Development Of Luciferase-linked Antibody Capture Assay(LACA)for Detection Of Antibodies Against Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS)is an infectious disease characterized by respiratory disorders in piglets and reproductive disorders in pregnant sows,which has caused serious economic loss to global swine-breeding industries in the past 30 years.PRRSV is one of the most rapidly mutating RNA viruses known,with considerable genetic variation within both PRRSV-1 and PRRSV-2.This feature makes the symptoms of the disease different in diverse regions and herd.And with the virus constantly changing,it is difficult to make an accurate diagnosis only from epidemiological and clinical manifestations.Currently,research on PRRS in countries around the world has been carried out and many vaccines have been developed,but there is still no reproducible immunological intervention that develops a broadly protective immune response against virulent PRRSV.Therefore,it is necessary to establish a rapid,simple,and effective early detection method for large-scale detection of PRRSV to prevent and control diseases.Early detection of porcine reproductive and respiratory syndrome virus(PRRSV)infection of swine is necessary to control this devastating disease.By monitoring host serum antibodies to viral antigens,early virus detection within herds is feasible.In this study,recombinant antigens were generated using recombinant DNA techniques to fuse PRRSV structural protein(N)or nonstructural protein 1α(nsp1α)with the Rellina luciferase gene.Next,fused genes were cloned into plasmids and transfected into HEK-293 T cells for transient expression.Upon co-incubation of lysates with pig sera,antigen-antibody complexes formed that bound to Protein-G coated onto microplates.By further measurement of luminance value,a modified form of Luciferase Immunoprecipitation Systems,namely luciferase-linked antibody capture assay(LACA)was developed for detection of PRRSV-specific antibodies.The main works and results of this study were as follows:1.Preparation of Renilla luciferase-fused PRRSV-N and PRRSV-nsp1α antigensThe recombinant plasmids coding for Renilla luciferase-fused PRRSV-N or nsp1α were constructed by overlap PCR.Then HEK293 T cells were transfected with recombinant plasmids and cell lysates were collected.The expression level of proteins were detected via western blotting,immunofluorescence assay and luciferase activities.The results showed that both N and nsp1α could colocalize with luciferase and had strong enzymatic activity,which indicated the detection antigen was successfully prepared.2.Establishment of the LACA testing processThe LACA detection process was optimized using detection antigen of N-Rluc and serum samples from either PRRSV-infected pig or SPF pig.Optimized reaction conditions were as follows: recombinant Protein G in PBS was used to coat polystyrene microplates;plates were blocked with PBS-T buffer containing 2.5% gelatin at 4℃ for 12 h;the optimal dilution of the test serum was identified as 1:20;the incubation method was decided as co-incubation of Protein G,serum,and detection antigen for 2 h at room temperature.3.Evaluation of the LACATo evaluate the validation of the LACA and compared it with IDEXX PRRS X3 ELISA,known anti-PRRSV antibody-positive or-negative serum samples(125 and 122 samples,respectively)and 107 serum samples from field pigs were tested.Statistical analysis showed that the cutoff value of N-Rluc LACA was 1.4608,with a diagnostic sensitivity of 98.4% and a diagnostic specificity of 100.0%;the cutoff value of nsp1α-Rluc LACA was 1.7537,with a diagnostic sensitivity of 93.6% and a diagnostic specificity of 100.0%.The CV values of intra-plate and between runs of N-Rluc LACA and nsp1α-Rluc LACA were both less than15%,which indicated that the repeatability of LACA was good.Based on the result,N-Rluc and nsp1α-Rluc LACA results were 95.3% and 94.4% in agreement with IDEXX PRRS X3 ELISA,suggested a similar specificity of LACA to IDEXX PRRS X3 ELISA.Moreover,when LACA,IFA and IDEXX PRRS X3 ELISA were used to evaluate sequential serum samples obtained from PRRSV experimentally infected pigs,the PRRSV-specific antibody response was detectable as early as 3 days post-inoculation(dpi)using N-Rluc LACA,but undetectable until 7 dpi using IDEXX PRRS X3 ELISA,suggesting an improved sensitivity of LACA.Meanwhile,antibodies specific for nsp1α were detected at higher levels overall,but were undetectable until 10 dpi.Furthermore,.Notably,one IDEXX PRRS X3 ELISA positive result was not confirmed by LACA or IFA and was thus considered a false-positive result.In conclusion,the LACA exhibited similar specificity but improved sensitivity to that of the commercial IDEXX PRRS X3 ELISA kit for detection of PRRSV-specific antibodies in pig serum.Importantly,LACA could be adapted for detecting antibodies against other PRRSV targets,such as nsp1α,to achieve earlier detection of PRRSV infection.