Infection of a child in Hong Kong by an influenza A H3N2 virus closely related to viruses circulating in European pigs

V. Gregory1, W. Lim2, K. Cameron1, M. Bennett1, S. Marozin1, A. Klimov3, H. Hall3, N. Cox3, A. Hay1 and Y. P. Lin1

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, People‘s 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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Influenza virus A/Hong Kong/1774/99, isolated from a young child with mild influenza, was shown to be similar in its antigenic and genetic characteristics to H3N2 viruses circulating in pigs in Europe during the 1990s and in particular to be closely related to viruses isolated from two children in the Netherlands in 1993. Similar viruses had previously not been identified outside Europe. Although there is little evidence as to how the child contracted the infection, it appears likely that pigs in southern China were the source of infection. Characteristics shared with the European swine viruses include resistance to the anti-influenza drugs amantadine and rimantadine. Thus not only does this incident once again highlight the potential of pigs as a source of novel human influenza viruses, but also indicates the potential for emergence of amantadine-resistant human viruses.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Interspecies transmission of influenza A viruses between humans and pigs is not uncommon and is reflected in the similarities of the two subtypes, H1N1 and H3N2, which have circulated in the two species, and in the notion that pigs may act as an intermediate host in the emergence of novel human subtypes (Scholtissek et al., 1985 ). The ‘classical’ H1N1 swine viruses, first isolated in 1931 (Shope, 1931 ), appear to have infected pigs in North America in 1918 at about the same time as the closely related H1N1 virus emerged to cause the 1918–19 pandemic (Gorman et al., 1991 ; Reid et al., 1999 ). Viruses similar to the H3N2 subtype viruses which caused the ‘Hong Kong’ influenza pandemic of 1968–69 and have since circulated in the human population became established in pig populations in various parts of the world and multiple introductions of human viruses into pigs have been described (Kundin, 1970 ; McFerran et al., 1972 ; Shortridge et al., 1977 ; Hinshaw et al., 1978 ; Tumova et al., 1980 ; Ottis et al., 1982 ; Mancini et al., 1985 ; Bikour et al., 1995 ; Nerome et al., 1981 , 1995 ; Brown et al., 1998 ; Zhou et al., 1999 ). A number of sporadic human infections by swine viruses of both subtypes have been reported, some of which have resulted in serious disease (Goldfield et al., 1977 ; Patriarca et al., 1984 ; Rota et al., 1989 ; Wells et al., 1991 ; Claas et al., 1994 ; Wentworth et al., 1994 ). On these occasions, person-to-person spread appears to have been limited and the viruses did not become established in the human population.

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 1976–78 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 1983–84 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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Viruses.
A/Hong Kong/1774/99 (A/HK/1774/99) was isolated by culture in MDCK cells from a nasopharyngeal aspirate taken from a 10-month-old girl admitted to hospital with fever and congested throat on 8 September, 1999. The illness was mild and the patient was discharged from hospital 2 days later. Viruses isolated from pigs which were analysed for this study are listed in Table 3. Isolates from Belgium were obtained from M. Pensaert, University of Gent, Belgium; isolates from France were obtained from M. Aymard, Université Claude Bernard, Lyon, France; isolates from northern Italy were obtained from G. Barigazzi, Istituto Zooprofilattico, Sperimentale della Lombardia e dell’ Emilia, Parma, Italy or I. Donatelli, Istituto Superiore di Sanitá, Rome, Italy. A/swine/Eire/471/96 (H3N2) was obtained from P. Lenihan, Central Veterinary Laboratory, Dublin, Ireland. These isolates were passaged in the allantoic cavity of 10-day-old fertile hen eggs.


