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Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus
Veterinary Microbiology 168 (2014) 34–40
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Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus Natalha Biondo a, Rejane Schaefer b,*, Danielle Gava b, Mauricio E. Canta˜o b, Simone Silveira b, Marcos A.Z. Mores b, Janice R. Ciacci-Zanella b, David E.S.N. Barcellos a a b
Federal University of Rio Grande do Sul/UFRGS, Porto Alegre, RS, Brazil Embrapa Swine and Poultry, Conco´rdia, SC, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 December 2012 Received in revised form 12 October 2013 Accepted 18 October 2013
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 realtime 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. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Captive wild boars Consolidation Influenza A virus H1N2 Reassortment
1. Introduction Swine influenza (SI) is an acute respiratory disease caused by influenza A virus (IAV) that affects different animal species, including humans. The IAVs are enveloped viruses, members of Orthomyxoviridae family virus and consist of a negative-sense, single-stranded segmented RNA genome. The influenza A viruses are classified into subtypes based on the antigenic properties of the surface
* Corresponding author at: BR153, Km 110, Vila Tamandua´, 89700-000, Conco´rdia, SC, Brazil. Tel.: +55 4934410423. E-mail addresses: [email protected], [email protected], [email protected] (R. Schaefer). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.10.010
glycoproteins hemagglutinin (HA) and neuraminidase (NA) (Van Reeth et al., 2012). Currently, seventeen different HAs and ten NAs have been described in the literature (Tong et al., 2012; Van Reeth et al., 2012). Swine have been implicated in the emergence of novel influenza viruses due to the dual expression of both sialic acid (alpha 2-3 and alpha 2-6 linkage) in the respiratory tract. When a single host cell is co-infected with two different influenza viruses, the exchange of viral RNA segments can occur, a process known as genetic reassortment, leading to the emergence of novel viruses (Van Reeth et al., 2012). Influenza is endemic in pig populations worldwide caused by genetically distinct lineages of H1N1, H3N2 and H1N2 IAV subtypes. In North America and in European countries, as well as in Asia, the circulating IAV in pigs are
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genetically different due to independent introductions and maintenance of avian, human and swine genomic lineages (Brown, 2000). In U.S, the ‘‘classical’’ H1N1 IAV (a cluster) was a major cause of swine influenza until the introduction of a H3N2 virus in 1998 (Vincent et al., 2008). Since then, there was an increase in the rate of genetic changes in swine influenza virus isolates in North America in H1 subtypes and different antigenic and genetic clusters began to evolve (Vincent et al., 2008). In Europe, three subtypes of influenza virus circulate in pigs as avian H1N1 (avian-like), the human H3N2 (human-like) and H1N2 viruses (Vincent et al., 2013). In Brazil, previous studies have detected the circulation of H1N1, H3N2 (Brentano et al., 2002; Rajao et al., 2013), 2009 pandemic H1N1 influenza virus (A(H1N1)pdm09) in commercial swine herds (Schaefer et al., 2011) and recently, a H1N2 IAV in commercial herds (Schaefer, unpublished data). In wild boars (Sus scrofa) and feral pigs, serological data from other countries showed the presence of antibodies to H3N2 and H1N1 IAV (Vicente et al., 2002) and reveals the absence of antibodies against H1N2 (Kaden et al., 2008). In Brazil, although the commercial swine herds are investigated for IAV infection through passive monitoring, the same does not occur in captive wild boars herds. Considering that wild boars are susceptible to IAV infection, the close contact with humans and possibly other animal species brings concern about the infection of captive wild boars with IAVs. Moreover, IAV is part of the porcine respiratory disease complex and, together with the Mycoplasma hyopneumoniae, are considered as primary pathogens in respiratory tract infections in swine, which can predispose to secondary infections usually caused by Pasteurella multocida. However, there are few studies about respiratory diseases in captive wild boars populations. Here we describe the histopathological, virological and bacteriological analyses of lungs from captive wild boars presenting pulmonary consolidation, suggestive of IAV infection. Moreover, the genomic sequencing and phylogenetic analysis of one virus sample revealed a human-like H1N2 IAV derived from the A(H1N1)pdm09 influenza virus. 2. Material and methods Sampling: In 2011, lung samples from captive wild boars (n = 60) presenting gross lesions of consolidation were harvested during the slaughter. All the animals used in this study were from two farms located in Rio Grande do Sul State, Southern region of Brazil. Farms had similar management (nutritional and vaccination schedule), following the requirements commonly applied to commercial swine herds in Brazil. Wild boars were slaughtered at seven months old with average weight of 42 kg. IAV detection: Viral RNA was extracted from lung tissue samples using MagMAXTM-96 Viral RNA Isolation Kit (AMB1836-5) according to the manufacturer’s instructions (Invitrogen1, Carlsbad, CA, USA). The resulting RNA was reverse-transcribed into cDNA using SuperScript II RT Kit (Invitrogen1, Carlsbad, CA, USA) and a complementary primer (Uni12) was designed to the conserved
