Preventive Veterinary Medicine 119 (2015) 172–178

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Preventive Veterinary Medicine

j ourna l ho me pa g e: www.elsev ier .com/ locate /prevetmed

Molecular epidemiology study of swine influenza virusrevealing a reassorted virus H1N1 in swine farms in Cuba

Lester J. Péreza,∗, Carmen Laura Pereraa, Liani Coronadoa, Liliam Riosa,Armando Vegaa, Maria T. Fríasa, Llilianne Gangesb, José Ignacio Núnezb,1,Heidy Díaz de Arcea,c,1

a Centro Nacional de Sanidad Agropecuaria (CENSA), La Habana, Cubab Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola delVallès, Spainc Hospital Italiano de Buenos Aires, Juan D. Perón 4190, C1181ACH Buenos Aires, Argentina

a r t i c l e i n f o

Article history:Received 20 November 2014Received in revised form 9 February 2015Accepted 16 February 2015

a b s t r a c t

In this report, we describe the emergence of reassorted H1N1 swine influenza virus,originated from a reassortment event between the H1N1 pandemic influenza virus(H1N1p/2009) and endemic swine influenza virus in Cuban swine population. In November2010, a clinical respiratory outbreak was reported on a pig fattening farm in Cuba. Phyloge-

Keywords:Swine influenza virusPhylogenetic analysisReassortant

netic analysis showed that all the genes of one of the isolate obtained, with the exception ofneuraminidase, belonged to the H1N1p/2009 cluster. This finding suggests that H1N1pdmhas been established in swine and has become a reservoir of reassortment that may producenew viruses with both animal and public health risks.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Influenza is a zoonotic viral disease that represents ahealth and economic threat to both humans and animalsworldwide. Influenza viruses present an enormous diver-sity due to their high mutation rate, short replication time,high number of replicates and the possibility of pseudorecombination or reassortment (Szewczyk et al., 2014).

In April 2009, a novel swine-lineage influenza virus

capable of rapid human transmission was reported,although infection with (H1N1) 2009 was not connected topig exposure or to a contemporary infection in the swine

∗ Corresponding author at: Centro Nacional de Sanidad Agropecuaria(CENSA), La Habana, Cuba. Tel.: +53 849133; fax: +53 47861104.

E-mail address: lesterjosue@gmail.com (L.J. Pérez).1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.prevetmed.2015.02.0130167-5877/© 2015 Elsevier B.V. All rights reserved.

population Neumann et al. (2009). Although the origin ofthe 2009 pandemic H1N1 virus is uncertain, genetically thevirus is closely related to triple reassortant swine influenzaviruses (SwIVs) currently circulating in pig populations(Smith et al., 2009).

Pigs are documented to allow productive replicationof human, avian, and swine influenza viruses. Swine havebeen shown to possess receptors for avian and humaninfluenza viruses in the tracheal epithelium, leading to thesuggestion that the pig is a mixing vessel. Thus it has beenconjectured that pigs are the “mixing vessel” enabling reas-sortment of genes from human, swine, and avian influenzaA viruses and thereby, raising the risks they pose to humanhealth (Hass et al., 2011).

Swine influenza is not presently an OIE listed disease,

so historical information on the global epidemiology of thisdisease is incomplete. The data of swine flu are generallyunderrepresented or absent in many geographical areas

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nd specially, whole virus genome sequence sets of swinenfluenza viruses are required (Pappaioanou and Gramer,010).

Hence, complete genomic sequencing and studying therocess and patterns of viral reassortment, especially inigs, is a key to better understanding of human influenzaandemics.

In a previous study, we carried out virological surveil-ance of pandemic H1N1/2009 influenza virus (H1N1pdm)n swine herds intending to determine whether the virus

as circulating among pig populations in Cuba (Pérez et al.,013). As result H1N1pdm was detected in 5 out of 1631.3%) swine farms located all across the country and com-lete genomic characterization of three viral isolates waserformed (Pérez et al., 2013).

