| Once virus invasion,host cells could detect virus nucleic acid sequence by pattern recognition receptors,and then it could produce interferon,chemokines and pro inflammatory factor to inhibit virus infection.During this process,interferon plays an important role.Previous studies found that the level of interferon increased in PCV2 subclinical infectionpiglets,but the regulation mechanism is unclear.In the study,a model of PCV2 subclinical infection in piglets was established,and then the alveolar macrophages cells(AMs)which are the natural immune cells and PCV2 target cells were collected.To understand the mechanism of PCV2 pathogenesis,the change and regulation of interferon in AMs were investigated.Fifteen 30-day-old piglets were selected as experiment animals,which were free of PCV2 and porcine reproductive and respiratory syndrome virus(PRRSV)antibody and antigen.After adaptation of one week,the piglets were divided into three groups:control group(0 dpi),PCV2 infection group(14 dpi)and PCV2 infection group(28 dpi).Each piglet of infected groups was treated with 2mL PCV2 suspension(5 × 105 TCID50ˇmL-1)by intranasal and intramuscular injection,respectively.And each piglet of control group was treated with 2mL PBS by same method.After infection,the piglets were euthanized,and then the plasma,lungs,lymph nodes and alveolar macrophages were collected.Part of the lungs and lymph nodes were fixed in formaldehyde solution and the rest were stored in-80? refrigerator.After experiment,rectal temperature,histopathological changes of lymph node and lung,serum antibody,PCV2 antigen in lung and lymph node,the mRNA transcription of interferon,pattern recognition receptors(PRRs)and pathogen associated molecular proteins(PAMPs)and the protein levels of pattern recognition receptors(TLR9,RIG-1,MDA-5,DAI)and the adapter molecule(MyD88,MAVS)and transcription factors(IRF3,IRF7)were measured.The result showed that the rectal temperature of control and experimental groups was normal;no obvious pathological changes were found except the slightly swollen of the lymph nodes at 14 dpi(day post infection)and 28 dpi;the serum antibody of the PCV2 antibody in PCV2-infected group was positive(the antibody blocking rate>38%),and the antibody blocking rate increased with increase of infetion time,while the srum antiboy of PCV2 in control group was negative(antibody blocking rate?30%);the PCV2 antigen of PCV2-infected group was positive while the control group was negative;the average copies of lung and lymph nodes were 7.0 ×103 copies/50 mg and 1.1×104 copies/50 mg at 14 dpi,and 1.9×104 copies/50 mg and 8.5×104 copies/50 mg at 28 dpi,respectively;these results demonstrated that the piglets were in PCV2 subclinical state.The mRNA levels of IFN-? and IFN-? were significantly higher at 14 dpi and 28 dpi(P<0.05);the mRNA expression of RIG-1,MDA-5,TLR4 and TLR9 were significantly increased at 14 dpi and 28 dpi(P<0.05);the mRNA level of DAI was significantly elevated at 14 dpi(P<0.05),and then decreased at 28 dpi;while there were no significant change in mRNA expression of TLR3,TLR7 and TLR8.Compared with control,the mRNA transcription of MAVS,MyD88,IRF3 and IRF7 were significantly increased at 14 dpi and 28 dpi(P<0.05).TheTLR9,RIG-1,DAI,MyD88,MAVS,IRF3 and IRF7 protein levels at 14 dpi and 28 dpi was significantly higher(P<0.05);while no change was found in MDA5 protein level at 14 dpi and 28 dpi(P>0.05).These results demonstrated that the up-regulations of IFN-? and IFN-? in alveolar macrophages of PCV2 subclinical infection were relative to TLR4/TLR9/MyD88 and RIG-1/MDA-5/DAI/MAVS/IRF3/7signaling. |
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