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Epidemiology of influenza virus in Korean poultry
International Congress Series 1263 (2004) 758 – 761
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Epidemiology of influenza virus in Korean poultry Sang Heui Seo *, Hyun Soo Kim Laboratory of Immunology, Division of Preventive Medicine, College of Veterinary Medicine, Chungnam National University, 220 Guang Dong, Yuseong Gu, Daejeon 305-764, South Korea
Abstract. Epidemiological information on currently circulating influenza viruses in poultry in Korea has not been available. We performed the surveillance of avian influenza viruses in the live poultry markets where chickens, ducks, geese, and doves are sold. H9N2, H3N2, and H6N1 influenza viruses were isolated from poultry in the Korean live bird markets. H9N2 influenza viruses were mainly isolated from chickens; H3N2 influenza viruses were isolated from ducks and a dove, and an H6N1 influenza virus was isolated from a duck. Serological surveillance in chickens showed that chickens were infected over 50% with H9N2 viruses. D 2004 Published by Elsevier B.V. Keywords: Poultry market; Surveillance; H9N2
1. Introduction Influenza virus pandemics occur in the interval of 10– 30 years and cause a devastating effect on the human population. They usually arise as a result of introducing the genes of avian influenza viruses into the human influenza viruses. In the 20th century, humans had experienced three major pandemics, starting in 1918 caused by an H1N1 subtype, in 1957 by an H2N1 subtype, and in 1968 by an H3N2 subtype. The 1968 H3N2 pandemic virus had emerged by introducing avian PB1 and hemagglutinin gene into the H2N2 human influenza viruses [1,2]. This indicates that the continuous surveillance of avian influenza viruses in poultry is very important for predicting a new pandemic. In 1997, the highly pathogenic H5N1 influenza viruses were directly transmitted from chickens in the Hong Kong live bird markets to humans and claimed six human lives out of 18 infected cases [3,4]. Considering that poultry in the live bird markets come into close contact with humans everyday, the surveillance of live bird markets can be very important for monitoring the new subtypes of influenza viruses in humans. In this study, the subtypes of avian influenza viruses were determined by surveying poultry in the live bird markets in Korea. The dominant subtype of avian influenza viruses
* Corresponding author. Tel.: +82-42-821-6762; fax: +82-42-821-6762. E-mail address: [email protected] (S.H. Seo). 0531-5131/ D 2004 Published by Elsevier B.V. doi:10.1016/j.ics.2004.02.062
S.H. Seo, H.S. Kim / International Congress Series 1263 (2004) 758–761
759
in chickens is an H9N2 influenza virus. Antigenically, the H9N2 subtypes isolated in Korea are different from those of H9N2 viruses isolated in Hong Kong. 2. Methods 2.1. Surveillance of poultry We visited the live poultry markets in Korea and picked samples from tracheas, cloacae, and feces of the poultry in PBS with antibiotics. The samples were shipped directly to the laboratory and were inoculated into 10-day-old embryonated eggs for the isolation of avian influenza viruses. 