This report describes the emergence of a novel H1N1wine influenza virus (SwIV) in pigs originated from a reas-ortment event between the H1N1 pandemic influenzairus (H1N1pdm) and endemic swine influenza virusSwIV) on a Cuban pig farm. This finding suggests that1N1pdm has been established in swine and has become

reservoir of reassortment that may produce new virusesith both animal and public health risks.

. Materials and methods

.1. Clinical specimens

In November 2010, a clinical outbreak was reported on pig fattening farm with 2000 pigs located in the provincef Pinar del Río (Western Cuba) with 15% morbidity and 1%ortality.A total of seven lung tissue samples were taken from

igs that showed respiratory clinical symptoms (coughing,neezing, nasal discharge, difficult breathing and depressedppetite) and evidence of interstitial pneumonia of variableeverity at necropsy.

.2. RNA isolation and swine influenza genome detection

Viral RNA was extracted from lung tissue homogenatesy using QIAamp Viral RNA Mini Kit (Qiagen GmbH) accord-

ng to the manufacturer’s directions. The samples werecreened for the presence of influenza A viruses by matrixene real time RT-PCR (Spackman et al., 2002). Positiveamples were further tested for the H1N1pdm by real timeT-PCR (Pérez et al., 2013).

.3. Virus isolation and identification

Five out of seven lung tissue samples were positive fornfluenza A and negative for H1N1pdm. One positive tis-ue suspension was selected and isolated in pathogen-free

SPF) embryonated chicken eggs and Madin Darby canineidney (MDCK) cells (OIE, 2008). The isolate was identifiedy the real time RT-PCR assay and confirmed by genomeequencing.

edicine 119 (2015) 172–178 173

2.4. Genome sequencing and sequence analysis

The eight gene segments of the SwIV isolate weresequenced as previously described (Pérez et al., 2013) andthe isolate was designated as A/swine/Pinar del Río/3/2010(H1N1). The nucleotide and amino acid sequences alig-ments as well as the prediction of N-linked glycosylationsites were performed as described Pérez et al. (2013).

2.5. Phylogenetic analysis

Phylogenetic relationships among SwIV sequences wereanalyzed using Bayesian Inference (BI). The best fittingnucleotide substitution model for each segment was testedby means of Akaike information criterion (AIC) imple-mented in Modeltest 3.07 software (Posada and Crandall,1998). The best fit models selected were used for phyloge-netic analysis. BI analysis was performed with the softwareMrBayes3.1 (Huelsenbeck et al., 2001; Ronquist andHuelsenbeck, 2003). The Bayesian Markov Chain MonteCarlo (MCMC) search was run with four chains for 5 milliongenerations, sampling the Markov chain every 100 genera-tions. At the end of run, the convergence of the chains wasinspected through the average standard deviation of splitfrequencies and the first 25% of the trees were discarded.After burn-in, the convergence was again assessed on thebasis of the effective sampling size (ESS) using Tracer soft-ware v1.4 (http://tree.bio.ed.ac.uk/software/tracer/). Onlya ESS’s of >400 for each tree was accepted. Eight treeswith clade credibility were constructed using the pos-terior probability distribution. Finally, for visualizationand printing of the trees, the FigTree program v1.2.3(http://tree.bio.ed.ac.uk/) was used.

3. Results

3.1. Sequence identity analysis

The eight gene segments of the SwIV isolate weresequenced and the isolate was designated as A/swine/Pinardel Río/3/2010 (H1N1). The nucleotide sequences obtainedin this study have been deposited in the EMBL/GenBankdatabase and the corresponding accession numberswere listed under the accession numbers HE589463 toHE589470.

The genetic origins of isolates were initially inferredfrom BLASTn analysis. Each of the eight genes of the novelisolate showed the highest nucleotide sequence identitiesto those of pandemic H1N1/2009 influenza virus for all thegenome segments except the neuraminidase which wasderived from N1 endemic SwIV.

