of risk, so a conservative cut-off value of HI titer was used. Historically, low pathogenic avian influenza (LPAI) viruses have not been the priority, however, LPAI H9N2 virus infections in poultry, mainly in chicken, have occurred in many countries since the mid 1990s.22 H9N2 viruses were isolated from 5 influenza like illness patients in southeastern China in 1998,23 from 2 children in Hong Kong in 1999,24 and one more child in 2003.25 It should be noted that H9N2 viruses isolated from poultry in Asia have human influenza virus-like receptor specificity, indicating an enhanced propensity for introduction into humans.26,27 If H9N2 viruses can succeed in transmitting between humans, these viruses will have more chance as the source of new influenza pandemic.6 Human infections with the H7 serotype have been reported. The earliest report of infection from avian source was in the United Kingdom in 1995.28 In 2003, 89 H7 human infections were reported in the Netherlands, of which one veterinarian died from respiratory distress.29 At the same time, the H7 virus was circulating in Italy, and 3.8% (7 out of 185) poultry workers had serological evidence of infection.30 One year later, two human cases of H7 infection were reported in Canada.8 Recently, a LPAI influenza A/H7N2 virus infection was reported in 4 exposed individuals during the poultry outbreak in UK.31 There has been only limited H7 virus isolated in China, and the first report was in 2002 when a routine surveillance was carried out in poultry in northern China32 . A previous seroepidemiological study on H7 infection was carried out in southern China, with a positive rate of 25%.33 In another study, HI assay was used to test antibodies against H9N2 avian influenza virus. The rate of positive HI titer ≥ 20 was about 13% in poultry workers in Shenzhen, a city in southern China.34 Until now, there was very limited information on H7 and H9 infections from the northern provinces. The current report identified possible H9 infections in humans in northern China. In Liaoning province, H9N2 virus was isolated from chicken in 1999 and 2000.14,15 Although there was no direct report of H9 virus isolated in Xinjiang, its adjacent regions, such as Pakistan and Central Asia countries, H9N2 viruses have been responsible for several outbreaks in poultry.35,36 These countries are part of an important bird migratory flyway, called the “East Africa–West Asia” flyway,37 making these birds a potential source for the rapid spread of influenza virus strains to other areas. In the study H5N1 and H9N2 influenza viruses were found cocirculating in the poultry markets in Hong Kong in 1997, and the six genes encoding the internal components of these H9N2 viruses were similar to those of the H5N1 genes isolated from both human and avian sources. This feature highlighted the potential mechanism for the emergence of avian influenza viruses that may infect humans.38,39 It is not clear whether the positive H9-specific HI antibodies had any relationship to the co-circulating of both H5 and H9 viruses in the study sites. The risk of human infections with avian influenza was thought greater through contact with outdoor-reared domestic poultry than with indoor commercial or industrial poultry where biosecurity and worker protection are generally higher.40 Our report confirmed that no serological evidence of human infection with H7 or H9 subtype avian influenza was detected in poultry workers. It is likely that reasonable protection measures and good working environment have efficiently prevented them from the infection. However, the use of gloves and respirators was inconsistent and infrequent, making it unlikely that we would be able to detect any effect of personal protective equipment using statistical methods. In conclusion, we have demonstrated H9 sero-positivity in northern China. Although no documented clinical cases of H9 infection were reported, it does not necessarily indicate a lower pandemic potential considering the possibility of mild or subclinical infections. The co-circulation of two influenza subtypes, H5 and H9, in these regions implies that there is a risk for virus
