Antigenic and genetic diversity among swine inf luenza viruses in Europe

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1 International Congress Series 1219 (2001) Antigenic and genetic diversity among swine inf luenza viruses in Europe S. Marozin a, *, V. Gregory a, K. Cameron a, M. Bennett a, M. Valette b, M. Aymard b, E. Foni c, G. Barigazzi c, Y. Lin a, A. Hay a a National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK b Laboratory of Virology, Université Lyon 1, 8 Avenue Rockefeller, Lyon Cedex 08, France c Istituto Zooprofilattico, Sperimentale della Lombardia e dell Emilia, Parma, Italy Abstract H3N2 and avian-like H1N1 subtypes have circulated in European pigs since the mid- to late 1970s. Following reassortment to acquire six internal genes of the H1N1 viruses in the early 1980s, the H3N2 viruses have evolved to produce antigenically distinguishable variants. Sw/Finistere/127/ 99, isolated from a pig in January 1999, was shown to be closely related in antigenic and genetic characteristics to contemporary human A/Sydney/5/97-like viruses, and may represent the emergence of another human-like virus in European pigs. The H1N1 viruses circulating in pigs in France have evolved gradually over the past 20 years, but are in general closely related to early isolates such as Sw/Finistere/2899/82. An antigenically distinct variant, represented by Sw/Ille et Vilaine/1455/99, represents a significant drift from other recent French H1N1 viruses. Most of the H1N2 reassortant viruses isolated in France and Italy since 1997 were shown to be genetically similar to the H1N2 viruses which have become prevalent in the U.K., but to exhibit significant variation in antigenicity. Genetic reassortment between H1N2 and H1N1 viruses has also contributed to the recent increase in diversity of viruses circulating in pigs in Europe, and emphasizes the potential difficulties in controlling influenza by vaccination. Since most of these swine viruses are resistant to amantadine, they provide an increasing pool of amantadine-resistant M genes for potential incorporation into future human viruses. D 2001 Elsevier Science B.V. All rights reserved. Keywords: Influenza; European swine; H1N1; H1N2; H3N2; Reassortment * Corresponding author. Tel.: x2152; fax: address: smarozi@nimr.mrc.ac.uk (S. Marozin) /01/$ see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S (01)

2 234 S. Marozin et al. / International Congress Series 1219 (2001) Introduction Similarities in the subtypes of influenza A, H1N1 and H3N2, which circulate in swine and human populations reflect the frequent transmission of viruses between the two species. Classical swine H1N1 viruses and H3N2 viruses similar to contemporary human H3N2 viruses were first isolated from pigs in Europe in the mid-1970s [1,2]. Avian-like H1N1 viruses, first isolated from European pigs in 1979 [3] replaced classical H1N1 swine viruses and have since co-circulated with H3N2 viruses. Genetic reassortment between viruses of the two subtypes in gave rise to H3N2 viruses which contain six internal genes (encoding PB2, PB1, PA, NP, M and NS proteins) corresponding to those of the avianlike H1N1 viruses [4]. The subsequent lack of divergence of these genes of the two subtypes indicates frequent exchange of genes between the H1N1 and H3N2 subtypes. Reassortment of the genes encoding the surface antigens to produce H1N2 viruses was observed [5], but these viruses did not become established in Europe. During the 1990s a novel H1N2 subtype emerged in pigs in the U.K, which possessed a haemagglutinin (HA) closely related to the HAs of H1N1 viruses circulating in the human population during the early 1980s [6]. This paper reports the results of studies of viruses recently isolated from pigs in Northern France (Brittany) and Northern Italy and focuses in particular on the characteristics of recent antigenic variants of H1N1 and H1N2 viruses emerging among pigs in continental Europe. 2. Viruses from France and Italy Of some 111 viruses isolated during 1997 to 2000, 26 were from France, including 2 H3N2, 19 H1N1 and 5 H1N2 viruses, and 85 were from Italy, including 52 H3N2, 29 H1N1 and 4 H1N2 viruses. Whereas H3N2 viruses were prevalent among viruses from Italy, few were obtained from Brittany. Since the initial H1N2 isolate from Brittany in 1997, this UK-like subtype appears to be increasing in prevalence. 3. H3N2 viruses Gradual evolution of the HA gene of H3N2 viruses has resulted in increased genetic heterogeneity with the appearance of antigenic variants, such as those represented by A/ swine/belgium/220/92 and A/swine/Italy/1523/98 (Sw/Italy/1523/98), which are distinguishable in haemagglutination inhibition (HI) tests using post infection ferret antisera. Most recent Italian isolates were antigenically and genetically similar to Sw/Italy/1523/ 98, although other variants have been identified. The only H3N2 virus from France during 1998 and 1999, A/swine/Finistere/127/99, was similar in its antigenic and genetic (see Fig. 2) characteristics to A/Sydney/5/97-like human viruses. Comparisons of the sequences of the HA and NA components showed that this swine virus was closely related to the contemporary human variant, consistent with the recent introduction of a human virus.

