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Chinese Medical Journal, 2008, Vol. 121 No. 20: 2050-2053
Potential infections of H5N1 and H9N2 avian influenza do exist in Guangdong populations of China
LU Ci-yong, LU Jia-hai, CHEN Wei-qing, JIANG Li-fang, TAN Bing-yan, LING Wen-hua, ZHENG Bo-jian, SUI Hong-yan
Abstract:
Background - Southeast China is one of the sites of influenza origin. During 2003?2004, nine avian influenza outbreaks took place in Guangdong Province. But no human case was reported. To examine the status of potential human infection by human influenza (H1N1, H3N2) and avian influenza (H5N1, H7N7, H9N2) in the avian influenza epidemic area of Guangdong Province, China, we conducted a seroepidemiologic survey in the people of this area from April to June of 2004.
Methods - Three out of 9 H5N1 avian influenza affected poultry areas in Guangdong were randomly selected, and the population living within 3 kilometers of the affected poultries were chosen as the survey subjects. One thousand two hundred and fourteen people were selected from 3 villages at random. Human and avian influenza antibody titers were determined by hemagglutination-inhibition (HI) test and microneutralization test (MNT).
Results - The positive rate of antibody to H5N1 was 3.03% in the occupational exposure group and 2.34% in general citizens group; that of H9N2 was 9.52% in the occupational exposure group and 3.76% in the general citizens group. Moreover one case in the occupational exposure group was positive for H7N7. One year later, all previously positive cases had become negative except for one H5N1-positive case.
Conclusion - The observations imply that H5N1 and H9N2 avian influenza silent infections exist in Guangdong populations.
Avian influenza is an infectious disease of birds caused by type A influenza virus. The epicenters of both Asian influenza pandemic of 1957 and Hong Kong influenza pandemic of 1968 were in Southeast Asia;<sup>1</sup> and multiple clades of H5N1 influenza virus have already emerged in this region. In 1996, the avian H5N1 virus was first detected in Guangdong Province, China. The HA gene is genetically similar to that of the H5N1 viruses isolated in Hong Kong in 1997, which killed 6 of 18 infected persons.<sup>2</sup> The culling of all poultry in Hong Kong ended the first wave of H5N1, but the virus continued to circulate among apparently healthy ducks in the coastal provinces of China.<sup>3</sup>
The human influenza H5N1 in Southeast Asia has paralleled large outbreaks of avian influenza H5N1. Most patients had well-documented exposure to sick or dead poultry.<sup>4</sup> The frequency of human infection has not been determined, and seroprevalence studies are urgently needed. The expanding geographic distribution of H5N1 avian influenza infections, with outbreaks in Kazakstan, Mongolia and Russia, indicates that more human populations are at risk.<sup>5</sup>
In addition to H5N1 subtypes, a H7N7 avian influenza outbreak was also reported in the Netherlands, causing 83 poultry workers and 3 family members to fall ill in 2003.<sup>6</sup> The H9N2 subtype capable of infecting human was isolated from throat swab specimens of the patients suffering from influenza-like symptoms in South China in 1998<sup>7</sup> and in Hong Kong (China) in 1999 and 2003.<sup>8</sup> The facts suggested that avian influenza virus could cross over the species barrier and infect human beings, which changed the traditional viewpoint that the receptor-specific avian influenza virus didn't directly transmit to human. Although there is no firm evidence of human-to-human transmission of avian influenza virus, it is nonetheless theoretically possible.
Incidents of human H5N1 avian influenza outbreaks have been reported in Fujian, Sichuan, Hunan, Jiangxi, Anhui, Shanghai, Zhejiang, Guangdong and Guangxi provinces in China from the end of 2003 to April 3, 2006.<sup>9</sup> However, it remains unclear how H5N1 avian influenza in China is transmitted to humans and whether infected persons will fall ill. Being close to Hong Kong, Guangdong Province has a similar climate and geographic environment. A prosperous poultry market gives rise to the potential of an epidemic or outbreak of human infection with H5N1 avian influenza virus. In order to better understand the status of H5N1 infection among people directly exposed to domestic poultry that were confirmed to be infected with the H5N1 avian influenza virus, a seroepidemiological study was carried out in this project from April to June of 2004 in Guangdong Province.
