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enomic analysis of influenza A virus from captive wild boars Brazil reveals a human-like H1N2 influenza virus

atalha Biondo a, Rejane Schaefer b,*, Danielle Gava b,auricio E. Cantao b, Simone Silveira b, Marcos A.Z. Mores b,nice R. Ciacci-Zanella b, David E.S.N. Barcellos a

ederal University of Rio Grande do Sul/UFRGS, Porto Alegre, RS, Brazil

mbrapa Swine and Poultry, Concordia, SC, Brazil

Introduction

Swine influenza (SI) is an acute respiratory diseaseused by influenza A virus (IAV) that affects differentimal species, including humans. The IAVs are enveloped

ruses, members of Orthomyxoviridae family virus andnsist of a negative-sense, single-stranded segmentedA genome. The influenza A viruses are classified into

btypes based on the antigenic properties of the surface

glycoproteins hemagglutinin (HA) and neuraminidase(NA) (Van Reeth et al., 2012). Currently, seventeendifferent HAs and ten NAs have been described in theliterature (Tong et al., 2012; Van Reeth et al., 2012). Swinehave been implicated in the emergence of novel influenzaviruses due to the dual expression of both sialic acid (alpha2-3 and alpha 2-6 linkage) in the respiratory tract. Whena single host cell is co-infected with two different influenzaviruses, the exchange of viral RNA segments can occur, aprocess known as genetic reassortment, leading to theemergence of novel viruses (Van Reeth et al., 2012).

Influenza is endemic in pig populations worldwidecaused by genetically distinct lineages of H1N1, H3N2 andH1N2 IAV subtypes. In North America and in Europeancountries, as well as in Asia, the circulating IAV in pigs are

R T I C L E I N F O

icle history:

ceived 20 December 2012

ceived in revised form 12 October 2013

cepted 18 October 2013

ywords:

ptive wild boars

nsolidation

uenza A virus

N2

assortment

A B S T R A C T

Influenza is a viral disease that affects human and several animal species. In Brazil, H1N1,

H3N2 and 2009 pandemic H1N1 A(H1N1)pdm09 influenza A viruses (IAV) circulate in

domestic swine herds. Wild boars are also susceptible to IAV infection but in Brazil until

this moment there are no reports of IAV infection in wild boars or in captive wild boars

populations. Herein the occurrence of IAV in captive wild boars with the presence of lung

consolidation lesions during slaughter was investigated. Lung samples were screened by

RT-PCR for IAV detection. IAV positive samples were further analyzed by quantitative real-

time PCR (qRRT-PCR), virus isolation, genomic sequencing, histopathology and

immunohistochemistry (IHC). Eleven out of 60 lungs (18.3%) were positive for IAV by

RT-PCR and seven out of the eleven were also positive for A(H1N1)pdm09 by qRRT-PCR.

Chronic diffuse bronchopneumonia was observed in all samples and IHC analysis was

negative for influenza A antigen. Full genes segments of H1N2 IAV were sequenced using

Illumina’s genome analyzer platform (MiSeq). The genomic analysis revealed that the HA

and NA genes clustered with IAVs of the human lineage and the six internal genes were

derived from the H1N1pdm09 IAV. This is the first report of a reassortant human-like

H1N2 influenza virus infection in captive wild boars in Brazil and indicates the need to

monitor IAV evolution in Suidae populations.

� 2013 Elsevier B.V. All rights reserved.

Corresponding author at: BR153, Km 110, Vila Tamandua, 89700-000,

ncordia, SC, Brazil. Tel.: +55 4934410423.

E-mail addresses: rejane.schaefer@embrapa.br,

talha_vet@yahoo.com.br, danielle.gava@embrapa.br (R. Schaefer).

