PHYLOGENETIC ANALYSIS OF THE FIRST ISOLATE OF POLISH H1N2 SWINE INFLUENZA VIRUS

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1 Bull Vet Inst Pulawy 56, , 2012 DOI: /v PHYLOGENETIC ANALYSIS OF THE FIRST ISOLATE OF POLISH H1N2 SWINE INFLUENZA VIRUS ANDRZEJ KOWALCZYK, KINGA URBANIAK, AND IWONA MARKOWSKA-DANIEL Department of Swine Diseases, National Veterinary Research Institute, 24- Pulawy, Poland Received: July 19, 2012 Accepted: November 22, 2012 Abstract Phylogenetic analysis of the genes determining influenza virus subtype haemagglutinin (HA) and neuraminidase (NA), was performed. The results showed that the Polish H1N2 isolate (A/Swine/Poland15817/2011) was reassortant of human-like swine H1N1 and human-like swine H3N2 origin. The novel isolate was presented to have a close phylogenic relationship with one of the latest European isolates of H1N2 (A/SW/Gent/102/2007 and A/SW/Hungary/13509/2007). Our evolutionary analyses also suggested that the HA and NA genes evolved in a significantly higher rate of synonymous substitutions after they were introduced from human to swine and established the European H1N2 swine lineage. Key words: H1N2 swine influenza virus, phylogenetic analysis, Poland. Intensive study of the molecular evolution of influenza A virus has provided an important insight into its seasonal genesis and spread in human and animal populations. The sequence-derived information obtained from the phylogenetic tree can assist in the design and implementation of public health and therapeutic interventions. Monitoring of influenza A viruses circulating among pigs is of substantial importance also to the swine industry. The main problems due to swine influenza are connected with weight losses (23). In this study, the genetic data of the first Polish H1N2 swine isolate and its closely related strains were examined. The aim was to determine spatial-temporal molecular evolution of H1N2 subtype isolated in Poland through phylogenetic analysis of haemagglutinin (HA) and neuraminidase (NA) genes. Material and Methods Viruses. In October 2011, an outbreak of influenza-like disease occurred in an intensive pig farm of Wielkopolska province in Poland, in which influenza has been never observed before. Many pigs showed similar clinical symptoms, including: cough, sneeze, runny nose. One pig was found dead and during the necropsy, lung was extracted and send to the National Veterinary Research Institute in Pulawy. The amount of 300 µl of tissue homogenate was directly used in RT-nested PCR based on M1 gene sequence and multiplex PCRs based on haemagglutinin (HA) and neuraminidase (NA) genes sequences. Subtype identification were conducted by RT-PCR assays (10). The influenza virus isolate was named: A/swine/Poland/15817/2011(H1N2). Three strains of swine influenza virus (SIV): H1N1 (A/Sw/Bel/1/), H1N2 (A/Sw/Eng/96), and H3N2 (A/Sw/Fl/1/) were used in all tests as reference material. Nucleotide sequence analysis. Viral RNA was extracted by means of RNA Total Isolation Kit (A&A Biotechnology, Poland). Reverse transcription was performed by mixing 5 µl of RNA with 15 µl of RT reaction mixture containing 50 mm Tris-HCl, ph 8.3, 75 mm KCl, 3 mm MgCl 2, 125 µm each dntp, 1 unit of RNaze, and units of M-MLV enzyme (Invitrogen, USA). cdna was synthesised using 50 ng of Random Primers (Invitrogen) in a single reaction. In the first step, a molecular test demonstrating the presence of influenza A virus genome was evaluated. Real-time PCR for detection of conservative region of matrix1 gene was evaluated. Then, RNA giving positive results in the first testing was examined with three sets of primers in PCR carried out to detect fragments of all segments of influenza virus. Each primer set was used at 12.5 pmol. The amplified PCR products were sequenced in GATCbiotech DNA analysis service (Germany). The nucleotide sequences were compared initially by using the clustal W alignment algorithm method. Phylogenetic tree of each gene was determined by Neighbour-Joining method, using MEGA 4 software (MEGA4, USA). Distance sequences were analysed with Megalign unit from Lasergene software. For evaluation of genetic homology among world-wild isolates of SIV, selected sequences of influenza virus genes accessible on-line in NCBI were added to phylogenetic analysis.