3.2. Phylogenetic relationship

To characterize the gene segments of the SwIV iso-late more precisely, eight phylogenetic trees using thenucleotide sequences of the eight genes were constructed

with sequences of reference viruses available in Gen-Bank consisting of viruses from avian-like swine, seasonalhuman, endemic swine, pandemic H1N1/2009 influenzaand novel emergent reassortant viruses.

174 L.J. Pérez et al. / Preventive Veterinary Medicine 119 (2015) 172–178

Fig. 1. Phylogenetic trees for M, HA and NA gene segments of influenza A viruses selected. Each gene is indicated in the upper-left side of each tree. Alltrees were constructed with MrBayes3.1. The best-fit model and the shape parameter of the gamma distribution (alpha) for all tree is also indicated inthe upper-left side of each tree. The numbers at a node are posterior probability values estimated. Different lineages are denoted and also marked with

ed usinterpret

different colors. The novel Cuban triple reassortant isolate was highlightH1N1/2009 influenza virus were denoted by an asterisk in HA-tree. (For into the web version of this article.)

Interestingly, phylogenetic analysis showed that theNA gene sequence grouped within the endemic swineinfluenza H1N1 clade (73% posterior probability), indi-cating that it was derived from endemic swine influenza

viruses. All of the seven remaining genes of the novelisolate clustered with the pandemic H1N1p/2009 viruses(data not shown). Representative results from haemaglu-tinin, matrix and neuraminidase gene segments are

g black letters. Others novel reassortant viruses derived from pandemication of the references to color in this figure legend, the reader is referred

showed (Fig. 1). Thus, the HA gene sequence of theCuban isolate A/swine/Pinar del Río/3/2010 (H1N1) wasclustered together with the Cuban isolate A/swine/LaHabana/130/2010 (H1N1) within the defined clade of

pandemic H1N1/2009 viruses with a slightly differencerepresented by the branch distance between them (Fig. 1).Meanwhile, the M gene sequence of the Cuban isolateA/swine/Pinar del Río/3/2010 (H1N1) was clustered in an

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ndependent cluster formed by all Cuban isolates withinhe defined clade of pandemic H1N1/2009 viruses (Fig. 1).onetheless, it is important to highlight that although

he M gene sequence of the Cuban isolate A/swine/Pinarel Río/3/2010 (H1N1) shared the same common ancestorith the remaining Cuban isolates, formed an independent

ranch into the cluster suggesting a slight divergence forhis viral isolate (Fig. 1).

.3. Molecular characterization

The deduced amino acid sequence of the HA gene ofhe Cuban isolate was aligned and compared with the oth-rs H1N1 SwIVs which were taken as reference sequencesFig. 2). These isolates contained an amino acid motifSIQSR↓G at the HA cleavage sites, which is characteristicf low pathogenic influenza viruses.

Of the amino acids that are components of receptor-inding sites of the HA1 domain just a small number ofutation were found (Fig. 2). Thus, similarly to the classi-

al H1N1 reference strain a G222 residue was kept in theeassortant isolate. Several mutations were found at thentigenic sites of the HA1 (Cb, Sb, Sa, and Ca) (Fig. 2). Sevenotential N-glycosylation sites (N–X–S/T) were found in theA1 protein of the Cuban isolate. Six of the N-glycosylation

ites at positions 10, 11, 23, 87, 276, 287 were conserved inhe Cuban isolate.

The M1 protein of the Cuban SwIV isolate showed autation pattern very similar to the pandemic H1N1/2009

nfluenza strain used (data not shown). Nonetheless, weave considered important to note the K101R replacement

ound in the Cuban isolate and the pandemic H1N1/2009nfluenza strain because of this mutation was located athe nuclear localization signal (NLS:101 RKLKR105). On thether hand, the mutation N31S was observed on the M2rotein of the Cuban isolate, which suggests that this iso-

ate is resistant to anti-influenza drugs amantadine andimantadine.