Contents lists available at ScienceDirect

Veterinary Microbiology

jou r nal h o mep ag e: w ww .e ls evier . co m/lo c ate /vetm i c

78-1135/$ – see front matter � 2013 Elsevier B.V. All rights reserved.

p://dx.doi.org/10.1016/j.vetmic.2013.10.010

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N. Biondo et al. / Veterinary Microbiology 168 (2014) 34–40 35

enetically different due to independent introductions andaintenance of avian, human and swine genomic lineagesrown, 2000). In U.S, the ‘‘classical’’ H1N1 IAV (a cluster)as a major cause of swine influenza until the introduction

f a H3N2 virus in 1998 (Vincent et al., 2008). Since then,ere was an increase in the rate of genetic changes in

wine influenza virus isolates in North America in H1ubtypes and different antigenic and genetic clustersegan to evolve (Vincent et al., 2008). In Europe, threeubtypes of influenza virus circulate in pigs as avian H1N1vian-like), the human H3N2 (human-like) and H1N2

iruses (Vincent et al., 2013).In Brazil, previous studies have detected the circulation

f H1N1, H3N2 (Brentano et al., 2002; Rajao et al., 2013),009 pandemic H1N1 influenza virus (A(H1N1)pdm09) inommercial swine herds (Schaefer et al., 2011) andecently, a H1N2 IAV in commercial herds (Schaefer,npublished data). In wild boars (Sus scrofa) and feral pigs,erological data from other countries showed the presencef antibodies to H3N2 and H1N1 IAV (Vicente et al., 2002)nd reveals the absence of antibodies against H1N2 (Kadent al., 2008). In Brazil, although the commercial swineerds are investigated for IAV infection through passiveonitoring, the same does not occur in captive wild boars

erds. Considering that wild boars are susceptible to IAVfection, the close contact with humans and possibly

ther animal species brings concern about the infection ofaptive wild boars with IAVs. Moreover, IAV is part of theorcine respiratory disease complex and, together withhe Mycoplasma hyopneumoniae, are considered as pri-

ary pathogens in respiratory tract infections in swine,hich can predispose to secondary infections usually

aused by Pasteurella multocida. However, there are fewtudies about respiratory diseases in captive wild boarsopulations.

Here we describe the histopathological, virological andacteriological analyses of lungs from captive wild boarsresenting pulmonary consolidation, suggestive of IAVfection. Moreover, the genomic sequencing and phylo-

enetic analysis of one virus sample revealed a human-like1N2 IAV derived from the A(H1N1)pdm09 influenzairus.

. Material and methods

Sampling: In 2011, lung samples from captive wild boars = 60) presenting gross lesions of consolidation were

arvested during the slaughter. All the animals used in thistudy were from two farms located in Rio Grande do Sultate, Southern region of Brazil. Farms had similaranagement (nutritional and vaccination schedule),llowing the requirements commonly applied to com-ercial swine herds in Brazil. Wild boars were slaughtered

t seven months old with average weight of 42 kg.IAV detection: Viral RNA was extracted from lung tissue

amples using MagMAXTM-96 Viral RNA Isolation KitMB1836-5) according to the manufacturer’s instructions

nvitrogen1, Carlsbad, CA, USA). The resulting RNA waseverse-transcribed into cDNA using SuperScript II RT Kitnvitrogen1, Carlsbad, CA, USA) and a complementaryrimer (Uni12) was designed to the conserved

12 nucleotides of the 30 end of the viral RNA (Hoffmannet al., 2001). Matrix (M) gene amplification was performedusing primers M52C and M253R (Fouchier et al., 2000).Reverse-transcription polymerase chain reaction (RT-PCR)positive samples were also tested for the presence of the Mgene of A(H1N1)pdm09 virus by quantitative real-time RT-PCR (qRRT-PCR), using primers and probe previouslydescribed (Lorusso et al., 2010).

Virus isolation (VI) was carried out in nine-day-oldSpecific Pathogen Free (SPF) embryonated chicken eggs(ECE). Four days post inoculation, chorioallantoic fluidswere collected and tested for the ability to agglutinate 0.5%suspensions of turkey red blood cells (hemagglutinationtest – HA) and for the presence of IAV nucleic acid by RT-PCR (Fouchier et al., 2000).

