Division of Virology, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK1
Government Virus Unit, Queen Mary Hospital, Hong Kong SAR of China, Peoples Republic of China2
Influenza Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA3
Author for correspondence: Yi Pu Lin. Fax +44 20 8906 4477. e-mail lyipu{at}nimr.mrc.ac.uk
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Abstract |
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Introduction |
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Studies of influenza viruses circulating in pigs in southern China between 1976 and 1982 and again during 1993 and 1994 indicated that classical H1N1 viruses predominated (Shu et al., 1994 ; Guan et al., 1996
). Only during 197678 and 1982 did H3N2 subtype viruses account for a significant proportion of the isolates. Of these, 29 of 32 were closely related to human H3N2 viruses, whereas three were reassortants possessing human-like H3 and N2 antigenic components and six internal genes (PB1, PB2, PA, NP, M and NS) corresponding to the genes of classical H1N1 swine viruses (Shu et al., 1994
; Nerome et al., 1995
). The genes of 11 H1N1 viruses isolated from pigs during September 1993 were more closely related to those of avian viruses and were distinct from the genes of classical H1N1 swine viruses (Guan et al., 1996
). Although more closely related to the genes of the avian-like H1N1 viruses circulating in pigs in Europe since 1979, the degree of difference indicated that these viruses were likely the result of a separate introduction of a related avian H1N1 virus into pigs. There have been no further published reports of swine viruses circulating in this region and prior to 1999 there were no reports of European-like H3N2 reassortant viruses in Asia.
Avian-like H1N1 viruses, distinct from classical swine viruses, were first isolated from European pigs in 1979 (Pensaert et al., 1981 ) and have since co-circulated with H3N2 viruses in European pigs. Genetic reassortment between viruses of these two subtypes in 198384 gave rise to H3N2 viruses which possess six internal genes corresponding to those of the avian-like H1N1 swine viruses, and these have continued to circulate in pigs in Europe (Castrucci et al., 1993
; Campitelli et al., 1997
).
In September 1999, an H3N2 influenza A virus isolated in Hong Kong SAR of China from a young child with mild influenza symptoms was antigenically distinct from H3N2 viruses recently circulating in the human population. In this report, we describe the antigenic and genetic properties of A/HK/1774/99, which show that it is closely related in all eight genes to H3N2 viruses recently circulating in pigs in Europe and in particular is similar to two viruses, A/Netherlands/5/93 and A/Netherlands/35/93, isolated from two children in the Netherlands during 1993 (Claas et al., 1994 ), further emphasizing the propensity of these swine viruses to infect people.
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Methods |
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Antisera.
Hyperimmune rabbit antisera and post-infection ferret antisera were prepared as previously described (Kendal et al., 1982 ). The post-infection ferret antiserum to A/HK/1774/99 was obtained from J. Wood, National Institute for Biological Standards, South Mimms, UK. Post-infection ferret antisera to A/Netherlands/5/93 and A/Netherlands/35/93 were obtained from A. Osterhaus. Rabbit antisera against reassortant viruses X15-HK (A/equine/Prague/1/56 (Eq/Prague/56)xA/Aichi/2/68), X-42 (Eq/Prague/56xA/PC/1/73) and Eq-Vic 75 (Eq/Prague/56xA/Vic/3/75) were used to compare the N2s of viruses with closely related H3 haemagglutinins.
Antigenic analyses.
Haemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests were performed as previously described (Kendal et al., 1982 ).
Amantadine/rimantadine sensitivity.
Susceptibility of virus replication to inhibition by amantadine or rimantadine was determined by ELISA, as described by Belshe et al. (1988) . MDCK cells were infected at different multiplicities of infection and incubated with different concentrations (0·011 µg/ml) of drug. HA expression was estimated using post-infection ferret antisera to antigenically similar reference viruses.
Gene sequencing and analyses.
Virus RNA was obtained from samples of infected cell culture fluid or allantoic fluid by phenol-chloroform extraction and ethanol precipitation. For RT-PCR primers (sequences are available on request) specific for each of the eight RNA segments were used. PCR products were purified by agarose gel electrophoresis and a Geneclean II kit (Bio101) and sequenced using an ABI Prism dye terminator cycle sequencing kit and an ABI model 377 DNA Sequencer (Perkin-Elmer, Applied Biosystems). Sequence data were edited and analysed using the Wisconsin Sequence Analysis Package version 8 (GCG). Phylogenetic analyses used PAUP (Phylogenetic Analysis Using Parsimony version 4.0; D. Swofford, Illinois Natural History Survey, Champaign, IL, USA).