The amino acid sequences of the NP protein of theuban SwIV isolate of the present study were highlyonserved on the non-conventional nuclear localizationignal (nNLS: 3SxGTKRSYxxM13) and on the nuclear accu-ulation signal/cytoplasmic retention signal (NAS/CRS:

36AAFEDLRVSS345). However, signatures related withhe host-range alteration were observed. In addition, aaline at position 313 was found in the isolate; this is an

mportant residue linked to the direct contact betweenP and other proteins of the polymerase complex. Theutations V100I and D211N were also found. It is impor-

ant to highlight the fact that the D211N mutation of thesolate A/swine/Pinar del Río/3/2010 (H1N1) was locatedn the classical bipartite nuclear localization signal (cNLS:98KRGINDRNFWRGENGRKTR216).

The PB1 protein and peptide PB1-F2 of the CubanwIV isolate showed the same pattern as the pandemic1N1/2009 influenza strain. The PB2 protein of the isolatebtained in the present study possessed E627K muta-

ion characteristic of the pandemic H1N1 strains. Likewise,mportant amino acid signatures such as 333T, 701D and14S were present in the Cuban SwIV isolate. In addition,he A271T and R591Q (SR motif) mutations which increase

edicine 119 (2015) 172–178 175

polymerase activity and viral replication in mammals andmight compensate the presence of 627E were also found.The essential residues of the PA protein linked to the syn-thesis of viral complementary RNA such as W88, K102,P103, D108, Y110, F117, K134, T157 and S186 were highlyconserved in the Cuban isolate.

The Cuban A/swine/Pinar del Río/3/2010 (H1N1) isolatepossessed D at position 92 of NS1, which has been linkedto an increase in the virulence of H5N1 viruses in pigs.

4. Discussion

Whereas the scientific community was prepared toface the expected pandemic influenza, which would becaused by highly pathogenic avian influenza from wild anddomestic birds subtype H5 (Capua and Alexander, 2006), inApril 2009, the World was surprised by an influenza out-break with pandemic behavior (WHO, 2010) coming frompigs (CDC, 2009). The novel H1N1p/2009 influenza virusemerged from the swine reservoir, carrying a constella-tion of influenza genes of avian, swine and human originsfrom North American and Eurasian lineages (Smith et al.,2009). The H1N1p/2009 influenza virus spread both widelyand rapidly and was transmitted to several countries in ashort term (Novel Swine-Origin Influenza A (H1N1) VirusInvestigation Team et al., 2009). This fact emphasized thecomplexity of influenza viral ecology and the difficulty ofpredicting influenza viral dynamics (Ozawa and Kawaoka,2013).

The current work describes the emergence of a novelH1N1 swine influenza virus (SwIV) in pigs originated from areassortment event between the H1N1 pandemic influenzavirus (H1N1p/2009) and endemic swine influenza virus(SwIV) on a Cuban pig farm. This finding was possible by theapplication of a molecular epidemiology approach usingcomplete genome sequence information obtained from theA/swine/Pinar del Río/3/2010 (H1N1) isolate. In fact, theuse of molecular epidemiology studies of infectious dis-eases has become, in the current day, a powerful methodto investigate the transmission, distribution, biology anddiversity of pathogens (reviewed in Field et al., 2014).This scientific discipline together with the development ofsequencing technologies have allowed to gain insight intothe origin, emergence and spread of viral agents (Pérezet al., 2011; Alfonso-Morales et al., 2013), support vacci-nation programs established for control of viral endemicdiseases (Pérez et al., 2012) and others. For influenza virus,in especial after the 2009/pandemic outbreak, the atten-tion to molecular epidemiology and evolutionary studies,revealing treatment resistance, virulence or the emergenceof novel strains, has been increased by the scientific com-munity.

