Diversity of influenza A virus subtypes isolated from domestic poultry in Hong Kong*

Bulkdn of the World Health Organizadon, 57 (3): 465-469 (1979) Diversity of influenza A virus subtypes isolated from domestic poultry in Hong Kong * K. F. SHORTRIDGE,1 W. K. BU1TERFIELD,2 R. G. WEBSTER,3 & C. H. CAMPBELL2 The second phase of a 2-year influenza virus surveillance programme of domestic avian species in Hong Kong (up to October 1977) yielded influenza A virus, Newcastle disease virus, and Hong Kong paramyxovirus, as well as unidentified haemagglutinating agents. These viruses were isolated from the trachea or cloaca of apparently healthy domestic ducks, geese, and chickens originating from China and Hong Kong. Twenty-five combinations of haemagglutinin and neuraminidase surface antigens were identified from the 136 influenza A viruses isolated. Eight of the combinations do not appear to have been previously reported - Hav3Nav2, Hav4Nav2, Hav4Nav4, Hav4NavS, Hav4Neql, Hav6Nav4, Hav6Nav6, and Hav9Navl. The existence of such a diverse pool of influenza virus genetic information may play a role in the emergence of new human pandemic strains. Studies fostered mainly by the World Health Organization have led to the recognition of many influenza viruses in lower animals and birds. Anti- genic and biochemical similarity between these viruses and those in man have given rise to the hypothesis that nonhuman reservoirs may be impor- tant in the origin of human strains (16). South-East Asia has been the point of origin of at least two of the recent pandemic strains of influenza (H2N2 and H3N2) and now a third one (HiN1) appears to have originated in China. In view of the geographical proximity of Hong Kong, surveillance studies were undertaken to evaluate the extent of influenza viruses in nature, with particular emphasis on domestic poultry. Fourteen different subtypes, seven of which were new, were isolated mainly from domestic ducks in the period November 1975 to June 1976 (2, 12). The completion of 2 years of surveillance studies to October 1977, reported here, * This work was supported in part by contract AI 52524 from the US National Institute of Allergy and Infectious Diseases and by a grant from the University of Hong Kong. 1 Member, Department of Microbiology, University of Hong Kong, Pathology Building, Queen Mary Hospital Compound, Hong Kong. 2 Mcrobiologist, Plum Island Animal Disease Center, US Department of Agriculture, P.O. Box 848, Greenport, NY 11944, USA. 3 Member, Division of Virology,WHO Collaborating Centre for the Ecology of Influenza Viruses in Animals, St Jude Children's Research Hospital, P.O. Box 318, Memphis, TN 38101, USA. consolidates those findings and highlights the anti- genic diversity and high prevalence of influenza viruses in this region. MATERIALS AND METHODS Sampling of domestic poultry was conducted from July 1976 to October 1977. The origin of the poultry, the protocol for sample collection, virus isolation, and identification procedures have been reported (12). A chicken antiserum to the newly recognized paramyxovirus duck/Hong Kong/3/76 was prepared by intranasal and intramuscular inocu- lation as previously described (13). RESULTS One hundred and thirty-six influenza viruses were isolated from the trachea or cloaca of 2785 indivi- dual ducks, geese, and chickens, the majority of these coming from ducks (Table 1). The isolation frequencies were similar from both sites for each type of bird. Newcastle disease virus (NDV) and the Hong Kong avian paramyxovirus comprised the remainder of the haemagglutinating agents with the exception of nine agents that remained unidentified. The overall isolation frequency of influenza vi- ruses was 7.8% in poultry from China and 2.3% in that from Hong Kong (Table 2). Ducks from China 3819 - 465 K. F. SHORTRIDGE ET AL. Table 1. Haemagglutinating (HA) agents isolated from domestic poultry at a Hong Kong dressing plant Source No. of No. positive Percentage No. serologically No. serologicallyType of Source No. of HA fo itive