Virus strain
The inactivated strains of influenza A virus were provided by the Center for Disease Control and Prevention (CDC) of Guangdong Province. The antigens used in this study were H1N1 (A/newcaledonia/20/99), H3N2 (A/panama/ 2007/99), H5N1 (A/goose/Guangdong/1/96), H7N7 (A/afican/starling/Englan-q/983/79) and H9N2 (A/chicken/ shanghai/10/01), which were all standard diagnosis antigens. Cock red cells were used in the hemagglutination inhibition (HI) test.
Survey sites selection
Three out of nine H5N1 avian influenza affected areas of Guangdong were randomly selected, and 1214 people living within 3 kilometers of the affected poultries were chosen as the survey subjects. According to these criteria, we selected subjects from 3 villages at random.
Subjects and grouping
The occupational exposure group included 231 people who were responsible for raising, selling, and slaughtering chicken and ducks in H5N1 outbreak areas. The general citizen group included 983 people who lived in H5N1 outbreak areas but did not engage in actives of handling living chicken and ducks. About 300 people (>3 years) were randomly selected as research subjects from each village in this group.
Sample collection
Blood samples (3 ml/person) were collected and the sera were separated by centrifugation and then frozen. All collected sera were examined antibodies against H1N1, H3N2, H5N1, H7N7 and H9N2 viruses. The H5N1-positive cases were followed up for 1 year.
HI test
The HI test was performed to detect antibodies specific for H1N1, H3N2, H5N1, H7N7, H9N2 viruses according to the operating procedures recommended by World Health Organization (WHO) in 2002.<sup>10 </sup>The sera were treated with receptor destroying enzyme (RDE) to remove non-specific inhibitors and excess hemagglutinin. HI was assessed using 25 ?l each of a series of serum dilutions 1:2, and 25 ?l of HA antigen, standardized at 4 hemagglutination units (HAU) by hemagglutination titration. The mixture was incubated for 1 hour at room temperature, 50 ?l of 1% chicken erythrocytes were added and the plate was gently shaken. The HI titer was recorded after incubation for 1 hour at room temperature and was expressed as the reciprocal of the serum dilution that inhibited hemagglutination. A titer of 1:20 or more was regarded as positive.
Microneutralization test
Microneutralization test (MNT) was carried out to verify the results of the HI test by the University of Hong Kong (Hong Kong, China). Ten HI test positive samples were randomly selected. The same reference virus was used in the HI assay. All microneutralization assays were performed with Madin-Darby canine kidney (MDCK) cells. The 50% tissue culture infectious dose (TCID<sub>50</sub>) of virus was calculated by the method of Reed and Muench.<sup>11</sup>
The serum was diluted initially to 1/5 and to a final dilution of 1:1280 by twofold serial dilution in culture medium. Controls for each microneutralization assay were performed and included back titration, inclusion of positive control antibody (i.e., serum from a convalescent H5N1 avian influenza patient) and a human serum negative for H5N1-specific antibody. The solutions were mixed in 1:1 (v/v) with suspensions containing 100 TCID<sub>50</sub> of highly purified virus particles. The virus-serum mix was then transferred onto MDCK cell monolayers. The plates were incubated until cytopathic effect (CPE) developed in virus back titration controls. Furthermore, all the positive cases of H5N1, H7N7 and H9N2 were confirmed using the HI test by Harbin Veterinary Research Institute, Agricultural Academy, China. A titer of 1:20 or more was regarded as positive. The microneutralization titer of test antibody was the reciprocal of the highest dilution of test antibody that showed inhibition in all triplicate wells.<sup>12</sup>
Statistical analysis
The collected data were analyzed using χ<sup>2</sup> test and correlation analysis with SPSS 10.0. A P value less than 0.05 was considered statistically significant.
Survey sites
The three outbreak areas were about 500 kilometers away from each other and did not connect directly. Two were chicken farms; birds were raised in houses open to the surrounding hills. The two chicken farms raised about 1000 chicken each. One was a duck farm, raising about 150 ducks, which were raised out of the house in a lake.