H1N1p/2009 appears to be relatively genetically stableand of relatively low virulence for humans since its detec-tion in humans in April 2009 (Graham et al., 2011; Octavianiet al., 2011). Nevertheless, it appears that the H1N1p/2009virus is currently displacing the preceding H1N1 strains

as the seasonal H1N1 influenza virus (reviewed in Potterand Jennings, 2011). In addition, the potential evolution ofH1N1p/2009 in multiple species like pigs is significant dueto the presence of multiple co-circulating viruses that may

176 L.J. Pérez et al. / Preventive Veterinary Medicine 119 (2015) 172–178

Fig. 2. Molecular analysis of HA amino acid sequences of the novel Cuban triple reassortant isolate and reference strains. Previously defined antigenic sitesare indicated: site Cb (blue line), site Sb (red line), site Sa (pink line), site Ca (brown line). The receptor binding sites of the HA1 domain are signed bygreen line. The HA cleavage sites is highlighted by dashed orange line and also by orange arrow. The potential glycosylation sites are underlined and also

raham e1N1pd

nd, the r

the N residues highlighted by bold letter. Other mutations reported by GThe novel Cuban triple reassortant isolate was denoted by an asterisk, a Hreference. (For interpretation of the references to color in this figure lege

favor the possibility of generating novel reassortants withthe potential to infect humans with increased virulence.

The results obtained in the current work evidence thatH1N1p/2009 has been established in swine and has becomea reservoir of reassortment that may produce new viruseswith both animal and public health risks. On the one hand,if it’s taken into account the current circulation in Cubaof different influenza A viruses in human such as A/H1N1,A/H1N1pdm2009 and emergent influenza viruses A/H3N2(Arencibia et al., 2014a,b), together with the circulation inpigs of H1N1p/2009 (Pérez et al., 2013) and endemic H1N1swine influenza virus (Barrera et al., 1983) then, the prob-ability that all these variants could be put together in theswine reservoir to mix and yield novel emergent viral vari-ants is really high. On the other hand, the lack of a controlprogram against influenza A virus affecting pigs in Cuba,the proximity among migratory birds settlements (Ferreret al., 2014) and porcine farms, the low efficiency of thecurrent surveillance program for avian influenza virus inCuban poultry (Ferrer et al., 2014) and the possibility ofescape of the current circulating strains in human to theestablished vaccination program caused by antigenic driftof these strains (Arencibia et al., 2014a,b), are importantaspects that can also contribute to emergence and fastspreading of a novel viral variant of influenza A virus.

From molecular epidemiology studies and molecularsurveillance networks for influenza virus in pigs, newemergent reassortants originated from H1N1p/2009 andendemic SwIV (Ducatez et al., 2011; Simon et al., 2014)and H1N1p/2009 and human like SwIV (Pereda et al.,

2011) have been identified. Even though, the preva-lence of the novel reassortants detected has been low,all these reports have highlighted the dynamic natureof influenza viruses in pig populations as well the fact

t al. (2011) as adaptive evolution mutations are signed by black arrows.m isolate previously reported by Pérez et al. (2013) was also included aseader is referred to the web version of this article.)

that a continue monitoring of viruses in pigs at thelevel of full genome sequencing is absolutely required(Ducatez et al., 2011; Pereda et al., 2011; Simon et al.,2014).

Our results show that the introduction of pandemicH1N1/2009 influenza virus to swine has provided the viruswith opportunities for reassortment and are consistentwith prior observations of the flexibility of the triple reas-sortant internal gene (TRIG) cassette to reassort with otherinfluenza viruses (Ducatez et al., 2011; Fan et al., 2012).This new viral variant also has the ability to escape thediagnostic rRT-PCR assays available for the early detectionof influenza viruses (Fig. 3). These findings suggest thatH1N1p/2009, although mild and apparently contained atpresent, could undergo further reassortment in swine withanimal and public health risks.

