Bull Vet Inst Pulawy 59, 451 455, 2015 DOI 10 1515/bvip 2015 0066 DE GRUYTER OPEN DE G Substitution in position 222 of haemagglutinin of pandemic influenza A (H1N1) viruses isolated from pigs in Polan[.]
DE G Bull Vet Inst Pulawy 59, 451-455, 2015 DOI:10.1515/bvip-2015-0066 DE GRUYTER OPEN Substitution in position 222 of haemagglutinin of pandemic influenza A (H1N1) viruses isolated from pigs in Poland Andrzej Kowalczyk, Kinga Urbaniak, Iwona Markowska-Daniel, Zygmunt Pejsak Department of Swine Diseases, National Veterinary Research Institute, 24-100 Pulawy, Poland andrzej.kowalczyk@piwet.pulawy.pl Received: June 30, 2015 Accepted: December 4, 2015 Abstract The aim of the study was to monitor genetic diversity and antigenic changes in the genome of influenza A(H1N1)pdm09 viral isolates detected during the post-pandemic period in Poland Clinical specimens obtained from three suspected cases of influenza were analysed by sequencing Among the differences identified in amino acids sequences, nine substitutions were located within the antigenic HA1 sites and in five residues forming receptor-binding pocket The HA(D222G) mutation was shown in the isolate Swine/Poland/134312/12 obtained from a mild case of the disease It must be emphasized that, in general, clinically mild cases are caused by the viruses in which that specific mutation, i.e haemagglutinin (D222G), does not occur Keywords: swine influenza virus, mutagenesis, pandemic influenza Introduction A novel influenza A virus, subtype H1N1, discovered in April 2009, spread globally in the human population causing a pandemic that was officially declared by the World Health Organisation on June 11, 2009 Phylogenetic analysis of the 2009 pandemic H1N1 virus (H1N1pdm09) indicated that the genome was a combination of gene segments that had not been previously identified in animal or human influenza viruses (5) The HA gene of H1N1pdm09 belongs to the classical swine lineage distinct from other viruses of human or avian origin that were introduced into pigs more recently, giving rise to ‘human-like’ and ‘avianlike’ swine influenza lineages (18) Its ancestry has been traced to the viruses similar to the human pandemic in 1918–1919 (1) Since 2009, the genetic script of influenza virus has revealed successive mutations Some of them result in amino acid substitutions at key locations in proteins, such as antigenic sites or a receptor-binding site of the HA Influenza viruses circulate among different host species including wild birds, poultry, pigs, horses, and humans The viruses usually favourably bind to two specific receptors: sialic acid (SA) α2,3 found in the epithelial cells of the gastrointestinal tract of wild aquatic birds, and SA α2,6 found in the epithelial cells of human respiratory tract, depending on the virus origin (1) A mutation at position 222 of the viral HA protein, causing replacement of aspartic acid (D) to glycine (G), might create a virus with a double preference, for both α2,3 SA and α2,6 SA The experimental data have shown that this virus can replicate in the lower respiratory tracts of infected mammals (16) as distinct from contemporary seasonal H1N1 viruses, which have a predilection for 2,6-linked sialic acid and mainly infect the upper respiratory tract Severity of the respiratory disease might be due to a deeper infection of the lungs The mutation appears to occur sporadically and spontaneously without spreading, and it is most importantly known for its altering properties, such as those associated with antigenicity or virulence Material and Methods A total of 29 nasal swabs were collected from two cases (farms A and C) with noticeable respiratory signs such as sneezing, coughing, conjunctivitis, and fever Lung tissues were collected from one 5-6-week-old © 2015 A Kowalczyk et al This is an open access article distributed under the Creative Commons Attribution- 