Construction Of Infectious Clone Of Porcine Epidemic Diarrhea Virus And Development Of Its Antiviral Drugs And Vaccines

Porcine epidemic diarrhea virus（PEDV）,an enterovirus Alphacoronavirus,causes vomiting,diarrhea,anorexia,dehydration even death in neonatal piglets.Variant PEDV strain（GⅡ）emerged in China in 2010,and rapidly spread to many American,European,and Asian countries.Since then,GⅡgroup PEDV has been considered one of the most devastating swine viruses due to its high morbidity and mortality.At present,the existing PEDV vaccines cannot provide adequate protective immunity against the prevalent PEDV strains,and the effective anti-PEDV drugs are absent,causing large-scale outbreaks and frequent recurrent of PEDV.Meanwhile,the co-infection of PEDV with other porcine enterovirus including porcine rotavirus（Po RV）,is common in piglet diarrhea,causing the disease aggravation,higher mortality and massive economic losses to the pig industry.Thus,the development of novel vaccines and antiviral agents are urgently required to prevent and control porcine epidemic diarrhea.Reverse genetics system is a powerful platform to determine the function of viral genes,develop novel vaccine candidates and evaluate the antiviral activity of drugs.In this study,a reverse genetic system of PEDV was successfully established.Based on the system,we constructed a stable reporter PEDV encoding Nanoluciferase（Nano Luc,NLuc）.Meanwhile,the role of non-structural protein1（nsp1）protein in PEDV replication and pathogenicity was determined by the reverse genetic system and animal infection studies.Based on the results,we deleted the functional domain of nsp1 and replaced the open reading frame 3（ORF3）gene with Po RV VP7 gene in YN150 genome,constructing a recombinant expressing Po RV VP7（r YN150-nsp1ΔC/ORF3^--Po RV-VP7）.We also evaluated the immunogenicity of the recombinant PEDV in piglets.Furthermore,employing the reporter PEDV,we screened for the anti-PEDV drugs from a natural product library containing 803 compounds and identified 25 compounds that could significantly inhibit PEDV replication.Next,the mechanism of 7 antioxidant drugs was further studied.Our data provides a theoretical basis and a potential novel vaccine for the prevention and control of piglet viral diarrhea.The main contents are as follows:1.Generation of a full-length infectious c DNA clone of PEDV strain YNThe full-length genome of the 150^th passage virus of the variant PEDV strain YN（YN150）was verified by sequencing and divided into six continuous fragments,as F1（6055 bp）,F2（3825 bp）,F3（5000 bp）,F4（4336 bp）,F5（4281 bp）and F6（4531 bp）.The full-length c DNA of PEDV was obtained by in vitro ligation.After in vitro transcription,electroporation,CPE observation,whole genome sequencing and IFA analysis,the YN150 was successfully rescued.Collectively,the reverse genetic system of PEDV based on in vitro ligation was successfully established,providing a powerful tool and platform for further researches on PEDV.To determine the operability and stability of the system,we replaced the ORF3 gene of YN150 with NLuc gene to engineer a recombinant PEDV expressing NLuc（r YN150-NLuc）.We found that r YN150-NLuc produced similar plaque morphology and showed similar growth kinetics compared with the parent PEDV in vitro.Remarkably,the foreign gene（NLuc）was effectively expressed within the genome of r YN150-NLuc,and the level of luciferase activity was stably detected in r YN150-NLuc-infected cells and exhibited a strong positive correlation with the viral titers.Moreover,luciferase activity was detectable as early as 4 hpi,while the titer of PEDV was detectable by TCID₅₀ assay at 8 hpi,which showed that the luciferase assay was more sensitive than TCID₅₀ assay,and the titer of PEDV could be measured by luciferase assay.The results showed the recombinant virus r YN150-NLuc could be used as an indicator virus for high-throughput screening（HTS）of