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Table 3. Gene sequences included in this study

 
Human virus isolates were passaged either in MDCK cells in medium containing 2·5 µg/ml trypsin (TPCK-treated) or in eggs. H3N2 viruses included A/Aichi/2/68, A/England/42/72 (A/Eng/42/72), A/Port Chalmers/1/73 (A/PC/1/73), A/Victoria/3/75 (A/Vic/3/75), A/Bangkok/1/79 (A/Bk/1/79), A/Beijing/32/92 (A/Beij/32/92), A/Sydney/5/97 (A/Syd/5/97), A/Wuhan/359/95 and A/swine/Taiwan/7310/70 (Sw/Tw/7310/70). A/Netherlands/5/93 (A/Neth/5/93) and A/Netherlands/35/93 (A/Neth/35/93) were obtained from A. Osterhaus, Erasmus University, Rotterdam, The Netherlands.

{blacksquare} 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.

{blacksquare} Antigenic analyses.
Haemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests were performed as previously described (Kendal et al., 1982 ).

{blacksquare} 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 (0·01–1 µg/ml) of drug. HA expression was estimated using post-infection ferret antisera to antigenically similar reference viruses.

{blacksquare} 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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Table 1. Antigenic relationships between the haemagglutinins of A/HK/1774/99 and human and swine H3N2 viruses

 

   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Influenza virus A/HK/1774/99 was isolated from a 10-month-old girl admitted to hospital with mild influenza symptoms and discharged 2 days later. An HI antibody titre of 160 to A/HK/1774/99 in serum obtained from the child confirmed infection by the virus. Antibody titres of 20 in serum from the mother and less than 10 in sera from other family members (brother, father and grandparents) did not demonstrate that other members of the girl‘s family had been infected by the same virus. Furthermore, since similar viruses had not previously been identified in Hong Kong and the laboratory was not handling such viruses, and since the virus from Hong Kong was subsequently identified independently in two laboratories, it is unlikely to have resulted from laboratory contamination by a swine virus.

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 (1968–75) 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 (1996–98) 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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Table 2. Antigenic characterization of the neuraminidase of A/HK/1774/99

 
Genetic relationships
Comparisons of the nucleotide sequences of the eight genes of A/HK/1774/99 with available sequence data (see Table 3) confirmed the close relationship between A/HK/1774/99 and recent European swine H3N2 viruses. Phylogenetic analyses (Fig. 1) showed that the HA gene of A/HK/1774/99 was most closely related to the HA genes of H3N2 viruses circulating in European pigs during the 1990s, such as Sw/Italy/1461/96 (95% similarity). The sequence encoding HA1 of A/HK/1774/99 is less closely related to those of earlier swine isolates, such as Sw/CA/3633/84 (90% similarity) or to the antigenically related early human H3N2 virus A/PC/1/73 (89% similarity). Sequence similarity to the HA genes of H3N2 viruses isolated from pigs in North America, Ireland, Japan or in Hong Kong between 1976 and 1982 is less than 90% and to current A/Sydney/5/97-like human H3N2 viruses is only 83%. Comparisons with the amino acid sequences of the H3 HAs of European swine viruses (including published and unpublished data) showed that the HA of A/HK/1774/99 possesses a number of amino acid residues which are typical of the HAs of swine viruses isolated in continental Europe during the 1990s when compared with similar H3N2 viruses circulating in pigs before 1987. These include asparagine-9, aspartic acid-31, asparagine-45, isoleucine-88, isoleucine-112, isoleucine-192 and isoleucine-202 of HA1, including an additional glycosylation site created by asparagine-45. The HA of A/HK/1774/99 possesses an extra glycosylation site at asparagine-122 (created by serine-124), which was observed to be present in the HAs of a few 1995–96 isolates, but lacks the site at asparagine-144, present in the HAs of many recent swine isolates from Italy and France (V. Gregory, K. Cameron, M. Bennett, Y. Lin & A. Hay, unpublished results). A number of variants have been distinguished by their HA sequences (unpublished data) but none showed a particularly close relationship to A/HK/1774/99. No differences were observed in the conserved amino acids constituting the receptor binding sites of human and swine H3 haemagglutinins.



