Antigenic and genetic diversity among swine influenza A H1N1 and H1N2 viruses in Europe

S. Marozin1, V. Gregory1, K. Cameron1, M. Bennett1, M. Valette2, M. Aymard2, E. Foni3, G. Barigazzi3, Y. Lin1 and A. Hay1

National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK1
Université Lyon 1, Laboratory of Virology, 8 Avenue Rockefeller, 69373 Lyon Cedex 08, France2
Istituto Zooprofilattico, Sperimentale della Lombardia e dell’Emilia, Parma, Italy3

Author for correspondence: Alan Hay. Fax +44 20 8906 4477. e-mail ahay{at}nimr.mrc.ac.uk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Three subtypes of influenza A viruses, H1N1, H1N2 and H3N2, co-evolve in pigs in Europe. H1N2 viruses isolated from pigs in France and Italy since 1997 were closely related to the H1N2 viruses which emerged in the UK in 1994. In particular, the close relationship of the neuraminidases (NAs) of these viruses to the NA of a previous UK H3N2 swine virus indicated that they had not acquired the NA from H3N2 swine viruses circulating in continental Europe. Moreover, antigenic and genetic heterogeneity among the H1N2 viruses appeared to be due in part to multiple introductions of viruses from the UK. On the other hand, comparisons of internal gene sequences indicated genetic exchange between the H1N2 viruses and co-circulating H1N1 and/or H3N2 subtypes. Most genes of the earlier (1997–1998) H1N2 isolates were more closely related to those of a contemporary French H1N1 isolate, whereas the genes of later (1999–2000) isolates, including the HAs of some H1N2 viruses, were closely related to those of a distinct H1N1 antigenic variant which emerged in France in 1999. In contrast, an H3N2 virus isolated in France in 1999 was closely related antigenically and genetically to contemporary human A/Sydney/5/97-like viruses. These studies reveal interesting parallels between genetic and antigenic drift of H1N1 viruses in pig and human populations, and provide further examples of the contribution of genetic reassortment to the antigenic and genetic diversity of swine influenza viruses and the importance of the complement of internal genes in the evolution of epizootic strains.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
The important relationship between influenza in pig and human populations is due to the relative ease of transmission of viruses between the two species and is reflected in the prevalent subtypes. Influenza A viruses of the H1N1 and H3N2 subtypes have co-circulated and caused outbreaks of disease among pigs in Europe since the mid 1970s. The initial H3N2 viruses were antigenically related to contemporary human H3N2 viruses, such as A/Port Chalmers/1/73 (Tumova et al., 1980 ; Ottis et al., 1982 ). The H1N1 viruses which emerged in pigs in 1979 were antigenically distinct from classical swine H1N1 viruses and apparently resulted from the introduction of an avian virus in toto (Pensaert et al., 1981 ). These ‘avian-like’ H1N1 viruses replaced the previously circulating ‘classical’ H1N1 swine viruses (Nardelli et al., 1978 ) and have since co-circulated with H3N2 viruses. Genetic reassortment between these two subtypes produced H3N2 reassortant viruses, possessing six internal genes (PB1, PB2, PA, NP, M and NS) corresponding to those of the ‘avian-like’ H1N1 swine viruses, which replaced the former ‘human-like’ H3N2 viruses around 1983–1984 (Castrucci et al., 1993 ; Campitelli et al., 1997 ). H1N2 subtype viruses, also derived by genetic reassortment between these H1N1 and H3N2 viruses, had only been isolated occasionally in European pigs (Gourreau et al., 1994 ). H1N2 viruses, derived from co-circulating classical H1N1 and H3N2 swine viruses did, however, become widespread in pig populations in Japan following the first outbreaks in the early 1980s (Sugimura et al., 1980 ; Nerome et al., 1985 ; Ouchi et al., 1996 ; Ito et al., 1998 ).