2 420 Results Haemagglutinin. In the branching of HA gene, three main lineages were formed: classical swine HA1 with prevalence in America, human-swine HA1, and avian-like SIV (Fig. 1). Phylogenetic analysis revealed that the HA gene of A/swine/Poland/15817/2011 is of human swine lineage located in the same clade with pandemic (H1N1) 2009A/California/04/2009 and classical-swine cluster virus strains. Classical-swine cluster is composed of SIV and human influenza isolates, including the first isolated SIV A/Swine/Iowa/15/ more closely located to the root of the tree. There were no contemporary IV strains directly linked with this virus but two separate lineage: classical and human-swine made two subgroups. In the human-swine subgroup strains, e.g. A/Swine/Gent/ 102/2007, A/Swine/Cotes d Armor/2433/, and A/Swine/Italy/25943/ almost 95% nucleotide similarity to A/Swine/Poland/15817/2011 strain was indentified. H1N2 SIV isolated in Poland formed close lineage with European human-like viruses, which have been isolated in the 90 s. The highest nucleotide similarity to Polish virus H1N1 was observed as regards the latest isolate HA1 - A/Swine/Poland/01311/2009 (66.9%). Comparing the similarity to pandemic H1N1 strain from 2009, the distance was equal to 69.9% for H1N2 Polish SIV. Neuraminidase. In neuraminidase, subtype 2, contrary to the HA phylogenetic tree, there were no main monophyletic lineages (Fig. 2). Polish H1N2 virus was included in human-like branch with European isolates, such as: A/Swine/Hungary/13509/2007, A/Swine/Damme/IDT5673/2006, A/Swine/Spain/54008/ 2004, and A/Swine/Poland/T2/2009 (.7%, %,.8%, and.5% of nucleotide identity, respectively). All Polish N2 strains were found in one subgroup. Among all selected viruses, a distinction of the phylogenetic soubgroups were selected and presented in the Fig. 2. One of the branches is provided with A/Port Chalmers/73 strain as the initial one in temporal-spatial tree; this virus was spread from human to pigs. In this branch, the majority of H3N2 and some of H1N2 viruses circulating in swine population since s till present were found. In this group temporal H3N2 and H1N2 SIV from Poland are located. In this European lineage, influenza viruses from Asia: H3N2 isolated from pigs (A/Swine/HongKong/72/00) and H1N2 from humans (A/Philippines/322/2004) were grouped. In the same cluster a vaccine strain A/Brisbane/59/2007 was found, indicating 83.1% nucleotide similarity to A/Swine/ Poland/15817/2011 virus. Discussion Sporadic cross-species transfer of swine and avian influenza viruses to humans has been documented repeatedly during the last decades. The first isolated influenza viruses were the strains derived from pigs in 1930 and they were called classical SIV (20). From this time to the late 10s, these viruses circulated in pigs in the USA and remained relatively stable. Novel pandemic H1N1 strain from 2009, containing a unique combination of gene segments from North American and Eurasian swine lineages, has continued circulating in humans. Swine has been known to be a suitable host for influenza A viruses. Several cases of human infection by SIVs have been described (24). Currently, three main subtypes of SIV are dominated in the pigs population throughout the world: H1N1, H1N2, and H3N2 (4). In North America, triple-reassortant H3N2 viruses that possess genes of classical swine H1N1, North American avian and human H3N2 viruses, and different lineages of H1N1, as well as, H1N2 viruses generated by reassortations from the afore-mentioned viruses co-circulate. In Asia, the situation is even more complex as, in addition to the above viruses, human H3N2 and Eurasian avian-like H1N1 SIVs are present (9). In Europe, the avian-like H1N1 virus, which was first detected in Belgium in 19, replaced the classical swine H1N1 viruses and became predominating (21). These avian-like SIVs reassorted with human H3N2 viruses and gave rise to human-like H3N2 SIVs, first detected in the mid-10s in Italy (6). At the same time, H1N2 SIVs, comprising HA of classical swine H1N1 and human-like neuraminidase (NA) from swine H3N2 viruses, were isolated in France (3). A novel reassortant H1N2 SIV was isolated during the influenza outbreaks in pig farms in the United Kingdom (UK) (2), which was distinct from the H1N2 swine viruses reported in Japan and North America (7). These reassortants have not become widespread among the European pig population. In 14, a further reassortant variant was identified in the UK, comprising HA and NA genes of human origin (human-like H1N2 viruses) (22). Since then, the HA of these viruses has evolved independently from their ancestor human viruses. The previous study of genome sequences data, obtained from field samples from pigs respiratory disorders, showed a single