3 Table 1 Antigenic characterization of swine H1N1 and H1N2 viruses Viruses a Subtype Haemagglutination inhibition titre b Rabbit sera Sw/Fin 2899/82 A/Brazil 11/78 Post-infection ferret sera Sw/Fin 2899/82 Sw/It 15131/98 Sw/IV 1482/99 Sw/CA 1455/99 Sw/It 2064/99 A/Braz 11/78 A/Chile 1/83 A/Taiw 1/86 Sw/Scot /94 Sw/It 1521/98 Sw/Fin/2899/82 H1N < < < < < < Sw/Italy/15131/98 H1N < < < < < < Sw/CA/1482/99 H1N < < < < < < Sw/IV/1455/99 H1N < < < < < < Sw/Italy/2064/99 H1N < < < < < < Sw/CA/1488/99 H1N < < < < < < Sw/IV/760/00 H1N1 nd nd nd nd nd nd nd nd nd A/Brazil/11/78 H1N < < < 160 < < A/Chile/1/83 H1N < < < 40 < < A/Taiwan/1/86 H1N1 < 2560 < < < < < < < < < Sw/Scot410440/94 H1N < < < < < Sw/Italy/1521/98 H1N2 < 5120 < < < < < < < < < 320 < Sw/CA/604/99 H1N < < < < < Sw/CA/790/97 H1N < < < < < Sw/CA/2433/98 H1N < < < < < < Sw/Italy/16541/99 H1N < < < < < 40 < < < Sw/Italy/1081/00 H1N2 < 5120 < < < < 40 < 40 < < a Reference viruses are in bold. b Homologous titres in bold; < indicates < 40. Sw/CA 604/99 S. Marozin et al. / International Congress Series 1219 (2001)

4 236 S. Marozin et al. / International Congress Series 1219 (2001) H1N1 viruses Although the H1N1 viruses have evolved gradually over the past 20 years, the majority are still closely related in their antigenic characteristics to viruses isolated in the Fig. 1. Phylogenetic relationships between N2 genes of human H3N2 viruses and swine H3N2 and H1N2 viruses. Nucleotides 70 to 1031 of the coding sequences were analysed with PAUP using a maximum parsimony algorithm. The lengths of the horizontal lines are proportional to the number of nucleotide differences (as indicated). Sequences not determined in this study were obtained from GenBank. Abbreviations used include: Beij, Beijing; Belg, Belgium; Bk, Bangkok; CA, Cotes d Armor; Dk, duck; Fin, Finistere; HK, Hong Kong; It, Italy; Mosc, Moscow; Ms, Mississippi; Nag, Nagasaki; Nc, Nanchang; Sc, Scotland; Sw, swine; Syd, Sydney; Tw, Taiwan; Ud, Uldorn; UK, United Kingdom; Vic, Victoria. H1N2 viruses are in grey.