10.1515/bvip-2015-0066 NonCommercial-NoDerivs license (http://creativecommons.org/licenses/by-nc-nd/3.0/) Downloaded from De Gruyter Online at 09/12/2016 02:37:46AM via free access 452 A Kowalczyk et al./Bull Vet Inst Pulawy/59 (2015) 451-455 necropsied pig (farm B) Seven virus isolates from these three cases with mild clinical outcomes were obtained from clinical material collected between March and October 2012, and analysed The isolates: A/Sw/1562021/Poland/12 (KJ191731), A/Sw/1562022/ Poland/12 (KJ191732), A/Sw/1562023/Poland/12 (KJ191733), A/Sw/1562024/Poland/12 (KJ191734), A/Sw/Poland/031951/12 (KJ191735), /Sw/Poland/ 031952/12 (KJ191736), and A/Sw/13431/Poland/12 (KJ191737) were grown on 9-day-old embryonated chicken eggs as described previously No more than one passage was performed Three outbreaks were assigned and they were termed as: case A - viruses with number 156202, case B - viruses with number 03195, and case C with virus number 13431 Total RNA was extracted using a commercial kit (QIAamp Viral RNA Mini Kit, Qiagen, Germany) as described by the manufacturer, and submitted for a routine reverse transcriptase polymerase chain reaction (RT-PCR) (9) Then, RNA that was positive in the first testing was examined with universal sets of primers in PCRs to detect the whole HA gene of influenza virus (6) The amplified PCR products were sequenced in Genomed (Poland) – a DNA analysis service The nucleotide sequences were initially compared using the ClustalW alignment algorithm method Sequence distances were analysed with MegAlign software (Lasergene, USA) For evaluation of genetic homology among the worldwild isolates of SIV, selected sequences of influenza virus genes accessible on-line in NCBI were added to phylogenetic analysis Results Polish pdmH1N109 strains were compared to the vaccine strain and to the first isolate of pdm virus A/California/7/09 (H1N1) (Fig 1) The lowest number of variations was detected in the viruses from farm A and this virus was approved as the most conserved The alignment of amino acid sequence of the HA of Polish isolates in 2012 was performed using H1 HA numbering of the mature protein; equivalent to 190 and 225 in H3 numbering or crystallographic annotation systems The results are summarised in Table The comparison of the sequences of viruses isolated from farms B and C to the isolate obtained from farm A revealed mutations at positions: A73V, S83P, N86D, D97N, S125D, S125N, T134S, T134A, A135S, K142R, N156D, P183S, S185T, D187N, L191I, T203S, D222G, K235E, V249M, S264G, P271S, A315V, and V321I (Table 1) Among the identified differences in amino acid sequences, nine substitutions were located within the antigenic HA1 sites (Cb, Ca1, Ca2, Sa, and Sb) and in five residues forming receptor-binding pocket In most of these variable places (86% of residues), the farm B viruses displayed antigenic substitutions Several HA residues contributed to the interaction by sidechains, including 68, 91, 150, 180, 183, 187, 191, 222, 223, and 225 In addition, residues 130, 133, and 134 also participated in the interactions with the main-chain amide and carbonyl oxygen atoms 134 ◊ 183 187 191 ◊ ◊ ◊ 222 ◊ Fig The most variable fragment of HA1 protein sequence alignment with critical amino acid changes between 2009 pandemic isolate A/California/2009 and the Polish isolates Sequence numbering according to HA1 The residues formed receptor-binding pocket are visible with numbers indicated with diamond (◊) - 10.1515/bvip-2015-0066 Downloaded from De Gruyter Online at 09/12/2016 02:37:46AM via free access A Kowalczyk et al./Bull Vet Inst Pulawy/59 (2015) 451-455 453 Table Amino acids analysis of the most variable region of HA including antigenic sites and receptor binding sites among all viruses isolated in this study Viruses strains belonged to one farm/case Farm A Farm B Farm C GenBank accession no Residue position in HA 73 Cb