Genetically (and antigenically) distinct H1N2 viruses which emerged in the UK in the early 1990s (Brown et al., 1995 , 1998 ) have become the predominant subtype causing influenza among UK pigs (Brown, 2000 ) and have since spread to pigs in continental Europe (Van Reeth et al., 2000 ; Marozin et al., 2001 ). These H1N2 viruses arose by genetic reassortment and possess a H1 haemagglutinin (HA) closely related to those of human H1N1 viruses circulating in the early 1980s, a human-like N2 neuraminidase (NA) and six internal genes (PB1, PB2, PA, NP, M and NS) corresponding to those of European avian-like H1N1 viruses. More recently, H1N2 viruses, responsible for an outbreak of influenza on a farm in the USA, were shown to be the result of reassortment between recently co-circulating classical H1N1 and swine–human–avian reassortant H3N2 viruses and, therefore, also to contain genes of swine, human and avian origin (Karasin et al., 2000a ). Frequent sporadic human infections by swine influenza viruses emphasize their potential importance in the emergence of novel epidemic or pandemic human viruses and the importance of understanding the nature and evolution of these viruses (Goldfield et al., 1977 ; Wells et al., 1991 ; Claas et al., 1994 ; Gregory et al., 2001 ).

In this paper we analyse the characteristics of H1N2 viruses isolated since 1997 from pigs in France and Italy, which suggest that they were derived from those previously circulating in pigs in the UK. Investigations of the genetic relationships between viruses of the three co-circulating subtypes, H1N1, H3N2 and H1N2, defined the recent emergence of an antigenic (and genetic) H1N1 variant, demonstrated reassortment between this H1N1 variant and H1N2 viruses, and identified an H3N2 swine virus closely related to contemporary A/Sydney/5/97-like human viruses. These data provide a clearer understanding of the genetic relationships among viruses of the different subtypes and further emphasize the importance of frequent genetic exchange in the evolution of swine influenza viruses.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Viruses.
These were isolated from nasal swabs or lung samples during outbreaks of respiratory disease in pigs on farms in the Brittany area of Northern France and Northern Italy, by passage in the amniotic and allantoic cavities of 10-day-old fertile hen eggs or in cultures of MDCK cells or newborn swine kidney (NSK) cells (Ferrari et al., 1989 ). A/swine/UK/119404/91 (H3N2) and A/swine/Scotland/410440/94 (H1N2) were obtained from I. Brown, Central Veterinary Laboratory, Weybridge, UK. A/swine/Belgium/74/95 was obtained from M. Pensaert, University of Ghent, Belgium.

{blacksquare} Antisera.
Hyperimmune rabbit antisera and post-infection ferret sera were prepared as described in Kendal et al. (1982) .

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

{blacksquare} Amantadine susceptibility.
Inhibition of virus replication by amantadine (or rimantadine) was determined by ELISA, as described by Belshe et al. (1988) .

{blacksquare} Sequence analyses.
These were done as described previously in Lin et al. (2000) . The sequences of the specific primers used for RT–PCR of the eight RNA segments are available on request. Sequence data were edited and analysed using the Wisconsin Sequence Analysis Package Version 10 (GCG). Phylogenetic analyses used PAUP (Phylogenetic Analysis Using Parsimony, Version 4.0, D. Swofford, Illinois Natural History Survey, Champaign, IL, USA). Nucleotide sequences obtained in this study, including those for swine viruses listed in Table 1, the HAs of the human viruses A/Wuhan/371/95 (H1N1; AJ344022) and A/New Caledonia/20/99 (A/NCal/20/99, H1N1;AJ344014) and the HA and NA of A/Lyon/2573/98 (AJ316063), are available from GenBank; accession numbers AJ306841–306855 (PB2); AJ306856–306869 (PB1); AJ307061–307074 (NP); AJ311203–311211 and AJ312836–312838 (PA); AJ316047–316061 (M); AJ344002–344023 and AJ412708–412712 (HA); AJ316046, AJ316048, AJ316062 and AJ344024–344041 (NS); AJ410875–410884 and AJ412689–412707 (NA).