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12 nucleotides of the 30 end of the viral RNA (Hoffmann et al., 2001). Matrix (M) gene amplification was performed using 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 M gene of A(H1N1)pdm09 virus by quantitative real-time RTPCR (qRRT-PCR), using primers and probe previously described (Lorusso et al., 2010). Virus isolation (VI) was carried out in nine-day-old Specific Pathogen Free (SPF) embryonated chicken eggs (ECE). Four days post inoculation, chorioallantoic fluids were collected and tested for the ability to agglutinate 0.5% suspensions of turkey red blood cells (hemagglutination test – HA) and for the presence of IAV nucleic acid by RTPCR (Fouchier et al., 2000). Pathological examination: Lung tissues were examined macroscopically and classified according to the lesions. The lungs affected area was quantified according to the methodology proposed by Madec and Kolbisch (1982). The affected lobes were fixed in 10% buffered formalin for histological analysis. Lung tissues were routinely processed and stained with hematoxilin and eosin (Luna et al., 1968). Immunohistochemistry (IHC): The formalin-fixed, paraffin embedded lung tissues were sectioned and processed for IHC. The influenza A and M. hyopneumoniae antigens were detected using a biotin-streptavidin-peroxidase kit (LSAB Kit + System HRP, Dako Carpinteria, CA, USA) employing, as primary antibodies, anti-influenza A nucleoprotein monoclonal antibody HB-65 (Vincent et al., 1997) and anti-lactate dehydrogenase protein (p36) (Ecco et al., 2009) for influenza A and M. hyopneumoniae antigen detection, respectively. Three-amino-9-ethyl-carbazole (AEC) was used as a chromogen in both IHC. Bacteriology: routine techniques applicable to the isolation of aerobic and microaerophilic pathogens were used and the biochemical characterization followed recommendations by Barrow and Feltham (1993). Genomic sequencing: Paired-end sequencing using Illumina Genome Analyzer was performed directly from the original sample. Viral RNA was extracted from 250 mL of lung homogenates using phenol-guanidine (TRIzol1, Ambion1, Carlsbad, CA, USA) and RNeasy Mini Kit (Qiagen1, Hilden, Germany), following the manufacturer’s instructions with minor modifications. The reverse transcription reaction and the amplification of the eight influenza virus gene segments were performed on 15 mL of RNA combined with 25 mL reaction mix, 1 mL of MBTUni12 and MBT-Uni13, 1 mL primer mix, 1 mL Superscript III Platinum kit (Invitrogen1, Carlsbad, CA, USA) and RNase/DNase free water (up to 50 mL). The primers were previously described by Hoffmann et al. (2001) and Zhou et 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 for 30 s, 68 8C for 7 min (for the amplification of PB2, PB1, PA and NS genes); and a final extension cycle of 68 8C for 5 min. The PCR products were purified using the QIAquick PCR purification kit (Qiagen1, Hilden, Germany). Preparation of DNA libraries: The amplified DNAs were tagged and fragmented by the Nextera XT transposome
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N. Biondo et al. / Veterinary Microbiology 168 (2014) 34–40
(http://supportres.illumina.com/documents/myillumina/ 9dc97885-3ae4-42f3-bee8 4d87fd72e2e3/nextera_xt_ sample_preparation_euc_15031943_c.pdf). After the amplification step via a limited-cycle PCR program, the PCR clean-up was performed with 25 mL of AMPure XP beads in order to capture longer library fragments for 2 250 bp MiSeq sequencing. Sequencing data analysis: The sequences generated from Illumina MiSeq were cleaned up for low quality reads and adapters using SeqyClean software V. 1.2.3 (https:// bitbucket.org/izhbannikov/seqyclean). All sequences with phred quality score < 30 and length < 220 bases were removed. The remaining sequences were compared against the influenza virus database from NCBI (http:// www.ncbi.nlm.nih.gov/genomes/FLU) to remove sequences that were not related to influenza viruses. After quality control steps, all sequences were assembled using the Newbler Assembler (Roche) V. 2.9. Multiple alignments of each influenza virus gene segments were carried out, and the evolutionary analyses and the distance-based
phylogenetic trees were generated using MEGA5 (Tamura et al., 2011). The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The complete genome of A/wild boar/Brazil/214-1113D/2011 (H1N2) was submitted to the GenBank with accession numbers KF572613–KF572620. 3. Results IAV detection by RT-PCR, qRRT-PCR and VI: Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR. When these samples were tested by qRRT-PCR, seven out of the
Fig. 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 study (indicated by black diamond) and other sequences available from GenBank. Open diamond = A/swine/Brazil/107-3A/2010 and A/swine/Brazil/12A/ 2010, representative of pandemic H1N1/2009 influenza viruses previously isolated from pigs in Brazil. Phylogenetic analyses were conducted in MEGA5.
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Table 1 Most 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 CY122700.1 CY055303.1 CY053625.1 KC833448.1 CY045233.2 CY125172.1 CY003696.1 CY045235.2 HQ728111.1 AF533744.1 AF533741.1 CY069644.1 CY069629.1 CY050371.1 GU108490.1
A/Singapore/GP1132/2009 A/Singapore/GP1146/2009 A/Singapore/GN285/2009 A/Russia/165/2009 A/swine/Thailand/UD400/2009 A/Taiwan/126/2009 A/New York/26/2002 A/New York/489/2003 A/Taiwan/126/2009 A/swine/Taiwan/CH-1204/2009 A/Neuquen/V690/98 A/Cordoba/V391/98 A/Singapore/527/2009 A/Singapore/471/2009 A/Korea/S1/2009 A/Zhejiang-Yiwu/11/2009
H1N1 H1N1 H1N1 H1N1 H1N1 H1N1 Mixed H1N2 H1N1 H1N1 H3N2 H3N2 H1N1 H1N1 H1N1 H1N1
99 99 99 99 99 99 97 97 99 99 96 96 99 99 100 100
PB1 PA HA NP NA M NS
eleven samples were positive to A(H1N1)pdm09 influenza virus with viral load ranging from 4.65 to 3863 copies/uL. Neither IAV was isolated on ECE nor was observed embryo death. HA titers were negative. Pathological examination: All 60 lung tissue samples had gross lesions of consolidation with a lung involvement of 28.6%. The location of main lesion was at the craniumventral position in all lobes. Consolidation affecting the ventral portion of caudal lobes was also observed in 13 lungs (21.6%) and extensive pleuritis in one lung (1.6%). IAV positive samples in RT-PCR (11/60) were submitted to histological analyses. The microscopic lesions were characteristic of chronic diffuse bronchopneumonia (11/11), hyperplasia of the bronchus-associated lymphoid tissue (BALT) (10/11), mild lymphohistioplasmocytic interstitial pneumonia (4/11) and chronic pleuritis (1/11). Immunohistochemistry: All eleven samples analyzed by IHC were negative to IAV nucleoprotein antigen but positive to M. hyopneumoniae. The M. hyopneumoniae antigen was found in cilia, in addition to bronchial and bronchiolar epithelium and exudates. Bacteriology: P. multocida was isolated in two of the 11 IAV positive samples. Sequencing data analysis: All eight gene segments of A/wild boar/Brazil/214-11-13D/2011 were completely sequenced with 704X average coverage (GenBank accession numbers KF572613–KF572620). For the phylogenetic analysis of the HA gene, we have included in the analysis IAVs representing the H1 clusters: a, b, g, d (Vincent et al., 2009) and A(H1N1)pdm09 cluster (Fig. 1). Influenza viruses representative of A(H1N1)pdm09 cluster isolated in pigs in Brazil (A/swine/Brazil/12A/2010 – JF421756.1 and A/swine/Brazil/107-3A/2010 – KF683614) and IAVs from wild boars from Italy (KC984944, H1 gene) and Germany (AM746618.1, N2 gene) were also included in the analysis. The phylogenetic analysis showed that A/wild boar/Brazil/214-11-13D/2011 is a novel reassortant H1N2 IAV carrying genes derived from human H1N2, H3N2 and A(H1N1)pdm09 viruses (Table 1, Figs. 1 and 2). In particular, the analysis of the H1 gene of A/wild boar/ Brazil/214-11-13D/2011 showed that it grouped with H1-d cluster and are closely related (97% of nucleotide