influenza A related to related to Unidentifiedpoultry sam les samples HAflor iflunza isolation Newcastle disease Hong Kong avian agentspoultry samples samp isolates A-nNP from swabs virus (NDV) paramyxovirus Duck trachea 1031 80 60 5.8 19 2a 0 cloaca 1002 126 66 6.6 52 12b 7 Goose trachea 229 5 3 1.3 2 0 0 cloaca 232 10 4 1.7 5 4c 1 Chicken trachea 118 1 1 0.8 0 0 0 cloaca 173 9 2 1.2 6 2d 1 a One agent was jointly detected with NDV. bEleven agents were jointly detected with NDV. cThree agents were jointly detected with NDV and one with influenza virus Hav4N2. dTwo agents were jointly detected with NDV. provided the largest number of influenza virus isolates with an isolation frequency of 10.6 %. Signi- ficantly more influenza viruses were isolated from ducks and chickens originating from China. All avian haemagglutinin subtypes except Hav8 and all neuraminidase subtypes except Nav3 were identified, yielding 25 different subtypes among the 136 influenza virus isolates (Table 3). The viruses most frequently isolated were Hav4Navl from 35 ducks and Hav2Nav5 from 23 ducks. The most frequently detected haemagglutinins were Hav4 and Hav6, which were isolated from 45 and 29 birds, respectively, each in combination with six different neuraminidase subtypes. Eight of the combinations of haemagglutinin and neuraminidase appear to be novel in that they have not previously been reported. These are Hav3Nav2, Hav4Nav2, Hav4Nav4, Hav4Nav5, Hav4Neql, Hav6Nav4, Hav6Nav6, and Hav9Navl. All were isolated from ducks; Hav6Nav4 and Hav6Nav6 were also isolated from a chicken and a goose, respectively. Table 2. Origin of influenza virus isolates Type Of o No. of No. influenza Percentage poultry Origin swabs virus isolates influenza virusisolation Duck China 880 93 10.6 Hong Kong 1153 33 2.9 Goose China 374 2 0.5 Hong Kong 87 1 1.2 Chicken China 62 7 11.3 Hong Kong 229 0 0 The most common isolate, Hav4Navl, occurred throughout the year whereas others such as Hav2Nav5 and Hav6N1 were more prevalent in the summer months. In contrast, the paramyxoviruses were more frequently isolated in winter (13,14). The haemagglutinin and neuraminidase antigens could not be identified on three non-avid isolates, namely D193/77, C14/76, and G8/76, and are under further investigation. DISCUSSION The knowledge that pandemic influenza A viruses of man first appeared in South-East Asia provided the logic behind the present 2-year surveillance study. Domestic poultry from China and Hong Kong was studied to determine which subtypes of influ- enza A viruses were circulating among avian species. A total of 136 influenza A viruses were isolated; 126 of these were from ducks. The majority of isolates (102) from ducks, geese, or chickens were from China. Newcastle disease virus, Hong Kong paramyxovirus, and unidentified haemagglutinating agents were also isolated, many as dual infections of the same bird. The 136 influenza A viruses were subtyped by haemagglutination-inhibition and neur- aminidase-inhibition into 25 combinations of the surface antigens. Eight of these combinations have not previously been described. New haemagglutinins or neuraminidases were not found. Each of the human (Ni and N2), equine (Neql and Neq2), and avian neuraminidase subtypes (Navl-Nav6), with the exception of Nav3, were detected among the isolates from avian species. All avian haemagglutinin subtypes except Hav8 were detected in these avian influenza virus isolates. 