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Table 1. Gene sequences of swine influenza isolates determined in this study

 

   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Antigenic characteristics
The antigenic properties of some 117 influenza A viruses isolated from pigs in France (Brittany) and Northern Italy during 1997 to 2000 were compared in haemagglutination inhibition (HI) and neuramindase inhibition (NI) assays. Whereas the H3N2 subtype predominated among viruses isolated in Italy (52 out of 86), few viruses of this subtype were isolated in Brittany (2 out of 31). Following the initial isolation of a ‘UK-like’ H1N2 virus, Sw/CA/790/97, in Brittany in 1997 (one of nine 1997 swine isolates characterized) the H1N2 viruses have increased in prevalence and accounted for half of the viruses isolated from pigs in Brittany during 2000, and have spread to Italy. The viruses, for which data are presented, are listed in Table 1.

H1N1 viruses.
The majority of swine influenza A H1N1 viruses circulating in France and Italy prior to 2000 were closely related antigenically to earlier avian-like H1N1 swine isolates such as Sw/Finistere/2899/82 (Table 2), even though their HA and NA sequences had drifted significantly (see below, Fig. 1). An antigenic variant, represented by Sw/IV/1455/99, which was initially isolated in France in 1999, was distinguishable from other contemporary isolates, such as Sw/CA/1482/99 and Sw/Italy/1513-1/98, using post-infection ferret sera (Table 2). Sw/IV/1455/99-like viruses have since increased in prevalence; they accounted for all three of the French H1N1 viruses isolated in 2000 and were present among H1N1 viruses isolated in Italy (Table 2). The NAs of Sw/IV/1455/99-like viruses were also distinguishable from the NAs of earlier isolates and other contemporary H1N1 swine viruses in NI tests using post-infection ferret sera (data not shown).


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Table 2. Antigenic relationships between the haemagglutinins of swine influenza AH1N1 viruses

 


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Fig. 1. Phylogenetic relationships between the HA and NA genes of H1N1 and H1N2 swine influenza viruses. Sequences encoding HA1 of H1 (A, nucleotides 1–957), N2 (B, nucleotides 70–986) and N1 (C, nucleotides 73–1359) were analysed with PAUP using a maximum parsimony algorithm; the H1 tree is rooted to the HA sequence of A/Japan/305/57 (H2N2). The lengths of the horizontal lines are proportional to the number of nucleotide differences (as indicated by the bar). Sequences of viruses not listed in Table 1 or Methods, or given in Gregory et al. (2001), were obtained from GenBank: A/Bangkok/1/79 (A/Bk/1/79, H3N2), A/Beijing/32/92 (A/Beij/32/92,H3N2), A/Brazil/11/78 (H1N1), A/England/333/80 (A/Eng/333/80, H1N1), A/Japan/305/57 (A/Jap/305/57, H2N2), A/Mississippi/1/85 (A/Ms/1/85,H3N2), A/Ohio/1/83 (H1N1), A/Taiwan/1/86 (A/Tw/1/86, H1N1), A/Texas/36/91 (A/Tx/36/91, H1N1), A/Udorn/72 (A/Ud/72,H3N2), A/USSR/90/77 (H1N1), A/Victoria/3/75 (A/Vic/3/75, H3N2), A/duck/Bavaria/2/77 (Dk/Bav/2/77, H1N1), A/parrot/Ulster/73 (Pt/Ulster/73,H7N1), A/swine/Ehine/1/80 (Sw/Ehime/1/80, H1N2), A/swine/England/195852/92 (Sw/Eng/195852/92, H1N1), A/swine/England/283902/93 (Sw/Eng/283902/93, H1N1), A/swine/England/690421/95 (Sw/Eng/690421/95, H1N2), A/swine/Germany/8533/91 (Sw/Ger/8533/91,H1N1), A/swine/Hong Kong/21/77 (Sw/HK/21/77, H3N2), A/swine/Indiana/9k035/99 (Sw/Ind/9k035/99, H1N2), A/swine/Iowa/15/30 (Sw/Iowa/15/30, H1N1), A/swine/Italy/v147/81 (Sw/It/v147/81, H1N1), A/swine/Nagasaki/1/89 (Sw/Nag/1/89, H1N2), A/swine/Nagasaki/1/90 (Sw/Nag/1/90, H1N2). H1N2 viruses are in bold type. Sw/IV/1455/99 (H1N1)-like viruses are underlined.