identity) to seasonal human influenza viruses from 2002 to 2003 (Fig. 1). In addition to that, the N2 gene is approximately 96% identical at the nucleotide level to N2 of the seasonally human H3N2 viruses from the late 1990s (1998–1999) (Fig. 2). The six remaining internal genes (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 virus infection in captive wild boars in Brazil. Although IAV infection is endemic in commercial pig herds, outbreaks of clinical swine influenza were only reported after the introduction of A(H1N1)pdm09 influenza virus in pigs (Schaefer et al., 2011). Nowadays, there are three IAV subtypes circulating in Brazilian pig herds, A(H1N1)pdm09, H3N2 and H1N2 viruses. Moreover, there is little information about IAV infection in captive wild boars. Studies on the wild boars or feral pigs populations are mainly based on serological surveys and antibodies to H1N1 and H3N2 have been detected in Spain (Vicente et 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 the genetic composition of IAV from wild boars. In our study, we performed the complete sequencing of the eight gene segments of IAV. The phylogenetic analysis of HA and NA genes of A/wild boar/214-11-13D/2011 revealed a human-like H1N2 influenza virus, whereas the six internal genes were derived from the A(H1N1)pdm09 influenza virus. The human-like H1 virus was first detected in pigs in Canada in 2004 (Karasin et al., 2006) and in the U.S in 2005 (Gramer, 2007). In recent years, the human-like H1 or d-cluster viruses have been recognized as the dominant 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 influenza human lineage. According to Lorusso et al. (2011) viruses belonging to the d-cluster were shown to be paired either with an N1 or an N2 gene of human lineage.
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Fig. 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 study (indicated by black diamond) and other sequences available from GenBank. Phylogenetic analyses were conducted in MEGA5.
Since the influenza pandemics in 2009, the monitoring of IAVs in pigs has increased worldwide and consequently the availability of IAV gene sequences in GenBank. Thus, reassortment between H1N1pdm09 virus and endemic SIVs was reported in pigs soon after pandemic strains emergence in humans (Ducatez et al., 2011; Vijaykrishna et al., 2010). Otherwise, there are few IAV nucleotide sequences from wild boars in Genbank database. The two available IAV sequences from wild boars were detected in
Italy, 2012 (KC984944, H1N1 subtype) and in Germany, 2006 (AM746617, H3N2 subtype) and they differ genetically (75% identity with H1 gene; 86% identity with N2 gene) from A/wild boar/214-11-13D/2011 (H1N2) described here (Figs. 1 and 2). Additionally, in this study, we did not detect viable infectious viral particles using the virus isolation on ECE, as demonstrated by the absence of viral titers on allantoic fluids from infected ECE, as well as by the lack of influenza
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virus antigen detection by IHC. According to Lange et al. (2009), viral excretion levels are higher during the acute phase of the disease, during the first five to seven days post infection. Moreover, viral particles can be detected in high concentration in bronchus before the development of the lung lesions (48–72 h post infection) (Howden et al., 2009; Lange et al., 2009). The lung samples analyzed in this study were collected from slaughtered animals; possibly a long time after IAV exposure. This fact could explain the negative results in VI and IHC and the positive results in RT-PCR and qRRT-PCR. We have also analyzed bacterial pathogens associated with IAV infection in wild boars. Gross lesions in IAV and M. hyopneumoniae infections are similar. Then, additional analyses by histopathology and IHC are necessary for a definitive diagnosis. The histological lesions observed in the lungs of the wild boars were chronic bronchopneumonia and marked BALT hyperplasia. It suggests that the IAV infection occurred earlier and it could explain the absence of IAV antigen by IHC. However, BALT hyperplasia is characteristic of M. hyopneumoniae infection (Redondo et al., 2009), which was confirmed by IHC. Moreover, different alveolar exudate is not characteristic of a single pathogen, but it can be detected in pneumonia caused by distinct pathogens, including bacterial pathogens such as M. hyopneumoniae and P. multocida and are related to the evolution of the lesion. The association of these pathogens has been described in pigs previously (Hansen et al., 2010). Southern Brazil is the most important region for swine production with the highest pig population in Brazil. Brazilian captive wild boars are commonly raised in semiintensive farms. This system allows the circulation of the wild boars to an open area, favoring the contact between wild boars and other animal species as avian and feral pigs. The nutrition management and vaccination schedule in captive wild boar production are similar to the commercial hog production system. However the vaccination schedule does not include immunization against influenza virus. Moreover, the Brazilian swine producers are not included in the Seasonal Flu Vaccination priority group, where only people over 60’s, health care workers, children, pregnant women or people with chronic diseases get free vaccination. These conditions are favorable for virus infection/ spreading and possibly an interspecies transmission (Forgie et al., 2011). In summary, a reassortant human-like H1N2 swine influenza virus as well as M. hyopneumoniae and P. multocida circulate in captive wild boar populations in the Southern region of Brazil. These pathogens are commonly found in respiratory infections in domestic pigs causing significant economic losses following chronic infections (Hansen et al., 2010; Redondo et al., 2009). Prevention and control of the transmission of influenza viruses, among captive wild boars, should be considered to minimize their impact on both production system and public health. Competing interests The authors have no competing interests.