Our study has several strengths. We were able todemonstrate the presence of a novel reassorted virus,which emerged from the pig reservoir containing NAgene from endemic SwIV and the remaining genes fromH1N1p/2009, associated to clinical signs in the field. Frommolecular characterization of this isolate, we revealed itspotentiality for the establishment in the Cuban pig popula-tion. This hypothesis could be supported by the possibilityof the A/swine/Pinar del Río/3/2010 (H1N1) isolate toescape to the immune response of the pig host (causedby the circulation of endemic SwIV strains) and othersadaptive advantages found. Nevertheless, the limitation ofour study is mainly given by the fact that the evidenceof the reassortment event was found in a single isolate,

which was obtained from a single outbreak. Because ofthis limitation, we were unable to determine the possibleestablishment and spreading of this reassorted strain or tocompare its prevalence to previous prevalence estimated

L.J. Pérez et al. / Preventive Veterinary Medicine 119 (2015) 172–178 177

Fig. 3. Sequence alignment showing the target region of the probe N1-F232 designed for the specific detection of theH1N1pdm viruses. The nucleotide sequence of the probe N1-F232 designed for the specific detection of the H1N1pdm viruses( 1n1 guil of the rv

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http://www.fao.org/ag/AGAinfo/programmes/en/empres/AH1N1/docs/hines. Mismatches were highlighted by red solid lines. (For interpretationersion of this article.)

or H1N1p/2009 in swine herds in Cuba (Pérez et al., 2013).evertheless, the results obtained from the molecular char-cterization study could support the hypothesis about aossible establishment and spreading of this reassortedtrain, since it was found that the isolate A/swine/Pinar delío/3/2010 (H1N1) possess several signatures related withhis characteristics. Despite the limitations, our study hasontributed to the literature describing the emergence of

novel reassorted influenza strain. In addition, consider-ng the significant role of swine in the ecology of influenzairuses, our results stress the need for greater monitoringn pig population and genetic characterization of all eightene segments so that reassortment events are rapidlydentified to prevent the emergence of viruses with theotential to infect humans and cause pandemics.

onflict of interest

The authors declare that they have no conflict of inter-st.

cknowledgements

Financial support for this study was provided by FAOroject TCP/RLA/3206. Work at CReSA was supported by acholarship from the MAEC-AECID program from Spanishovernment.

eferences

lfonso-Morales, A., Martínez-Pérez, O., Dolz, R., Valle, R., Perera, C.L.,Bertran, K., Frías, M.T., Majó, N., Ganges, L., Pérez, L.J., 2013. Spa-tiotemporal phylogenetic analysis and molecular characterisation ofinfectious bursal disease viruses based on the VP2 hyper-variableregion. PLoS ONE 8, e65999.

rencibia, A., Acosta, B., Muné, M., Valdés, O., Fernández, L., Savón, C.,Oropesa, S., Gonzalez, G., Gonzalez, G., Hernández, B., Roque, R.,Goyenechea, A., Pinón, A., 2014a. Genetic drift of hemagglutinin (HA)of influenza A(H3N2) viruses circulating in Cuba between 2011 and2013. Infect. Genet. Evol. 28, 58–61.

rencibia, A., Pinon, A., Muné, M., Acosta, B., Valdés, O., Savón, C., Oropesa,S., Gonzalez, G., Soto, Y., Guilarte, E., Gonzalez, G., Hernández, B.,Goyenechea, A., 2014b. Molecular characterization of influenza Aviruses circulating in Cuba between April 2009 and August 2010. J.Infect. Dev. Ctries. 8, 929–932.

arrera, M., González, R., Gutiérrez, E., 1983. Influenza porcina: Pesquisaserológica en 2 Provincias en Cuba: Porcine Influenza: serologicalinquest from 2 province in Cuba. Rev. Salud Anim. 5, 433–436.

apua, I., Alexander, D.J., 2006. The challenge of avian influenza to theveterinary community. Avian Pathol. 35, 189–205.

delines fao.pdf) was highlighted in yellow and framed using boldeferences to color in this figure legend, the reader is referred to the web

CDC, 2009. Swine influenza A (H1N1) infection in two children – SouthernCalifornia, March–April 2009. Morb. Mortal. Wkly. Rep. (MMWR) 58,400–402.