 
H1N2 viruses.
With two exceptions, the HAs of the swine H1N2 viruses were, like that of the UK virus Sw/Scot/410440/94, closely related to the HA of the human H1N1 virus A/Brazil/11/78 and were distinct from the HAs of the Sw/Fin/2899/82 (H1N1)-like viruses circulating since 1979 (Table 3). Post-infection ferret antisera to Sw/CA/790/97, Sw/Italy/1521/98 and Sw/CA/604/99 distinguished between the French and Italian isolates and the earlier UK isolate, Sw/Scot/410440/94; furthermore Sw/CA/604/99 was not representative of the other French isolates.


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Table 3. Antigenic relationships between the haemagglutinins of swine influenza AH1N2 viruses

 
In contrast, the HAs of Sw/Italy/2064/99 (H1N2) and Sw/Italy/66734/00 (H1N2) were more closely related to the HAs of European swine H1N1 viruses (Table 2). Ferret antisera to Sw/Italy/2064/99 and Sw/IV/1455/99 showed little cross-reactivity in the HI tests.

NI tests using hyperimmune rabbit antisera against A/Port Chalmers/1/73 (H3N2), A/Victoria/3/75 (H3N2) and A/Bangkok/1/79 (H3N2) showed that the NAs of all the H1N2 viruses were related antigenically to the NAs of the early human H3N2 virus A/Port Chalmers/1/73 and were clearly distinguishable from the NAs of later H3N2 viruses, such as A/Bangkok/1/79 (data not shown).

H3N2 viruses.
The majority of H3N2 swine viruses isolated in Italy were antigenically closely related to recent reference strains, descendants of A/Port Chalmers/1/73-like viruses which were introduced into European pigs in the early 1970s (Gregory et al., 2001 ). In contrast, one French isolate, A/swine/Finistere/127/99, was distinguishable from these viruses and was shown, by HI and NI tests, to be closely related to recent A/Sydney/5/97-like human viruses (data not shown).

Genetic relationships
Haemagglutinin.
Phylogenetic comparisons of the sequences of HA genes of the H1N2 viruses (with two exceptions) confirmed that they are closely related to the HA genes of earlier UK H1N2 viruses and indicated that viruses such as Sw/Scot/410440/94 and Sw/Eng/690421/95 were intermediates in evolution from the HAs of earlier human H1N1 viruses, such as A/Ohio/1/83 (Fig. 1A). Whereas most were more closely related to Sw/Eng/690421/95, the HA gene of Sw/CA/604/99 was more closely related to that of Sw/Scot/410440/94, suggesting that Sw/CA/604/99 may have resulted from a separate introduction of a UK H1N2 virus into pigs in France. This notion is supported by similar genetic relationships between Sw/CA/604/99 and the other H1N2 swine viruses apparent in comparisons of the sequences of N2 and other genes (see below).

Although the accumulation of mutations in the HA genes of the swine H1N2 viruses (relative to the HA of A/Ohio/1/83) was comparable to that of the related human H1N1 viruses (3–4x10-3 substitutions per nucleotide per year), this was associated with less change in the amino acid sequences of the swine H1N2 virus HAs; 22% of nucleotide substitutions caused amino acid changes (Sw/Italy/1521/98) compared with about 39% for human H1N1 viruses between 1983 and 1998. Only two common amino acid changes, G239R and G264E/R (numbered according to H3 HA), distinguish HA1 of the French (with the exception of Sw/CA/604/99) and Italian H1N2 isolates from HA1 of the two earlier UK isolates. Variation in glycosylation at asparagine-158 may contribute to differences in antigenicity of the HAs of the swine H1N2 viruses, evident in the HI tests. A two amino acid deletion, residues 133 and 134 (H3 numbering), in the HA of Sw/Italy/1521/98, which is not present in the HAs of other H1N2 viruses, does not, however, appear to account for differences in antigenicity.

