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

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1 Journal of General Virology (2002), 83, Printed in Great Britain... Antigenic and genetic diversity among swine influenza A H1N1 and H1N2 viruses in Europe S. Marozin, 1 V. Gregory, 1 K. Cameron, 1 M. Bennett, 1 M. Valette, 2 M. Aymard, 2 E. Foni, 3 G. Barigazzi, 3 Y. Lin 1 and A. Hay 1 1 National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK 2 Universite Lyon 1, Laboratory of Virology, 8 Avenue Rockefeller, Lyon Cedex 08, France 3 Istituto Zooprofilattico, Sperimentale della Lombardia e dell Emilia, Parma, Italy 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 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 ( ) H1N2 isolates were more closely related to those of a contemporary French H1N1 isolate, whereas the genes of later ( ) isolates, including the HAs of some H1N2 viruses, were closely related to those of a distinct H1N1 antigenic variant which emerged in France in 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 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 Author for correspondence: Alan Hay. Fax ahay nimr.mrc.ac.uk 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 (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 SGM HDF

2 S. Marozin and others Table 1. Gene sequences of swine influenza isolates determined in this study Virus Abbreviated name Gene sequences H1N2 A swine Scotland Sw Scot PB1, PB2, PA, NP, NA, M, NS A swine Co tes d Armor Sw CA PB1, PB2, PA, HA, NP, NA, M, NS A swine Co tes d Armor Sw CA PB1, PB2, PA, HA, NP, NA, NS A swine Italy Sw It PB1, PB2, PA, HA, NP, NA, M, NS A swine Co tes d Armor Sw CA PB1, PB2, PA, HA, NP, NA, M, NS A swine Italy Sw It NP, NA, M, NS A swine Italy Sw It PB1, PB2, PA, HA, NP, NA, M, NS A swine Co tes d Armor Sw CA HA, NA, NS A swine Co tes d Armor Sw CA PB1, PB2, PA, HA, NP, NA, M, NS A swine Co tes d Armor Sw CA PB1, NS A swine Italy Sw It PB1, PB2, PA, HA, NP, NA, M, NS A swine Italy Sw It HA H1N1 A swine Finistere Sw Fin HA, M, NS A swine Lot Sw Lot NA A swine Finistere Sw Fin NA A swine Belgium Sw Belg NA A swine France Sw Fr HA A swine Italy Sw It HA A swine Italy Sw It NA A swine Italy Sw It HA A swine Italy Sw It NA A swine Italy Sw It HA A swine Italy Sw It HA, NA A swine Italy Sw It HA A swine Italy Sw It PB1, PB2, NP, NS A swine Italy Sw It NA A swine Italy Sw It M A swine Italy Sw It HA, M A swine Co tes d Armor Sw CA PB1, PB2, PA, HA, NA, M, NS A swine Co tes d Armor Sw CA PB1, PB2, PA, HA, NP, NA, NS A swine Co tes d Armor Sw CA HA, NA A swine Ille et Vilaine Sw IV PB1, PB2, PA, HA, NP, NA, M, NS A swine Italy Sw It PB2 A swine Italy Sw It PB2, M A swine Co tes d Armor Sw CA HA A swine Co tes d Armor Sw CA HA, NA, NS A swine Italy Sw It HA, NS H3N2 A swine Co tes d Armor Sw CA NS A swine UK Sw UK NA, NS A swine Italy Sw It NA A swine Italy Sw It NP A swine Italy Sw It NS A swine Finistere Sw Fin PB1, PB2, PA, HA, NP, NA, NS A swine Italy Sw It NA 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 humanlike N2 neuraminidase (NA) and six internal genes (PB1, PB2, PA, NP, M and NS) corresponding to those of European avianlike 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 HDG