rather than multiple viral lineage of H1N1. The H1N1 virus was introduced and has been spread across Poland (15). Given the extent of the period of SIV isolation in Poland, the amount of genetic diversity observed over one season is relatively small, maybe in some cases even lack any pattern of influenza virus evolution. To obtain a strong indication of the spatial- a temporal pattern of the spread of different lineages, it would clearly entail a large increase in sampling. The same situation was observed among French SIVs from Cotes de Armor, isolated from the end of the 10s. Novel H1N2 virus could be, however, a result of the circulation of contemporary European lineage and the formation of a similar pathway of evolution was observed among these SIVs in Europe. External genes of Polish H1N2 isolate indicate that they are most similar to a subgroup, which evolved at the beginning of 10s and formed a new antigenic subcluster with the novel European and the Asian human influenza viruses and SIV isolates: H1N2 and H3N2 around the year 2000 (8). A new subcluster made separate branch with A/Swine/Get/1/84 as an ancestor. In the same cluster, an independent branch formed by H1N2 isolates introduced in 14 in England to pig was observed. This indicates

3 421 that a new genetic pattern of mutation in HA1 in H1N2 SIV in Europe and Asia has occurred. The new subgroup is a complex of two sublineages mixed among: HA3 human isolates from Europe in the s and mostly world-wild porcine viruses H1N2, that emerged at the beginning of 90 s. There was host and geographic region association between SIV in these groups. Among European H1N2 SIV, there was a relation between year and place of isolation represented by almost 95% nucleotide similarity among these strains, including A/Swine/Poland/15817/2011 virus. In the same subclade, viruses like A/Swine/England/72685/96 and A/Swine/Scotalnd/410440/94 isolated in different time points out, showed about 88% similarity, but formed a separate branch. In opposition to HA phylogenies, NA of European H1N2 swine viruses did not establish a monophyletic lineage, but sparsely distributed the similarity over the lineage with avian-like European H3N2 and H1N1 swine viruses, though three separate and distinguishable groups of European H1N2 swine viruses could be observed. This reveals our phylogenetic analyses are in accordance with those reported previously (16). From the early 10s onwards, an H3N2 reassortant strain, which acquired genome segments from the H3N2 human viruses and avian-like H1N1 swine viruses, replaced the former human-like H3N2 viruses circulating in the European swine populations, and established a persistent lineage (14), here denoted as European and Asian H3N2 swine lineage. NA gene from the H3N2 human virus was initially introduced into swine population in 12 19, and launched the lineage that eventually evolved to the European H1N2 genotype (12). The frequent human-to-pig transmissions of H3N2 human viruses at that time support the speculation that this NA2 introduction into the H1N2 lineage may be the result of a direct transmission of H3N2 human viruses to swine. Remarkably, the NA phylogeny shows that UK H3N2 swine virus, Swine/UK/119404/, clusters within the H1N2 lineage, and is genetically (NA gene) closer to European H1N2 swine viruses than other European H3N2 viruses do (19). Therefore, the next route of antigenic escape of novel H1N2 virus started with introduction of NA2 gene from human to pig. This was also observed in case of Polish H1N2 virus. Here, the term initial strain was used to indicate an early ancestor of European H1N2 swine viruses (e.g. direct progenitor from H3N2 human viruses) before it acquired the HA gene of H1 type. The HA gene of the H1N1 human virus was introduced into the initial strain in through reassortments, and generated the first antigenic precursor of the European H1N2 swine virus (1). Based on the observation that NA genes of European H1N2 swine viruses diverged since the 10s, which was prior to the first time of novel HA (H1 subtype) introduction, we speculated that an additional reassortment, either the second introduction of NA from the diverged initial strain to the antigenic precursor, or another HA introduction from the H1N1 human viruses to the diverged initial strain may have occurred, and gave rise to the strain Swine/Scotland/410440/94 (H1N2). The phylogenetic analysis of the group A European H1N2 swine virus suggests that the internal genes from European avian-like H3N2 and H1N2 swine viruses were initially transferred to the precursor of European H1N2 swine virus around 16-14, and generated the modern European H1N2 swine virus strains (group A), that consequently caused disease outbreaks in UK since 14. Multiple transmissions of early