2.2. Hemagglutination inhibition (HI) assay for serological surveillance In 96-well plates, 50 Al of sera (1:10) were added to a well and then two-fold dilutions were performed in 25 Al of PBS; 25 Al of H9N2, H6N1, or H3N2 influenza viruses were added to each well in plates and plates were incubated at room temperature for 15 min before 50 Al of 0.5% chicken RBC in PBS were added to wells. HI titers were recorded at 40 min after RBC was added. 2.3. Transmission study Three chickens were intranasally (i.n.) infected with 103 EID50 of A/Chicken/Korea/ S1/03 (H9N2) and then one infected chicken was mixed with five uninfected chickens in each cage. Tracheal swabs were performed in infected chickens 3 days post infection (p.i.) and in uninfected chickens 6 days after chickens were mixed. The virus titers were determined in the embryonated eggs by log egg infectious dose 50 (logEID50). 3. Results 3.1. Subtypes of avian influenza viruses from poultry in Korean live bird markets There has been limited information on avian influenza viruses from poultry in Korean live bird markets. To understand what subtypes of viruses are circulating among poultry in Korea, we surveyed poultry in the live bird markets. We could isolate eight H9N2 influenza viruses from 95 chicken samples, five H3N2 and one H6N1 influenza viruses from 26 ducks, and one H3N2 virus from five dove samples (Table 1). Table 1 Subtypes of avian influenza viruses isolated from poultry in Korean live bird markets Subtypes
Poultry (# positive/total isolates) Chickens
Ducks
Geese
Dove
H9N2 H6N1 H3N2
8/8 – –
– 1/1 5/5
– – –
– – 1/5
760
S.H. Seo, H.S. Kim / International Congress Series 1263 (2004) 758–761
Table 2 Serological surveillance in chickens Subtypes
Numbers of chickens (positive #/total samples)
H9N2 H6N1 H3N2
52/100 0/100 0/100
3.2. Serological surveillance in chickens We are interested in determining the serological prevalence in chickens in Korea. One hundred sera were collected from chickens from the farms in Korea before HI assays were performed. Over 50% of chicken sera were positive for H9N2 influenza viruses. No reactivity was detected for an H6N1 or H3N1 influenza virus. HI titers over 40 were regarded as positive samples (Table 2). 3.3. Transmission study of H9N2 influenza viruses in chickens Based on serological data, it seems that H9N2 influenza viruses are widespread in chickens in Korea. We decided to study the transmission ability of H9N2 influenza viruses among chickens. One infected chicken per five uninfected chickens were mixed and tracheal swabs were performed in inoculated chickens 3 days p.i and in uninfected chickens 6 days after mixing. All uninfected chickens were infected with H9N2 influenza viruses by aerosol. Eight, six and one out of 15 chickens showed viral titers of 2 logEID50, 3 logEID50, and 4 logEID50, respectively. The mean of three inoculated chickens was 3 EID50 (Table 3). 3.4. Antigenic comparison To study the serological similarity of H9N2 influenza viruses isolated from the Korean live bird markets to other H9N2 isolates from other countries, we made immune sera Table 3 Transmission study of H9N2 influenza virus among chickens Virus titers (logEID50)
Numbers of positive chickens/total chickens
– 1 EID50 2 EID50 3 EID50 4 EID50
0/15 0/15 8/15 6/15 1/15
– : no virus isolation. The mean titer of three inoculated chickens was 3 EID50.
Table 4 Serological similarity among H9N2 influenza viruses Viruses A/Chicken/Korea/S1/03
A/Turkey/W1/66
A/Hong Kong/1073/99
A/Duck/Hong Kong/Y280/97
640
80
10
20
Sera against A/Chicken/Korea/S1/03 were used for HI.
