Reappearance of H1N1 influenza virus in man: evidence for the persistence of the virus in domestic chickens*

BRIEF COMMUNICATIONS Bulletin of the World Health Organization, 57 (3): 475-477 (1979) Reappearance of HlNl influenza virus in man: evidence for the persistence of the virus in domestic chickens * K. F. SHORTRIDGE,' R. G. WEBSTER,2 S. L. KAM,3 & J. M. GARDNER4 Abstract Serological studies on domestic poultry originating from China and Hong Kong suggest that the HJNJ virus may have persisted in domestic chickens prior to its reappearance in man in 1977. Experimental infection of young, local strains of poultry provided equivocal evidence that 4-day-old chickens could be infected; virus was not recovered from infected 1-month-old chickens. Strains of influenza virus similar to the HlNl subtype that affected man from 1947 to 1956 reappeared in man in 1977 in the far east of the Asian mainland. The virus was first observed in northern China in May 1977 and its presence was recorded in Nahodka and Habarovsk in the USSR and in Hong Kong in November 1977. The reappearance of this HlNl virus prompted an investigation in Hong Kong into its possible origin in lower animals for two reasons: (a) this region of the world has been the point of origin of at least two of the previous pandemic strains of influenza and (b) many influenza viruses, some of which are closely related to the major human subtypes, have been isolated in Hong Kong from pigs and domestic poultry, particularly ducks (4-7). Surveillance studies conducted on a weekly basis from November 1975 to April 1978 on domestic poultry (2880 ducks, 702 geese, and 771 chickens), miscellaneous birds, pigs, cattle, and dogs did not yield HlNl virus, although there were numerous * This work was supported in part by contract Al 52424 from the US National Institute for Allergy and Infectious Diseases. I Member, Department of Microbiology, University of Hong Kong, Pathology Building, Queen Mary Hospital Compound, Hong Kong. 2 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, Tennessee 38101, USA. 3 Technician, Department of Microbiology, University of Hong Kong. 4 Research Assistant, Department of Microbiology, University of Hong Kong. isolations of viruses containing the Ni neuramini- dase subtype. At a recent World Health Organization/US Nat- ional Institutes of Health Workshop (8), Dr Chu Chi-ming of the Academy of Sciences, Peking pro- vided serological evidence of HlNl infection in chickens and goats. We report here the results of serological studies in avian species, which are in accord with Dr Chu's findings and suggest that the virus may have persisted in chickens. Findings Examination of RDE-treated avian sera by haemagglutination inhibition (HI) using A/HKU/ 10/77, a human HlNl isolate similar to A/USSR/ 90/77 (A.P. Kendal, personal communication), re- vealed the presence of HI activity exclusively in sera collected from domestic chickens as far back as 1968 and as recently as April 1978 (Table 1). A Table 1. Serological reactivity of chicken seraa for A/HKU/10/77 (Hi Ni) virus by haemagglutination inhibition Serab with HI titre > 10 originating Year of serum from: collection China Hong Kong 1968 1/5 1971 0/1 1972 1/2 1975 8/21 6/30 1976 1/11 13/91 1977 (pre-May) 3/11 1977 (May onwards) 3/28 1978 1/8 9/29 Total 10/40 36/197 aThe following sera were also examined and were of titre <10: duck, 120 from China and Hong Kong collected 1975 to post-May 1977; goose, 101 from China and Hong Kong collected 1975 to post-May 1977; pigeon, 51'from China and Hong Kong collected 1975; bamboo chicken, 24 from China collected pre-May 1977; quail, 45 of unknown origin collected 1976 and post-May 1977. Number of sera positive over number tested. 3821 - 475 - BRIEF COMMUNICATIONS total of 341 domestic ducks, geese, pigeons, bamboo chickens (Bambusicola thoracica), and quail (Cotur- nix coturnix japonica), collected at various times from 1975 to after May 1977, were all seronegative at a titre of 10. Of the 237 chicken sera examined, 46 had HI titres ranging from 10 to 80 (geometric mean 18). The overall frequencies of seropositive chickens originating from China and Hong Kong were 25 % and 18 %, respectively, and there was no significant difference between chickens collected prior to May 1977 (20%) and those collected from May 1977 onwards (19 %). All the sera were negative at titre 10 for the antigenically related viruses A/PR/8/34 (HON1), A/duck/HK/23/76 (Hav5Nav2), A/NJ/8/ 76 (HswlN1), and A/duck/Alberta/35/76 (avian HswlNl isolate) (V.S. Hinshaw, personal communi- cation), suggesting that the HI reactivity for HlNl virus was due to antibody rather than to a nonspec- ific inhibitor. This view is supported by the finding that all the other avian sera examined, particularly those of ducks (which are known to have a high frequency of influenza A virus infection) (5, 7) and of geese, were seronegative. Furthermore, when two seropositive samples were submitted to density grad- ient centrifugation (3), HI activity for A/HKU/10/ 77 and A/USSR/90/77 viruses was detected in the 7S position in both cases whereas it was absent in this position in a scronegative sample; nonspecific inhibitory activity in