Pathological examination: Lung tissues were examinedmacroscopically and classified according to the lesions.The lungs affected area was quantified according to themethodology proposed by Madec and Kolbisch (1982). Theaffected lobes were fixed in 10% buffered formalin forhistological analysis. Lung tissues were routinely processedand stained with hematoxilin and eosin (Luna et al., 1968).

Immunohistochemistry (IHC): The formalin-fixed, par-affin embedded lung tissues were sectioned and processedfor IHC. The influenza A and M. hyopneumoniae antigenswere detected using a biotin-streptavidin-peroxidase kit(LSAB Kit + System � HRP, Dako Carpinteria, CA, USA)employing, as primary antibodies, anti-influenza A nucleo-protein monoclonal antibody HB-65 (Vincent et al., 1997)and anti-lactate dehydrogenase protein (p36) (Ecco et al.,2009) for influenza A and M. hyopneumoniae antigendetection, respectively. Three-amino-9-ethyl-carbazole(AEC) was used as a chromogen in both IHC.

Bacteriology: routine techniques applicable to theisolation of aerobic and microaerophilic pathogens wereused and the biochemical characterization followedrecommendations by Barrow and Feltham (1993).

Genomic sequencing: Paired-end sequencing usingIllumina Genome Analyzer was performed directly fromthe original sample. Viral RNA was extracted from 250 mLof lung homogenates using phenol-guanidine (TRIzol1,Ambion1, Carlsbad, CA, USA) and RNeasy Mini Kit(Qiagen1, Hilden, Germany), following the manufacturer’sinstructions with minor modifications. The reverse tran-scription reaction and the amplification of the eightinfluenza virus gene segments were performed on 15 mLof RNA combined with 25 mL reaction mix, 1 mL of MBT-Uni12 and MBT-Uni13, 1 mL primer mix, 1 mL SuperscriptIII Platinum kit (Invitrogen1, Carlsbad, CA, USA) andRNase/DNase free water (up to 50 mL). The primers werepreviously described by Hoffmann et al. (2001) and Zhouet al. (2009). The following cycling conditions were used:42 8C for 60 min, 94 8C for 2 min, 20 cycles of 94 8C for 30 s,45 8C for 30 s, 68 8C for 7 min (for the amplification of HA,NP, NA and M genes); or 20 cycles of 94 8C for 30 s, 57 8C for30 s, 68 8C for 7 min (for the amplification of PB2, PB1, PAand NS genes); and a final extension cycle of 68 8C for5 min. The PCR products were purified using the QIAquickPCR purification kit (Qiagen1, Hilden, Germany).

Preparation of DNA libraries: The amplified DNAs weretagged and fragmented by the Nextera XT transposome

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N. Biondo et al. / Veterinary Microbiology 168 (2014) 34–4036

ttp://supportres.illumina.com/documents/myillumina/c97885-3ae4-42f3-bee8 4d87fd72e2e3/nextera_xt_mple_preparation_euc_15031943_c.pdf). After theplification step via a limited-cycle PCR program, the

R clean-up was performed with 25 mL of AMPure XPads in order to capture longer library fragments for

250 bp MiSeq sequencing.Sequencing data analysis: The sequences generatedm Illumina MiSeq were cleaned up for low quality readsd adapters using SeqyClean software V. 1.2.3 (https://tbucket.org/izhbannikov/seqyclean). All sequences withred quality score < 30 and length < 220 bases were

moved. The remaining sequences were compared againste influenza virus database from NCBI (http://ww.ncbi.nlm.nih.gov/genomes/FLU) to remove sequencesat were not related to influenza viruses. After qualityntrol steps, all sequences were assembled using thewbler Assembler (Roche) V. 2.9. Multiple alignments ofch influenza virus gene segments were carried out,d the evolutionary analyses and the distance-based

phylogenetic trees were generated using MEGA5 (Tamuraet al., 2011). The evolutionary history was inferred using theNeighbor-Joining method. The percentage of replicate treesin which the associated taxa clustered together in thebootstrap test (1000 replicates) is shown next to thebranches. The tree is drawn to scale, with branch lengths inthe same units as those of the evolutionary distances used toinfer the phylogenetic tree. The evolutionary distances werecomputed using the Maximum Composite Likelihoodmethod and are in the units of the number of basesubstitutions per site.