In contrast, the HA genes of two Italian H1N2 isolates, Sw/Italy/2064/99 and Sw/Italy/66734/00, were more closely related to those of recent Sw/IV/1455/99-like H1N1 swine viruses, in particular Sw/Italy/3364/00 (95% sequence similarity; Fig. 1A). The HA genes of H1N1 viruses antigenically similar to Sw/IV/1455/99, including Sw/Italy/3364/00, were clearly distinguishable phylogenetically from the HA genes of other contemporary French and Italian isolates, represented by, e.g., Sw/CA/1482/99 and Sw/Italy/1511/98, respectively (Fig. 1A). Sixteen amino acids common to the HA1s of the Sw/IV/1455/99-like viruses, one of which removed a potential glycosylation site at asparagine-94 (H3 numbering), distinguished them from the HAs of Sw/Fin/2899/82-like viruses, such as Sw/CA/1482/99. The HA1 sequences of Sw/Italy/2064/99 and Sw/Italy/66734/00 possess only six of these amino acid changes, including the loss of glycosylation at residue 94, and appear to be derived from an intermediate in the emergence of Sw/IV/1455/99-like viruses (Fig. 1A). Thus sequence differences can readily account for the antigenic differences between the HAs of Sw/Italy/2064/99 and Sw/IV/1455/99 (Table 2).

The sequence of the H3 HA of Sw/Fin/127/99 (H3N2), in contrast to those of the other H3N2 swine viruses recently isolated in Europe, was similar to the HA sequences of human A/Sydney/5/97-like viruses, in particular the French isolate A/Lyon/2573/98 (isolated about 2 months prior to Sw/Fin/127/99) (data not shown). It possessed the amino acid changes, relative to A/Sydney/5/97, typical of the human variant prevalent at that time.

Neuraminidase.
The N2 genes of the H1N2 viruses (including Sw/Italy/2064/99) were more closely related (93–95% sequence similarity) to the N2 gene of the UK H3N2 virus Sw/UK/11904/91 than to the N2 genes of H3N2 viruses isolated from pigs in continental Europe between 1984 and 2000 (86–91% similarity) or H1N2 viruses circulating in pigs in Japan (Fig. 1B). It is apparent, therefore, that the N2 of these H1N2 viruses, was, like the HA, derived from previously identified UK H1N2 viruses and not acquired, by reassortment, from local swine H3N2 viruses. The phylogenetic comparisons also show that the N2 gene of Sw/CA/604/99 was more closely related to that of Sw/UK/119404/91 (H3N2; 95% similarity) than to the NA genes of other French and Italian H1N2 isolates (92–93% similarity), emphasizing the difference between Sw/CA/604/99 and the others H1N2 isolates. These differences were also apparent in the NA sequences where some 13 amino acids distinguished the former from the latter viruses. The extent of drift in the NA genes of these H1N2 viruses relative to those of early human H3N2 viruses, e.g. A/Udorn/72, was comparable to that observed for human H3N2 viruses and H3N2 viruses circulating in European pigs (8–10% over 28 years).

Fig. 1(B) also illustrates the similarity between the NA of Sw/Fin/127/99 (H3N2) and the NA of a contemporary human A/Sydney/5/97 (H3N2)-like virus, A/Lyon/2573/98. Comparisons of the amino acid sequences showed that both the HA and NA were typical of H3N2 viruses circulating in the human population in France at the time of isolation of Sw/Fin/127/99.

Phylogenetic relationships between the NA gene sequences of French and Italian H1N1 swine viruses were comparable to those observed for the corresponding HA gene sequences (Fig. 1). The N1 sequences of the Sw/IV/1455/99-like viruses were also clearly distinguishable (93% similarity) from those of other contemporary French H1N1 viruses, such as Sw/CA/1482/99 and Sw/CA/1515/99 and were, in fact, more closely related to some earlier H1N1 isolates from Belgium (Sw/Belgium/74/95) or the UK (Sw/England/195852/92) (Fig. 1C). Eighteen amino acid differences, including eight characteristic of Sw/IV/1455/99-like viruses and four characteristic of Sw/CA/1482/99-like viruses, distinguished the NAs of the two phylogenic groups.