KJ191731 KJ191732 KJ191733 KJ191734 A KJ191735 KJ191736 KJ191737 V - 83 S - P 86 N D - D N - S D N 97 125 Sa 134 RBS 135 T S A A S - 142 Ca2 K R - 156 Sa N - D 183 RBC P S S 185 Sb S T - 187 RBS D N - 191 RBS/Sb L - I 203 Ca1 T - S 222 RBS/Ca2 D - G 235 Ca1 E K E 249 V M - 264 S G G 271 P S - 315 A V - 321 V - I RBS – receptor binding site; Ca1, Ca2, Cb, Sa, Sb – antigenic sites Residues 164, 206, and 159 were located at the bottom of the receptor-binding pocket and probably created the hydrophobic environment for the binding In residue 68, Polish viruses were represented by glutamic acid (E), similarly as in the majority of the pandemic strains isolated from pigs or humans, in contrast to Polish avian-like isolates in which this position was represented by aspartic acid (D) In 134 amino acid residues, three different mutations were discriminated In the farm A virus, this position was replaced by T, corresponding to human pandemic viruses isolated during post-pandemic period and pandemic SIV circulating mostly in 2009 In this residue in the virus Swine/Poland/134312/12, an antigenic drift corresponded mostly with pandemic H1N1 isolated from humans and with avian H1N1, H1N2, H1N3, and H1N9 viruses circulating in Europe It was declared as a precursor of avian-like SIV The substitution in 134 position with serine (S) was identified in farm B viruses, unlike in the other influenza viruses already deposited in the Gene Bank In the 187 residue, the substitutions in farm B viruses were only found in six sequences of pandemic human viruses deposited in Gene Bank and in two SIVs, one from 2004 and one from 2011 The residue 191 in farm C virus was substituted by I, in contrast to L in most viruses presented in the alignment This amino acid was recognised in some of the isolates from late 70’s identified in North and South America Other unique amino acid changes observed in farm B isolates were situated in 125 position with asparagine (N) amino acid substitution, which were also present in human post pandemic (2011-2013) and some American isolates This residue was identified within antigenic or RBS regions Amino acids residues 91, 130, 133, 150, 159, 164, 180, 183, 206, 223, and 225, described as RBS, remained stable in all the viruses Substitutions noted in the position 222 of HA gene were observed in Swine/Poland/134312/12 virus sequences Discussion A specific mutation in the viral haemagglutinin (D222G) was found with a considerable frequency in fatal and severe cases in humans Currently, it is - 10.1515/bvip-2015-0066 Downloaded from De Gruyter Online at 09/12/2016 02:37:46AM via free access 454 A Kowalczyk et al./Bull Vet Inst Pulawy/59 (2015) 451-455 identified in isolates from mild cases (as observed by veterinarians) in pigs This study demonstrates that pandemic-like influenza A (H1N1) 2009 viruses with the D222G substitutions in HA circulating in Poland were isolated from farm C where mild course of the disease was observed The HA protein has a tendency to undergo antigenic changes, due to most commonly occurring antigenic drift, i.e the accumulation of point mutations over time These frequent genomic changes result in phenotypic changes, which add to the challenge in developing treatments and vaccines for this disease It is important in regard to health status of swine herds In addition, a more precise tracing of the formation, transmission, and spread of new reassortant influenza A viruses with unknown zoonotic potential is urgently required Several substitutions in HA gene were identified in this study They have been previously reported by Ledesma et al (10), who demonstrated that Spanish viruses carried D97N, S83P, S185T, and V321I mutations, which were also found in the viruses circulating in Poland Remarkably, P83 and I321 points diagnosed only in the HA gene of 134312 virus were present in the vaccine A/H1 component, and the