The complete genome of A/wild boar/Brazil/214-11-13D/2011 (H1N2) was submitted to the GenBank withaccession numbers KF572613–KF572620.

3. Results

IAV detection by RT-PCR, qRRT-PCR and VI: Eleven out of60 lungs (18.3%) were positive for IAV by RT-PCR. Whenthese samples were tested by qRRT-PCR, seven out of the

. 1. Phylogenetic tree for the HA gene segment based on nucleotide sequences (1697 bp) from the A/wild boar/Brazil/214-11-13D/2011 analyzed in this

dy (indicated by black diamond) and other sequences available from GenBank. Open diamond = A/swine/Brazil/107-3A/2010 and A/swine/Brazil/12A/

10, representative of pandemic H1N1/2009 influenza viruses previously isolated from pigs in Brazil. Phylogenetic analyses were conducted in MEGA5.

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N. Biondo et al. / Veterinary Microbiology 168 (2014) 34–40 37

leven samples were positive to A(H1N1)pdm09 influenzairus with viral load ranging from 4.65 to 3863 copies/uL.either IAV was isolated on ECE nor was observed embryoeath. HA titers were negative.

Pathological examination: All 60 lung tissue samples hadross lesions of consolidation with a lung involvement of8.6%. The location of main lesion was at the cranium-entral position in all lobes. Consolidation affecting theentral portion of caudal lobes was also observed in 13ngs (21.6%) and extensive pleuritis in one lung (1.6%).V positive samples in RT-PCR (11/60) were submitted to

istological analyses. The microscopic lesions were char-cteristic of chronic diffuse bronchopneumonia (11/11),yperplasia of the bronchus-associated lymphoid tissueALT) (10/11), mild lymphohistioplasmocytic interstitial

neumonia (4/11) and chronic pleuritis (1/11).Immunohistochemistry: All eleven samples analyzed

y IHC were negative to IAV nucleoprotein antigen butositive to M. hyopneumoniae. The M. hyopneumoniae

ntigen was found in cilia, in addition to bronchial andronchiolar epithelium and exudates.

Bacteriology: P. multocida was isolated in two of the1 IAV positive samples.

Sequencing data analysis: All eight gene segments of/wild boar/Brazil/214-11-13D/2011 were completelyequenced with 704X average coverage (GenBank acces-ion numbers KF572613–KF572620). For the phylogeneticnalysis of the HA gene, we have included in the analysisVs representing the H1 clusters: a, b, g, d (Vincent et al.,

009) and A(H1N1)pdm09 cluster (Fig. 1). Influenzairuses representative of A(H1N1)pdm09 cluster isolated

pigs in Brazil (A/swine/Brazil/12A/2010 – JF421756.1nd A/swine/Brazil/107-3A/2010 – KF683614) and IAVsom wild boars from Italy (KC984944, H1 gene) andermany (AM746618.1, N2 gene) were also included in thenalysis. The phylogenetic analysis showed that A/wildoar/Brazil/214-11-13D/2011 is a novel reassortant H1N2V carrying genes derived from human H1N2, H3N2 and

(H1N1)pdm09 viruses (Table 1, Figs. 1 and 2). Inarticular, the analysis of the H1 gene of A/wild boar/razil/214-11-13D/2011 showed that it grouped with

identity) to seasonal human influenza viruses from 2002 to2003 (Fig. 1). In addition to that, the N2 gene isapproximately 96% identical at the nucleotide level toN2 of the seasonally human H3N2 viruses from the late1990s (1998–1999) (Fig. 2). The six remaining internalgenes (PB2, PB1, PA, NP, M and NS) were almost identical(99–100%) to A(H1N1)pdm09 lineage viruses (Table 1).