3 Swine influenza A H1N2 viruses 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 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 (H3N2) and A swine Scotland (H1N2) were obtained from I. Brown, Central Veterinary Laboratory, Weybridge, UK. A swine Belgium was obtained from M. Pensaert, University of Ghent, Belgium. Antisera. Hyperimmune rabbit antisera and post-infection ferret sera were prepared as described in Kendal et al. (1982). Antigenic characterization. Haemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests were performed as described in Kendal et al. (1982). Amantadine susceptibility. Inhibition of virus replication by amantadine (or rimantadine) was determined by ELISA, as described by Belshe et al. (1988). 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 (H1N1; AJ344022) and A New Caledonia (A NCal 20 99, H1N1;AJ344014) and the HA and NA of A Lyon (AJ316063), are available from GenBank; accession numbers AJ (PB2); AJ (PB1); AJ (NP); AJ and AJ (PA); AJ (M); AJ and AJ (HA); AJ316046, AJ316048, AJ and AJ (NS); AJ and AJ (NA). Results 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 , 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 (Table 2), even though their HA and NA sequences had drifted significantly (see below, Fig. 1). An antigenic variant, represented by Sw IV , which was initially isolated in France in 1999, was distinguishable from other contemporary isolates, such as Sw CA and Sw Italy , using post-infection ferret sera (Table 2). Sw IV 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 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). H1N2 viruses. With two exceptions, the HAs of the swine H1N2 viruses were, like that of the UK virus Sw Scot , closely related to the HA of the human H1N1 virus A Brazil and were distinct from the HAs of the Sw Fin (H1N1)-like viruses circulating since 1979 (Table 3). Post-infection ferret antisera to Sw CA , Sw Italy and Sw CA distinguished between the French and Italian isolates and the earlier UK isolate, Sw Scot ; furthermore Sw CA was not representative of the other French isolates. In contrast, the HAs of Sw Italy (H1N2) and Sw Italy (H1N2) were more closely related to the HAs of European swine H1N1 viruses (Table 2). Ferret antisera to Sw Italy and Sw IV 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 HDH

4 S. Marozin and others Table 2. Antigenic relationships between the haemagglutinins of swine influenza AH1N1 viruses Reference viruses and homologous titres are in bold. Haemagglutination inhibition titre Hyperimmune rabbit serum Post-infection ferret sera Sw/Fin/ Sw/Fin/ Sw/It/ Sw/CA/ Sw/IV/ Sw/It/ Virus Subtype 2899/ / / / / /99 Sw/Fin/2899/82 H1N Sw/Italy/1513-1/98 H1N Sw/CA/1482/99 H1N Sw/IV/1455/99 H1N Sw/Italy/2064/99 H1N Sw Italy H1N Sw Italy H1N Sw CA H1N Sw CA H1N Sw IV H1N Sw CA H1N Sw CA H1N Sw Italy H1N Sw Italy H1N 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 , 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 and Sw Eng 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 , the HA gene of Sw CA was more closely related to that of Sw Scot , suggesting that Sw CA 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 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 ( 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 ) compared with about 39% for human H1N1 viruses between 1983 and 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 ) 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 , 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 and Sw Italy , were more closely related to those of recent Sw IV like H1N1 swine viruses, in particular Sw Italy (95% sequence similarity; Fig. 1A). The HA genes of H1N1 viruses antigenically similar to Sw IV , including Sw Italy , were clearly distinguishable phylogenetically from HDI

5 HDJ 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 ) and N1 (C, nucleotides ) 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. Swine influenza A H1N2 viruses

6 S. Marozin and others Table 3. Antigenic relationships between the haemagglutinins of swine influenza AH1N2 viruses, 40. Reference viruses and homologous titres are in bold. Haemagglutination inhibition titre Hyperimmune rabbit sera Post-infection ferret sera Sw/Fin/ A/Bz/ Sw/Fin/ A/Bz/ A/Ch/ Sw/Sc/ Sw/CA/ Sw/It/ Sw/CA/ Virus Subtype 2899/82 11/ /82 11/78 1/83 440/94 790/ /98 604/99 Sw/Finistere/2899/82 H1N A/Brazil/11/78 H1N A/Chile/1/83 H1N Sw/Scotland/410440/94 H1N Sw/CA/790/97 H1N Sw/Italy/1521/98 H1N Sw/CA/604/99 H1N Sw CA H1N Sw Italy H1N Sw Italy H1N Sw CA H1N Sw CA H1N Sw CA H1N Sw Italy H1N the HA genes of other contemporary French and Italian isolates, represented by, e.g., Sw CA and Sw Italy , respectively (Fig. 1A). Sixteen amino acids common to the HA1s of the Sw IV like viruses, one of which removed a potential glycosylation site at asparagine-94 (H3 numbering), distinguished them from the HAs of Sw Fin like viruses, such as Sw CA The HA1 sequences of Sw Italy and Sw Italy 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 like viruses (Fig. 1A). Thus sequence differences can readily account for the antigenic differences between the HAs of Sw Italy and Sw IV (Table 2). The sequence of the H3 HA of Sw Fin (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 (isolated about 2 months prior to Sw Fin ) (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 ) were more closely related (93 95% sequence similarity) to the N2 gene of the UK H3N2 virus Sw UK 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 was more closely related to that of Sw UK (H3N2; 95% similarity) than to the NA genes of other French and Italian H1N2 isolates (92 93% similarity), emphasizing the difference between Sw CA 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 (H3N2) and the NA of a contemporary human A Sydney 5 97 (H3N2)-like virus, A Lyon 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 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 like viruses were HEA