first pandemic A(H1N1)2009 isolates were all classified in one pattern group (15) the S185T mutation, which occurred in A/Poland/031925/2012 virus, was identical as in the previously identified Northern hemisphere pdm SIV and the SIVs widely spread in Spain (10) This fact, widely reported in human cases of the disease, suggests that the receptor specificity of pdm SIV circulating in Poland prior to the emergence of 2009 pandemic H1N1 was sufficient for sustained transmission in the human population A successive introduction of the H1N1pdm09 virus to swine population demonstrates a new evolutionary adaptable pattern of mutations which occurred after 2009 Numerous studies have identified mutations within the HA and the polymerase subunits as mediators of increased pathogenicity due to passaging in mice (3, 23) Genetic and antigenic site analysis of isolates from the post-pandemic phase of the 2009 pandemic has shown that the Polish pandemic H1N1 2012 viruses possess the human virus type residue at positions 183, 187, and 222, supporting the efficient transmissibility of these viruses among humans The most mutual set of the genetic substitution in HA sequence was discovered in Turkish isolates, where between 75% up to 100% isolates shared the same mutations: S83P, D97N, S203T, V249I, I321V as the Polish isolate from farm A (5) However, no evidence of D222G mismatch was confirmed in the study by Guldemir et al (5) An in vitro assay demonstrated that pandemic H1N1 virus has a wider specificity for binding to the α2,3- and α2,6-linked sialic acid receptors The human-type 187D and D222G substitutions in the receptor-binding site of HA may provide this pandemic H1N1 virus with a unique tissue tropism for human infection It is possible that current pandemic H1N1 virus is still in the process of adaptation to the human host and that the receptorbinding site is under selection, as it has been observed during the early phases of other pandemics (1) It is generally accepted that the receptor-binding sites of HA have different configurations for the influenza viruses adapted for avian and mammalian hosts Numerous studies have documented that the host cells supporting influenza replication in vivo or in vitro can select variant viruses with amino acid substitutions near the receptor-binding site of the HA (2, 20) For example, human H3N2 clinical specimens can be the source of different sequence variants when propagated in MDCK cells or in embryonated eggs (17) Similarly, classical swine H1N1 viruses isolated in eggs have changes at positions 187 and/or 222 and modulate their receptor specificity (4, 18) The first detected case might be insufficient to estimate whether this mutation was a new occurrence or accumulated consolidation The only certain conclusion which can be formed is that the mutation which increases the pathogenicity of the virus, in our case D222G mutation, is always related to a severe course of influenza However, the severity of the disease can be affected by a number of other factors In humans, several specimens possessing D222G substitution were also obtained from mild H1N1pdm cases during the first (May 2009) and second (Dec 2010) waves of the epidemic in Japan (21) In addition to the D222G substitution, two other changes D187E and Q223R were recognised These mutations are known to be critical for receptor binding, and they cause a shift from α2,6 to α2,3 sialic acid receptor specificity However, their presence was associated with a moderate course of the disease In the genetic study on pdm isolates in Tunis, three different viruses with D222G substitution were isolated from three different clinical observations: mild, severe, and fatal (14) Several reports have shown that the D222G substitution was only associated with severe, and sometimes fatal, cases of H1N1pdm (12, 13) Kilander et al (8) reported the occurrence of an amino