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Fig. 1. Phylogenetic comparisons of nucleotide sequences encoding H3 haemagglutinins (HA, nucleotides 1–994) and N2 neuraminidases (NA, nucleotides 69–1416). Sequences of viruses not listed in Table 1 or Methods were obtained from GenBank: A/Aichi/2/68, A/BK/1/79, A/Beij/32/92, A/Eng/42/72, A/Hong Kong/8/68 (A/HK/8/68), A/Leningrad/134/47/57 (A/Len/134/57, H2N2), A/PC/1/73, A/Udorn/72, A/Vic/3/75, A/chicken/Pennsylvania/1370/83 (Ck/Pen/1370/83, H5N2), A/duck/Hong Kong/7/75 (Dk/HK/7/75), A/duck/Hong Kong/24/76 (Dk/HK/24/76), A/duck/Hong Kong/Y280/97 (Dk/HK/280/97, H9N2), A/duck/Hong Kong/940/80 (Dk/HK/940/80), A/duck/Hokkaido/5/77 (Dk/Hok/5/77), A/duck/Ukraine/1/63 (Dk/Ukr/1/63), A/swine/Breugel/97 (Sw/Breu/97), A/swine/Hong Kong/3/76 (Sw/HK/3/76), A/swine/Hong Kong/4/76 (Sw/HK/4/76), A/swine/Hong Kong/82/78 (Sw/HK/82/78), A/swine/Hong Kong/127/82 (Sw/HK/127/82), A/swine/Kanagawa/2/78 (Sw/Kan/2/78), A/swine/Nagasaki/1/90 (Sw/Nag/1/90, H1N2), A/swine/North Carolina/35922/98 (Sw/NC/98) and A/swine/Obihiro/1/94 (Sw/Ob/1/94). Unless indicated otherwise viruses were of the H3N2 subtype.

 
Similar relationships were observed among the N2 genes of these viruses (Fig. 1). Sequence similarity between the N2 genes of A/HK/1774/99 and H3N2 viruses isolated from pigs in Belgium (e.g. Sw/Gent/70/84) and Italy (e.g. Sw/Italy/1461/96) of 94–95% was greater than that between the N2 genes of A/HK/1774/99 and early human H3N2 viruses, such as A/Udorn/72 (92% similarity), swine viruses isolated more recently in Hong Kong, Japan, Ireland, UK or USA (86–90% similarity) or current A/Sydney/5/97-like human viruses (88% similarity). These relationships were also apparent in the amino acid sequences of these N2 neuraminidases. The NA of A/HK/1774/99 possessed a set of nine amino acids (methionine-20, lysine-60, valine-77, alanine-113, lysine-210, lysine-249, isoleucine-263, isoleucine-360 and serine-455) which are characteristic of the European swine H3N2 viruses and two residues, glycine-331 and phenylalanine-390, which are more typical of later 1990s isolates.

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 (95–97% 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 (81–87% similarity), ‘avian-like’ H1N1 swine viruses, such as Sw/HK/168/93 (86–91 % similarity), human H5N1 and H9N2 viruses, represented by A/HK/1073/99 (86–90% similarity) and other genetically distinct, avian H9N2 viruses, represented by Dk/HK/280/97 (86–91% similarity).



