INFLUENZA IN SWINE HERDS FOLLOWING THE INTRODUCTION OF PANDEMIC 2009 H1N1
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1 INFLUENZA IN SWINE HERDS FOLLOWING THE INTRODUCTION OF PANDEMIC 2009 H1N1 Janice Reis Ciacci Zanella PAHO Webinar December 18, 2015
2 INFLUENZA IN SWINE IN BRAZIL Janice Reis Ciacci Zanella Brazilian Agricultural Research Corporation EMBRAPA Embrapa Swine and Poultry Research Center
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5 EMBRAPA Swine and Poultry (176 ha): 211 employees Animal Health laboratory
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7 The big challenge km inhabitants 27 States cities pigs 22 million pigs slaughter / year pig farms Official Veterinarians 180 countries import Brazilian pork
8 Distribution per Region
9 Influenza in swine in Brazil»Since 2009: frequent outbreaks of H1N1pdm associated with respiratory illness (first outbreaks observed in different ages; more recent outbreaks mainly observed in growing pigs).»since 2011: human-like H1N2 influenza virus detected in swine and in captive wild pigs in four Brazilian states.»h3n2 IAV detected in pigs in five Brazilian states.»h1n2 and H3N2 viruses have the internal gene segments of H1N1pdm origin.
10 CURRENT AND RETROSPECTIVE SEROLOGY STUDY OF INFLUENZA A VIRUSES ANTIBODIES IN BRAZILIAN PIG POPULATIONS Janice Reis Ciacci Zanella, Rejane Schaefer, Marisete Fracasso Schiochet, Simone Silveira, Luizinho Caron, Ubiratan Piovezan 100% 80% 60% 40% 20% Log 2 Titer 0% / / / /2007 HI - H1N1 Swine Herd ID/ Year 176/09/ /09/ b/10/ /10/ c/10/ /10/2010 Log 2 Titer Log 2 Titer / / / / / /2007 HI - H3N2 Swine Herd ID/ Year HI - ph1n / / /09/ /09/2009 Swine Herd ID/ Year 176/09/ /09/ b/10/ /10/ c/10/ /10/ b/10/ /10/ c/10/ /10/ commercial farms and 01 feral swine herd (176/09/2009) from Pantanal wetland in Brazil. 359 serum samples collected between HI assays: classic H1N1- A/sw/IA/31(AAF6/19/92) or H1N1, H3N2-A/sw/IA/ or H3N2, both purchased from NVSL-ARS- USDA and ph1n1/107b/10-3a (H1N1) or ph1n1 isolated from Embrapa. Lack of specific antibodies to the ph1n1, which suggests Brazilian pigs were not fully protected against the ph1n1 from previous exposure. Besides commercial swine herds, feral swine population (176/09/2009) resulted positive to IAV antibodies by Elisa (5/31) and HI.
11 Project funded by CNPq/MAPA: Diagnostic, molecular characterization and pathogenesis studies of infectious agents economically important for the Brazilian Swine Production. 02/ /2012 Comercial farms 2009 CNPq Project 2009 to 2012 Comercial farms * Quarantine Boars Captive wild pig Monteiro Feral Pigs Diagnostic Lab - CEDISA Plasma, OF 1 protocol 1000 plasma samples 257 OF samples 51 farms Serum, NS, OF 16 farms 87 pools 435 samples Serum, SN, FO 49 farms 288 pools 1440 samples Serum, OF 1 protocol 52 OF samples 471 serum samples Lungs, NS 2 protocols 60 lung samples Serum, NS, GS 2 protocols 152 samples Lungs 86 protocols Nasal Swab (NS) Serum Oral Fluid (OF) Lung tissue qrt-pcr IAV ELISA SIV qrt-pcr IAV Subtyping H1, H3, N1, N2 Genome Sequencing Virus Isolation Histopathology - IHC HI *Comercial farms : 8 (PR), 7 (RS), 3 (SP), 11 (SC), 10 (MG), 5 (MS) e 5 (MT)= 49 Total: 62 farms 11
12 Serological analysis A. Sera collected from all 48 commercial farms were positive by NP- ELISA test. B. E. Specific antibodies to different subtypes
13 1881 nasal swabs and 89 lung tissue samples were collected from swine in the southern, midwest and southeast regions of Brazil ( ); RNA extraction, RT-qPCR (M gene of IAV and H1N1pdm); Virus isolation (MDCK cells or ECE); Genetic sequencing (ABI 3130xl and Illumina MiSeq). Brasil
14 Results Assay Nasal swabs Lung RT-qPCR/ IAV 59/1881 (3.3%) 58/89 (65.2%) Virus isolation* 14/59 27/58 *Parcial sequencing (HA, NA and M gene) of 35 IAVs, eight gene segments of 11 IAVs.
15 INFLUENZA A VIRUS DETECTION IN NASAL SWABS AND ORAL FLUIDS FROM PIGS BY QUANTITATIVE REAL-TIME RT-PCR 15
16 Phylogenetic analysis Nucleotide alignments were generated for discrete data sets: H1 (human seasonal virus-like, H1s, n = 209) H1 (pandemic virus-like, H1p, n = 451) H3 (n = 463) N2 (n = 682) N1 (pandemic virus-like, N1p, n = 311) Each data set was comprised of: (a) Brazilian swiavs (16 IAVs: five H1N2, four H3N2 and seven H1N1pdm); (b) Related human and swine viruses, collected globally, available at NCBI s GenBank. Nelson M, Schaefer R, Gava D, Cantão M, Ciacci-Zanella J. Influenza A Viruses of Human Origin in Swine, Brazil. Emerging Infectious Diseases. 2015;21(8).