Internal genes.
The six internal genes (PB1, PB2, PA, NP, M and NS) of the H1N2 viruses were closely related to the corresponding genes of co-circulating H1N1 and/or H3N2 swine viruses. Fig. 2(A, B) shows that genes of different viruses fall into phylogenetically distinguishable groups, in particular those represented by the antigenically distinguishable H1N1 variants Sw/IV/1455/99 and Sw/CA/1482/99. Differences between the genes of these two viruses ranged from 2·6% for PB2 to 5·8% for NS, compared with 8% and 7%, respectively, for the HA and NA genes.




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Fig. 2. Phylogenetic relationships between the genes of H1N1, H1N2 and H3N2 swine influenza viruses. Coding sequences of A, PB2 (nucleotides 53–894), PB1 (nucleotides 25–1479) and PA (nucleotides 19–887) genes, and B, NP (nucleotides 449–1372), M (nucleotides 127–871) and NS (nucleotides 35–813) genes were analysed using PAUP. The trees were rooted: PB2 to A/chicken/Rostock/1/34 (Ck/Rost/34, H7N1), NP to A/teal/Iceland/29/80 (Tl/Ice/29/80, H7N7) M to A/oystercatcher/Germany/87 (Oy/Ger/87, H1N1) NS to A/equine/Alaska/1/91 (Eq/Ak/1/91, H3N8). The lengths of the horizontal lines are proportional to the number of nucleotide differences (as indicated by the bars). Sequences of viruses not listed in Table 1 or Methods, or given in Gregory et al. (2001) , were obtained from GenBank. Names (and abbreviations) not given elsewhere include: A/Fukushima/140/96 (A/Fuk/140/96, H3N2). A/Nagasaki/76/98 (A/Nag/76/98, H3N2), A/duck/Hong Kong/412/78 (Dk/HK/412/78, H4N2), A/duck/Hong Kong/Y280/97 (Dk/HK/Y280/97, H9N2), A/duck/Hokkaido/8/80 (Dk/Hok/8/80, H3N8), A/swine/Tennessee/24/77 (Sw/Tn/24/77, H1N1), A/swine/Tennessee/26/77 (Sw/Tn/26/77, H1N1), A/turkey/Minnesota/833/80 (Ty/Mn/833/80, H4N2). H1N2 subtype viruses are in bold type. Sw/IV/1455/99-like viruses are underlined.

 
The genes of early 1997–1998 H1N2 viruses, Sw/CA/790/97, Sw/CA/2433/98 and Sw/Italy/1521/98 (including some genes of Sw/Italy/16541/99), were similar to each other and to the genes of Sw/CA/1482/99 (H1N1), which, in terms of HA and NA sequences, is representative of most of the 1999 French H1N1 isolates (sequence similarity of 98–99%, data not shown). An exception was PB1 of Sw/CA/2433/98, which was more closely related to the PB1 of Sw/Italy/15131/98 (H1N1) (Fig. 2A).

With the exception of the NS gene, the internal genes of other later H1N2 isolates, Sw/Italy/2064/99, Sw/Italy/1081/00 and Sw/CA/800/00, were phylogenetically distinguishable from those of the earlier H1N2 isolates and clustered with the corresponding sequences of the Sw/IV/1455/99(H1N1)-like viruses. In particular, differences between the six genes of Sw/CA/800/00 (H1N2) and Sw/IV/1455/99 (H1N1) of less than 1% contrasted with differences of 3–5% between the genes of Sw/CA/800/00 and Sw/CA/790/97. The NS genes of Sw/Italy/2064/99 and Sw/Italy/1081/00 were more closely related to those of the earlier H1N2 virus isolates and grouped most closely with the sequence of the H3N2 swine virus Sw/Italy/1523/98. The PB2 and NP genes of these three viruses were also closely related. The lack of divergence between the genes of recent H3N2 and H1N1 (and H1N2) viruses is illustrated by the closer relationship between the NP genes of Sw/Italy/1477/96 (H3N2) and Sw/Italy/15096/97 (H1N1) than between the NP genes of contemporary viruses of the same subtype (Fig. 2). It is apparent, therefore, that genetic reassortment between co-circulating H1N2 and H1N1 (and/or H3N2) viruses is responsible for changes in the genetic makeup of the H1N2 viruses.