HI test results
In the 1214 serum samples, HI test results showed that the positive rates of H1N1, H3N2, H5N1, H9N2 and H7N7 influenza viruses were 31.22%, 71.75%, 2.47%, 4.86% and 0.08%, respectively (Table 1). The results indicated that H1N1 and H3N2 influenza viruses were endemic in South China, and the potential infection of avian influenza H5N1 and H9N2 existed in the population directly exposed to H5N1-infected poultry.
<table class="MsoNormalTable" style="width: 100%; margin-left: 4.5pt; background: none repeat scroll 0% 0% rgb(51, 102, 51);" border="0" cellpadding="0" cellspacing="1" width="100%"> <tbody><tr style=""> <td style="width: 75pt; background: none repeat scroll 0% 0% white; padding: 4.5pt;" width="100">
</td> <td style="background: none repeat scroll 0% 0% rgb(244, 250, 244); padding: 0.75pt;"> Table 1. Distributions of human influenza and avian influenza strains among the 1214 cases from the H5N1 avian influenza epidemic areas in Guangdong Province
</td> </tr> </tbody></table> The age of the 30 H5N1-positive cases ranged from 17 to 74 years old, mean 41.2?16.3. The age of the 59 H9N2-positive cases ranged from 19 to 74 years old, mean 41.7?12.8. The single H7N7-positive case was a 39-year-old man.
After following the H5N1-, H9N2-, and H7N7-positive cases for one year, one case remained H5N1-positive (antibody titer 1:40), while others became negative.
Positive rates between occupational exposure population and general citizens
The positive rate of H5N1 was slightly higher in the occupational exposure group than in the general citizens group, 3.03% vs 2.34% (P >0.05, Table 2), but the positive rate of H9N2 was significantly higher in the occupational exposure group than in the general citizens group, 9.52% vs 3.76% (P <0.01, Table 3). The single H7N7 positive case was found in the occupational exposure group.
<table class="MsoNormalTable" style="width: 100%; margin-left: 4.5pt; background: none repeat scroll 0% 0% rgb(51, 102, 51);" border="0" cellpadding="0" cellspacing="1" width="100%"> <tbody><tr style=""> <td style="width: 75pt; background: none repeat scroll 0% 0% white; padding: 4.5pt;" width="100">
</td> <td style="background: none repeat scroll 0% 0% rgb(244, 250, 244); padding: 0.75pt;"> Table 2. Distribution of H5N1 stain in the two groups
</td> </tr> <tr style=""> <td style="width: 75pt; background: none repeat scroll 0% 0% white; padding: 4.5pt;" width="100">
</td> <td style="background: none repeat scroll 0% 0% rgb(244, 250, 244); padding: 0.75pt;"> Table 3. Distribution of H9N2 strain in the two groups
</td> </tr> </tbody></table> MNT test results
Ten HI test positive samples were randomly selected to be assayed by MNT to verify the results of the HI test. Compared with the MNT test, the HI test was also reliable (Table 4). The MNT titer was positively correlated to the HI titer (r=0.760, P <0.05). After one year the remaining H5N1 positive case also tested as positive in the MNT assay.
<table class="MsoNormalTable" style="width: 100%; margin-left: 4.5pt; background: none repeat scroll 0% 0% rgb(51, 102, 51);" border="0" cellpadding="0" cellspacing="1" width="100%"> <tbody><tr style=""> <td style="width: 75pt; background: none repeat scroll 0% 0% white; padding: 4.5pt;" width="100">
</td> <td style="background: none repeat scroll 0% 0% rgb(244, 250, 244); padding: 0.75pt;"> Table 4. Comparison between HI titer and MNT titer
</td> </tr> </tbody></table>
DISCUSSION This study showed that the positive rates of H1N1 and H3N2 subtypes were as high as 31.22% and 71.75%, respectively in Guangdong populations. Chen et al<sup>7</sup> also showed that H3N2 made up 79.8% (193/242) of the all viruses isolated among the influenza viruses. In September, 2006, WHO reported that H3N2 influenza viruses predominated and caused an outbreak in North America and some Eastern European countries.<sup>13</sup> In Asia, influenza H1N1, H3N2 and B viruses co-circulated. This study implied that the predominant strains were still H3N2 and H1N1 in Guangdong Province in 2004.