S.H. Seo, H.S. Kim / International Congress Series 1263 (2004) 758–761
761
against A/Chicken/Korea/S1/03 (H9N2) by inoculating chickens before HI assay was carried out. HI titers were 640 against A/Chicken/Korea/S1/03, 80 against A/Turkey/WI/ 66, 10 against A/Hong Kong/1073/99, and 20 against A/Duck/Hong Kong/Y280/97. The results indicate that H9N2 influenza viruses isolated from chickens in Korea are serologically different from those of Hong Kong isolates (Table 4). 4. Discussion Live bird markets seem to play an important role in transmission of avian influenza viruses to humans since live bird markets are ideal places where humans can closely contact live birds. In 1997, the highly pathogenic H5N1 viruses were transmitted to humans, killing six humans [3,4]. This incident indicates that avian influenza viruses can infect humans and cause the disease. In Korea, there are several hundred live bird markets scattered throughout eight provinces. There are two types of live bird markets, 5-day interval and sentinel markets. The live birds are sold when markets open every 5 days in 5day markets and sentinel markets sell live birds everyday. Antigenic study indicates that H9N2 Korean isolates are different from those of Hong Kong. In 1997, G1 and Y280 lineage H9N2 viruses circulated in the Hong Kong live bird markets [5,6]. Results indicate that the origins of Korean isolates are different from those of Hong Kong H9N2 isolates. In Korea, H9N2 viruses are widespread in chickens. In China, H9N2 viruses infected humans with flu symptoms [7]. A pandemic can occur anywhere in the world. Since humans have only immunity to H1N1 and H3N2 influenza A viruses, it is very important to monitor H9N2 viruses and to prepare a vaccine for protecting humans from H9N2 infections when these viruses become a pandemic strain. Acknowledgements This work was supported by Ellison Medical Foundation, USA. We greatly appreciate Dr. Robert G. Webster for providing us with standard sera for avian influenza viruses. References [1] R. Fang, et al., Complete structure of A/duck/Ukraine/63 influenza haemagglutinin gene: animal virus as progenitor of human H3 Hong Kong 1968 influenza haemagglutinin, Cell 25 (1981) 315 – 323. [2] Y. Kawaoka, S. Krauss, R.G. Webster, Avian to human transmission of the PB1 gene of influenza A virus in the 1957 and 1968 pandemics, J. Virol. 63 (1989) 4603 – 4608. [3] K. Subbarao, et al., Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness, Science 279 (1998) 393 – 396. [4] S.H. Seo, R.G. Webster, Cross-reactive cell-mediated immunity and protection of chickens from lethal H5N1 influenza virus infection in the Hong Kong poultry markets, J. Virol. 75 (2001) 2516 – 2525. [5] Y. Guan, et al., H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in poultry in Southeastern China, J. Virol. 74 (2000) 9372 – 9380. [6] K.S. Li, 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. 77 (2003) 6988 – 6994. [7] Y.P. Lin, et al., PNAS 97 (2000) 9654 – 9658.
www.ics-elsevier.com
Epidemiology of influenza virus in Korean poultry Sang Heui Seo *, Hyun Soo Kim Laboratory of Immunology, Division of Preventive Medicine, College of Veterinary Medicine, Chungnam National University, 220 Guang Dong, Yuseong Gu, Daejeon 305-764, South Korea
Abstract. Epidemiological information on currently circulating influenza viruses in poultry in Korea has not been available. We performed the surveillance of avian influenza viruses in the live poultry markets where chickens, ducks, geese, and doves are sold. H9N2, H3N2, and H6N1 influenza viruses were isolated from poultry in the Korean live bird markets. H9N2 influenza viruses were mainly isolated from chickens; H3N2 influenza viruses were isolated from ducks and a dove, and an H6N1 influenza virus was isolated from a duck. Serological surveillance in chickens showed that chickens were infected over 50% with H9N2 viruses. D 2004 Published by Elsevier B.V. Keywords: Poultry market; Surveillance; H9N2
1. Introduction Influenza virus pandemics occur in the interval of 10– 30 years and cause a devastating effect on the human population. They usually arise as a result of introducing the genes of avian influenza viruses into the human influenza viruses. In the 20th century, humans had experienced three major pandemics, starting in 1918 caused by an H1N1 subtype, in 1957 by an H2N1 subtype, and in 1968 by an H3N2 subtype. The 1968 H3N2 pandemic virus had emerged by introducing avian PB1 and hemagglutinin gene into the H2N2 human influenza viruses [1,2]. This indicates that the continuous surveillance of avian influenza viruses in poultry is very important for predicting a new pandemic. In 1997, the highly pathogenic H5N1 influenza viruses were directly transmitted from chickens in the Hong Kong live bird markets to humans and claimed six human lives out of 18 infected cases [3,4]. Considering that poultry in the live bird markets come into close contact with humans everyday, the surveillance of live bird markets can be very important for monitoring the new subtypes of influenza viruses in humans. In this study, the subtypes of avian influenza viruses were determined by surveying poultry in the live bird markets in Korea. The dominant subtype of avian influenza viruses
