International Congress Series 1263 (2004) 209 213 Emergence of distinct avian-like influenza A H1N1 viruses in pigs in Ireland and their reassortment with cocirculating H3N2 viruses Y.P. Lin a, *, M. Bennett a, V. Gregory a, S. Grambas a, V. Ragazzoli b, P. Lenihan b, A. Hay a www.ics-elsevier.com a Virology Division, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK b Virology Division, Department of Agriculture and Food, Central Veterinary Research Laboratory, Abbotstown, Castleknock, Dublin 15, Ireland Abstract. The influenza A H1N1 viruses, initially isolated from pigs in Ireland in 1991, were genetically distinct from previously identified human and swine H1N1 viruses, and appear to be the result of introduction of an avian virus distinct from those circulating in pigs in other European countries. The first Irish H3N2 swine isolates were genetically closely related to human H3N2 viruses. In contrast, H3N2 viruses isolated during the latter half of the 1990s were reassortants possessing internal genes similar to those of the H1N1 viruses, and both subtypes acquired amantadine resistance. These changes mimic those which occurred among swine influenza viruses in continental Europe during the 1980s, and emphasize the apparent advantage of properties associated with the avian internal genes and amantadine resistance for maintenance of the viruses in pigs. D 2003 Elsevier B.V. All rights reserved. Keywords: Swine influenza; H1N1; Reassortment; Amantadine resistance 1. Introduction The characteristics of influenza A viruses circulating in pigs in different parts of the world have shown how readily pigs can be infected with avian and human viruses and the importance of genetic reassortment between swine, avian and human viruses in the emergence of viruses causing disease in pigs. The emergence of avian-like H1N1 viruses in European pigs in 1979 [1], infection of pigs in Hong Kong with distinct avian-like H1N1 viruses in 1993 [2] and H9N2 viruses in 1998 [3], and the emergence of reassortant H3N2 viruses, containing human and avian as well as swine virus genes, in pigs in North America in 1998 [4] provide examples of the introduction of avian viruses into pigs. * Corresponding author. Tel.: +44-20-8816-2152; fax: +44-20-8906-4477. E-mail address: lyipu@nimr.mrc.ac.uk (Y.P. Lin). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.039
210 Y.P. Lin et al. / International Congress Series 1263 (2004) 209 213 This paper describes the characteristics of influenza A H1N1 viruses first isolated in Ireland in 1991, H3N2 viruses isolated from 1993, reassortment between these H1N1 and H3N2 viruses and acquisition of resistance to amantadine. 2. H1N1 viruses The influenza A H1N1 viruses were isolated during outbreaks of disease in pigs in Ireland between December 1991 and August 1998. Haemagglutination inhibition (HI) tests, using post-infection ferret antisera, distinguished them antigenically from avianlike H1N1 viruses circulating in other parts of Europe since the early 1980s. Comparisons of HA gene sequences showed that HAs of the Irish isolates, such as A/swine/ Ireland/756/92, were phylogenetically distinguishable from the HAs of viruses isolated in continental Europe, such as A/swine/Finistere/2899/82 (sequence similarity approximately 88%), and, although more closely related to the HA of the Hong Kong isolate, A/swine/ Hong Kong/168/93, the sequences differed by 9% (Fig. 1A). The N1 sequences of the Irish isolates were similarly differentiated from those of other European swine viruses (sequence similarity, approximately 88%) and were more closely related to the N1 sequences of certain avian viruses, such as A/goose/Guangdong/1/96 (H5N1; 90% similarity) (Fig. 1B). Comparisons of the sequences of the six internal genes of selected viruses also distinguished the Irish swine isolates from other swine viruses, as shown for the M gene in Fig. 2B. For example, the differences in sequence between A/swine/Ireland/756/92 and A/swine/Finistere/2899/82 ranged from 6 7% for PB1, NP, M and NS genes to 11 12% for PB2, PA and HA genes, similar to the differences between the genes of the latter virus and A/swine/Hong Kong/168/93. It is apparent, therefore, that the H1N1 viruses were most likely introduced into Irish pigs from an avian source and not directly related to previously identified swine viruses. Distinctive features of the HAs of these viruses, which may be associated with adaptation to the new host, include the presence of leucine at residue 226 in contrast to glutamine, identified in the sequences of most H1 HAs of swine, human and avian origin, and a change in residue 190 from glutamic acid in 1992 isolates, typical of avian viruses, to aspartic acid in later isolates, typical of swine and human viruses. 