Among all influenza viruses that circulate in birds, the H5N1 virus has caused by far the greatest number of human cases of very severe disease and the greatest number of deaths and it could develop the characteristics to start an influenza pandemic if given enough opportunities.<sup>14</sup> WHO only reports the diagnosed cases, while there are seldom reports about the potential infected cases. The potential infected cases are important for H5N1 control in public health view. In our study, 30 H5N1-positive cases are detected in H5N1 avian influenza epidemic areas in Guangdong Province. It implies that the population in Guangdong Province may have a potential infection of H5N1 avian influenza. However it remains unknown why none of the H5N1-positive cases got sick. In addition, we do not know when these H5N1-infected cases were infected. In the H5N1 avian influenza outbreak area, there was no difference of H5N1 seroreactivity between the occupationally exposed and general citizens, but the infection of H9N2 subtype was predominant in the occupational exposure group. The result indicated that the avian influenza virus infecting humans came from the poultry. It is important to block the transmission of avian influenza virus from poultry to human.
We found that the youngest of all avian influenza-infected cases is aged from 17 to 74 years old. The fact that all infected cases were adults is different from that of Hong Kong (China), Thailand and Vietnam where the majority of the infected cases were children. Whether this is a major cause for the absence of avian influenza patients in the mainland of China or the age distribution difference between PCR confirmed patients and asymptomatic persons with only an antibody response is not known; it requires further study.
In recent years, H9 subtype avian influenza outbreaks in poultry, usually H9N2, have been widespread. In the mainland of China, H9N2 have been circulating in domestic poultry since 1994,<sup>15</sup> swine influenza surveillance indicated that H9N2 influenza viruses had been introduced into pig herds in several provinces in the last few years.<sup>16</sup> Human infections of avian influenza H9N2 were detected in both Guangdong and Hong Kong areas from 1997 to 1999 and detected in Hong Kong in 2003.<sup>13,17,18</sup> In this study, the H9N2 infection rate is higher in the occupational exposure group than in the general citizens group (P <0.01). Although the source of A (H9N2) was unknown in our study, the result suggests that H9N2 avian influenza virus might be a potential candidate pandemic strain in South China.
Antibody to H7N7 was positive only in one case. It is the first time that an H7N7-infected case was discovered in Guangdong, China.
One year after detection, all previously positive cases became negative except for one H5N1-positive case. None developed a clinical H5N1 infection. It suggests that antibody to H5N1 avian influenza has a limited effect to protect humans. When avian influenza breaks out, people will be susceptible to infection again. We were not certain why the antibody became negative so fast.
So far, an unprecedented epizootic avian influenza A virus that is highly pathogenic has crossed the species barrier in Asia and cause many human fatalities. Therefore, avian influenza is extremely important to public hygiene. These observations imply that the infections by H5N1 and H9N2 avian influenza do exist in Guangdong populations. But the virus has not acquired effective human-to-human transmission ability. The situation may change if the virus continues to mutate and assort during the epidemic. In order to prevent future pandemic avian influenza, we should establish an active market poultry virologic surveillance system in Guangdong Province. International collaboration will also be very helpful to combat this threat.
Acknowledgements: We thank Harbin Veterinary Research Institute, Agricultural Academy for providing the kits for analyzing the serum and reproving the positive cases.
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2. Xu X, Subbarao, Cox NJ, Guo Y. Genetic characterization of the pathogenic influenza A/Goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology 1999; 261: 15-19.
3. Cauthen AN, Swayne DE, Schultz-Cherry S, Perdue ML, Suarez DL. Continued circulation in China of highly pathogenic avian influenza viruses encoding the hemagglutinin gene associated with the 1997 H5N1 outbreak in poultry and humans. J Virol 2000; 74: 6592-6599.
4. Lu JH, Zhang DM, Wang GL. Highlight the significance of genetic evolution of H5N1 avian flu. Chin Med J 2006; 119: 1458-1464.
5. Update of avian influenza in animals. Geneva: World Health Organization, 2007. (Accessed at http://www.oie.int/downld/ AVIAN%20INFLUENZA/A_AI-Asia.htm)
6. Koopmans M, Wilbrink B, Conyn M, Natrop G, van der Nat H, Vennema H, et al. Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet 2004; 363: 587-593.