H3N2 human variants to pigs took place in different areas (11). This finding is consistent with the hypothesis of multiple reassortments proposed by Brown et al. (3). In addition, it is also suggested that the H1N2 virus from UK might have been introduced to continental Europe near to the period of UK outbreaks. A HA gene phylogenetic tree analysis demonstrated that the A/Swine/Poland/15817/2011 shows origins closely associated with H1N2 A/Swine/Gent/102/2007, which was located in a sublineage distinguished from the rest of Polish SIV H1N1. In this sublineage, SIV and AIV from the previous decade were grouped and were close to the ancestors of the whole tree - European H1N2 SIV isolated from 14. In China, in 2010, H1N2 reassortant was reported as a HA, more closely related to swine origin viruses circulating in USA, whereas NA related to human like European strains (13). In HA phylogenetic tree, Asian human H1N1 viruses were grouped with H1N2 swine reassortant together with A/Swine/Poland15817/2011. In the same subcluster triple reassortant from USA H1N2 were found. This could be stressful according to the rapid way of mutagenesis of novel SIV in America. Polish isolates co-circulating in both, time and region, or during the last 12 years were included in small clades with very high nucleotide homology. There were no planned statistic examinations, which could provide a general picture of all the viruses circulating in Poland, due to the limited sampling and occasional prophylactic vaccination and partial protection of piglets by maternal antibodies (17). The spatial model presented on all phylogenetic trees was simplified to a single lineage spread in a unidirectional manner. In Polish SIV, there was no strong indication of viral migration. They all displayed a parallel nucleotide changes with Spanish, French, and Hungarian viruses isolated within the last ten years. A clear datum indicating any peak of genetic diversity among Polish isolates was not presented. To sum up, the first generation of European H1N2 antigenic precursor was established after the initial strain acquired from human-like NA2 and HA1 from human H1N1 viruses circulating between 19 and 16. During this unnoticeable period, the virus underwent enormous genetic turnovers, such as multiple reassortment. Swine play an important role in the interspecies transmissions of influenza viruses (18, 24), and a stringent surveillance s role should be to keep us better informed and prepared, so that proper policies can be set up, such as setting up barriers, applications of vaccine, or eradication, to reduce the probability of outbreaks caused by the novel subtypes before they emerge into virulent forms and a large population would be affected.

4 A/Swine/Poland/15817/2011 A/swine/Gent/102/2007H1N A/swine/Cotes darmor/2433/h1n2 A/swine/Italy/259543/2003H1N2 53 sw/gent/108/01 A/swine/Italy/1521/H1N swine/italy/320546/2009h1n2 sw/england/72685/ A/swine/England/438207/94H1N2 sw/scotland/410440/94 Finland/5/14 A/sw/Obihiro/5/12 A/Bayern/7/95H1N1 A 88 A/swine/Illinois/02066/2008H1N2 A/swine/Illinois/A /2011H1N A/Niedersachsen/217/2005H1N1 A/Sapporo/186/2002H1N1 57 A/Korea/AF18/2008H1N A/Singapore/41/2006H1N1 A/Denmark/4/2008H1N1 A/Brisbane/59/2007 A/Alaska/1935H1N1 A/Tokyo/3/1967H1 A/swine/Cambridge/39 swine/iowa/15/1930h1n1 swine/jamesburg/1942h1n1 A/swine/Thailand/HF6/ sw/england/283902/93 sw/england/117316/86 87 sw/tennessee/25/17 66 sw/wisconsin/1/ A/swine/Hong Kong/294/2009 A/swine/Shanghai/1/2007 swine/iowa/17672/18h1n1 Swine/North Carolina/93523/01H1N2 B swine/korea/jl02/2005h1n2 swine/korea/s11/2005h1n2 75 swine/ohio/24366/07h1n1 swine/north Carolina/A /2010H1N1 16 Turkey/MO/24093/H1N2 23 A/California/07/2009 swine/brazil/20/2010h1n1 A/duck/Hokkaido/55/96(H1N1 turkey/germany/2482/90 66 sw/belgium/117/96 sw/belgium/1/83 60 A/swine/Parma/17(H1N1 duck/bavaria/2/77 62 A/swine/Netherlands/3/(H1N 22 Sw/Netherlands/386/86 64 Sw/Netherlands/477/ sw/england/195852/92 sw/italy/france/2111/95 65 Sw/Belgium/1/ 40 sw/italy/13260/ sw/spain/50047/2003 Sw/Poland/01311/2009 sw/spain/53207/2004 A/Switzerland/5165/2010H1N1 A/Swine/Poland/W1/2005 C 60 sw/cotes darmor/736/ A/Swine/Poland/R4/2005 Sw/Switzerland/88/2002 A/swine/Spain/515/2003H1N1 A/swine/Poland/16318/ swine/hong Kong/20/2009H1N1 swine/guangdong/1616/ Sw/Poland/KPR9/04 Sw/Swine/Poland/LU1/ swine/germany/siv05/2007h1n swine/sweden/1021/2009h1n2 swine/italy/58769/2010h1n Fig. 1. Phylogenetic tree of the nucleotide sequence of the HA portion gene. Panel A shows HA genes of the humanswine European-Asian subgroup and related novel H1N2 Polish virus - A/Swine/Poland/15817/2011 ( ). Panel B shows HA genes of USA classical swine and triple reassortants prevailing in USA and Asia. Panel C shows HA genes of avian-like H1N1 viruses.