* Corresponding author. Tel.: +82-42-821-6762; fax: +82-42-821-6762. E-mail address: [email protected] (S.H. Seo). 0531-5131/ D 2004 Published by Elsevier B.V. doi:10.1016/j.ics.2004.02.062
S.H. Seo, H.S. Kim / International Congress Series 1263 (2004) 758–761
759
in chickens is an H9N2 influenza virus. Antigenically, the H9N2 subtypes isolated in Korea are different from those of H9N2 viruses isolated in Hong Kong. 2. Methods 2.1. Surveillance of poultry We visited the live poultry markets in Korea and picked samples from tracheas, cloacae, and feces of the poultry in PBS with antibiotics. The samples were shipped directly to the laboratory and were inoculated into 10-day-old embryonated eggs for the isolation of avian influenza viruses. 2.2. Hemagglutination inhibition (HI) assay for serological surveillance In 96-well plates, 50 Al of sera (1:10) were added to a well and then two-fold dilutions were performed in 25 Al of PBS; 25 Al of H9N2, H6N1, or H3N2 influenza viruses were added to each well in plates and plates were incubated at room temperature for 15 min before 50 Al of 0.5% chicken RBC in PBS were added to wells. HI titers were recorded at 40 min after RBC was added. 2.3. Transmission study Three chickens were intranasally (i.n.) infected with 103 EID50 of A/Chicken/Korea/ S1/03 (H9N2) and then one infected chicken was mixed with five uninfected chickens in each cage. Tracheal swabs were performed in infected chickens 3 days post infection (p.i.) and in uninfected chickens 6 days after chickens were mixed. The virus titers were determined in the embryonated eggs by log egg infectious dose 50 (logEID50). 3. Results 3.1. Subtypes of avian influenza viruses from poultry in Korean live bird markets There has been limited information on avian influenza viruses from poultry in Korean live bird markets. To understand what subtypes of viruses are circulating among poultry in Korea, we surveyed poultry in the live bird markets. We could isolate eight H9N2 influenza viruses from 95 chicken samples, five H3N2 and one H6N1 influenza viruses from 26 ducks, and one H3N2 virus from five dove samples (Table 1). Table 1 Subtypes of avian influenza viruses isolated from poultry in Korean live bird markets Subtypes
Poultry (# positive/total isolates) Chickens
Ducks
Geese
Dove
H9N2 H6N1 H3N2
8/8 – –
– 1/1 5/5
– – –
– – 1/5
760
S.H. Seo, H.S. Kim / International Congress Series 1263 (2004) 758–761
Table 2 Serological surveillance in chickens Subtypes
Numbers of chickens (positive #/total samples)
H9N2 H6N1 H3N2
52/100 0/100 0/100
3.2. Serological surveillance in chickens We are interested in determining the serological prevalence in chickens in Korea. One hundred sera were collected from chickens from the farms in Korea before HI assays were performed. Over 50% of chicken sera were positive for H9N2 influenza viruses. No reactivity was detected for an H6N1 or H3N1 influenza virus. HI titers over 40 were regarded as positive samples (Table 2). 3.3. Transmission study of H9N2 influenza viruses in chickens Based on serological data, it seems that H9N2 influenza viruses are widespread in chickens in Korea. We decided to study the transmission ability of H9N2 influenza viruses among chickens. One infected chicken per five uninfected chickens were mixed and tracheal swabs were performed in inoculated chickens 3 days p.i and in uninfected chickens 6 days after mixing. All uninfected chickens were infected with H9N2 influenza viruses by aerosol. Eight, six and one out of 15 chickens showed viral titers of 2 logEID50, 3 logEID50, and 4 logEID50, respectively. The mean of three inoculated chickens was 3 EID50 (Table 3). 3.4. Antigenic comparison To study the serological similarity of H9N2 influenza viruses isolated from the Korean live bird markets to other H9N2 isolates from other countries, we made immune sera Table 3 Transmission study of H9N2 influenza virus among chickens Virus titers (logEID50)
Numbers of positive chickens/total chickens
– 1 EID50 2 EID50 3 EID50 4 EID50
0/15 0/15 8/15 6/15 1/15
– : no virus isolation. The mean titer of three inoculated chickens was 3 EID50.