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8. Recommended composition of influenza virus vaccines for use in the 2007 influenza season. Geneva: World Health Organization, 2007. (Accessed at http://www.who.int/entity/ csr/disease/influenza/recommendationfinal.pdf)
9. Cumulative number of confirmed human cases of avian influenza A/(H5N1) reported to WHO. Geneva: World Health Organization, 2006. (Accessed at http://www.who.int/csr/ disease/avian_influenza/country/en/index.html)
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13. Recommended composition of influenza virus vaccines for use in the 2007 influenza season. Geneva: World Health Organization, 2006. (Accessed at http://www.who.int/entity/ csr/disease/influenza/recommendationfinal.pdf )
14. Avian influenza (?bird flu?) ? Fact sheet. Geneva: World Health Organization, 2006. (Accessed at http://www.who.int/ mediacentre/factsheets/avian_influenza/en/index.html)
15. Tang XY, Tian GB, Zhao CH, Zhou JF, Yu KZ. Isolation and characterization of prevalent strains of avian influenza viruses in China. J Animal Poultry Infect Dis (Chin) 1998; 20: 1-5.
16. Ninomiya A, Takada A, Okazaki K, Shortridge KF, Kida H. Seroepidemiological evidence of avian H4, H5, and H9 influenza A virus transmission to pigs in southeastern China. Vet Microbiol 2002; 88: 107-114.
17. Guo Y, Li J, Cheng X. Discovery of men infected by avian influenza A (H9N2) virus. Chin J Exp Clin Virol (Chin) 1999; 13: 105-108.
18. Lin YP, Shaw M, Gregory V, Cameron K, Lim W, Klimov A, et al. Avian-to-human transmission of H9N2 subtype influenza A viruses: relationship between H9N2 and H5N1 human isolates. Proc Natl Acad Sci U S A 2000; 97: 9654-9658.
For full tables see original article at: http://www.cmj.org/periodical/PaperL...17316792408201
Potential infections of H5N1 and H9N2 avian influenza do exist in Guangdong populations of China
LU Ci-yong, LU Jia-hai, CHEN Wei-qing, JIANG Li-fang, TAN Bing-yan, LING Wen-hua, ZHENG Bo-jian, SUI Hong-yan
Abstract:
Background - Southeast China is one of the sites of influenza origin. During 2003?2004, nine avian influenza outbreaks took place in Guangdong Province. But no human case was reported. To examine the status of potential human infection by human influenza (H1N1, H3N2) and avian influenza (H5N1, H7N7, H9N2) in the avian influenza epidemic area of Guangdong Province, China, we conducted a seroepidemiologic survey in the people of this area from April to June of 2004.
Methods - Three out of 9 H5N1 avian influenza affected poultry areas in Guangdong were randomly selected, and the population living within 3 kilometers of the affected poultries were chosen as the survey subjects. One thousand two hundred and fourteen people were selected from 3 villages at random. Human and avian influenza antibody titers were determined by hemagglutination-inhibition (HI) test and microneutralization test (MNT).
Results - The positive rate of antibody to H5N1 was 3.03% in the occupational exposure group and 2.34% in general citizens group; that of H9N2 was 9.52% in the occupational exposure group and 3.76% in the general citizens group. Moreover one case in the occupational exposure group was positive for H7N7. One year later, all previously positive cases had become negative except for one H5N1-positive case.
Conclusion - The observations imply that H5N1 and H9N2 avian influenza silent infections exist in Guangdong populations.