5 423 A/England/1/02 H1N2 96 A/Egypt/84/2001H1N2 A/Israel/7/2002 A/Oman/16353/2001 Denmark/208/ Berlin/43/02 Brisbane/10/ A/swine/British Columbia/28103/2005H3N2 swine/minnesota/66743/2006h1n swine/texas/050593/2008h1n2 swine/oklahoma/032726/2008h1n2 sw/shizuoka/110/ 86 Thessaloniki/23/18 Greece/109/ Triple reassortants swine/korea/cy10/2007 A/Swine/Iowa/533/H3N2 swine/korea/hongsong2/2004h1n2 swine/hong Kong/1479/2009H1N2 Turkey/MO/24093/H1N2 A/Buenos Aires/40/95H3N Parma/2/17 A/Eng/23/16H3N Finland/211/92 sw/obihiro/1/14 Siena/10/10H1N2 swine/cotes darmor/43394/2002h1n swine/morbihan/0028/2007h1n2 swine/italy/1521/h1n2 swine/cotesdarmor/2433/h1n2 swine/germany/sek1178/2000h1n2 European H1N2 SIV 77 swine/granstedt/idt3475/2004h1n2 sw/gent/24/00h1n2 swine/scotland/410440/94h1n2 sw/gent/1/ swine/hong Kong/72/00H3N2 swine/hong Kong/5190/H3N2 swine/hong Kong/1144/02 swine/sweden/1021/2009h1n2 A/Swine/Poland/BAL9/2009 A/Philippines/344/2004H1N2 swine/spain/33601/2001h3n2 62 swine/italy/1510/h3n Swine/Belgium/220/92 swine/lohne/1/h3n2 swine/bakum/909/93 wild boar/germany/ws169/2006h3n2 75 sw/flanders/1/h3n2 European H3N2 SIV swine/italy/1942/ swine/bakum/idt1769/2003h3n2 swine/doetlingen/idt4735/2005h1n2 A/swine/Spain/42386/2002H3N2 swine/spain/54008/2004h3n2 A/Poland/Swine/T2/2008/ swine/damme/idt5673/ A/Port Chalmers/73H3N2 swine/hungary/13509/2007 A/Swine/Poland/15817/11 62 A/Memphis/102/72H3N2 A/Bilthoven/9459/14H3N2 18 swine/hong Kong/82/78 A/sw/Hong Kong/4/16H3N swine/guangdong/111/2002 Human H3N2 A/Swine/Colorado/1/77H3N2 swine/hong Kong/127/12 A/England/72 Hungary/2/71 94 Swine/Taiwan/7310/70 swine/ehime/1/10h1n2 88 A/USSR/039/68 83 A/Czech Republic/1/1966H2N2 41 Tokyo/3/1967H1N2 Human H2N2 North Carolina/1/1963H2N2 72 Berlin/3/1964H2N A/Singapore/1/57H2N2 El Salvador/2/1957H2N2 A/X-135Puerto Rico/8/1934 H2N2 A/turkey/England/69H1N Teal/Norway/10 3/2007H1N2 mallard/sweden/3/2002h1n2 85 mallard/netherlands/1/2007h1n A/swine/Korea/C12/2008H5N2 swine/korea/s190/2004h9n2 duck/italy/194659/2006h1n2 swine/yunnan/simao2/2007h9n2 A/swine/Hebei/012/2008H9N A/swine/Shandong/w4/2003H9N2 Swine/Hong Kong/10/H9N2 A/swine/KU/16/2001H7N2 duck/hokkaido/95/01h2n2 northern pintail/california/ /20 AIV canine/korea/gcvp01/2007h3n2 A/turkey/Massachusetts/3740/1965H6N2 A/whiskered tern/egypt/04/2004h6n A/turkey/CO/1188/12H5N2 mallard duck/new York/170/12H1N A/avian/New York/Sg-00425/2004H2N2 A/mallard/Minnesota/27/18H2N2 A/chicken/New York/ /15H2N Fig. 2. Phylogenetic tree of nucleotide sequences of NA2 genes related to novel Polish H1N2 SIV A/Swine/Poland/15817/2011 ( ) grouped with European H3N2 SIV

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