Risk factors for exposure to influenza a viruses, including subtype H5 viruses, in Thai free-grazing ducks.

Free-grazing ducks (FGD) have been associated with highly pathogenic avian influenza (HPAI) H5N1 outbreaks and may be a viral reservoir. In July-August 2010, we assessed influenza exposure of Thai FGD and risk factors thereof. Serum from 6254 ducks was analysed with enzyme-linked immunosorbent assay (ELISA) to detect antibodies to influenza A nucleoprotein (NP), and haemagglutinin H5 protein. Eighty-five per cent (5305 ducks) were seropositive for influenza A. Of the NP-seropositive sera tested with H5 assays (n = 1423), 553 (39%) were H5 ELISA positive and 57 (4%) suspect. Twelve per cent (74 of 610) of H5 ELISA-positive/suspect ducks had H5 titres ≥ 1 : 20 by haemagglutination inhibition. Risk factors for influenza A seropositivity include older age, poultry contact, flock visitors and older purchase age. Study flocks had H5 virus exposure as recently as March 2010, but no HPAI H5N1 outbreaks have been identified in Thailand since 2008, highlighting a need for rigorous FGD surveillance.

The antibody responses to swine influenza virus (SIV) recombinant matrix 1 (rM1), matrix 2 (M2), and hemagglutinin (HA) proteins in pigs with different SIV exposure.

The influenza invariant matrix 2 (M2) protein is a potential subunit vaccine candidate to induce protective immunity against broader strains of influenza A viruses (IAV). Antibodies to M2 protein have not been well characterized in IAV natural hosts. To characterize M2-specific antibodies in pigs, an ELISA to the extracellular region of the M2 (M2e) protein was developed. Sera from pigs experimentally infected with three different swine influenza virus (SIV) subtypes, immunized with an SIV inactivated vaccine, or positive for SIV maternally derived antibodies (MDA) in the absence of SIV infection were tested in assay. Confirmation of antibody titer status of pigs, was determined using a hemagglutination-inhibition (HI) test and the presence of antibodies to matrix 1 (M1) protein was measured by a recombinant M1 (rM1)-based ELISA. The antibody titers to the HA and M2e proteins but not to the rM1 were directly correlated to the dose of virus used to infect the pigs and the level of antibodies detected by the HI assay varied according to SIV subtype. Pigs experimentally infected with SIV produced low levels of M2e antibodies compared to antibodies detected by the HI and rM1 assays. Vaccination alone followed by infection did not increase the levels of M2e antibodies in contrast to HA and rM1 antibodies. Pigs with MDA had different levels of HA antibodies and were positive to M2e antibodies, but results were not correlated to HA antibodies levels and inconsistently present.

Quantification of PCV2 capsid transcript in peripheral blood mononuclear cells (PBMCs) in vitro.

The presence of PCV2 DNA or spliced capsid mRNA (Cap mRNA) for viral replication was assessed following addition of PCV2 to resting or concanavalin A (ConA) stimulated peripheral blood mononuclear cells (PBMCs). Real-time PCR or real-time RT-PCR assays were used to measure viral DNA or Cap mRNA, respectively. The study demonstrated that PCV2 replication increased in infected PBMCs over time. Replication within infected PBMCs was significantly (P<0.05) increased when PBMCs were stimulated with ConA, compared to unstimulated PBMCs. The data showed a strong correlation between the level of PCV2 Cap mRNA and the level of viral DNA in the ConA stimulated PBMCs. Replication of PCV2 was also assessed in T lymphocyte- and monocyte/macrophage-enriched or monocyte/macrophage-depleted PBMC populations which had been stimulated with ConA for 3 days. It was demonstrated that the enriched T lymphocytes and the monocyte/macrophage-depleted PBMCs had significantly higher Cap mRNA and viral DNA levels (P<0.05) compared to the monocyte/macrophage-enriched population, indicating that in addition to monocytes/macrophages, PCV2 replicates in lymphocytes, particularly T lymphocytes following stimulation. These results suggest that the presence of activated T lymphocytes may play an important role in PCV2 replication and potentially the development of clinical disease.

Porcine circovirus type 2 (PCV2) distribution and replication in tissues and immune cells in early infected pigs.

Replication of porcine circovirus type 2 (PCV2) in pigs, as measured by spliced capsid mRNA (Cap mRNA) and viral DNA, was investigated following experimental infection. Peripheral blood mononuclear cells (PBMCs), and tissue from bronchial lymph nodes (BLN), inguinal lymph nodes (ILN), tonsils, lungs, liver, kidneys, spleen and thymus from infected pigs on different days post-infection (DPI) were assessed. PCV2 replication differed dramatically between tissues from the same infected pig. The virus actively replicated in most tested tissues at 14DPI in association with increased PCV2 associated lesions and PCV2 antigen levels, although no clinical signs correlated with PCV2 associated disease were observed in infected pigs during the course of the study. The PCV2 Cap mRNA was detected only at 13DPI in PBMCs from infected pigs, suggesting replication of the virus in circulating blood is transient and not a major site for PCV2 replication in vivo. Evaluation of the Cap mRNA and viral DNA synthesis in T and B lymphocyte and monocyte populations from PBMCs and BLN at various intervals post-inoculation revealed replication of PCV2 in all cell subpopulations; however, viral replication in B lymphocytes was greater than observed in mononuclear cells isolated from BLN at 14DPI indicating that B lymphocytes may be an important cell population for PCV2 replication. These findings further our understanding of the cell types permissive for PCV2 replication and the pathogenesis of PCV2 infection in vivo.