Differences in the sequences of NS1 proteins were more marked than for other gene products. Twelve amino acids differentiated the NS1 proteins encoded by ‘Sw/CA/1482/99-like’ and ‘Sw/IV/1455/99-like’ genes; 50% of the nucleotide changes caused amino acid substitutions. There were differences in the lengths of NS1 (normally 230 amino acids) of particular viruses. The NS1 of Sw/Italy/2064/99 (H1N2) (220 residues) was 10 amino acids shorter, whereas NS1 of Sw/Italy/1523/98 (H3N2) was 7 amino acids longer. Of particular note was the truncated NS1 (119 amino acids) of Sw/CA/604/99 (H1N2). This was due to a deletion of 17 nucleotides which caused a frame-shift after codon 116 and termination at residue 119. The significance of these sporadic differences in NS1 is unclear, although they are not uncharacteristic of this virus protein. Four amino acids in the NS1 protein and two in the nuclear export protein (NS2) were characteristic of the Sw/IV/1455/99-like viruses.

The lower rate of accumulation of mutations in the M genes of H1N1 and H1N2 viruses of about 1·4x10-3 nucleotide substitutions per site per year (between 1984 and 2000) is reflected in the high degree of conservation in the M1 proteins of these viruses. All M2 proteins of the recent H1N2 and H1N1 swine viruses had asparagine at position 31, indicating that like other European swine viruses isolated since 1987, including those of the H3N2 subtype, they are resistant to the anti-influenza drugs amantadine and rimantadine (Gregory et al., 2001 ). This was confirmed for several viruses, including Sw/IV/1455/99 and Sw/Italy/1521/98, by an ELISA for drug susceptibility.

As for HA and NA, the PB1, PB2, PA, NP and NS genes of Sw/CA/604/99 were more closely related to the corresponding genes of the earlier UK H1N2 isolate, Sw/Scot/410440/94 than to those of the other French or Italian H1N2 isolates (Fig. 2A,B). The differences from the latter viruses were emphasized further by the close relationship of the NP genes of Sw/CA/604/99 and the earlier H1N1 virus Sw/Eng/195852/92.

Nucleotide sequences of the PB1, PB2, PA, NP and NS genes of Sw/Fin/127/99 (H3N2) were closely related to corresponding genes of recent human H3N2 viruses, such as A/Fukushima/140/96 and A/Nagasaki/76/98 (Fig. 2A, B).


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Recent changes in the viruses causing influenza in French and Italian pigs include the following. (1) The introduction, in 1997 or before, of H1N2 subtype viruses similar to those initially detected in the UK. (2) The emergence in 1999 of Sw/IV/1455/99 (H1N1)-like viruses, antigenically and genetically distinguishable from previously circulating H1N1 swine viruses. (3) Genetic reassortment between H1N2 and Sw/IV/1455/99 (H1N1)-like viruses to generate H1N2 viruses possessing five or six genes (including HA) more closely related to those of the recent H1N1 variant. (4) The isolation in 1999 of a H3N2 swine virus antigenically and genetically closely related to contemporary human A/Sydney/5/97-like viruses.

The H1N2 viruses have become established in pig populations in continental Europe and have increased in prevalence during the past 4 years. The antigenic and genetic relationships between the HAs and NAs of viruses isolated from pigs in France, Italy and the UK indicated that H1N2 viruses were introduced into continental Europe from the UK, probably on more than one occasion. In particular, the N2s of all the H1N2 viruses were closely related to the N2s of H3N2 viruses previously circulating in pigs in the UK and distinct from the N2s of contemporary continental H3N2 swine viruses, which have diverged following the introduction of human H3N2 viruses into pigs in the early 1970s. The NAs of the viruses were therefore not acquired from continental European H3N2 swine viruses.