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Acknowledgement This work was supported by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq Proc.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 Joa˜o Xavier Oliveira-Filho for technical assistance and Dr. Ricardo Zanella for critically reading the manuscript. Natalha Biondo has a scholarship from CAPES. References Barrow, G.I., Feltham, R.K.A., 1993. Cowan and Steel’s Manual for The Identification of Medical Bacteria, 3rd ed. Cambridge University Press, Cambridge; New York. Brentano, L., Zanella, J.R.C., Mores, N., Piffer, I.A., 2002. Levantamento soroepidemiolo´gico para coronavı´rus respirato´rio e da gastroenterite transmissı´vel e dos vı´rus de influenza H3N2 e H1N1 em rebanhos suı´nos no Brasil. In: In Comunicado te´cnico (Conco´rdia, Embrapa Suı´nos e Aves). pp. 1–6. Brown, I.H., 2000. The epidemiology and evolution of influenza viruses in pigs. Vet. Microbiol. 74, 29–46. Corn, J.L., Cumbee, J.C., Barfoot, R., Erickson, G.A., 2009. Pathogen exposure in feral swine populations geographically associated with high densities of transitional swine premises and commercial swine production. J. Wildl. Dis. 45, 713–721. 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. Multiple reassortment between pandemic (H1N1) 2009 and endemic influenza viruses in pigs, United States. Emerg. Infect. Dis. 17, 1624–1629. Ecco, R., Lazzari, A.M., Guedes, R.M.C., 2009. Pneumonia enzoo´tica em javalis (Sus scrofa). Pesquisa. Vet. Brasil. 29, 461–468. Forgie, S.E., Keenliside, J., Wilkinson, C., Webby, R., Lu, P., Sorensen, O., Fonseca, K., Barman, S., Rubrum, A., Stigger, E., Marrie, T.J., Marshall, F., Spady, D.W., Hu, J., Loeb, M., Russell, M.L., Babiuk, L.A., 2011. Swine outbreak of pandemic influenza A virus on a Canadian research farm supports human-to-swine transmission. Clin. Infect. Dis. 52, 10–18. Fouchier, R.A., Bestebroer, T.M., Herfst, S., Van Der Kemp, L., Rimmelzwaan, G.F., Osterhaus, A.D., 2000. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J. Clin. Microbiol. 38, 4096–4101. Gramer, M., 2007. SIV: an update on circulating strains, advances in diagnostic tests and interpretation of test results. In: In: 38th Annual Meeting of the American Association of swine Veterinarians, Orlando, FL. Hansen, M.S., Pors, S.E., Jensen, H.E., Bille-Hansen, V., Bisgaard, M., Flachs, E.M., Nielsen, O.L., 2010. An investigation of the pathology and pathogens associated with porcine respiratory disease complex in Denmark. J. Comp. Pathol. 143, 120–131. Hoffmann, E., Stech, J., Guan, Y., Webster, R.G., Perez, D.R., 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146, 2275–2289. Howden, K.J., Brockhoff, E.J., Caya, F.D., McLeod, L.J., Lavoie, M., Ing, J.D., Bystrom, J.M., Alexandersen, S., Pasick, J.M., Berhane, Y., Morrison, M.E., Keenliside, J.M., Laurendeau, S., Rohonczy, E.B., 2009. An investigation into human pandemic influenza virus (H1N1) 2009 on an Alberta swine farm. Can. Vet. J. 50, 1153–1161. Kaden, V., Lange, E., Starick, E., Bruer, W., Krakowski, W., Klopries, M., 2008. Epidemiological survey of swine influenza A virus in selected wild boar populations in Germany. Vet. Microbiol. 131, 123–132. Karasin, A.I., Carman, S., Olsen, C.W., 2006. Identification of human H1N2 and human-swine reassortant H1N2 and H1N1 influenza A viruses among pigs in Ontario, Canada (2003–2005). J. Clin. Microbiol. 44, 1123–1126. Lange, E., Kalthoff, D., Blohm, U., Teifke, J.P., Breithaupt, A., Maresch, C., Starick, E., Fereidouni, S., Hoffmann, B., Mettenleiter, T.C., Beer, M., Vahlenkamp, T.W., 2009. Pathogenesis and transmission of the novel swine-origin influenza virus A/H1N1 after experimental infection of pigs. J. Gen. Virol. 90, 2119–2123. Lorusso, A., Faaberg, K.S., Killian, M.L., Koster, L., Vincent, A.L., 2010. Onestep real-time RT-PCR for pandemic influenza A virus (H1N1) 2009 matrix gene detection in swine samples. J. Virol. Meth. 164, 83–87. Lorusso, A., Vincent, A.L., Harland, M.L., Alt, D., Bayles, D.O., Swenson, S.L., Gramer, M.R., Russell, C.A., Smith, D.J., Lager, K.M., Lewis, N.S., 2011.