Avian influenza is an infectious disease of birds caused by type A influenza virus. The epicenters of both Asian influenza pandemic of 1957 and Hong Kong influenza pandemic of 1968 were in Southeast Asia;<sup>1</sup> and multiple clades of H5N1 influenza virus have already emerged in this region. In 1996, the avian H5N1 virus was first detected in Guangdong Province, China. The HA gene is genetically similar to that of the H5N1 viruses isolated in Hong Kong in 1997, which killed 6 of 18 infected persons.<sup>2</sup> The culling of all poultry in Hong Kong ended the first wave of H5N1, but the virus continued to circulate among apparently healthy ducks in the coastal provinces of China.<sup>3</sup>
The human influenza H5N1 in Southeast Asia has paralleled large outbreaks of avian influenza H5N1. Most patients had well-documented exposure to sick or dead poultry.<sup>4</sup> The frequency of human infection has not been determined, and seroprevalence studies are urgently needed. The expanding geographic distribution of H5N1 avian influenza infections, with outbreaks in Kazakstan, Mongolia and Russia, indicates that more human populations are at risk.<sup>5</sup>
In addition to H5N1 subtypes, a H7N7 avian influenza outbreak was also reported in the Netherlands, causing 83 poultry workers and 3 family members to fall ill in 2003.<sup>6</sup> The H9N2 subtype capable of infecting human was isolated from throat swab specimens of the patients suffering from influenza-like symptoms in South China in 1998<sup>7</sup> and in Hong Kong (China) in 1999 and 2003.<sup>8</sup> The facts suggested that avian influenza virus could cross over the species barrier and infect human beings, which changed the traditional viewpoint that the receptor-specific avian influenza virus didn't directly transmit to human. Although there is no firm evidence of human-to-human transmission of avian influenza virus, it is nonetheless theoretically possible.
Incidents of human H5N1 avian influenza outbreaks have been reported in Fujian, Sichuan, Hunan, Jiangxi, Anhui, Shanghai, Zhejiang, Guangdong and Guangxi provinces in China from the end of 2003 to April 3, 2006.<sup>9</sup> However, it remains unclear how H5N1 avian influenza in China is transmitted to humans and whether infected persons will fall ill. Being close to Hong Kong, Guangdong Province has a similar climate and geographic environment. A prosperous poultry market gives rise to the potential of an epidemic or outbreak of human infection with H5N1 avian influenza virus. In order to better understand the status of H5N1 infection among people directly exposed to domestic poultry that were confirmed to be infected with the H5N1 avian influenza virus, a seroepidemiological study was carried out in this project from April to June of 2004 in Guangdong Province.
METHODS
The inactivated strains of influenza A virus were provided by the Center for Disease Control and Prevention (CDC) of Guangdong Province. The antigens used in this study were H1N1 (A/newcaledonia/20/99), H3N2 (A/panama/ 2007/99), H5N1 (A/goose/Guangdong/1/96), H7N7 (A/afican/starling/Englan-q/983/79) and H9N2 (A/chicken/ shanghai/10/01), which were all standard diagnosis antigens. Cock red cells were used in the hemagglutination inhibition (HI) test.
Survey sites selection
Three out of nine H5N1 avian influenza affected areas of Guangdong were randomly selected, and 1214 people living within 3 kilometers of the affected poultries were chosen as the survey subjects. According to these criteria, we selected subjects from 3 villages at random.
Subjects and grouping
The occupational exposure group included 231 people who were responsible for raising, selling, and slaughtering chicken and ducks in H5N1 outbreak areas. The general citizen group included 983 people who lived in H5N1 outbreak areas but did not engage in actives of handling living chicken and ducks. About 300 people (>3 years) were randomly selected as research subjects from each village in this group.
Sample collection
Blood samples (3 ml/person) were collected and the sera were separated by centrifugation and then frozen. All collected sera were examined antibodies against H1N1, H3N2, H5N1, H7N7 and H9N2 viruses. The H5N1-positive cases were followed up for 1 year.
HI test
The HI test was performed to detect antibodies specific for H1N1, H3N2, H5N1, H7N7, H9N2 viruses according to the operating procedures recommended by World Health Organization (WHO) in 2002.<sup>10 </sup>The sera were treated with receptor destroying enzyme (RDE) to remove non-specific inhibitors and excess hemagglutinin. HI was assessed using 25 ?l each of a series of serum dilutions 1:2, and 25 ?l of HA antigen, standardized at 4 hemagglutination units (HAU) by hemagglutination titration. The mixture was incubated for 1 hour at room temperature, 50 ?l of 1% chicken erythrocytes were added and the plate was gently shaken. The HI titer was recorded after incubation for 1 hour at room temperature and was expressed as the reciprocal of the serum dilution that inhibited hemagglutination. A titer of 1:20 or more was regarded as positive.