17 H3 Time-scaled Bayesian MCC tree for the H3 gene
18 H1
19 N2 Three H1N2, two H3N2 IAVs: belong to clade N2a; Two H1N2 IAVs: belong to clade N2b
20 H1N1pdm
21 Virus Subtype HA NA M A/swine/Brazil/ S1/2009 H1N1 H1pdm N1pdm pdm A/swine/Brazil/ S7/2009 H1N1 H1pdm N1pdm pdm A/swine/Brazil/ S8/2009 H1N1 H1pdm N1pdm pdm A/swine/Brazil/106-09/2009 H1N1 H1pdm N1pdm pdm A/swine/Brazil/170h-10/2009 H1N1 H1pdm N1pdm pdm A/swine/Brazil/170e-10/2009 H1N1 H1pdm N1pdm pdm A/swine/Brazil/132-09/2009 H1N1 H1pdm N1pdm pdm A/swine/Brazil/107-3A/2010 H1N1 H1pdm N1pdm pdm A/swine/Brazil/12a10/2010 H1N1 H1pdm N1pdm pdm A/swine/Brazil/136-10/2010 H1N1 H1pdm N1pdm pdm A/swine/Brazil/ /2011 H1N1 H1pdm N1pdm pdm A/swine/Brazil/66-11/2011 H1N1 H1pdm N1pdm pdm A/swine/Brazil/95-11/2011 H1N1 v H1pdm N1pdm pdm A/swine/Brazil/ /2011 H1N1 H1pdm N1pdm pdm A/swine/Brazil/263-12/2012 H1N1 H1pdm N1pdm pdm A/swine/Brazil/18-12/2012 H1N1 H1pdm N1pdm pdm A/swine/Brazil/ /2011 H1N2 H1 N2 pdm A/swine/Brazil/ /2011 H1N2 H1 N2 pdm A/swine/Brazil/ /2011 H1N2 H1 N2 pdm A/wild boar/brazil/ d/2011 H1N2 H1 N2 pdm A/swine/Brazil/ /2011 H1N2 H1 N2 pdm A/swine/Brazil/ /2011 H1N2 H1 N2 pdm A/swine/Brazil/ /2011** H3N2 H3 N2 pdm A/swine/Brazil/ /2011 H3N2 H3 N2 pdm A/swine/Brazil/ /2011 H3N2 H3 N2 pdm A/swine/Brazil/ /2011 H3N2 H3 N2 pdm **Identified H3N2, H1N1pdm09 and H1N2 at the same farm
22 Feral pigs and Captive wild boar N. Biondo, 2012
23 Captive wild boars HA and NA δ cluster Internal genes derived from H1N1pdm09
24 Research at Embrapa Antibody profile and Subtypes Farm (n=13) Farrowing (n=193) Weaning (n=253) Growing (n=504) Finishing (n=404) Subtypes A 66,7 60,0 73,3 53,3 H1N1pdm09 + H3N2 B 85,7 28,6 75,0 100,0 H1N1pdm09 + H3N2 C 93,3 60,0 91,1 73,3 H1N1pdm09 + H3N2 D 100,0 93,3 28,9 46,7 H1N1pdm09 + H1N1 + H3N2 E 93,3 46,7 34,2 96,6 H3N2 F 6,7 13,3 52,3 50,0 H3N2 G 93,3 66,7 13,3 36,7 H3N2 H 26,7 33,3 33,3 82,8 H3N2 I 0,0 23,3 80,0 81,8 H1N2 + H3N2 J 21,4 20,0 86,7 69,0 H1N1pdm09 + H3N2 K 46,7 60,0 88,6 83,3 H1N1pdm09 + H1N1 + H3N2 L 66,8 66,8 76,7 90,0 H1N1pdm09 + H3N2 M 80,0 35,7 58,6 20,0 H1N1 + H1N2 + H3N2 Avg (%) 60,0 46,7 60,9 70,0
25 13 Farrow-to-finish farms (HI results): Antibodies against H3N2: 4 farms Gene M Antibodies against H1N1pdm09 and H3N2: 5 farms Antibodies against H1N1, H1N1pdm09 and H3N2: 2 farms swh 1α Gene M Antibodies against H1N2 and H3N2: 1 farm Antibodies against H1N1, H1N2 and H3N2; 1 farm swh 1α
26 Before 2010 After 2010 swh1α swh1α Gene M Gene M
27 Conclusions H1N1pdm, H1N2 and H3N2 IAVs are widespread in Brazilian pig herds. We identified multiple previously uncharacterized clades of viruses that are most closely related to human seasonal H3N2 and H1N2 viruses that circulated in the late 1990s and early 2000s, respectively. In contrast to the five genetically distinct HA and NA identified in Brazilian swine (H3, seasonal H1, pandemic H1, pandemic N1, and two seasonal N2 segments), the genetic diversity of the internal gene segments was restricted to only the pandemic lineage.
28 Acknowledgements
29 df
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