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Fig. 2. Phylogenetic comparisons of nucleotide sequences encoding PB2, PB1, PA, NP, M and NS proteins. Sequences of viruses not included in Table 1 and Methods were obtained from GenBank; virus names and abbreviations not included in the legend to Fig. 1 are A/Ann Arbor/6/60 (A/AA/6/60, H2N2), A/Hong Kong/1/68 (A/HK/1/68, H3N2), A/Northern Territories/60/68 (A/NT/60/68, H3N2) A/Puerto Rico/8/34 (A/PR/8/34, H1N1), A/Shiga/25/97 (H3N2), A/Singapore/1/57 (A/Sing/1/57, H2N2), A/budgerigar/Hokkaido/1/77 (Bud/Hok/1/77, H4N6), A/gull/Maryland/704/77 (Gul/MD/704/77, H13N6), A/mallard/New York/6750/78 (Mal/NY/6750/78, H2N2), A/ruddy turnstone/New Jersey/47/85 (RT/NJ/47/85, H4N6), A/swine/Germany/2/81 (Sw/Ger/2/81, H1N1), A/swine/Germany/8533/91 (Sw/Ger/8533/91, H1N1), A/swine/Hong Kong/6/76 (Sw/HK/6/76, H1N1), A/swine/Hong Kong/168/93 (Sw/HK/168/93, H1N1), A/swine Hong Kong/273/94 (Sw/HK/273/94, H1N1), A/swine/Iowa/15/30 (Sw/Iowa/15/30, H1N1), A/swine/Schleswig-Holstein/1/93 (Sw/SH/1/93, H1N1), A/swine/Tennessee/24/77 (Sw/TN/24/77, H1N1), A/swine/Tennessee/26/77 (Sw/TN/26/77, H1N1) and A/turkey/Minnesota/833/80 (Ty/MN/833/80, H4N2).

 

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Table 4. Comparisons of the internal genes of A/HK/1774/99 with the internal genes of recent human H3N2 viruses, European swine viruses and viruses circulating recently in pigs and avian species in Hong Kong

 
Although there are few complete coding sequences available for the PB1, PB2 and PA polymerase components, several amino acid residues were identified which differentiated the proteins of recent (1997–99) H1N1 and H3N2 isolates from those of earlier isolates. Of these four of four, two of three and five of seven were common to the PB1, PB2 and PA proteins of A/HK/1774/99, respectively (Table 5). The NP, M and NS genes of A/HK/1774/99 were most closely related to the corresponding genes of Sw/Ger/8533/91. The NP and NS1 proteins of Sw/Ger/8533/91 were representative of the proteins of subsequent H3N2 and H1N1 European swine isolates and A/HK/1774/99, and were distinguishable from those of pre-1988 isolates (Table 5). Chronological differences in the amino acid sequences of the M1 proteins of European swine viruses were less apparent. In addition to an amino acid residue more typical of post-1990 isolates, glutamic acid-16 in place of glycine in the proteins of earlier isolates, the M2 protein of A/HK/1774/99 possesses asparagine at position 31 which accounts for the resistance of A/HK/1774/99 replication to inhibition by amantadine and rimantadine (Fig. 3), a characteristic feature of H1N1 and H3N2 European swine viruses isolated since about 1987 (M. Bennett, S. Grambas & A. Hay, unpublished results).


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Table 5. Amino acid changes which differentiate the proteins of A/HK/1774/99 and recent European swine viruses from those of earlier isolates

 


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Fig. 3. Inhibition by rimantadine of the expression of HA on virus-infected cells, detected by ELISA. Absorbance at 450 nm (A450) in the presence of different concentrations of rimantadine is expressed as a percentage of the value in the absence of drug. {bullet}, Sw/CA/3633/84; {triangleup}, A/HK/1774/99.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
A/HK/1774/99 was shown to be closely related in its antigenic and genetic characteristics to H3N2 viruses prevalent in pigs in Europe during the 1990s and not to have acquired, by reassortment, any genes from other, genetically distinguishable, viruses reported to be circulating recently in local human, pig or domestic poultry populations in South-east Asia. Although prior to isolation of this virus there were no reports of the isolation of similar viruses from pigs in South-east Asia, subsequent investigations have identified related viruses in pigs in Hong Kong SAR of China (K. Shortridge, personal communication), consistent with local pigs as the likely source of infection. The genetic relationships between A/HK/1774/99 and European swine viruses is consistent with these viruses entering the pig population in southern China during the 1990s.

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.


   Footnotes
 
The GenBank accession numbers of the sequences reported in this paper are AJ293920-AJ293943 and AJ311454-AJ311466.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 12 January 2001; accepted 20 February 2001.