466 INFLUENZA A VIRUS SUBTYPES FROM POULTRY Table 3. Antigenic subtypes of influenza A viruses isolated from avian species in Hong Kong Virus Number of Influenza A previously subtype Influenza A isolates isolates isolated, includingprototype strains Havl N2 A/duck/HK/47/76 Hav2N2 A/duck/HK/135n77 Hav2Nav5 A/duck/HK/129, 130,132,133,137,138,158,159,160,162,163,165, 167, 169, 170, 171, 172, 176, 177, 178, 179, 180, 195/77 Hav3N2 A/duck/HK/62/76 A/duck/HKI/125f77 Hav3Nav2 A/duck/HK/44/76 Hav3Nav6 A/duck/HK/197, 214, 239/77' Nav4N2 A/duck/HK/40, 41, 42, 66/76 A/goose/HKI7/76 Hav4Navl A/duck/HK/67, 72, 78, 84, 85, 88, 96, 97/76 A/duck/HK/131, 136, 141, 142, 143, 144, 146, 148, 164,187, 188, 189, 190, 192, 194, 200, 204, 207, 210, 211, 213, 216, 219, 223, 230, 234, 237M Hav4Nav2 A/duck/HK/229/77 Hav4Nav4 A/duck/HK/224, 228/77 Hav4Nav5 A/duck/HKI/191/77 Hav4Neql A/duck/HK/174/77 Hav5Nav2 A/duck/HK/205/77 Hav6N1 A/duck/HK/56, 60, 61, 68, 69, 70, 71, 73, 75, 76, 79, 95/76 A/duck/HK/175, 202, 232, 233n7b A/goose/HK/17M Hav6N2 A/duck/HK/48/76 A/duck/HK/114, 134/77 A/goose/HK/6n6 Hav6Nav4 A/duck/HK/58/76 A/chicken/HK/17M Hav6Nav5 A/duck/HK/108/76 Hav6Nav6 A/duck/HK/182/77 A/goose/HK/9/76 Hav6Neq2 A/duck/HK/151, 212, 221/77 Hav7N2 A/duck/HK/59, 64/76 A/duck/HK/115, 122/77 A/goose/HKI1O/76 A/chicken/HK/8/76 Hav7Navl A/duck/HK/145, 155, 157, 198, 231/ Hav7Nav2 A/duck/HK/1 16/77 Hav7Neq2 A/duck/HK/43, 49, 55, 57, 74/76 AJduck/1 19, 120, 124, 242n7 A/goose/HK/1 1/76 Hav9N2 A/duck/HK/86, 92/76 A/duck/HK/149, 168/77 Hav9Nav1 A/duck/HK/147n7 23 A/chicken/FPV/Bresica/1902 (5) A/duck/ltaly/66 (10) A/duck/HK/15/76 (12) 2 A/duck/Ukraine/1/60 (15) 3 5 None A/duck/Memphis/546/74 (18) A/duck/HK/37/76 (12) 35 A/duck/Czech/56 (7) 1 None 2 None 17 None None A/tern/S. Africa/61 (1) A/duck/Germany/1868/68 (11) 4 A/turkey/Mass./65 (9) 2 None 1 Alshearwater/E. Australia/1/72 (3) 2 None 3 A/turkey/Canada/63 (8) 6 A/turkey/England/69 (4) 5 A/duck/HK/22B/76 (12) 1 A/duck/Khabarovsk/1574/72; A/tern/Turk- menistan/18/73 (17) 10 A/duck/Ukraine/1/63 (15) 4 A/turkey/Wisconsin/66 (19) 1 None ' A/duck/HK/239/77 has a non-avid haemagglutinin. b A/duck/HK/232 and 233/77 have non-avid neuraminidames- 467 468 K. F. SHORTRIDGE ET AL. The present data show that ducks from South- East Asia harbour a diverse population of influenza A viruses and it can be postulated that recombina- tion is probably occurring in nature to produce such a diversity of virus subtypes. Studies by other investigators have shown that wild ducks also har- bour a diverse population of influenza virus subtypes (6) and it can be postulated that wild waterfowl may be involved in the transmission of these viruses. The isolation of influenza viruses from lake water and from faecal samples on river banks (V. Hinshaw, personal communication) indicates that this trans- mission probably occurs through the water supply. Although these viruses are not known to be patho- genic for man, the possibility exists that domestic poultry harbour a vast pool of influenza virus genetic information that may play a role in the emergence of new pandemic strains of man. NOTE ADDED IN PROOF Seven isolates previously classified as unidentified agents because of their failure to react with reference antiserum to influenza A RNP and with prototype antisera in HI and NI tests were subsequently shown by Dr D. Alexander to have typical myxovirus morphology. The isolates were found to be non-avid viruses: duck H2Nav2, Hav6Neq2(3), Hav9N2, and HswlN2; goose Hav6Nav4. Identification was made after passage in eggs and, in the case of the Hswl antigen, after antiserum production. ACKNOWLEDGEMENTS This study was made possible by the cooperation of the Health Inspectors, Urban Services Department, Hong Kong. The authors acknowledge the excellent technical assistance of Miss L. Y. Hu, S. L. Kam, and E. V. Kramer, Jr. RtSUMS DIVERS1TE DES SOUS-TYPES DE VIRUS GRIPPAL A ISOLES A PARTIR DE VOLAILLES ELEVEES A HONG KONG II s'agissait de la deuxieme phase d'un programme prevoyant la surveillance pendant deux ans de virus grip- paux chez les especes aviaires domestiques a Hong Kong. La surveillance exercee jusqu'en octobre 1977 a mis en 6vidence des virus grippaux A, le virus de la maladie de Newcastle et d'autres paramyxovirus aviaires de Hong Kong, et des agents hemagglutinants non identifies. Les isolements ont et6 operes a partir de pr6levements dans la trachee ou le cloaque d'oies, canards et poulets eleves en Chine et a Hong Kong et apparemment sains. Les virus grippaux isol6s etaient au nombre de 136 et appartenaient au groupe A. 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