Interleukin-dependent modulation of the expression of MHC class I and MHC class II genes in chicken HD11 cells.

Interleukins (ILs) regulate cell surface antigens known as activation markers, which have distinct functional roles. However, the regulation of major histocompatibility complex (MHC) class I, MHC class II, and related genes by cytokines in chickens is not well understood. In the present study, we evaluated the influence of certain recently discovered chicken interleukins-i.e., IL-11, IL-12B, IL-17A, IL-17B, IL-26, and IL-34-on the expression and regulation of genes related to MHC class I, MHC class II, and the associated proteins in an HD11 chicken macrophage cell line. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunocytochemical, and flow cytometric analyses to assess dose- and time-dependent expression in the HD11 cell line and found that the ILs induced MHC class I, MHC class II, and associated protein. As NF-κB is actively involved in cell activation and is constitutively activated in many immune cells, we also determined whether NF-κB regulates MHC class I, MHC class II, and related gene expression in the HD11 cell line. The NF-κB inhibitor sulfasalazine (Sz) dose-dependently inhibited MHC class I and MHC class II in the HD11 cell line. Sz also downregulated the expression of MHC class I, MHC class II, and the associated proteins in the IL-induced HD11 cell line. The expression of MHC class I, MHC class II, and associated genes was accompanied by the Sz-sensitive degradation of the p65 (RelA) and p50 subunits of NF-κB and IκBα. Our results indicate that the different effects of each IL on the expression of genes related to MHC class I, MHC class II, and the associated proteins are involved with the regulation of the dose and duration of antigenic peptide presentation and, thus, also influence Th1, Th2, and Th17 production.

An improvement of real-time polymerase chain reaction system based on probe modification is required for accurate detection of African swine fever virus in clinical samples in Vietnam.

OBJECTIVE: The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including realtime polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam. METHODS: Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE. RESULTS: Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78). CONCLUSION: We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.

The potential efficacy of the E2-subunit vaccine to protect pigs against different genotypes of classical swine fever virus circulating in Vietnam.

PURPOSE: To date, many kinds of classical swine fever (CSF) vaccines have been developed to protect against this disease. However, the efficacy of these vaccines to protect the pig against field CSF strains needs to be considered, based on circulating strains of classical swine fever virus (CSFV). MATERIALS AND METHODS: Recombinant E2-CSFV protein produced by baculovirus/insect cell system was analyzed by western blots and immunoperoxidase monolayer assay. The effect of CSFV-E2 subunit vaccines was evaluated in experimental pigs with three genotypes of CSFV challenge. Anti-E2 specific and neutralizing antibodies in experimental pigs were analyzed by blocking enzyme-linked immunosorbent assay and neutralization peroxidize-linked assay. RESULTS: The data showed that CSFV VN91-E2 subunit vaccine provided clinical protection in pigs against three different genotypes of CSFV without noticeable clinical signs, symptoms, and mortality. In addition, no CSFV was isolated from the spleen of the vaccinated pigs. However, the unvaccinated pigs exhibited high clinical scores and the successful virus isolation from spleen. These results showed that the E2-specific and neutralizing antibodies induced by VN91-E2 antigen appeared at day 24 after first boost and a significant increase was observed at day 28 (p<0.01). This response reached a peak at day 35 and continued until day 63 when compared to controls. Importantly, VN91-E2 induced E2-specific and neutralizing antibodies protected experimental pigs against high virulence of CSFVs circulating in Vietnam, including genotype 1.1, 2.1, and 2.2. CONCLUSION: These findings also suggested that CSFV VN91-E2 subunit vaccine could be a promising vaccine candidate for the control and prevention of CSFV in Vietnam.