On the other hand, the similarities between the internal genes of the earliest H1N2 isolates and those of the contemporary French H1N1 virus Sw/CA/1482/99 suggest that genetic reassortment with H1N1 viruses occurred following introduction of H1N2 viruses into continental Europe. This was particularly evident from subsequent changes in 1999 and 2000 H1N2 isolates, which apparently resulted from reassortment with recently emergent H1N1 variants, antigenically and genetically distinguishable from H1N1 viruses isolated from pigs prior to 1999. For example, the recent French isolate Sw/CA/800/00, which was closely related in HA (98·6%) and NA (99·0%) to the earlier H1N2 isolate Sw/CA/790/97, possessed a complement of six internal genes similar (99·0–99·8% sequence similarity) to that of the Sw/IV/1455/99 (H1N1) variant. The genomes of Italian H1N2 viruses were also mixtures of genes from the two phylogenetic groups; in particular two possessed HAs closely related to those of Sw/IV/1455/99 (H1N1)-like viruses. Thus, antigenic heterogeneity among H1N2 viruses, previously observed among viruses circulating in the UK (Brown et al., 1998 ) and which may in part be due to multiple introductions of different H1N2 viruses from the UK, has also resulted from reassortment with contemporary H1N1 subtype viruses. Regarding the potential significance of the latter viruses, it may be noted that previous examples of H1N2 viruses, deriving genes from co-circulating H1N1 and H3N2 viruses (Gourreau et al., 1994 ), did not become prevalent in the swine population. The antigenic heterogeneity among H1N2 viruses contrasts with the antigenic similarity among H1N1 viruses circulating between 1980 and 1999.

Sw/IV/1455/99–like viruses represent the first clearly definable antigenic variant to have emerged among H1N1 viruses circulating in pigs in France and Italy since the early 1980s. Within a year of detection Sw/IV/1455/99-like viruses have become the predominant H1N1 virus in France (of the few viruses characterized) and are present in Italy. They were distinguished in all eight genes from previously circulating H1N1 viruses. The degree of genetic divergence of the HA and NA genes of Sw/IV/1455/99 from contemporary Sw/Fin/2899/82 (H1N1)-like viruses, such as Sw/CA/1482/99, of 8% and 7%, respectively, is comparable to the divergence between the HA (7%) and NA (6%) genes of recent (2000) representatives of the two antigenically distinct lineages of H1N1 viruses co-circulating in the human population, at a time when the newer variant (A/New Caledonia/20/99-like) became predominant (Hay et al., 2001 ). Parallels between the evolution of H1N1 viruses in swine and human populations are therefore apparent in both the circulation of antigenically similar viruses for extended periods of time (in contrast to human H3N2 viruses) and the slow emergence of antigenic variants as distinct lineages.

The emergence of H1N2 viruses deriving some or all of their internal genes from Sw/IV/1455/99 (H1N1)-like viruses emphasizes the contribution of these genes to the greater ‘epizootic’ potential of recent H1N1 and H1N2 viruses and the importance of exchange of internal genes in the evolution of co-circulating subtypes. The latter feature has been evident from the lack of divergence of the internal genes of H1N1 and H3N2 viruses circulating in European pigs between 1984 and 1999. (Fig. 2; Gregory et al., 2001 ).

Sw/Fin/127/99 provides another example of the relatively frequent introduction of human H3N2 viruses into swine populations. Recently, H3N2 viruses with HAs genetically similar to contemporary human viruses (1995–1997) have been responsible for serious outbreaks of disease in North American pigs. Although some have been wholly human viruses, the predominant epizootic strains have been human–swine–avian reassortants (Zhou et al., 1999 ; Karasin et al., 2000b ; Webby et al., 2000 ). Recent studies of influenza in pigs in various parts of the world have thus demonstrated the importance of the introduction of human and avian virus genes and genetic reassortment among co-circulating subtypes in the emergence and evolution of an increasingly diverse population of swine influenza viruses. This also highlights the possibility of the emergence in pigs of novel viruses with the potential to cause major human epidemics.


   Acknowledgments
 
We acknowledge the major contribution of Dr H. Guilmoto, Cooperl Hunaudaye, Lamballe, 22403, and Dr J. P. Buffereau, LDA 22, Ploufragan, 22440, to surveillance of swine influenza and isolation of viruses.


   References
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Abstract
Introduction
Methods
Results
Discussion
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Received 23 October 2001; accepted 14 December 2001.