4. Discussion

This is the first report of a H1N2 influenza virusinfection in captive wild boars in Brazil. Although IAVinfection is endemic in commercial pig herds, outbreaks ofclinical swine influenza were only reported after theintroduction of A(H1N1)pdm09 influenza virus in pigs(Schaefer et al., 2011). Nowadays, there are threeIAV subtypes circulating in Brazilian pig herds,A(H1N1)pdm09, H3N2 and H1N2 viruses. Moreover, thereis little information about IAV infection in captive wildboars. Studies on the wild boars or feral pigs populationsare mainly based on serological surveys and antibodies toH1N1 and H3N2 have been detected in Spain (Vicenteet al., 2002), Slovenia (Vengust et al., 2006), Germany(Kaden et al., 2008), United States (Corn et al., 2009),France (Vittecoq et al., 2012) and Croatia (Roic et al., 2012).However, not much information is available about thegenetic composition of IAV from wild boars.

In our study, we performed the complete sequencing ofthe eight gene segments of IAV. The phylogenetic analysisof HA and NA genes of A/wild boar/214-11-13D/2011revealed a human-like H1N2 influenza virus, whereas thesix internal genes were derived from the A(H1N1)pdm09influenza virus. The human-like H1 virus was first detectedin pigs in Canada in 2004 (Karasin et al., 2006) and in theU.S in 2005 (Gramer, 2007). In recent years, the human-likeH1 or d-cluster viruses have been recognized as thedominant genotype in the U.S pigs (Lorusso et al., 2011).The analysis of the NA gene of A/wild boar/214-11-13D/2011 showed that the N2 gene belong to the influenzahuman lineage. According to Lorusso et al. (2011) virusesbelonging to the d-cluster were shown to be paired either

able 1

ost closely relatives to A/wild boar/Brazil/214-11-13D/2011 determined by BLAST search at NCBI (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).

Gene GenBank accession number Isolate Subtype Identity (%)

PB2 CY122668.1 A/Singapore/GP1132/2009 H1N1 99

CY122700.1 A/Singapore/GP1146/2009 H1N1 99

PB1 CY055303.1 A/Singapore/GN285/2009 H1N1 99

CY053625.1 A/Russia/165/2009 H1N1 99

PA KC833448.1 A/swine/Thailand/UD400/2009 H1N1 99

CY045233.2 A/Taiwan/126/2009 H1N1 99

HA CY125172.1 A/New York/26/2002 Mixed 97

CY003696.1 A/New York/489/2003 H1N2 97

NP CY045235.2 A/Taiwan/126/2009 H1N1 99

HQ728111.1 A/swine/Taiwan/CH-1204/2009 H1N1 99

NA AF533744.1 A/Neuquen/V690/98 H3N2 96

AF533741.1 A/Cordoba/V391/98 H3N2 96

M CY069644.1 A/Singapore/527/2009 H1N1 99

CY069629.1 A/Singapore/471/2009 H1N1 99

NS CY050371.1 A/Korea/S1/2009 H1N1 100

GU108490.1 A/Zhejiang-Yiwu/11/2009 H1N1 100

ith an N1 or an N2 gene of human lineage.

1-d cluster and are closely related (97% of nucleotide w

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N. Biondo et al. / Veterinary Microbiology 168 (2014) 34–4038

Since the influenza pandemics in 2009, the monitoring IAVs in pigs has increased worldwide and consequentlye availability of IAV gene sequences in GenBank. Thus,assortment between H1N1pdm09 virus and endemicVs was reported in pigs soon after pandemic strains

ergence in humans (Ducatez et al., 2011; Vijaykrishna al., 2010). Otherwise, there are few IAV nucleotidequences from wild boars in Genbank database. The twoailable IAV sequences from wild boars were detected in

Italy, 2012 (KC984944, H1N1 subtype) and in Germany,2006 (AM746617, H3N2 subtype) and they differ geneti-cally (75% identity with H1 gene; 86% identity with N2gene) from A/wild boar/214-11-13D/2011 (H1N2)described here (Figs. 1 and 2).

