Caspase-8-mediated Apoptosis Pathway And Devoloped A Real-time RT-PCR Method Of QH-08PRRS Isolate

Porcine Porcine reproductive and respiratory syndrome(PRRS)Causes in pregnant sows and respiratory disease in any age of pig.And,this viral disease has been identified bio threats to the global swine-industry and the economy.The causative agent,PRRS virus(PRRSV),is a spherical enveloped virus containing a genome of single-stranded positive-sense RNA and belonging to the genus Arterivirus,family Arteriviridae,order Nidovirales.Furthermore,equine arteritis virus(EAV),simian hemorrhagic fever virus(SHFV),and lactate dehydrogenase elevating virus(LDV)are included in this arteriviruses genus.PRRSV strains are divided into two species: the European isolates,and North American isolates,formerly known as genotype I(Eu strain)and genotype II(NA strain),respectively.The PRRSV genome is approximately 15 kb in size and consists of nine or ten open reading frames(ORFs),designated as ORFs 1a,1b,2a,2b,and 3-7.Both ORF1 a and ORF1 b encode the non-structural proteins,replicase and polymerase,that are believed to be involved in viral replication.The ORFs 2-7 are postulated to encode structural proteins.In this study,a high pathogenic(HP)-PRRSV 2 strain was isolated,which is denominated QH-08,from the Qinghai province of China.Complete genome sequencing of QH-08 showed that it shares 97.2% and 97% nucleotide identities with PRRSV 2 HP-PRRSV strains of HUN4 and JXA1,respectively.Moreover,it also shared 95.7%,93.8% and 95.9% nucleotide identities with PRRSV 2 strains of HP/Thailand19500LL/2010(Thailand,KF735060),PRRSV/MZ/IND/1A/18(India,MK287894)and BH58/10(Laos,JN626287),respectively.The genome-based phylogenetic analysis revealed that these 5 PRRSV type 2 isolates are clustered in a same lineage.And the sequence analysis revealed that QH-08 is closely related to the HUN4 isolates.Additionally,analyzing the complete genome sequencing and each ORF genome sequencing of 37 PRRSV type2 to obtain a hypothesis that the difference of ORF1a1 b of PRRSV type 2 could play be an important role in influencing the efficacy of PRRS vaccines in pigs against PRRSV challenge.These findings are critically beneficial for developing the strategy of PRRSV control and prevention.For host antiviral pathways,such as the TLR 3 induced apoptosis pathway,can be activated by a number of stimulating agents to inhibit the replication of PRRSV.In addition,we want to evaluate a novel insight for prevention and control PRRS by choosing some vaccine to prevent QH-08 PRRSV infection.Meanwhile,we set up a real-time PCR that cover and specifically detect the classic PRRSV,HP-PRRSV and variant PRRSV for prevention and control of PRRS.Based on the sequence analysis of different diverse of PRRSV-2 type,such as QH-08,a series of studies have been carried out are flowing.Firstly,The present study confirmed that Virus spirit(Yansuanmalingua)can inhibit PRRSV type 2 replication using plaque assay,qPCR and immuno-fluorescence assay(IFA)in Marc-145,and the mechanism of inhibition of PRRSV replication was shown to be related to TLR3-dependent apoptosis induction by YASML in the PRRSV infected MARC-145,and TLR3-dependent apoptosis-induction by YASML was found to suppress PRRSV replication via the activation of caspase-8 and caspase-3 pathways,respectively.The activation of the caspase-3pathway seemed to be related to the downregulation of myeloid cell leukemia 1(Mcl-1)expression.Our results showed that YASML-induced TLR3-dependent apoptosis could be blocked by a pan-caspase inhibitor and small interfering RNA against TLR3.In conclusion,the present study demonstrates that YASML exerts its anti-PRRSV effect by activating the caspase-8/caspase-3signaling pathway and by negatively regulating Mcl-1 expression.These findings may provide new insights to develop the new antiviral drugs by expressing caspase-3 or down expressing Mcl-1 for inhibiting viruses replication using medicines.Secondly,we used four vaccines,such as Ingelvac PRRS MLV,CH-1a,JXA1 and JXA1-RMLV,the sequence identities of ORF1a1 b of these vaccine is 25.3-96.3%,the ORF 2-ORF7 is 85.8-99.6%,respectively.Theses vaccines applied to 3 weeks old piglets.Group-1 and Group-4(n=5)was immunized with Ingelvac PRRS MLV,CH-1a,JXA1 and JXA1-RMLV,respectively.The group-5 and-6(n = 3)were injected with PBS as a positive and negative control.At 28 days,all groups,except group-6 which was categorized as the negative control,were challenged by the QH-08 PRRSV.All piglets were monitored daily and necropsied at 14 days post challenge(dpc).The results revealed that vaccinated pigs showed significantly lower(p < 0.05)mean temperatures,lung lesion scores,levels of virus load in serum and lung tissue compared with the group-5 pigs(positive control).Moreover,our findings indicated that JXA1 and JXA1-R MLV vaccines can provide 100% protection more than Ingelvac PRRS MLV and CH-1a vaccines by the QH-08 PRRSV challenge.According to the above results the PRRS MLV and inactivated vaccine could protect the identitity of ORF1a1 bs in more than 90% homologous strains infection.Finally,a one-step real-time RT-PCR was developed for detecting type 2 PRRSV based on the sequence characteristics of the QH-08 isolate and other 37 PRRSV strains,and its specificity and sensitivity were evaluated subsequently.The results indicated that this method could detecte the limit of 10 copies/u L of 2 type PRRSV,its sensitivity is 10 times than multiplex RT-PCR.No cross-reactivity was shown among type 1 PRRSV,PCV2,PRV and CSF.Clinical samples were detected using this method indicated that the positive rates and negative rates of 2 type PRRSV is100%.Furthermore,the specificity,sensitivity,repeatability and stability of the three batches kits were also evaluated.In conclusion,the results indicated that the specificity,sensitivity and stability of the kits are promising and will be a perfect choice to use in PRRS endemic countries in their prevention and control schemes.