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Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus Natalha Biondo a, Rejane Schaefer b,*, Danielle Gava b, Mauricio E. Canta˜o b, Simone Silveira b, Marcos A.Z. Mores b, Janice R. Ciacci-Zanella b, David E.S.N. Barcellos a a b
Federal University of Rio Grande do Sul/UFRGS, Porto Alegre, RS, Brazil Embrapa Swine and Poultry, Conco´rdia, SC, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 December 2012 Received in revised form 12 October 2013 Accepted 18 October 2013
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 realtime 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. ß 2013 Elsevier B.V. All rights reserved.
Keywords: Captive wild boars Consolidation Influenza A virus H1N2 Reassortment
1. Introduction Swine influenza (SI) is an acute respiratory disease caused by influenza A virus (IAV) that affects different animal species, including humans. The IAVs are enveloped viruses, members of Orthomyxoviridae family virus and consist of a negative-sense, single-stranded segmented RNA genome. The influenza A viruses are classified into subtypes based on the antigenic properties of the surface
* Corresponding author at: BR153, Km 110, Vila Tamandua´, 89700-000, Conco´rdia, SC, Brazil. Tel.: +55 4934410423. E-mail addresses: [email protected], [email protected], [email protected] (R. Schaefer). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.10.010
glycoproteins hemagglutinin (HA) and neuraminidase (NA) (Van Reeth et al., 2012). Currently, seventeen different HAs and ten NAs have been described in the literature (Tong et al., 2012; Van Reeth et al., 2012). Swine have been implicated in the emergence of novel influenza viruses due to the dual expression of both sialic acid (alpha 2-3 and alpha 2-6 linkage) in the respiratory tract. When a single host cell is co-infected with two different influenza viruses, the exchange of viral RNA segments can occur, a process known as genetic reassortment, leading to the emergence of novel viruses (Van Reeth et al., 2012). Influenza is endemic in pig populations worldwide caused by genetically distinct lineages of H1N1, H3N2 and H1N2 IAV subtypes. In North America and in European countries, as well as in Asia, the circulating IAV in pigs are
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genetically different due to independent introductions and maintenance of avian, human and swine genomic lineages (Brown, 2000). In U.S, the ‘‘classical’’ H1N1 IAV (a cluster) was a major cause of swine influenza until the introduction of a H3N2 virus in 1998 (Vincent et al., 2008). Since then, there was an increase in the rate of genetic changes in swine influenza virus isolates in North America in H1 subtypes and different antigenic and genetic clusters began to evolve (Vincent et al., 2008). In Europe, three subtypes of influenza virus circulate in pigs as avian H1N1 (avian-like), the human H3N2 (human-like) and H1N2 viruses (Vincent et al., 2013). In Brazil, previous studies have detected the circulation of H1N1, H3N2 (Brentano et al., 2002; Rajao et al., 2013), 2009 pandemic H1N1 influenza virus (A(H1N1)pdm09) in commercial swine herds (Schaefer et al., 2011) and recently, a H1N2 IAV in commercial herds (Schaefer, unpublished data). In wild boars (Sus scrofa) and feral pigs, serological data from other countries showed the presence of antibodies to H3N2 and H1N1 IAV (Vicente et al., 2002) and reveals the absence of antibodies against H1N2 (Kaden et al., 2008). In Brazil, although the commercial swine herds are investigated for IAV infection through passive monitoring, the same does not occur in captive wild boars herds. Considering that wild boars are susceptible to IAV infection, the close contact with humans and possibly other animal species brings concern about the infection of captive wild boars with IAVs. Moreover, IAV is part of the porcine respiratory disease complex and, together with the Mycoplasma hyopneumoniae, are considered as primary pathogens in respiratory tract infections in swine, which can predispose to secondary infections usually caused by Pasteurella multocida. However, there are few studies about respiratory diseases in captive wild boars populations. Here we describe the histopathological, virological and bacteriological analyses of lungs from captive wild boars presenting pulmonary consolidation, suggestive of IAV infection. Moreover, the genomic sequencing and phylogenetic analysis of one virus sample revealed a human-like H1N2 IAV derived from the A(H1N1)pdm09 influenza virus. 2. Material and methods Sampling: In 2011, lung samples from captive wild boars (n = 60) presenting gross lesions of consolidation were harvested during the slaughter. All the animals used in this study were from two farms located in Rio Grande do Sul State, Southern region of Brazil. Farms had similar management (nutritional and vaccination schedule), following the requirements commonly applied to commercial swine herds in Brazil. Wild boars were slaughtered at seven months old with average weight of 42 kg. IAV detection: Viral RNA was extracted from lung tissue samples using MagMAXTM-96 Viral RNA Isolation Kit (AMB1836-5) according to the manufacturer’s instructions (Invitrogen1, Carlsbad, CA, USA). The resulting RNA was reverse-transcribed into cDNA using SuperScript II RT Kit (Invitrogen1, Carlsbad, CA, USA) and a complementary primer (Uni12) was designed to the conserved
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12 nucleotides of the 30 end of the viral RNA (Hoffmann et al., 2001). Matrix (M) gene amplification was performed using 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 M gene of A(H1N1)pdm09 virus by quantitative real-time RTPCR (qRRT-PCR), using primers and probe previously described (Lorusso et al., 2010). Virus isolation (VI) was carried out in nine-day-old Specific Pathogen Free (SPF) embryonated chicken eggs (ECE). Four days post inoculation, chorioallantoic fluids were collected and tested for the ability to agglutinate 0.5% suspensions of turkey red blood cells (hemagglutination test – HA) and for the presence of IAV nucleic acid by RTPCR (Fouchier et al., 2000). Pathological examination: Lung tissues were examined macroscopically and classified according to the lesions. The lungs affected area was quantified according to the methodology proposed by Madec and Kolbisch (1982). The affected lobes were fixed in 10% buffered formalin for histological analysis. Lung tissues were