acid substitution of aspartic acid (D) to glycine (G) in position 222 (D222G) in the HA1 subunit of the viral samples derived from 11 out of 61 cases with severe outcome analysed in Norway Such mutants were not observed in any of the 205 mild cases investigated Thus, the frequency of this mutation was significantly higher in severe (including fatal) cases Moderate but not severe course of the disease in the case from farm C could be the result of an insufficient group of mutations in the presented viral genome The aspartic acid to glycine substitution D222G within the HA of 2009 H1N1V viruses could be epidemiologically correlated to severe cases of illness in several countries In mice, the D222G mutation is related to higher virus titres, elevated proinflammatory cytokine release in the lungs, and enhanced lethality (22) Similar H1N1v mice - 10.1515/bvip-2015-0066 Downloaded from De Gruyter Online at 09/12/2016 02:37:46AM via free access A Kowalczyk et al./Bull Vet Inst Pulawy/59 (2015) 451-455 adaptation mechanisms matched the HA(D222G) mutation as well (7), possibly indicating the necessity of an α 2,3-linked sialic receptor-binding specificity for efficient replication in mice (19) In order to explore swine receptor specificity and pathogenesis for pdm H1N1 with D222G, a clinical study might be required Conflict of Interests Statement: The authors declare that they have no conflict of interests regarding the publication of this article References Chen L.M., Rivailler P., Hossain J., Carney P., Balish A., Perry I., Davis C.T., Garten R., Shu B., Xu X., Klimov A., Paulson J.C., Cox N.J., Swenson S., Stevens J., Vincent A., Gramer M., Donis R.O.: Receptor specificity of subtype H1 influenza A viruses isolated from swine and humans in the United States Virology 2011, 412, 401–410 Couceiro J.N., Paulson J.C., Baum L.G.: Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity Virus Res 1993, 29, 155–165 Gabriel G., Dauber B., Wolff T., Planz O., Klenk H.D., Stech J.: The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host Proc Natl Acad Sci USA 2003, 102, 18590–18595 Gambaryan A.S., Boravleva E.Y., Matrosovich T.Y., Matrosovich M.N., Klenk H.D., Moiseeva E.V., Tuzikov A.B., Chinarev A.A., Pazynina G.V., Bovin N.V.: Polymer-bound 6' sialyl-N-acetyllactosamine protects mice infected by influenza virus Antiviral Res 2005, 68, 116–123 Guldemir D., Kalaycioglu A.T., Altas A.B., Korukluoglu G., Durmaz R.: Monitoring genetic diversity of influenza A(H1N1)pdm09 virus circulating during the post–pandemic period in Turkey Jpn J Infect Dis 2013, 66, 299–305 Hoffmann E., Stech J., Guan Y., Webster R.G., Perez D.R: Universal primer set for the full-length amplification of all influenza A viruses Arch Virol 146, 2275–2289 Ilyushina N.A., Khalenkov AM., Seiler J.P., Forrest H.L., Bovin N.V., Marjuki H., Barman S., Webster R.G., Webby R.J.: Adaptation of pandemic H1N1 influenza viruses in mice Virology 2010, 84, 8607–8616 Kilander A., Rykkvin R., Dudman S.G., Hungnes O.: Observed association between the HA1 mutation D222G in the 2009 pandemic influenza A(H1N1) virus and severe clinical outcome, Norway 2009–2010 Euro Surveill 2010, 15, 19498 Kowalczyk A., Markowska-Daniel I., Rasmussen T.B.: Development of a primer-probe energy transfer based real–time PCR for the detection of Swine influenza virus J Virol Methods 2013, 187, 228–233 10 Ledesma J., Pozo F., Reina G., Blasco M., Rodríguez G., Montes M., López-Miragaya I., Salvador C., Reina J., Ortíz de Lejarazu R., Egido P., López Barba J., Delgado C., Cuevas M.T., Casas I.: Genetic diversity of influenza A(H1N1)2009 virus circulating during the season 2010–2011 in Spain J Clin Virol 2012, 53, 16–21 455 11 Maines T.R., Jayaraman A., Belser, J.A., Wadford D.A., Pappas C., Zeng H., Gustin K.M., Pearce M.B., Viswanathan K., Shriver Z.H., Raman R, Cox N.J., Sasisekharan R., Katz J.M., Tumpey T.M.: Transmission