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Janice Reis Ciacci Zanella
PAHO Webinar
December 18, 2015
INFLUENZA IN SWINE
HERDS FOLLOWING
THE INTRODUCTION OF
PANDEMIC 2009 H1N1
INFLUENZA IN SWINE IN BRAZIL
Janice Reis Ciacci Zanella
Brazilian Agricultural Research Corporation – EMBRAPA Embrapa Swine and Poultry Research Center
EMBRAPA Swine and Poultry (176 ha): 211 employees
Animal Health
laboratory
• 8.514.876 km2
• 189.612.814 inhabitants
• 27 States
• 5.565 cities
• 36.819.017 pigs
• 22 million pigs slaughter / year
• 1.496.107 pig farms
• 5.335 Official Veterinarians
•180 countries import Brazilian pork
The big challenge…
Distribution per Region
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.
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
• 09 commercial farms and 01 feral swine herd (176/09/2009) from Pantanal wetland in Brazil.
• 359 serum samples collected between 2006 – 2010.
• HI assays: classic H1N1-A/sw/IA/31(AAF6/19/92) or H1N1, H3N2-A/sw/IA/8548-2 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.
0%
20%
40%
60%
80%
100%
HI - H1N1
10-0
6-11
/200
6
0064
-07/
2007
0071
-07/
2007
0094
-07/
2007
176/
09/2
009
110/
09/2
009
12b/1
0/20
10
51/1
0/20
10
83c/
10/2
010
92/1
0/20
10
0
1
2
3
4
5
Swine Herd ID/ Year
Lo
g2 T
iter
HI - pH1N1
10-0
6-11
/200
6
0064
-07/
2007
0071
-07/
2007
0094
-07/
2007
176/
09/2
009
110/
09/2
009
12b/1
0/20
10
51/1
0/20
10
83c/
10/2
010
92/1
0/20
10
0
1
2
3
4
5
Swine Herd ID/ Year
Lo
g2 T
iter
HI - H3N2
10-0
6-11
/200
6
0064
-07/
2007
0071
-07/
2007
0094
-07/
2007
176/
09/2
009
110/
09/2
009
12b/1
0/20
10
51/1
0/20
10
83c/
10/2
010
92/1
0/20
10
0
2
4
6
Swine Herd ID/ YearL
og
2 T
iter
Comercial farms 2009
•Plasma, OF
•1 protocol
•1000 plasma samples
•257 OF samples
•51 farms
CNPq Project
2009 to 2012
•Serum, NS, OF
•16 farms
•87 pools
•435 samples
Comercial farms
2011-2012*
•Serum, SN, FO
•49 farms
•288 pools
•1440 samples
Quarantine Boars
•Serum, OF
•1 protocol
•52 OF samples
•471 serum samples
Captive wild pig
•Lungs, NS
•2 protocols
•60 lung samples
Monteiro Feral Pigs
•Serum, NS, GS
•2 protocols
•152 samples
Diagnostic Lab - CEDISA
•Lungs
•86 protocols
Nasal Swab
(NS)
Subtyping H1, H3, N1, N2
Genome Sequencing Virus Isolation
HI
qRT-PCR IAV
Serum
ELISA SIV
Oral Fluid
(OF)
qRT-PCR IAV
Lung tissue
Histopathology - IHC
*Comercial farms 2011-2012: 8 (PR), 7 (RS), 3 (SP), 11 (SC), 10 (MG), 5 (MS) e 5 (MT)= 49
Total: 62 farms
Project funded by CNPq/MAPA: “Diagnostic, molecular characterization and
pathogenesis studies of infectious agents economically important for the Brazilian
Swine Production”. 02/2009 – 02/2012
11
Serological analysis
• A. Sera collected from all 48 commercial farms were positive by NP-ELISA test.
• B. – E. Specific antibodies to different subtypes
1881 nasal swabs and 89 lung tissue samples were collected from
swine in the southern, midwest and southeast regions of Brazil (2009-
2012);
RNA extraction, RT-qPCR (M gene of IAV and H1N1pdm);
Virus isolation (MDCK cells or ECE);
Genetic sequencing (ABI 3130xl and Illumina MiSeq).
Brasil
Assay Nasal swabs Lung
RT-qPCR/ IAV 59/1881 (3.3%) 58/89 (65.2%)
Virus isolation* 14/59 27/58
Results
*Parcial sequencing (HA, NA and M gene) of 35 IAVs,
eight gene segments of 11 IAVs.
INFLUENZA A VIRUS DETECTION IN NASAL SWABS AND ORAL
FLUIDS FROM PIGS BY QUANTITATIVE REAL-TIME RT-PCR
15
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)
N1 (pandemic virus-like, ‘N1p’, n = 311)
N2 (n = 682)
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).
H3
Time-scaled Bayesian
MCC tree for the H3 gene
H1
•Three H1N2, two H3N2 IAVs:
belong to clade N2a;
•Two H1N2 IAVs: belong to clade
N2b
N2
H1N1pdm
v
Virus Subtype HA NA M
A/swine/Brazil/104-09-S1/2009 H1N1 H1pdm N1pdm pdm
A/swine/Brazil/104-09-S7/2009 H1N1 H1pdm N1pdm pdm
A/swine/Brazil/104-09-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/72-11-507/2011 H1N1 H1pdm N1pdm pdm
A/swine/Brazil/66-11/2011 H1N1 H1pdm N1pdm pdm
A/swine/Brazil/95-11/2011 H1N1 H1pdm N1pdm pdm
A/swine/Brazil/173-11-4/2011 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/31-11-1/2011 H1N2 H1 N2 pdm
A/swine/Brazil/31-11-3/2011 H1N2 H1 N2 pdm
A/swine/Brazil/232-11-13/2011 H1N2 H1 N2 pdm
A/wild boar/Brazil/214-11-13D/2011 H1N2 H1 N2 pdm
A/swine/Brazil/185-11-7/2011 H1N2 H1 N2 pdm
A/swine/Brazil/232-11-14/2011 H1N2 H1 N2 pdm
A/swine/Brazil/231-11-1/2011** H3N2 H3 N2 pdm
A/swine/Brazil/365-11-6/2011 H3N2 H3 N2 pdm
A/swine/Brazil/355-11-6/2011 H3N2 H3 N2 pdm
A/swine/Brazil/365-11-7/2011 H3N2 H3 N2 pdm
**Identified
H3N2,
H1N1pdm09
and H1N2 at
the same
farm
Feral pigs and Captive wild boar
N. Biondo, 2012
Captive wild boars
• HA and NA δ cluster
• Internal genes derived from H1N1pdm09
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
Research at Embrapa– Antibody profile and Subtypes
13 Farrow-to-finish farms (HI results):
Antibodies against H3N2: 4 farms
Antibodies against H1N1pdm09 and H3N2: 5 farms
Antibodies against H1N1, H1N1pdm09 and H3N2: 2 farms
Antibodies against H1N2 and H3N2: 1 farm
Antibodies against H1N1, H1N2 and H3N2; 1 farm
Gene M
swH
1α
Gene M
swH
1α
swH1α
Gene M
Before 2010 After 2010
Gene M
swH1α
Conclusions
H1N1pdm, H1N2 and H3N2 IAVs are widespread in Brazilian pig herds.
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.
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.
Acknowledgements
www.cnpsa.embrapa.br/sgc/sgc_publicacoes/publicacao_p1e97z4d.p
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