Effects On TNF-α And IFN-β Gene Transcription Of Porcine Pulmonary Alveolar Macrophages By PRRSV ADE Infection

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease characterized by respiratory disorders and abortion in pregnant sows, resulting in important economic losses to the swine industry. It’s causative agent, Porcine reproductive and respiratory syndrome virus (PRRSV), belongs to Arterivirus family in the order of Nidovirales, infects primarily the porcine alveolar macrophage (PAM). Virus entry either via natural virus receptor or antibody-Fc receptor mediated antibody-dependent enhancement of infection (ADE). ADE of PRRSV infection is a major obstacle in PRRS prevention and control.Macrophage can secrete IFN-β、TNF-α upon infection, IFN-β plays a major role in antiviral immunology, and TNF-a is a pro-inflammational cytokine and has regulatory activity. NF-κB locates the center of signaling pathway network, can regulate the transcription of many genes, including TNF-a, IFN-P and other cytokines. IRF-1and IRF-3is another important transcription factor, functioned with NF-κB and regulate IFN-β gene transcription. In order to investigate TNF-a and IFN-β gene transcription of porcine pulmonary alveolar macrophages by PRRSV ADE infection, PAMs was infected with PRRSV BJ-4strain via ADE and normal pathway in vitro. The infected and control cells were collected and the transcription level of TNF-a and IFN-P gene were analyzed by Real-time RT-PCR.PAMs were isolated from fresh lung and cultured properly. Total RNA was extracted and cDNA was synthesized by RT-PCR. Fragments of TNF-α, IFN-β, NF-κB, IRF-1, IRF-3and β-actin gene were amplified using specific primers and cDNA as template. The PCR products were detected by agarose electrophoresis, purified, ligated into PMD19-T vector and transformed into E.coli DH5a. Plasmids were extracted and verified by PCR and sequencing. A series of diluted recombinant plasmids were used as standard of SYBR Green-1real-time quantitative PCR. The standard curves showed Ct value of TNF-α, IFN-β, NF-κB, IRF-1, IRF-3and p-actin gene has a good linear relationship. The real-time RT-PCR detection of porcine cytokine TNF-α, IFN-β and related transcription factor NF-κB, IRF-1, IRF-3and β-actin gene were successfully established.PAMs were infected with PRRSV BJ-4strain through ADE pathway, Cells were collected after24h infection. The mRNA transcriptions of TNF-α, IFN-β mRNA were analyzed using established real-time RT-PCR. The results showed that the mRNA transcriptions of TNF-α and IFN-β in PAMs by PRRSV ADE infection were down regulated and related trscription factor NF-κB, IRF-1and IRF-3were also down regulated.The study demonstrate the innate intracelluler immunity was altered by PRRSV ADE infection and may be a clue to further understanding the mechanisim of ADE.