routinely processed and stained with hematoxilin and eosin (Luna et al., 1968). Immunohistochemistry (IHC): The formalin-fixed, paraffin embedded lung tissues were sectioned and processed for IHC. The influenza A and M. hyopneumoniae antigens were detected using a biotin-streptavidin-peroxidase kit (LSAB Kit + System HRP, Dako Carpinteria, CA, USA) employing, as primary antibodies, anti-influenza A nucleoprotein monoclonal antibody HB-65 (Vincent et al., 1997) and anti-lactate dehydrogenase protein (p36) (Ecco et al., 2009) for influenza A and M. hyopneumoniae antigen detection, respectively. Three-amino-9-ethyl-carbazole (AEC) was used as a chromogen in both IHC. Bacteriology: routine techniques applicable to the isolation of aerobic and microaerophilic pathogens were used and the biochemical characterization followed recommendations by Barrow and Feltham (1993). Genomic sequencing: Paired-end sequencing using Illumina Genome Analyzer was performed directly from the original sample. Viral RNA was extracted from 250 mL of lung homogenates using phenol-guanidine (TRIzol1, Ambion1, Carlsbad, CA, USA) and RNeasy Mini Kit (Qiagen1, Hilden, Germany), following the manufacturer’s instructions with minor modifications. The reverse transcription reaction and the amplification of the eight influenza virus gene segments were performed on 15 mL of RNA combined with 25 mL reaction mix, 1 mL of MBTUni12 and MBT-Uni13, 1 mL primer mix, 1 mL Superscript III Platinum kit (Invitrogen1, Carlsbad, CA, USA) and RNase/DNase free water (up to 50 mL). The primers were previously described by Hoffmann et al. (2001) and Zhou et 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 for 30 s, 68 8C for 7 min (for the amplification of PB2, PB1, PA and NS genes); and a final extension cycle of 68 8C for 5 min. The PCR products were purified using the QIAquick PCR purification kit (Qiagen1, Hilden, Germany). Preparation of DNA libraries: The amplified DNAs were tagged and fragmented by the Nextera XT transposome
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(http://supportres.illumina.com/documents/myillumina/ 9dc97885-3ae4-42f3-bee8 4d87fd72e2e3/nextera_xt_ sample_preparation_euc_15031943_c.pdf). After the amplification step via a limited-cycle PCR program, the PCR clean-up was performed with 25 mL of AMPure XP beads in order to capture longer library fragments for 2 250 bp MiSeq sequencing. Sequencing data analysis: The sequences generated from Illumina MiSeq were cleaned up for low quality reads and adapters using SeqyClean software V. 1.2.3 (https:// bitbucket.org/izhbannikov/seqyclean). All sequences with phred quality score < 30 and length < 220 bases were removed. The remaining sequences were compared against the influenza virus database from NCBI (http:// www.ncbi.nlm.nih.gov/genomes/FLU) to remove sequences that were not related to influenza viruses. After quality control steps, all sequences were assembled using the Newbler Assembler (Roche) V. 2.9. Multiple alignments of each influenza virus gene segments were carried out, and the evolutionary analyses and the distance-based
phylogenetic trees were generated using MEGA5 (Tamura et al., 2011). The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The complete genome of A/wild boar/Brazil/214-1113D/2011 (H1N2) was submitted to the GenBank with accession numbers KF572613–KF572620. 3. Results IAV detection by RT-PCR, qRRT-PCR and VI: Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR. When these samples were tested by qRRT-PCR, seven out of the
Fig. 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 study (indicated by black diamond) and other sequences available from GenBank. Open diamond = A/swine/Brazil/107-3A/2010 and A/swine/Brazil/12A/ 2010, representative of pandemic H1N1/2009 influenza viruses previously isolated from pigs in Brazil. Phylogenetic analyses were conducted in MEGA5.
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Table 1 Most 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 CY122700.1 CY055303.1 CY053625.1 KC833448.1 CY045233.2 CY125172.1 CY003696.1 CY045235.2 HQ728111.1 AF533744.1 AF533741.1 CY069644.1 CY069629.1 CY050371.1 GU108490.1
A/Singapore/GP1132/2009 A/Singapore/GP1146/2009 A/Singapore/GN285/2009 A/Russia/165/2009 A/swine/Thailand/UD400/2009 A/Taiwan/126/2009 A/New York/26/2002 A/New York/489/2003 A/Taiwan/126/2009 A/swine/Taiwan/CH-1204/2009 A/Neuquen/V690/98 A/Cordoba/V391/98 A/Singapore/527/2009 A/Singapore/471/2009 A/Korea/S1/2009 A/Zhejiang-Yiwu/11/2009
H1N1 H1N1 H1N1 H1N1 H1N1 H1N1 Mixed H1N2 H1N1 H1N1 H3N2 H3N2 H1N1 H1N1 H1N1 H1N1
99 99 99 99 99 99 97 97 99 99 96 96 99 99 100 100
PB1 PA HA NP NA M NS
eleven samples were positive to A(H1N1)pdm09 influenza virus with viral load ranging from 4.65 to 3863 copies/uL. Neither IAV was isolated on ECE nor was observed embryo death. HA titers were negative. Pathological examination: All 60 lung tissue samples had gross lesions of consolidation with a lung involvement of 28.6%. The location of main lesion was at the craniumventral position in all lobes. Consolidation affecting the ventral portion of caudal lobes was also observed in 13 lungs (21.6%) and extensive pleuritis in one lung (1.6%). IAV positive samples in RT-PCR (11/60) were submitted to histological analyses. The microscopic lesions were characteristic of chronic diffuse bronchopneumonia (11/11), hyperplasia of the bronchus-associated lymphoid tissue (BALT) (10/11), mild lymphohistioplasmocytic interstitial pneumonia (4/11) and chronic pleuritis (1/11). Immunohistochemistry: All eleven samples analyzed by IHC were negative to IAV nucleoprotein antigen but positive to M. hyopneumoniae. The M. hyopneumoniae antigen was found in cilia, in addition to bronchial and bronchiolar epithelium and exudates. Bacteriology: P. multocida was isolated in two of the 11 IAV positive samples. Sequencing data analysis: All eight gene segments of A/wild boar/Brazil/214-11-13D/2011 were completely sequenced with 704X average coverage (GenBank accession numbers KF572613–KF572620). For the phylogenetic analysis of the HA gene, we have included in the analysis IAVs representing the H1 clusters: a, b, g, d (Vincent et al., 2009) and A(H1N1)pdm09 cluster (Fig. 1). Influenza viruses representative of A(H1N1)pdm09 cluster isolated in pigs in Brazil (A/swine/Brazil/12A/2010 – JF421756.1 and A/swine/Brazil/107-3A/2010 – KF683614) and IAVs from wild boars from Italy (KC984944, H1 gene) and Germany (AM746618.1, N2 gene) were also included in the analysis. The phylogenetic analysis showed that A/wild boar/Brazil/214-11-13D/2011 is a novel reassortant H1N2 IAV carrying genes derived from human H1N2, H3N2 and A(H1N1)pdm09 viruses (Table 1, Figs. 1 and 2). In particular, the analysis of the H1 gene of A/wild boar/ Brazil/214-11-13D/2011 showed that it grouped with H1-d cluster and are closely related (97% of nucleotide