and pathogenesis of swine–origin 2009 A(H1N1) influenza viruses in ferrets and mice Science 2009, 325, 484–487 12 Mak D.B., Daly A.M., Armstrong P.K., Effler P.V.: Pandemic (H1N1) 2009 influenza vaccination coverage in Western Australia Med J Aust 2010, 193, 401–404 13 Melidou A., Gioula G., Exindari M., Chatzidimitriou D., Diza E., Malisiovas N.: Molecular and phylogenetic analysis of the haemagglutinin gene of pandemic influenza H1N1 2009 viruses associated with severe and fatal infections Vet Res 2010, 151, 192–199 14 Moussi A., Kacem M.A., Pozo F., Ledesma J., Cuevas M.T., Casas I., Slim A.: Genetic diversity of HA1 domain of heammaglutinin gene of influenza A(H1N1)pdm09 in Tunisia J Virol 2013, 10, 150 15 Nelson M., Spiro D., Wentworth D., Beck E., Fan J., Ghedin E., Halpin R., Bera J., Hine E., Proudfoot K., Stockwell T., Lin X., Griesemer S., Kumar S., Bose M., Viboud C., Holmes E., Henrickson K.: The early diversification of influenza A/H1N1pdm 2009, PLoS Curr 1:RRN1126 16 Schaefer R., Zanella R.C., Brentano L., Vincent A., Ritterbusch G.A., Silveira S., Luizinho Caron L., Mores G.: Isolation and characterization of a pandemic H1N1 influenza virus in pigs in Brazil Pesq Vet Bras 2011, 31, 761–767 17 Stevens J., Chen L.M., Carney P.J., Garten R., Foust A., Le J., Pokorny B.A., Manojkumar R., Silverman J., Devis R., Rhea K., Xu X., Bucher D.J., Paulson J.C., Cox N.J., Klimov A., Donis R.O.: Receptor specificity of influenza A H3N2 viruses isolated in mammalian cells and embryonated chicken eggs J Virol 2010, 84, 8287–8299 18 Takemae N., Ruttanapumma R., Parchariyanon S., Yoneyama S., Hayashi T., Hiramatsu H., Sriwilaijaroen N., Uchida Y., Kondo S., Yagi H., Kato K., Suzuki Y., Saito T.: Alterations in receptor-binding properties of swine influenza viruses of the H1 subtype after isolation in embryonated chicken eggs J Gen Virol 2010, 91, 938–948 19 Tate M.D., Brooks A.G., Reading P.C.: Receptor specificity of the influenza virus hemagglutinin modulates sensitivity to soluble collections of the innate immune system and virulence in mice Virology 2011, 413, 128–138 20 Vines A., Wells K., Matrosovich M., Castrucci M.R., Ito T., Kawaoka Y.: The role of influenza A virus hemagglutinin residues 226 and 228 in receptor specificity and host range restriction J Virol 1998, 72, 7626–7631 21 Yasugi M., Nakamura S., Daidoji T., Kawashita N., Ramadhany R., Yang C.S., Yasunaga T., Iida T., Horii T., Ikuta K., Takahashi K., Nakaya T.: Frequency of D222G and Q223R hemagglutinin mutants of pandemic (H1N1) 2009 influenza virus in Japan between 2009 and 2010 PLoS One 2012, 7, 30946 22 Zheng B., Chan K.H., Zhang A.J., Zhou J., Chan C.C., Poon V.K., Zhang K., Leung V.H., Jin D.Y., Woo P.C., Chan J.F., To K.K., Chen H, Yuen K.Y.: D225G mutation in hemagglutinin of pandemic influenza H1N1 (2009) virus enhances virulence in mice Exp Biol Med 2010, 235, 981–988 23 Zhou B., Li Y., Halpin R., Hine E., Spiro D.J., Wentworth D.E.: PB2 residue 158 is a pathogenic determinant of pandemic H1N1 and H5 influenza a viruses in mice J Virol 2011, 85, 357–365 - 10.1515/bvip-2015-0066 Downloaded from De Gruyter Online at 09/12/2016 02:37:46AM via free access - 10.1515/bvip-2015-0066 Downloaded from De Gruyter Online at 09/12/2016 02:37:46AM via free access ... M., Nakamura S., Daidoji T., Kawashita N., Ramadhany R., Yang C.S., Yasunaga T., Iida T., Horii T., Ikuta K., Takahashi K., Nakaya T.: Frequency of D222G and Q223R hemagglutinin mutants of pandemic. .. Molecular and phylogenetic analysis of the haemagglutinin gene of pandemic influenza H1N1 2009 viruses associated with severe and fatal infections Vet Res 2010, 151, 192–199 14 Moussi A. , Kacem M .A. ,... pandemic- like influenza A (H1N1) 2009 viruses with the D222G substitutions in HA circulating in Poland were isolated from farm C where mild course of the disease was observed The HA protein has a tendency