7 Swine influenza A H1N2 viruses also clearly distinguishable (93% similarity) from those of other contemporary French H1N1 viruses, such as Sw CA and Sw CA and were, in fact, more closely related to some earlier H1N1 isolates from Belgium (Sw Belgium 74 95) or the UK (Sw England ) (Fig. 1C). Eighteen amino acid differences, including eight characteristic of Sw IV like viruses and four characteristic of Sw CA 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 and Sw CA 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. The genes of early H1N2 viruses, Sw CA , Sw CA and Sw Italy (including some genes of Sw Italy ), were similar to each other and to the genes of Sw CA (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 , which was more closely related to the PB1 of Sw Italy (H1N1) (Fig. 2A). With the exception of the NS gene, the internal genes of other later H1N2 isolates, Sw Italy , Sw Italy and Sw CA , were phylogenetically distinguishable from those of the earlier H1N2 isolates and clustered with the corresponding sequences of the Sw IV (H1N1)-like viruses. In particular, differences between the six genes of Sw CA (H1N2) and Sw IV (H1N1) of less than 1% contrasted with differences of 3 5% between the genes of Sw CA and Sw CA The NS genes of Sw Italy and Sw Italy 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 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 (H3N2) and Sw Italy (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 like and Sw IV 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 (H1N2) (220 residues) was 10 amino acids shorter, whereas NS1 of Sw Italy (H3N2) was 7 amino acids longer. Of particular note was the truncated NS1 (119 amino acids) of Sw CA (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 like viruses. The lower rate of accumulation of mutations in the M genes of H1N1 and H1N2 viruses of about 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 and Sw Italy , by an ELISA for drug susceptibility. As for HA and NA, the PB1, PB2, PA, NP and NS genes of Sw CA were more closely related to the corresponding genes of the earlier UK H1N2 isolate, Sw Scot 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 and the earlier H1N1 virus Sw Eng Nucleotide sequences of the PB1, PB2, PA, NP and NS genes of Sw Fin (H3N2) were closely related to corresponding genes of recent human H3N2 viruses, such as A Fukushima and A Nagasaki (Fig. 2A, B). Discussion 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 (H1N1)-like viruses, antigenically and genetically distinguishable from previously circulating H1N1 swine viruses. (3) Genetic reassortment between H1N2 and Sw IV (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. HEB

8 S. Marozin and others Fig. 2. For legend see facing page. Fig. 2(A) HEC

9 Fig. 2(B) HED Fig. 2. Phylogenetic relationships between the genes of H1N1, H1N2 and H3N2 swine influenza viruses. Coding sequences of A, PB2 (nucleotides ), PB1 (nucleotides ) and PA (nucleotides ) genes, and B, NP (nucleotides ), M (nucleotides ) and NS (nucleotides ) 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. Swine influenza A H1N2 viruses

10 S. Marozin and others 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 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 For example, the recent French isolate Sw CA , which was closely related in HA (98 6%) and NA (99 0%) to the earlier H1N2 isolate Sw CA , possessed a complement of six internal genes similar ( % sequence similarity) to that of the Sw IV (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 (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 Sw IV 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 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 from contemporary Sw Fin (H1N1)-like viruses, such as Sw CA , 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 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 (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 (Fig. 2; Gregory et al., 2001). Sw Fin 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 ( ) 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). 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Genetic reassortment of avian, swine and human influenza A viruses in American pigs. Journal of Virology 73, Received 23 October 2001; Accepted 14 December 2001 HEF