Additionally, in this study, we did not detect viableinfectious viral particles using the virus isolation on ECE, asdemonstrated by the absence of viral titers on allantoicfluids from infected ECE, as well as by the lack of influenza

. 2. Phylogenetic tree for the NA gene segment based on nucleotide sequences (1358 bp) from the A/wild boar/Brazil/214-11-13D/2011 analyzed in this

dy (indicated by black diamond) and other sequences available from GenBank. Phylogenetic analyses were conducted in MEGA5.

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N. Biondo et al. / Veterinary Microbiology 168 (2014) 34–40 39

irus antigen detection by IHC. According to Lange et al.009), viral excretion levels are higher during the acute

hase of the disease, during the first five to seven days postfection. Moreover, viral particles can be detected in high

oncentration in bronchus before the development of theng lesions (48–72 h post infection) (Howden et al., 2009;

ange et al., 2009). The lung samples analyzed in this studyere collected from slaughtered animals; possibly a longme after IAV exposure. This fact could explain theegative results in VI and IHC and the positive results inT-PCR and qRRT-PCR.

We have also analyzed bacterial pathogens associatedith IAV infection in wild boars. Gross lesions in IAV and. hyopneumoniae infections are similar. Then, additional

nalyses by histopathology and IHC are necessary for aefinitive diagnosis. The histological lesions observed inhe lungs of the wild boars were chronic bronchopneu-

onia and marked BALT hyperplasia. It suggests that theV infection occurred earlier and it could explain the

bsence of IAV antigen by IHC. However, BALT hyper-lasia is characteristic of M. hyopneumoniae infectionRedondo et al., 2009), which was confirmed by IHC.

oreover, different alveolar exudate is not characteristicf a single pathogen, but it can be detected in pneumoniaaused by distinct pathogens, including bacterial patho-ens such as M. hyopneumoniae and P. multocida and areelated to the evolution of the lesion. The association ofhese pathogens has been described in pigs previouslyHansen et al., 2010).

Southern Brazil is the most important region for swineroduction with the highest pig population in Brazil.razilian captive wild boars are commonly raised in semi-tensive farms. This system allows the circulation of theild boars to an open area, favoring the contact betweenild boars and other animal species as avian and feral pigs.

he nutrition management and vaccination schedule inaptive wild boar production are similar to the commercialog production system. However the vaccination scheduleoes not include immunization against influenza virus.oreover, the Brazilian swine producers are not included

the Seasonal Flu Vaccination priority group, where onlyeople over 60’s, health care workers, children, pregnantomen or people with chronic diseases get free vaccina-on. These conditions are favorable for virus infection/preading and possibly an interspecies transmissionorgie et al., 2011).

In summary, a reassortant human-like H1N2 swinefluenza virus as well as M. hyopneumoniae and P.

ultocida circulate in captive wild boar populations ine Southern region of Brazil. These pathogens are

ommonly found in respiratory infections in domesticigs causing significant economic losses following chronicfections (Hansen et al., 2010; Redondo et al., 2009).

revention and control of the transmission of influenzairuses, among captive wild boars, should be considered toinimize their impact on both production system and

ublic health.

ompeting interests

Acknowledgement

This work was supported by the Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPqProc.578102/2008-0 and Proc.578376/2008-3, Brazil).The authors acknowledge Marisete F. Schiochet, Neide L.Simon, Franciele Ianiski, Camila S. Rocha, Karine Takeuti,Alana Motta and Joao Xavier Oliveira-Filho for technicalassistance and Dr. Ricardo Zanella for critically reading themanuscript. Natalha Biondo has a scholarship from CAPES.

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The authors have no competing interests.

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