identity) to seasonal human influenza viruses from 2002 to 2003 (Fig. 1). In addition to that, the N2 gene is approximately 96% identical at the nucleotide level to N2 of the seasonally human H3N2 viruses from the late 1990s (1998–1999) (Fig. 2). The six remaining internal genes (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 virus infection in captive wild boars in Brazil. Although IAV infection is endemic in commercial pig herds, outbreaks of clinical swine influenza were only reported after the introduction of A(H1N1)pdm09 influenza virus in pigs (Schaefer et al., 2011). Nowadays, there are three IAV subtypes circulating in Brazilian pig herds, A(H1N1)pdm09, H3N2 and H1N2 viruses. Moreover, there is little information about IAV infection in captive wild boars. Studies on the wild boars or feral pigs populations are mainly based on serological surveys and antibodies to H1N1 and H3N2 have been detected in Spain (Vicente et 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 the genetic composition of IAV from wild boars. In our study, we performed the complete sequencing of the eight gene segments of IAV. The phylogenetic analysis of HA and NA genes of A/wild boar/214-11-13D/2011 revealed a human-like H1N2 influenza virus, whereas the six internal genes were derived from the A(H1N1)pdm09 influenza virus. The human-like H1 virus was first detected in pigs in Canada in 2004 (Karasin et al., 2006) and in the U.S in 2005 (Gramer, 2007). In recent years, the human-like H1 or d-cluster viruses have been recognized as the dominant 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 influenza human lineage. According to Lorusso et al. (2011) viruses belonging to the d-cluster were shown to be paired either with an N1 or an N2 gene of human lineage.
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Fig. 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 study (indicated by black diamond) and other sequences available from GenBank. Phylogenetic analyses were conducted in MEGA5.
Since the influenza pandemics in 2009, the monitoring of IAVs in pigs has increased worldwide and consequently the availability of IAV gene sequences in GenBank. Thus, reassortment between H1N1pdm09 virus and endemic SIVs was reported in pigs soon after pandemic strains emergence in humans (Ducatez et al., 2011; Vijaykrishna et al., 2010). Otherwise, there are few IAV nucleotide sequences from wild boars in Genbank database. The two available IAV sequences from wild boars were detected in
Italy, 2012 (KC984944, H1N1 subtype) and in Germany, 2006 (AM746617, H3N2 subtype) and they differ genetically (75% identity with H1 gene; 86% identity with N2 gene) from A/wild boar/214-11-13D/2011 (H1N2) described here (Figs. 1 and 2). Additionally, in this study, we did not detect viable infectious viral particles using the virus isolation on ECE, as demonstrated by the absence of viral titers on allantoic fluids from infected ECE, as well as by the lack of influenza
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virus antigen detection by IHC. According to Lange et al. (2009), viral excretion levels are higher during the acute phase of the disease, during the first five to seven days post infection. Moreover, viral particles can be detected in high concentration in bronchus before the development of the lung lesions (48–72 h post infection) (Howden et al., 2009; Lange et al., 2009). The lung samples analyzed in this study were collected from slaughtered animals; possibly a long time after IAV exposure. This fact could explain the negative results in VI and IHC and the positive results in RT-PCR and qRRT-PCR. We have also analyzed bacterial pathogens associated with IAV infection in wild boars. Gross lesions in IAV and M. hyopneumoniae infections are similar. Then, additional analyses by histopathology and IHC are necessary for a definitive diagnosis. The histological lesions observed in the lungs of the wild boars were chronic bronchopneumonia and marked BALT hyperplasia. It suggests that the IAV infection occurred earlier and it could explain the absence of IAV antigen by IHC. However, BALT hyperplasia is characteristic of M. hyopneumoniae infection (Redondo et al., 2009), which was confirmed by IHC. Moreover, different alveolar exudate is not characteristic of a single pathogen, but it can be detected in pneumonia caused by distinct pathogens, including bacterial pathogens such as M. hyopneumoniae and P. multocida and are related to the evolution of the lesion. The association of these pathogens has been described in pigs previously (Hansen et al., 2010). Southern Brazil is the most important region for swine production with the highest pig population in Brazil. Brazilian captive wild boars are commonly raised in semiintensive farms. This system allows the circulation of the wild boars to an open area, favoring the contact between wild boars and other animal species as avian and feral pigs. The nutrition management and vaccination schedule in captive wild boar production are similar to the commercial hog production system. However the vaccination schedule does not include immunization against influenza virus. Moreover, the Brazilian swine producers are not included in the Seasonal Flu Vaccination priority group, where only people over 60’s, health care workers, children, pregnant women or people with chronic diseases get free vaccination. These conditions are favorable for virus infection/ spreading and possibly an interspecies transmission (Forgie et al., 2011). In summary, a reassortant human-like H1N2 swine influenza virus as well as M. hyopneumoniae and P. multocida circulate in captive wild boar populations in the Southern region of Brazil. These pathogens are commonly found in respiratory infections in domestic pigs causing significant economic losses following chronic infections (Hansen et al., 2010; Redondo et al., 2009). Prevention and control of the transmission of influenza viruses, among captive wild boars, should be considered to minimize their impact on both production system and public health. Competing interests The authors have no competing interests.