Table 4 Serological similarity among H9N2 influenza viruses Viruses A/Chicken/Korea/S1/03
A/Turkey/W1/66
A/Hong Kong/1073/99
A/Duck/Hong Kong/Y280/97
640
80
10
20
Sera against A/Chicken/Korea/S1/03 were used for HI.
S.H. Seo, H.S. Kim / International Congress Series 1263 (2004) 758–761
761
against A/Chicken/Korea/S1/03 (H9N2) by inoculating chickens before HI assay was carried out. HI titers were 640 against A/Chicken/Korea/S1/03, 80 against A/Turkey/WI/ 66, 10 against A/Hong Kong/1073/99, and 20 against A/Duck/Hong Kong/Y280/97. The results indicate that H9N2 influenza viruses isolated from chickens in Korea are serologically different from those of Hong Kong isolates (Table 4). 4. Discussion Live bird markets seem to play an important role in transmission of avian influenza viruses to humans since live bird markets are ideal places where humans can closely contact live birds. In 1997, the highly pathogenic H5N1 viruses were transmitted to humans, killing six humans [3,4]. This incident indicates that avian influenza viruses can infect humans and cause the disease. In Korea, there are several hundred live bird markets scattered throughout eight provinces. There are two types of live bird markets, 5-day interval and sentinel markets. The live birds are sold when markets open every 5 days in 5day markets and sentinel markets sell live birds everyday. Antigenic study indicates that H9N2 Korean isolates are different from those of Hong Kong. In 1997, G1 and Y280 lineage H9N2 viruses circulated in the Hong Kong live bird markets [5,6]. Results indicate that the origins of Korean isolates are different from those of Hong Kong H9N2 isolates. In Korea, H9N2 viruses are widespread in chickens. In China, H9N2 viruses infected humans with flu symptoms [7]. A pandemic can occur anywhere in the world. Since humans have only immunity to H1N1 and H3N2 influenza A viruses, it is very important to monitor H9N2 viruses and to prepare a vaccine for protecting humans from H9N2 infections when these viruses become a pandemic strain. Acknowledgements This work was supported by Ellison Medical Foundation, USA. We greatly appreciate Dr. Robert G. Webster for providing us with standard sera for avian influenza viruses. References [1] R. Fang, et al., Complete structure of A/duck/Ukraine/63 influenza haemagglutinin gene: animal virus as progenitor of human H3 Hong Kong 1968 influenza haemagglutinin, Cell 25 (1981) 315 – 323. [2] Y. Kawaoka, S. Krauss, R.G. Webster, Avian to human transmission of the PB1 gene of influenza A virus in the 1957 and 1968 pandemics, J. Virol. 63 (1989) 4603 – 4608. [3] K. Subbarao, et al., Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness, Science 279 (1998) 393 – 396. [4] S.H. Seo, R.G. Webster, Cross-reactive cell-mediated immunity and protection of chickens from lethal H5N1 influenza virus infection in the Hong Kong poultry markets, J. Virol. 75 (2001) 2516 – 2525. [5] Y. Guan, et al., H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in poultry in Southeastern China, J. Virol. 74 (2000) 9372 – 9380. [6] K.S. Li, 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. 77 (2003) 6988 – 6994. [7] Y.P. Lin, et al., PNAS 97 (2000) 9654 – 9658.