Microneutralization test
Microneutralization test (MNT) was carried out to verify the results of the HI test by the University of Hong Kong (Hong Kong, China). Ten HI test positive samples were randomly selected. The same reference virus was used in the HI assay. All microneutralization assays were performed with Madin-Darby canine kidney (MDCK) cells. The 50% tissue culture infectious dose (TCID<sub>50</sub>) of virus was calculated by the method of Reed and Muench.<sup>11</sup>
The serum was diluted initially to 1/5 and to a final dilution of 1:1280 by twofold serial dilution in culture medium. Controls for each microneutralization assay were performed and included back titration, inclusion of positive control antibody (i.e., serum from a convalescent H5N1 avian influenza patient) and a human serum negative for H5N1-specific antibody. The solutions were mixed in 1:1 (v/v) with suspensions containing 100 TCID<sub>50</sub> of highly purified virus particles. The virus-serum mix was then transferred onto MDCK cell monolayers. The plates were incubated until cytopathic effect (CPE) developed in virus back titration controls. Furthermore, all the positive cases of H5N1, H7N7 and H9N2 were confirmed using the HI test by Harbin Veterinary Research Institute, Agricultural Academy, China. A titer of 1:20 or more was regarded as positive. The microneutralization titer of test antibody was the reciprocal of the highest dilution of test antibody that showed inhibition in all triplicate wells.<sup>12</sup>
Statistical analysis
The collected data were analyzed using χ<sup>2</sup> test and correlation analysis with SPSS 10.0. A P value less than 0.05 was considered statistically significant.
RESULTS
The three outbreak areas were about 500 kilometers away from each other and did not connect directly. Two were chicken farms; birds were raised in houses open to the surrounding hills. The two chicken farms raised about 1000 chicken each. One was a duck farm, raising about 150 ducks, which were raised out of the house in a lake.
HI test results
In the 1214 serum samples, HI test results showed that the positive rates of H1N1, H3N2, H5N1, H9N2 and H7N7 influenza viruses were 31.22%, 71.75%, 2.47%, 4.86% and 0.08%, respectively (Table 1). The results indicated that H1N1 and H3N2 influenza viruses were endemic in South China, and the potential infection of avian influenza H5N1 and H9N2 existed in the population directly exposed to H5N1-infected poultry.
<table class="MsoNormalTable" style="width: 100%; margin-left: 4.5pt; background: none repeat scroll 0% 0% rgb(51, 102, 51);" border="0" cellpadding="0" cellspacing="1" width="100%"> <tbody><tr style=""> <td style="width: 75pt; background: none repeat scroll 0% 0% white; padding: 4.5pt;" width="100">
</td> <td style="background: none repeat scroll 0% 0% rgb(244, 250, 244); padding: 0.75pt;"> Table 1. Distributions of human influenza and avian influenza strains among the 1214 cases from the H5N1 avian influenza epidemic areas in Guangdong Province
</td> </tr> </tbody></table> The age of the 30 H5N1-positive cases ranged from 17 to 74 years old, mean 41.2?16.3. The age of the 59 H9N2-positive cases ranged from 19 to 74 years old, mean 41.7?12.8. The single H7N7-positive case was a 39-year-old man.
After following the H5N1-, H9N2-, and H7N7-positive cases for one year, one case remained H5N1-positive (antibody titer 1:40), while others became negative.
Positive rates between occupational exposure population and general citizens
The positive rate of H5N1 was slightly higher in the occupational exposure group than in the general citizens group, 3.03% vs 2.34% (P >0.05, Table 2), but the positive rate of H9N2 was significantly higher in the occupational exposure group than in the general citizens group, 9.52% vs 3.76% (P <0.01, Table 3). The single H7N7 positive case was found in the occupational exposure group.
<table class="MsoNormalTable" style="width: 100%; margin-left: 4.5pt; background: none repeat scroll 0% 0% rgb(51, 102, 51);" border="0" cellpadding="0" cellspacing="1" width="100%"> <tbody><tr style=""> <td style="width: 75pt; background: none repeat scroll 0% 0% white; padding: 4.5pt;" width="100">
</td> <td style="background: none repeat scroll 0% 0% rgb(244, 250, 244); padding: 0.75pt;"> Table 2. Distribution of H5N1 stain in the two groups
</td> </tr> <tr style=""> <td style="width: 75pt; background: none repeat scroll 0% 0% white; padding: 4.5pt;" width="100">
</td> </tr> </tbody></table> MNT test results
Ten HI test positive samples were randomly selected to be assayed by MNT to verify the results of the HI test. Compared with the MNT test, the HI test was also reliable (Table 4). The MNT titer was positively correlated to the HI titer (r=0.760, P <0.05). After one year the remaining H5N1 positive case also tested as positive in the MNT assay.