Ducatez, M.F., Hause, B., Stigger-Rosser, E., Darnell, D., Corzo, C., Juleen,K., Simonson, R., Brockwell-Staats, C., Rubrum, A., Wang, D., Webb,A., Crumpton, J.C., Lowe, J., Gramer, M., Webby, R.J., 2011. Multiplereassortment between pandemic (H1N1) 2009 and endemic influenzaviruses in pigs, United States. Emerg. Infect. Dis. 17, 1624–1629.

Fan, X., Zhub, H., Zhou, B., Smith, D.K., Chen, X., Lam, T.Y., Poon, L.L.M.,Peiris, M., Guan, Y., 2012. Emergence and dissemination of a swineH3N2 reassortant with 2009 pandemic H1N1 genes in pigs in China.J. Virol. 86, 2375–2378.

Ferrer, E., Alfonso, P., Ippoliti, C., Abeledo, M., Calistri, P., Blanco, P.,Conte, A., Sánchez, B., Fonseca, O., Percedo, M., Pérez, A., Fernán-dez, O., Giovannini, A., 2014. Development of an active risk-basedsurveillance strategy for avian influenza in Cuba. Prev. Vet. Med. 116,161–167.

Field, N., Cohen, T., Struelens, M.J., Palm, D., Cookson, B., Glynn, J.R., Gallo,V., Ramsay, M., Sonnenberg, P., Maccannell, D., Charlett, A., Egger, M.,Green, J., Vineis, P., Abubakar, I., 2014. Strengthening the Reportingof Molecular Epidemiology for Infectious Diseases (STROME-ID): anextension of the STROBE statement. Lancet Infect. Dis. 14, 341–352.

Graham, M., Liang, B., Van Domselaar, G., Bastien, N., Beaudoin,C., Tyler, S., Kaplen, B., Landry, E., Yang, L., 2011. Nation-wide molecular surveillance of pandemic H1N1 influenzaA virus genomes: Canada, 2009. PLoS ONE 6, e16087,http://dx.doi.org/10.1371/journal.pone.0016087.

Hass, J., Matuszewski, S., Cieslik, D., Haase, M., 2011. The role of swineas ‘mixing vessel’ for interspecies transmission of the influenza Asubtype H1N1: a simultaneous Bayesian inference of phylogeny andancestral hosts. Infect. Genet. Evol. 11, 437–441.

Huelsenbeck, J.H., Ronquist, F., Nielsen, R., Bollback, J., 2001. Bayesianinference of phylogeny and its impact on evolutionary biology. Science294, 2310–2314.

Neumann, G., Noda, T., Kawaoka, Y., 2009. Emergence and pandemicpotential of swine-origin H1N1 influenza virus. Nature 459, 931–939.

Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team,Dawood, F.S., Jain, S., Finelli, L., Shaw, M.W., Lindstrom, S., Garten, R.J.,Gubareva, L.V., Xu, X., Bridges, C.B., Uyeki, T.M., 2009. Emergence ofa novel swine-origin influenza A (H1N1) virus in humans. N. Engl. J.Med. 360, 2605–2615.

Octaviani, C.P., Goto, H., Kawaoka, Y., 2011. Reassortment between sea-sonal H1N1 and pandemic (H1N1) 2009 influenza viruses is restrictedby limited compatibility among polymerase subunits. J. Virol. 85,8449–8452.

OIE, 2008. Swine influenza. In: Manual of Diagnostic Tests and Vaccines forTerrestrial Animals, 6th ed. Office International des Epizooties, Paris,France, pp. 1128–1138, Chapter 2.8.8.

Ozawa, M., Kawaoka, Y., 2013. Cross talk between animal and humaninfluenza viruses. Annu. Rev. Anim. Biosci. 1, 21–42.

Pappaioanou, M., Gramer, M., 2010. Lessons from pandemic H1N1 2009 toimprove prevention, detection, and response to influenza pandemicsfrom a one health perspective. ILAR J. 51, 268–280.