Sequences, other than those for viruses mentioned above or listed in Table 1 were obtained for A/Hong Kong/156/97 (A/HK/156/97, H5N1; Bender et al., 1999
) and A/Hong Kong/1073/99(A/HK/1073/99, H9N2; Lin et al., 2000
). The nucleotide sequences determined in this study are available from GenBank under accession numbers AJ293920-AJ293943 and AJ311454-AJ311466.
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Results |
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Antigenic characteristics
The results of HI tests using hyperimmune rabbit antisera against avian, swine and human H3 viruses showed that A/HK/1774/99 was antigenically related to early (196875) human and swine H3N2 viruses but was clearly distinguishable from human H3N2 viruses isolated since 1979 and avian H3 viruses such as A/duck/Ukraine/1/63. Comparisons including representative European swine H3N2 viruses and using post-infection ferret antisera demonstrated a close relationship with A/PC/1/73 and Sw/CA/3633/84 and in particular the similarity between A/HK/1774/99 and two Dutch viruses, A/Netherlands/5/93 and A/Netherlands/35/93 (Table 1). The lower HI titres (4-fold lower than with the homologous viruses) with antisera to antigenically distinguishable recent (199698) European swine isolates, such as Sw/Eire/471/96, Sw/Italy/1477/96 and Sw/Italy/1523/98, suggests a closer antigenic relationship of the Hong Kong isolate to the Dutch viruses than to more recent swine viruses.
NI tests using hyperimmune rabbit antisera against various N2 viruses showed that the NA of A/HK/1774/99 was related to the N2s of early human H3N2 viruses such as A/Aichi/2/68 and A/PC/1/73 and was clearly distinguishable from the N2s of more recent human H3N2 viruses such as A/Bangkok/1/79 and A/Beijing/32/92, the H2N2 virus A/Singapore/1/57 and the avian virus A/turkey/Wisconsin/1/66 (H9N2) (Table 2). Although interpretation of corresponding results with post-infection ferret antisera is complicated by potential interference of antibody to the homologous H3 haemagglutinin, the data (not shown) supported these conclusions.
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Phylogenetic relationships between the six internal genes of A/HK/1774/99 and the corresponding genes of selected viruses are shown in Fig. 2 and indicate the closer relationship to the genes of European swine viruses, H1N1 viruses circulating since 1981 and H3N2 reassortant viruses isolated since 1984 (9597% similarity, Table 4
). As for the HA and NA genes, differences between corresponding genes of A/HK/1774/99 and European swine viruses were comparable to the genetic diversity observed among recent European swine viruses. The genetic comparisons given in Table 4
stress the more distant relationship to viruses recently isolated in Hong Kong, including human H3N2 viruses (8187% similarity), avian-like H1N1 swine viruses, such as Sw/HK/168/93 (8691 % similarity), human H5N1 and H9N2 viruses, represented by A/HK/1073/99 (8690% similarity) and other genetically distinct, avian H9N2 viruses, represented by Dk/HK/280/97 (8691% similarity).
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Discussion |
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How the child contracted the infection is unclear. Neither the child nor her immediate family had any recent history of contact with pigs inside or outside Hong Kong. The virus is similar to A/Netherlands/5/93 and A/Netherlands/35/93, which infected two children in the Netherlands; on that occasion serological studies pointed to the father as a possible intermediary in transmission (Claas et al., 1994 ). Serological studies of the Hong Kong family failed to demonstrate conclusively whether or not certain other family members, mother and brother, may also have been infected. A survey of sera from some 100 children failed to reveal evidence of further infections and no A/HK/1774/99-like viruses were detected among some 1500 A and B viruses isolated from people in Hong Kong since October 1999 (W. Lim, unpublished results). Thus this incident represents another example of sporadic human infection by a swine virus with little evidence of significant human-to-human transmission.
It is not known whether spread within the human population may be restricted by immunity to H3N2 viruses or by incompatible features of the avian internal genes. Together with the recent human infections by avian H5N1 and H9N2 viruses (Claas et al., 1998 ; Subbarao et al., 1998
; Lin et al., 2000
), it is evident, however, that viruses with genetically divergent avian internal genes can replicate effectively in the human respiratory tract and cause disease. A recent serological survey, which concluded that as many as 20% of people under 20 years of age who had contact with pigs in Italy had been infected with H3N2 swine viruses, emphasizes the possible frequency with which these infections may occur (Campitelli et al., 1997
).
Of significance in regard to the wider geographical spread of these swine viruses is the spread of amantadine-resistant viruses to a part of the world considered to be an epicentre for the emergence of novel human viruses (Shortridge & Stuart-Harris, 1982 ). The role of the pig as a potential intermediate host and the possible involvement of genetic reassortment in the emergence of such viruses indicate the greater potential for the emergence of amantadine-resistant human viruses.
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Footnotes |
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References |
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Received 12 January 2001;
accepted 20 February 2001.