5 S. Marozin et al. / International Congress Series 1219 (2001) early 1980s, such as A/swine/Finistere/2899/82 (Sw/Fin/2899/82). A/swine/Ille et Vilaine/ 1455/99 (Sw/IV/1455/99) provided an example of a more clearly distinguishable antigenic variant (Table 1). Two later French isolates A/swine/Cotes d Armor/1488/99 (Sw/CA/1488/99) and Sw/IV/760/00 were antigenically similar in HI and NI tests to Sw/ IV/1455/99, whereas an Italian variant, Sw/Italy/2064/99, was distinguishable in HI tests (Table 1). Comparisons of the HA gene sequences of these viruses showed that Sw/IV/ 1455/99 and Sw/CA/1488/99 shared 99.5% homology but were distantly related (92% homology) to the majority of other recent French H1N1 swine viruses, represented by, e.g. Sw/CA/1482/99. They were in fact more closely related genetically (94 95% homology) to some of the recent Italian H1N1 isolates and to earlier isolates such as Sw/Germany/8533/91. Sequences of the NA genes of these two variants exhibited a similar divergence from the NA gene sequences of other recent French H1N1 viruses. Although Sw/Italy/2064/99 was antigenically distinguishable in HI tests, its HA gene, which shares only 95% sequence homology, groups phylogenetically with the HA genes of the two French variants. This virus is however of the H1N2 subtype and possesses a NA gene similar to those of several H1N2 viruses recently isolated in France and Italy (Fig. 1). 5. H1N2 viruses Eight H1N2 viruses isolated in Brittany and Italy were shown to be similar to H1N2 viruses previously isolated in the U.K. HI tests (using rabbit antisera) showed that their HAs were antigenically related to human H1N1 viruses, such as A/Brazil/11/78 (Table 1). The results obtained using post-infection ferret antisera indicated that the French isolates, like A/swine/Scotland/410440/94 (Sw/Sc/410440/94), were more closely related to the early human H1N1 viruses than were the Italian isolates and distinguished the Scottish and French isolates from the three closely related Italian isolates (Table 1). These relationships were to some extent reflected in comparisons of HA sequences. Phylogenetic relationships showed that the HA genes of two of the French and two of the Italian isolates were more closely related (93 97% sequence homology) to the HA gene of Sw/England/690421/95 (H1N2), while the isolate Sw/CA/604/99 was more closely related to Sw/Sc/41440/94. Phylogenetic comparisons of the NA genes (Fig. 1) showed that the N2s of these viruses were closely related to the N2 of the U.K. H3N2 virus Sw/UK/119404/91 (93 95% homology) and distantly related (86 91% homology) to the N2s of swine H3N2 viruses circulating in continental Europe between 1984 and 2000, indicating that, like the HA, the NA of these viruses was derived from the previously identified U.K. H1N2 viruses. Whereas the N2s of most of the French and Italian isolates were closely related to each other and had diverged somewhat from those of U.K. isolates, as for the HA, the N2 of Sw/CA/604/99 was more closely related to the N2s of certain U.K. swine isolates, indicating separate introduction of this, or a related, virus into French pigs. Comparisons of the sequences of the six internal genes showed that they were closely related to the internal genes of co-circulating H1N1 and H3N2 viruses. Differences in sequence homology between subtypes were comparable to differences between corresponding genes of viruses within the same subtype. This is illustrated in Fig. 2 for the NP

6 238 S. Marozin et al. / International Congress Series 1219 (2001) Fig. 2. Phylogenetic relationships between NP genes of human H3N2 viruses and swine H1N1, H1N2 and H3N2 viruses. Nucleotides of the coding sequences were analysed as indicated in Fig. 1. Abbreviations not explained in the legend to Fig. 1 include: Eng, England; Ger, Germany; IV, Ille et Vilaine; Ne, Nebraska. H1N2 viruses are in boxed and H3N2 viruses are in black. The asterisks indicate the antigenically distinguishable H1N1 viruses. gene where the similarity between the NP genes of Sw/Italy/15096/97 (H1N1) and Sw/ Italy/1477/97 (H3N2) contrasts with the differences between each of these genes and another of a virus of the same subtype, Sw/Italy/15131/98 (H1N1) and Sw/Italy/1523/98 (H3N2), respectively. The NP genes of most of the H1N2 viruses, represented by Sw/Italy/ 1521/98, were similar to each other and distinguishable from the corresponding genes of the two 1999 H1N1 variants, represented by Sw/IV/1455/99. In contrast, as for the HA, NA and other five internal genes, the NP gene of Sw/CA/604/99 was more closely related to the NP gene of Sw/Sc/410440/94.

