OFFLU Technical Meeting Coordinating world-wide

surveillance for influenza in swine

OIE headquarters, Paris, France March 27-28, 2012

Brazil update

Janice Reis

Ciacci Zanella

Brazilian Agricultural Research Corporation –

EMBRAPA

Embrapa Swine and Poultry Research Center

Approach and Organization for our project

• 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

• The objective of this work is to perform and implement diagnostic methods for economical important infectious agents for swine production. The election of these pathogens was based in several reasons, mainly on the difficulty to do an investigation of agents considered up this date exotic in Brazil, such as the virus of porcine reproductive and respiratory syndrome (PRRSV) and the swine influenza virus (SIV). Other agents include the Aujeszky’s disease virus or Pseudorabies virus (PRV), knowingly present in domestic swine herds in some Brazilian states, but of unknown epidemiology and virulence in wild pig population.

Influenza A virus strains isolated from Brazilian pigs

• Number of herds from 2009 to 2011: 106

farms, 7 Brazilian states (PR,RS, SP, SC, MG, MS and MT),

• Sampling (nasal swabs and sera) considered 95% confidence and 95% sensibility of the test and a minimal prevalence of 10% in the herd. Thus, 30 pigs (60-85 days old) were sampled per farm.

http://www.brasil-turismo.com/mapas.htm

Viral samples

• 646 NS or lung samples from pigs of various ages and raised in commercial herds in Brazil were analyzed

• A total of 111 (17.18%) samples were positive to influenza A by RT-PCR

• 46 (41.44%) viruses were isolated.

Viral sequence

• 25 influenza viruses sequences were obtained.

• Based on the sequence analyses of HA, NA, M and PB1 genes, 16 influenza viruses showed a high identity (98-100%) with pH1N1 influenza virus that has been circulated in humans and in pigs since 2009.

• Five influenza viruses were closely related to an American H3N8 equine influenza virus (EIV).

Viral sequence

• Four virus isolates, based on the sequence analyses of HA, NA, M, NP, PB1, PB2 and PA genes proved to be a new H1N2 IAV which had not been described in Brazil yet. In addition, the sequence analyses of the new H1N2 virus has proved to be an arrangement where the genes for glycoprotein (H1 and N2) are of human origin (delta cluster) and the internal genes (M, NP, PB1, PB2 and PA) are derivatives the pH1N1 that has been circulating in pigs in Brazil over the past two years.

Serological analysis

• Sera collected from 106 commercial farms in the period July 2009 to December 2011 showed a prevalence of 60% of positive sera (positive in 1889/3150 sera) by commercial ELISA test (Avian Influenza MultiS-Screen Idexx ELISA).

• Furthermore, the analysis of sera collected HI pigs in Brazil, before and after 2009, 2009 revealed that before the presence of antibodies against the influenza pandemic (H1N1) was not detected in pigs in Brazil .

Comercial farms

Number of herds total samples influenza +

2010 14 350 190 2011 60 1801 1309 2009 31 999 390

Subtotal 105 3150 1889

Feral pigs farms 2009 5 31 5

2010 - 2011 5 110 100 Subtotal 5 141 105

Quarantine 1 471 8

Total geral 106 3762 2002

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.

HI - H1N1

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Fig. 1 – Elisa

Feral Pigs or Wild Boars

• The population of wild Suidae, both captive wild boars reared under intensive management or free range feral pigs (feral pigs of the Pantanal Region in Brazil) were also evaluated.

• Lung samples were collected from wild boars at slaughter in order to investigate the involvement of the IAV in symptoms of pneumonia, along with other agents such as Mycoplasma hyopneumoniae. IAV were detected by quantitative PCR in 11 out of 60 lungs with macroscopic lesions typical of pneumonia. Of these samples, the M gene sequencing showed 98-99% identity with the pH1N1 (Biondo, 2012).

• Regarding the population of feral pigs analyzed, samples of blood serum indicated the presence of antibodies to IAV in 105 out of 141 samples tested (74.5%).

Table 1. Influenza A pH1N1 viruses with the higher nucleotide sequence identity detected in this study, as determined by BLAST search in NCBI.

SI1 Gene Pb2 Identity E-value Virus designation Subtype GenBank acess n°

11D M 221 98% 0.0 A/Kenya/0066/2009/(H1N1) pH1N1 HQ214432.1

M 98% 0.0 A/California/VRDL133/2009(H1N1) pH1N1 CY066440.1

13D M 224 99% 0.0 A/Kenya/0066/2009/(H1N1) pH1N1 HQ214432.1

M 99% 0.0 A/California/VRDL133/2009(H1N1) pH1N1 CY066440.1

14D M 221 99% 0.0 A/Kenya/0066/2009/(H1N1) pH1N1 HQ214432.1

M 99% 0.0 A/California/VRDL133/2009(H1N1) pH1N1 CY066440.1

Feral Pigs or Wild Boars

UFMG (Rajão et. al, 2012).

• A study of detection of IAV infection in pigs in six Brazilian states, but mainly in the state of MG was held recently.

• This study covered the serological detection, virus isolation, genomic sequencing and study of the dynamics of infection.

• It was concluded that the IAV is circulating in at least 64.7% of the studied farms.

• The sequencing of the HA gene of the isolates showed high identity of VI with pH1N1.

• Serology indicated that samples collected before 2009 showed IAV infection was present in those farms, but not the pH1N1.

• Serologic studies indicated a decrease in antibody titers of pigs in the nursery phase, which may cause serious respiratory disease and losses