INFLUENZA IN SWINE HERDS FOLLOWING THE INTRODUCTION …€¦ · swine (H3, seasonal H1, pandemic H1, pandemic N1, and two seasonal N2 segments), the genetic diversity of the internal

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/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/107-3A/2010 H1N1 H1pdm N1pdm pdm

A/swine/Brazil/12a10/2010 H1N1 H1pdm N1pdm pdm


A/swine/Brazil/72-11-507/2011 H1N1 H1pdm N1pdm pdm



A/swine/Brazil/173-11-4/2011 H1N1 H1pdm N1pdm pdm



A/swine/Brazil/31-11-1/2011 H1N2 H1 N2 pdm



A/wild boar/Brazil/214-11-13D/2011 H1N2 H1 N2 pdm



A/swine/Brazil/231-11-1/2011** 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

[email protected]

www.cnpsa.embrapa.br/sgc/sgc_publicacoes/publicacao_p1e97z4d.p

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Documents

INFLUENZA IN SWINE HERDS FOLLOWING THE INTRODUCTION …€¦ · swine (H3, seasonal H1, pandemic H1, pandemic N1, and two seasonal N2 segments), the genetic diversity of the internal