<table class="MsoNormalTable" style="width: 100%; margin-left: 4.5pt; background: none repeat scroll 0% 0% rgb(51, 102, 51);" border="0" cellpadding="0" cellspacing="1" width="100%"> <tbody><tr style=""> <td style="width: 75pt; background: none repeat scroll 0% 0% white; padding: 4.5pt;" width="100">
</td> <td style="background: none repeat scroll 0% 0% rgb(244, 250, 244); padding: 0.75pt;"> Table 4. Comparison between HI titer and MNT titer
</td> </tr> </tbody></table>
DISCUSSION
Among all influenza viruses that circulate in birds, the H5N1 virus has caused by far the greatest number of human cases of very severe disease and the greatest number of deaths and it could develop the characteristics to start an influenza pandemic if given enough opportunities.<sup>14</sup> WHO only reports the diagnosed cases, while there are seldom reports about the potential infected cases. The potential infected cases are important for H5N1 control in public health view. In our study, 30 H5N1-positive cases are detected in H5N1 avian influenza epidemic areas in Guangdong Province. It implies that the population in Guangdong Province may have a potential infection of H5N1 avian influenza. However it remains unknown why none of the H5N1-positive cases got sick. In addition, we do not know when these H5N1-infected cases were infected. In the H5N1 avian influenza outbreak area, there was no difference of H5N1 seroreactivity between the occupationally exposed and general citizens, but the infection of H9N2 subtype was predominant in the occupational exposure group. The result indicated that the avian influenza virus infecting humans came from the poultry. It is important to block the transmission of avian influenza virus from poultry to human.
We found that the youngest of all avian influenza-infected cases is aged from 17 to 74 years old. The fact that all infected cases were adults is different from that of Hong Kong (China), Thailand and Vietnam where the majority of the infected cases were children. Whether this is a major cause for the absence of avian influenza patients in the mainland of China or the age distribution difference between PCR confirmed patients and asymptomatic persons with only an antibody response is not known; it requires further study.
In recent years, H9 subtype avian influenza outbreaks in poultry, usually H9N2, have been widespread. In the mainland of China, H9N2 have been circulating in domestic poultry since 1994,<sup>15</sup> swine influenza surveillance indicated that H9N2 influenza viruses had been introduced into pig herds in several provinces in the last few years.<sup>16</sup> Human infections of avian influenza H9N2 were detected in both Guangdong and Hong Kong areas from 1997 to 1999 and detected in Hong Kong in 2003.<sup>13,17,18</sup> In this study, the H9N2 infection rate is higher in the occupational exposure group than in the general citizens group (P <0.01). Although the source of A (H9N2) was unknown in our study, the result suggests that H9N2 avian influenza virus might be a potential candidate pandemic strain in South China.
Antibody to H7N7 was positive only in one case. It is the first time that an H7N7-infected case was discovered in Guangdong, China.
One year after detection, all previously positive cases became negative except for one H5N1-positive case. None developed a clinical H5N1 infection. It suggests that antibody to H5N1 avian influenza has a limited effect to protect humans. When avian influenza breaks out, people will be susceptible to infection again. We were not certain why the antibody became negative so fast.
So far, an unprecedented epizootic avian influenza A virus that is highly pathogenic has crossed the species barrier in Asia and cause many human fatalities. Therefore, avian influenza is extremely important to public hygiene. These observations imply that the infections by H5N1 and H9N2 avian influenza do exist in Guangdong populations. But the virus has not acquired effective human-to-human transmission ability. The situation may change if the virus continues to mutate and assort during the epidemic. In order to prevent future pandemic avian influenza, we should establish an active market poultry virologic surveillance system in Guangdong Province. International collaboration will also be very helpful to combat this threat.
Acknowledgements: We thank Harbin Veterinary Research Institute, Agricultural Academy for providing the kits for analyzing the serum and reproving the positive cases.
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For full tables see original article at: http://www.cmj.org/periodical/PaperL...17316792408201