Pereda, A., Rimondi, A., Cappuccio, J., Sanguinetti, R., Angel, M., Ye, J., Sut-

ton, T., Dibárbora, M., Olivera, V., Craig, M.I., Quiroga, M., Machuca,M., Ferrero, A., Perfumo, C., Perez, D.R., 2011. Evidence of reassort-ment of pandemic H1N1 influenza virus in swine in Argentina: are wefacing the expansion of potential epicenters of influenza emergence?Influenza Other Respir. Viruses 5, 409–412.

erinary M

178 L.J. Pérez et al. / Preventive Vet

Pérez, L.J., Perera, C.L., Vega, A., Frías, M.T., Rouseaux, D., Ganges, l., Nunez,J.I., Díaz de Arce, H., 2013. Isolation and complete genomic character-ization of pandemic H1N1/2009 influenza viruses from Cuban swineherds. Res. Vet. Sci. 94, 781–788.

Pérez, L.J., Díaz de Arce, H., Cortey, M., Domíngez, P., Percedo, M.I.,Perera, C.L., Tarradas, J., Frías, M.T., Segalés, J., Ganges, L., Núnez,J.I., 2011. Phylogenetic networks to study the origin and evolutionof porcine circovirus type 2 (PCV2) in Cuba. Vet. Microbiol. 151,245–254.

Pérez, L.J., Díaz de Arce, H., Perera, C.L., Rosell, R., Frías, M.T., Percedo,M.I., Tarradas, J., Dominguez, P., Núnez, J.I., Ganges, L., 2012. Positiveselection pressure on the B/C domains of the E2-gene of classical swinefever virus in endemic areas under C-strain vaccination. Infect. Genet.Evol. 12, 1405–1412.

Posada, D., Crandall, K., 1998. ModelTest: testing the model of DNA sub-stitution. Bioinformatics 14, 817–818.

Potter, C.W., Jennings, R., 2011. A definition for influenza pandemics basedon historical records. J. Infect. 63, 252–259.

Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phy-logenetic inference under mixed models. Bioinformatics 19,1572–1574.

edicine 119 (2015) 172–178

Simon, G., Larsen, L.E., Dürrwald, R., Foni, E., Harder, T., Van Reeth, K.,Markowska-Daniel, I., Reid, S.M., Dan, A., Maldonado, J., Huovilainen,A., Billinis, C., Davidson, I., Agüero, M., Vila, T., Hervé, S., Breum, S.Ø.,Chiapponi, C., Urbaniak, K., Kyriakis, C.S., ESNIP3 consortium, Brown,I.H., Loeffen, W., 2014. European surveillance network for influenzain pigs: surveillance programs, diagnostic tools and Swine influenzavirus subtypes identified in 14 European countries from 2010 to 2013.PLOS ONE 9, e115815.

Smith, G.J., Vijaykrishna, D., Bahl, Lycett, S.J., Worobey, M., Pybus,O.G., Ma, S.K., Cheung, C.L., Raghwani, J., Bhatt, S., Peiris, J.S.M.,Guan, Y., Rambaut, A., 2009. Origins and evolutionary genomicsof the 2009 swineorigin H1N1 influenza A epidemic. Nature 459,1122–1125.

Spackman, E., Senne, D.A., Myers, T.J., Bulaga, L.L., Garber, L.P., Perdue, M.L.,Lohman, K., Suarez, D.L., 2002. Development of a real time reversetranscriptase PCR assay for type A influenza virus and the avian H5

and H7 haemagglutinin subtypes. J. Clin. Microbiol. 40, 3256–3260.

Szewczyk, B., Bienkowska-Szewczyk, K., Król, E., 2014. Introduction tomolecular biology of influenza a viruses. Acta Biochim. Pol. 61,397–401.

WHO, 2010. Weekly Epidemiological Record, 85., pp. 229–236.