7 6. Genetic reassortment S. Marozin et al. / International Congress Series 1219 (2001) It is apparent that, as for the HA, the NP of Sw/Italy/2064/99 (H1N2) is more closely related to that of the H1N1 virus Sw/IV/1455/99 (Fig. 2); a similar relationship was observed for the PB2, PB1, PA and M genes (Table 2). The NS gene of Sw/Italy/2064/99 was, however, like the N2, more closely related to the NS genes of the other H1N2 viruses (Table 2) indicating that this virus is the product of genetic reassortment between a H1N2 virus and a virus similar to Sw/IV/1455/99. Fig. 2 also indicates that contrary to the other H1N2 viruses, Sw/Italy/1081/00 has an NP gene more closely related to that of Sw/IV/ 1455/99 than to that of, e.g. Sw/Italy/1521/98. Table 2 indicates the relationships of the various genes of Sw/Italy/1081/00 (H1N2) with the corresponding genes of Sw/Italy/ 1521/98 (H1N2) and Sw/IV/1455/99 (H1N1). It is apparent that Sw/Italy/1081/00 possesses five genes, PB2, PB1, PA, NP and M, which are more closely related to those of the H1N1 virus than to those of the H1N2 virus (the degree of sequence homology reflects the results of phylogenetic comparisons, not shown) and is the product of genetic reassortment. 7. Amatadine resistance M gene sequences showed that the M2 proteins of the different subtypes (with the exception of Sw/Fin/127/99), like H1N1 and H3N2 viruses circulating since 1987 [7], possessed asparagine at position 31, indicating resistance to the anti-influenza drugs, amantadine and rimantadine. For several viruses, including Sw/IV/1455/99 and Sw/Italy/ 1521/98, this was confirmed by drug sensitivity tests. Table 2 Evidence for genetic reassortment between H1N1 and H1N2 viruses Gene Sequence difference (%) a Sw/IV/1455/99 (H1N1) Sw/It/1521/98 (H1N2) Sw/It/2064/99 Sw/It/1081/00 Sw/It/2064/99 Sw/It/1081/00 PB PB PA H NP N M NS a Percent difference in sequence between the corresponding genes of reassortant viruses Sw/Italy/2064/99 and Sw/Italy/1081/00 and reference viruses (bold) Sw/IV/1455/99 (H1N1) and Sw/Italy/1521/98 (H1N2). Numbers in bold indicate the closer relationship.

8 240 S. Marozin et al. / International Congress Series 1219 (2001) Conclusions The results of these studies (1) showed that the majority of H3N2 and H1N1 influenza A viruses recently circulating in pigs in continental Europe are closely related to previously circulating variants, (2) identified a swine A/Sydney/5/97 (H3N2)-like virus, and (3) revealed the emergence of antigenically distinguishable H1N1 variants and antigenically heterogeneous H1N2 subtype viruses. The genetic similarities and differences between the H1N2 viruses isolated in France and the U.K. indicate that introductions from the U.K. may have occurred on more than one occasion. Two of the H1N2 viruses were shown to be reassortants, apparently deriving genes from H1N2 and H1N1 viruses; Sw/Italy/1081/00 (H1N2) had acquired five internal genes more closely related to the H1N1 viruses, whereas Sw/Italy/2064/99 (H1N2) possessed two genes, NA and NS, of the H1N2 virus. These viruses thus contribute to a significant increase in the diversity of swine influenza viruses in Europe. References [1] L. Nardelli, S. Pascucci, G.L. Gualandi, P. Loda, Zentralbl. Veterinaermed., Reihe B 25 (1978) [2] B. Tumova, D. Veznikova, J. Mensik, A. Stumpa, Zentralbl. Veterinaermed., Reihe B 27 (1980) [3] M. Pensaert, K. Ottis, J. Vandeputte, M.M. Kaplan, P.A. Bachmann, Bull. W. H. O. 59 (1981) [4] M.R. Castrucci, I. Donatelli, L. Sidoli, G. Barigazzi, Y. Kawaoka, R.G. Webster, Virology 193 (1993) [5] J.M. Gourreau, C. Kaiser, M. Valette, A.R. Douglas, J. Labie, M. Aymard, Arch. Virol. 135 (1994) [6] I.H. Brown, P.A. Harris, J.W. McCauley, D.J. Alexander, J. Gen. Virol. 79 (1998) [7] M. Bennett, S. Grambas, Y. Lin, A. Hay, submitted for publication.