vertebrate sera resides in the 18S and 12S fractions (2). Of the 46 HI-positive sera screened at a 1:2 dilution, only four inhibited the neuraminidase activity of the recombinant virus A/Eq1(H)-A/NJ/68(N). These sera also had, the highest HI titres (20-80), suggesting that the im- mune response of chickens to infection by HlNl virus was weak and that it was directed mainly at the haemagglutinin. Fourteen of the sera were also screened for their ability to neutralize the infectivity of A/HKU/10/77 virus and this was observed in four samples. None of them did so with the antigen- ically related A/swine/HK/37/77 virus (HswlNl) (6) nor with the unrelated A/duck/HK/276/78 vi- rus, a recent avian H2N2 isolate (K. F. Shortridge, unpublished data). Four 1-month-old chickens, four 1-month-old ducklings, and eight 1-week-old goslings, all local strains, that were virus negative were each inocu- lated with approximately 4.5 log1OEID50 of A/HKU/ 10/77 via the cloaca or via the nostrils and throat. None of the birds yielded virus from tracheal or cloacal swabs taken daily for 14 days nor did they seroconvert after this time. In contrast, when four virus-negative 4-day-old chickens of a local strain were inoculated with the same dose of virus via the nostrils and throat, virus confirmed as HlNl was isolated from the tracheal swabs of two birds after 1 day and from tracheal and cloacal swabs of all birds after 2 days. Thereafter, the chickens were virus negative in spite of subsequent attempts to reinfect them 10, 17, and 27 days later. Only one chicken seroconverted after the original infection. None of the chickens showed overt signs of infec- tion. Three contact 4-day-old chickens introduced 1 day after infection remained virus negative through- out this period and did not seroconvert. In view of the fact that HswlNl and HlN1 viruses are antigenically related and that HswlNl virus has been isolated from pigs in this region (6), sera collected from 924 pigs originating fronm China, Hong Kong, and Singapore from 1971 to 1978 were examined by HI for evidence of HlNl virus infec- tion using A/HKU/10/77, A/USSR/90/77, and A/FM/1/47 viruses; none was found. Discussion The HlNl virus is a notable absentee from the large variety of influenza A viruses isolated from domestic pouftry in Hong Kong in recent years (5, 7; K. F. Shortridge, unpublished data). Nevertheless, there is good serological evidence in this study to suggest that the virus was present in chickens in China and Hong Kong for a number of years before its reappearance in man in 1977. That chickens rather than ducks should be implicated in the persistence of the virus in nature -is surprising since the isolation frequencies of influenza viruses in Hong Kong during the period November 1975 to April 1978 were 6.1 %, 1.3 %, and 0.6% for ducks, geese, and chickens, respectively (K. F. Shortridge, unpublished data). The detection of low-titre HlNl antibody in chickens in the absence of virus isolation would suggest a low level of infectivity, a view which is partially supported by our inability to infect young poultry. However, 4-day-old chickens may have been subject to a transient infection of the respira- tory and intestinal tracts that was not transmitted to contact chickens, but there was no conclusive evi- dence that the virus isolated was not residual input virus. It may be of interest to note that in studies to date on natural avian influenza infections in poultry antibody, if and when detected, is invariably of low titre similar to that observed in this investigation (H. S. Cheung & K. F. Shortridge, unpublished data). 476 BRIEF COMMUNICATIONS 477 It is difficult to explain the apparent association of the virus with chickens and it is therefore tempting to speculate that the virus might alternatively be transmitted vertically rather than horizontally, a phenomenon which has been reported to occur with Newcastle disease virus (1). Such a hypothesis implies that the virus would need to multiply in the tissues or organs of the chicken, although experi- mental studies to date in ducks with human HON1, H3N2, and HswlNl subtypes indicate that these viruses replicate in, and are confined to, the respira- tory tract (9). ACKNOWLEDGEMENTS The authors are grateful to F. S. F. Wong, Department of Agriculture and Fisheries, Hong Kong, for supplying pre- 1975 chicken and pig sera and to Miss L. Y. Hu for tech'nical assistance. REFERENCES 1. LANCASTER, J. E. & ALEXANDER, D. J. Newcastle disease virus and spread. Canada Department of Agri- culture, Monograph No. 11, 1975, p. 60. 2. PEPPER, D. Biochimica et biophysica acta, 156: 327- 339 (1968). 3. SHORTRIDGE, K. F. Journal of clinical pathology, 30: 409-416 (1977). 4. SHORTRIDGE, K. F. ET AL. Science, 196: 1454-1455 (1977). 5. SHORTRIDGE, K. F. ET AL. Bulletin of the World Health Organization, 55: 15-20 (1977). 6. SHORTRIDGE, K. F. & WEBSTER, R. G. Intervirology, 11: 9-15 (1979). 7. SHORTRIDGE, K. F. ET AL. Bulletin of the World Health Organization, 57: 465-469 (1979). 8. Summary of the International Workshop on the Eco- logy of Influenza Viruses. Journal of infectious disea- ses, 138: 110-113 (1978). 9. WEBSTER, R. G. ET AL. Virology, 84: 268-278 (1978).