3. H3N2 viruses The H3N2 viruses, isolated from pigs in Ireland between 1993 and 1998, were antigenically distinguishable in HI tests, using post-infection ferret antisera, from contemporary H3N2 swine viruses isolated in other European countries. Their HA gene sequences were more closely related to those of UK isolates, such as A/swine/UK/119404/91, than to those of contemporary viruses isolated in continental European countries, such as A/swine/ Belgium/220/92 (Fig. 2A). The N2 sequences of the Irish isolates were also phylogenetically distinguishable from those of other European swine viruses and, like the HAs, have evolved independently. The six internal genes of earlier isolates, e.g. A/swine/Ireland/933/93, were phylogenetically closely related to the corresponding genes of human H3N2 viruses, as shown for the M gene in Fig. 2B, reflecting their origin. In contrast, the later isolate, A/swine/Ireland/
Fig. 1. Phylogenetic comparisons of the HA and NA genes of H1N1 viruses. The sequences of HA genes (A, nucleotides 84 1027) and NA genes (B, nucleotides 34 1116) were compared using PAUP. The lengths of horizontal lines are proportional to the numbers of nucleotide differences (indicated by the bar). The avian-like genes of swine and avian viruses are distinguished from the genes of classical swine and human (and swine H1N2) viruses. Y.P. Lin et al. / International Congress Series 1263 (2004) 209 213 211
212 Y.P. Lin et al. / International Congress Series 1263 (2004) 209 213 Fig. 2. Phylogenetic comparisons of H3 HA and M genes. The sequences of HA (A, nucleotides 29 1035) and M (B, nucleotides 403 822) genes were compared using PAUP. Sequences of the Irish H3N2 viruses are in boldface. The avian-like M gene sequences of swine and avian viruses are distinguished from those of classical swine and human (and swine) viruses.
832/98, possessed six internal genes closely related to those of the H1N1 viruses (Fig. 2B), indicating that it was the result of reassortment between H1N1 and H3N2 viruses cocirculating in the pig population in Ireland. 4. Amatandine resistance Sequences of the M genes showed that residue 31 of the M2 proteins of early isolates, including A/swine/Ireland/750/93 (H1N1) and A/swine/Ireland/933/93 (H3N2), was serine, whereas in viruses isolated in 1996 or later it was asparagine. Drug susceptibility tests [5] confirmed that this change correlated with resistance of virus replication to inhibition by amantadine. 5. Discussion These studies of influenza A viruses circulating in pigs in Ireland during the 1990s have identified novel swine H1N1 viruses and revealed changes in the viruses associated with their adaptation in pigs. The H1N1 viruses were genetically distinct from other swine viruses and, therefore, appear to have resulted from direct avian to swine transmission. Features of the HAs of these viruses, leucine at position 226 and the glutamic acid 190 to aspartic acid change, are likely to influence receptor binding [6] and may be important in adaptation to the swine host. The emergence of reassortant H3N2 viruses deriving a complement of avian genes from the H1N1 viruses and the acquisition of amantadine resistance mimic changes which occurred among other European swine viruses in the mid 1980s [5,7], and point to the apparent advantages of avian internal genes and of changes in the M2 protein associated with amantadine resistance for replication of influenza viruses in pigs. References Y.P. Lin et al. / International Congress Series 1263 (2004) 209 213 213 [1] M. Pensaert, et al., Evidence for the natural transmission of influenza A virus from wild ducks to swine and its potential importance for man, Bull. W.H.O. 59 (1981) 75 78. [2] Y. Guan, et al., Emergence of avian H1N1 influenza viruses in pigs in China, J. Virol. 70 (1996) 8041 8046. [3] J.S.M. Peiris, et al., Cocirculation of avian H9N2 and contemporary human H3N2 influenza A viruses in pigs in southeastern China: potential for genetic reassortment? J. Virol. 75 (2001) 9679 9686. [4] N.N. Zhou, et al., Genetic reassortment of avian, swine and human influenza A viruses in American pigs, J. Virol. 73 (1999) 8851 8856. [5] V. Gregory, et al., Infection of a child in Hong Kong by an influenza A H3N2 virus closely related to viruses circulating in European pigs, J. Gen. Virol. 82 (2001) 1397 1406. [6] S.J. Gamblin, et al., The structure and receptor-binding properties of the 1918 influenza hemagglutinin, Science (2004) (in press). [7] M.R. Castrucci, et al., Genetic reassortment between avian and human influenza A viruses in Italian pigs, Virology 193 (1993) 503 506.