reassortment. Thus, extensive public health surveillance needs to be established and maintained in areas where low-level avian influenza infections may exist. Competing interests The authors declare that they have no competing interests. Acknowledgements We thank Dr. Jill M. Grimes-Serrano (UMass Medical School) for editing the manuscript. This study was supported by the grants of Natural Science Fund for Distinguished Young Scholars (30725032), Natural Science for Major International Joint Research Project (30810103903), China’s National High-tech 863 Program (2006AA02Z457), China’s National Scientific and Technical Supporting Program (2006BAD06A02), the Foundation for State Key Laboratory of Pathogen and Biosecurity (200701), a Jiangsu Province Natural Science Foundation Major Research Grant (BK2006728), and Jiangsu Province ID Key Laboratory Program. References 1. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiol Rev 1992;56:152–79. 2. Fouchier RA, Munster V, Wallensten A, Bestebroer TM, Herfst S, Smith D, et al. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 2005;79:2814–22. 3. Claas EC, Osterhaus AD. New clues to the emergence of flu pandemics. Nat Med 1998;4:1122–3. 4. Monto AS. The threat of an avian influenza pandemic. N Engl J Med 2005;352:323–5. 5. Abdel-Ghafar AN, Chotpitayasunondh T, Gao Z, Hayden FG, Nguyen DH, de Jong MD, et al. Update on avian influenza A (H5N1) virus infection in humans. N Engl J Med 2008;358:261–73. 6. Lipatov AS, Govorkova EA, Webby RJ, Ozaki H, Peiris M, Guan Y, et al. Influenza: emergence and control. J Virol 2004;78:8951–9. 7. Wong SS, Yuen KY. Avian influenza virus infections in humans. Chest 2006;129:156–68. 8. Tweed SA, Skowronski DM, David ST, Larder A, Petric M, Lees W, et al. Human illness from avian influenza H7N3, British Columbia. Emerg Infect Dis 2004;10:2196–9. 9. Li KS, Xu KM, Peiris JS, Poon LL, Yu KZ, Yuen KY, et al. Characterization of H9 subtype influenza viruses from the ducks of southern China: a candidate for the next influenza pandemic in humans? J Virol 2003;77:6988–94. 10. World Health Organisation. Avian influenza A (H5N1) – update 15: additional confirmed human case in Thailand; China announces suspected spread of infection in poultry; investigation of possible human-to-human transmission; 2004 February 2. http://www.who.int/csr/don/2004 02 02/en/index.html [accessed April 2006]. 11. World Health Organisation. Avian influenza – situation in China, Thailand – update 47. http://www.who.int/csr/don/2005 12 09/en/index.html [accessed April 2006]. 12. World Health Organisation. Avian influenza – situation in China – update 14. http://www.who.int/csr/don/2006 08 14/en/index.html [accessed October 2007]. 13. Cong YL, Pu J, Liu QF, Wang S, Zhang GZ, Zhang XL, et al. Antigenic and genetic characterization of H9N2 swine influenza viruses in China. J Gen Virol 2007;88:2035–41. 14. Liu H, Liu X, Cheng J, Peng D, Jia L, Huang Y. Phylogenetic analysis of the hemagglutinin genes of twenty-six avian influenza viruses of subtype H9N2 isolated from chickens in China during 1996–2001. Avian Dis 2003;47:116–27. 15. Liu JH, Okazaki K, Mweene A, Shi WM, Wu QM, Su JL, et al. Genetic conservation of hemagglutinin gene of H9 influenza virus in chicken population in Mainland China. Virus Genes 2004;29:329–34. 16. Meijer A, Bosman A, van de Kamp EE, Wilbrink B, van Beest Holle Mdu R, Koopmans M. Measurement of antibodies to avian influenza virus A(H7N7) in humans by hemagglutination inhibition test. J Virol Methods 2006;132:113– 20. 17. Jia N, Wang SX, Liu YX, Zhang PH, Zuo SQ, Lin Z, et al. Increased sensitivity for detecting avian influenza-specific antibodies by a modified hemagglutination inhibition assay using horse erythrocytes. J Virol Methods 2008;153:43– 8. 18. World Health Organization. Recommendations and laboratory procedures for detection of avian influenza A(H5N1) virus in specimens from suspected human cases. http://www.who.int/csr/disease/avian influenza/guidelines/labtests/ en/index.html [accessed December 2007]. 19. Couch RB. An overview of serum antibody responses to influenza virus antigens. Dev Biol (Basel) 2003;115:25–30.
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