Effects Of Variable PRRSV Infection On The Transcriptions Of Cytokines In Porcine Pulmonary Alveolar Macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which is a transmissible disease characterized with either reproductive failure in pregnant sows, or respiratory tract distress particularly in sucking pigs, causing great economic losses worldwide each year.PRRSV has a high mutant rate. Mutant PRRSV is the cause of the large-scale devastating outbreak of PRRS in China during the summer of 2006, known locally as''high fever''. Genetic variation analysis indicates that there exist large mutations in Nsp2 gene, compared with classic PRRSV strain.Transcription levels of PAM secreted cytokines were compared in order to indicate the difference between mutant strain and attenuated strain in the effects on PAM immunologic function, which will help to reveal the relationship between mutant strain PRRSV infection and severe pathological reaction in host organism.RT-PCR and sequencing were used to screening a mutant strain PRRSV named FZ06A from several strains, which has a 30 aa. deletion in Nsp2 gene.Then animal experiments were conducted using am attenuated strain VA besides FZ06A.The result is that all the animals died in 10 days and the pathological changes were similar with the 2006 prevalenced strain.RT-PCR was used to amplify partial sequences of PRRSV N gene , proinflammatory cytokines (IL-1 and IL-6), innative cytokines(TNF-α,TNF-βand IFN-γ) ,Th1 cytokines (IFN-α,IL-2,IL-12p40), Th2 cytokines(IL-4), regulatory cytokines(IL-8) and inhibitory cytokines(TGF-βand IL-10).Then these sequences were cloned into pEASY-T1 vector. After the identification by PCR and restriction enzyme digestion, recombinant standard plasmids were selected. Real-time FQ-PCR was used to acquire their standard curves and the amplification efficiency were calculated.The result is that all the amplification efficiency were between 90%-105% and the correlation coefficient( R2 )were all above 0.99,which showed that the real-time FQ-PCR system fit the demand of this experiment.Thirty 30-day-old piglets were infected with mutant strain FZ06A and attenuated strain VA, then were euthanized at 2, 4, 8, 10DPI. The bronchoalveolar lavage (BAL) ?uids were collected and used to separate PAMs. Then ,the transcriptional levels of proinflammatory cytokines (IL-1 and IL-6), innative cytokines(TNF-α,TNF-βand IFN-γ) ,Th1 cytokines (IFN-α,IL-2,IL-12p40),Th2 cytokines(IL-4),regulatory cytokines(IL-8) and inhibitory cytokines(TGF-βand IL-10) were detected and analyzed by real-time FQ-PCR.The results showed that the transcriptions of proinflammatory cytokines mRNA decreased before 8DPI in mutant strain FZ06A group, and reached the lowest level at 4 DPI. For the attenuated strain VA group, the transcription levels of proinflammatory cytokines mRNA decreased before 4 DPI,also reached the lowest level at 4 DPI,then increased at 8DPI and increased rapidly at 10 DPI,especially, the IL-1βincreased with significant levels (P<0.05) at 10 DPI. The discrepancy between mutant strain FZ06A group and attenuated strain VA group is significant(P<0.05). After the infection of FZ06A, the transcriptions of innative cytokines were suppressed, while attenuated strain VA group showed a lower suppression. After the infection of attenuated strain VA,the transcriptions of IFN-γ,TNF-αand TNF-βmRNAs increased with significant level(P<0.05 or P<0.001),while for mutant strain FZ06A group, the transcription of TNF-αmRNA increased with significant level(P<0.05) at 10DPI. The transcription of IFN-γwas different .It was slightly increased after 4DPI in FZ06A group, while in VA group, the IFN-γlevel was decreased before 8DPI seriously.The transcriptions of Th1 cytokones mRNAs of mutant strain FZ06A group exhibited a state of suppression,while attenuated strain VA group showed a lower suppression.After the infection of attenuated strain VA,the transcriptions of IFN-αand IL-12 mRNAs increased at 10 DPI ,especially IFN-αincreased with significant leve(lP<0.05). The transcription of IL-2 mRNA of attenuated strain VA group exhibited an increased level.For mutant strain FZ06A group, the transcriptions of Th1 cytokones mRNA were decreased or increased slightly. The transcription of IL-2 mRNAs has the same tendency with VA group, but only increased at 8DPI while decreased at other phases,which further indicated that FZ06A has a stronger suppressive effect on the transcription of Th1 cytokones than VA.The transcription of IL-4 mRNAs of the mutant strain FZ06A group increased at 2DPI, decreased at 4-8 DPI ,then increased again at 10 DPI. The transcription of IL-4 mRNAs had no significant decrease in the attenuated strain VA group before 8 DPI, but increased more rapidly than mutant strain FZ06A group at 10 DPI.It indicated that the transcription of Th2 cytokines mRNAs of both groups were suppressed. The transcription of IL-8 mRNAs of the mutant strain FZ06A group was suppressed before 8 DPI and reached the lowest level at 4 DPI, while the VA group was suppressed and reached the lowest level at 4 DPI, then increased at 8 DPI.The difference between two groups was significant at 10 DPI(P<0.05).These results showed that after the infection of PRRSV, the transcription of IL-8 mRNA was suppressed, and FZ06A has a stronger suppressive effect than VA.The transcription of TGF-βmRNAs of the mutant strain FZ06A group was depressed during the experiment.There was a significant decrease at 4 DPI(P<0.05),and the level increased slightly at 8 DPI,then decreased at 10 DPI ,reached the similar level of control group.While, for the attenuated strain VA group, the transcription of TGF-βmRNAs decreased at 2-4 DPI ,but increased significantly at 8 and DPI(P<0.05). The transcriptions of IL-10 mRNAs of the two groups were suppresse before 8 DPI, then increased at 10 DPI, but the level of VA group was higher than FZ06A group, which indicated that FZ06A has a stronger suppressive effect than VA.All the results above indicate that mutant strain FZ06A has a stronger suppressive effect on the transcriptions of proinflammatory cytokines (IL-1 and IL-6), innative cytokines(TNF-α,TNF-βand IFN-γ) ,Th1 cytokines (IFN-α,IL-2,IL-12p40),Th2 cytokines(IL-4),regulatory cytokines(IL-8) and inhibitory cytokines(TGF-βand IL-10) mRNAs, compared with attenuated strain VA .So the mutant strain FZ06A has serious effects on the immune responses against PRRSV and other pathogens ,and will cause severe immunosupression.