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Acknowledgement This work was supported by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq Proc.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 Joa˜o Xavier Oliveira-Filho for technical assistance and Dr. Ricardo Zanella for critically reading the manuscript. Natalha Biondo has a scholarship from CAPES. References Barrow, G.I., Feltham, R.K.A., 1993. Cowan and Steel’s Manual for The Identification of Medical Bacteria, 3rd ed. Cambridge University Press, Cambridge; New York. Brentano, L., Zanella, J.R.C., Mores, N., Piffer, I.A., 2002. Levantamento soroepidemiolo´gico para coronavı´rus respirato´rio e da gastroenterite transmissı´vel e dos vı´rus de influenza H3N2 e H1N1 em rebanhos suı´nos no Brasil. In: In Comunicado te´cnico (Conco´rdia, Embrapa Suı´nos e Aves). pp. 1–6. Brown, I.H., 2000. The epidemiology and evolution of influenza viruses in pigs. Vet. Microbiol. 74, 29–46. Corn, J.L., Cumbee, J.C., Barfoot, R., Erickson, G.A., 2009. Pathogen exposure in feral swine populations geographically associated with high densities of transitional swine premises and commercial swine production. J. Wildl. Dis. 45, 713–721. 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. Multiple reassortment between pandemic (H1N1) 2009 and endemic influenza viruses in pigs, United States. Emerg. Infect. Dis. 17, 1624–1629. Ecco, R., Lazzari, A.M., Guedes, R.M.C., 2009. Pneumonia enzoo´tica em javalis (Sus scrofa). Pesquisa. Vet. Brasil. 29, 461–468. Forgie, S.E., Keenliside, J., Wilkinson, C., Webby, R., Lu, P., Sorensen, O., Fonseca, K., Barman, S., Rubrum, A., Stigger, E., Marrie, T.J., Marshall, F., Spady, D.W., Hu, J., Loeb, M., Russell, M.L., Babiuk, L.A., 2011. Swine outbreak of pandemic influenza A virus on a Canadian research farm supports human-to-swine transmission. Clin. Infect. Dis. 52, 10–18. Fouchier, R.A., Bestebroer, T.M., Herfst, S., Van Der Kemp, L., Rimmelzwaan, G.F., Osterhaus, A.D., 2000. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J. Clin. Microbiol. 38, 4096–4101. Gramer, M., 2007. SIV: an update on circulating strains, advances in diagnostic tests and interpretation of test results. In: In: 38th Annual Meeting of the American Association of swine Veterinarians, Orlando, FL. Hansen, M.S., Pors, S.E., Jensen, H.E., Bille-Hansen, V., Bisgaard, M., Flachs, E.M., Nielsen, O.L., 2010. An investigation of the pathology and pathogens associated with porcine respiratory disease complex in Denmark. J. Comp. Pathol. 143, 120–131. Hoffmann, E., Stech, J., Guan, Y., Webster, R.G., Perez, D.R., 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146, 2275–2289. Howden, K.J., Brockhoff, E.J., Caya, F.D., McLeod, L.J., Lavoie, M., Ing, J.D., Bystrom, J.M., Alexandersen, S., Pasick, J.M., Berhane, Y., Morrison, M.E., Keenliside, J.M., Laurendeau, S., Rohonczy, E.B., 2009. An investigation into human pandemic influenza virus (H1N1) 2009 on an Alberta swine farm. Can. Vet. J. 50, 1153–1161. Kaden, V., Lange, E., Starick, E., Bruer, W., Krakowski, W., Klopries, M., 2008. Epidemiological survey of swine influenza A virus in selected wild boar populations in Germany. Vet. Microbiol. 131, 123–132. Karasin, A.I., Carman, S., Olsen, C.W., 2006. Identification of human H1N2 and human-swine reassortant H1N2 and H1N1 influenza A viruses among pigs in Ontario, Canada (2003–2005). J. Clin. Microbiol. 44, 1123–1126. Lange, E., Kalthoff, D., Blohm, U., Teifke, J.P., Breithaupt, A., Maresch, C., Starick, E., Fereidouni, S., Hoffmann, B., Mettenleiter, T.C., Beer, M., Vahlenkamp, T.W., 2009. Pathogenesis and transmission of the novel swine-origin influenza virus A/H1N1 after experimental infection of pigs. J. Gen. Virol. 90, 2119–2123. Lorusso, A., Faaberg, K.S., Killian, M.L., Koster, L., Vincent, A.L., 2010. Onestep real-time RT-PCR for pandemic influenza A virus (H1N1) 2009 matrix gene detection in swine samples. J. Virol. Meth. 164, 83–87. Lorusso, A., Vincent, A.L., Harland, M.L., Alt, D., Bayles, D.O., Swenson, S.L., Gramer, M.R., Russell, C.A., Smith, D.J., Lager, K.M., Lewis, N.S., 2011.
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