antiviral drugs,and the exogenous genes could be stably inserted into PEDV and achieved effective expression via the reverse genetic system.2.The role of PEDV NSP1 protein in viral replication and virulence and functional domain of nsp1Nsp1 is an important virulent factor of PEDV that played an essential role in host gene expression and innate immune response.Nevertheless,its role in PEDV replication and pathogenicity has been undetermined.We found four conserved domains,as 4-17 amino acids（A domain）,38-53 amino acids（B domain）,59-67 amino acids（C domain）and 87-107 amino acids（D domain）,of PEDV nsp1 by a bioinformatics analysis.The four domains of YN17 were respectively deleted to construct the mutant via the reverse genetic system of PEDV.The r YN17-nsp1?C and r YN17-nsp1?D were rescued successfully,while CPE and genome were not detected in r YN17-nsp1?A-and r YN17-nsp1?B-electrotransfected Vero cells,which suggested that r YN17-nsp1?A and r YN17-nsp1?B were failure to rescue.The study revealed the A and B domain of nsp1 played an indispensable role in PEDV replication.The phenotype characterization in vitro and animal infection experiments showed that nsp1 D domain had no effects on viral replication and pathogenicity,while r YN17-nsp1?C showed a significant decrease in viral titer.Meanwhile,the levels of viral replication and cytokines,as TNF-α,IL-6,IL-8 and IL-1β,in the intestinal tissue from r YN17-nsp1?C-inoculated piglets were significantly decreased compared with the piglets inoculated with wild-type PEDV.Pathological damage degree of intestinal villi and diarrhea of piglets were also significantly reduced.These results demonstrated that the C domain of PEDV nsp1 played a role in viral replication,pathogenicity and inflammatory response.Further studies showed that the C domain of nsp1 inhibited host gene expression including IFN-β,IFN-λ3 and ISGs in the early stage of PEDV infection,suggesting that nsp1 was critical for viral immune evasion and pathogenicity.This study was the first to identify the role of PEDV nsp1 in immune escape,viral replication and pathogenicity at the viral level,providing a potential candidate strain for PEDV live attenuated vaccine.3.Construction and immunogenicity evaluation of a recombinant PEDV expressing Po RV VP7 geneThe co-infection of PEDV-Po RV has become the main cause of piglet diarrhea,resulting in disease aggravation and massive economic losses to pig industry.It is a promising approach to develop a novel bivalent vaccine against the co-infection of PEDV with Po RV.Here,we replaced the ORF3 of PEDV YN150 with Po RV VP7 to engineered a recombinant PEDV expressing Po RV VP7 gene（r YN150-Po RV-VP7）using the reverse genetics platform.The phenotypes characteristic exhibited that the exogenous VP7 gene was stably expressed in the genome of r YN150-Po RV-VP7 in vitro,and r YN150-Po RV-VP7 showed a similar replication kinetics to r YN150.After the inoculation with r YN150-Po RV-VP7,piglets did not display any clinical symptoms such as diarrhea,vomiting,dehydration.Meanwhile,the specific Ig G and Ig A antibodies against PEDV S protein and Po RV VP7protein,and the neutralizing antibodies against PEDV and Po RV could be also detectable in inoculated piglets.There results suggested that r YN150-Po RV-VP7 showed an excellent immunogenicity and safety in piglets.In order to further attenuate r YN150-Po RV-VP7,the nsp1 C domain of the recombinant virus was deleted employing the reverse genetic system to constructed the recombinant PEDV,r YN150-nsp1?C/ORF3^--Po RV-VP7.As expected,the expression of Po RV VP7,and PEDV S were detectable in r YN150-nsp1?C/ORF3^--Po RV-VP7-infected cells,and the virus showed similar growth kinetics compared to parent virus.Our study provides a novel bivalent vaccine candidate with a potential application in prophylaxis against the co-infection of PEDV-Po RV.4.HTS of PEDV antiviral drugsUsing the reporter PEDV（r YN150-NLuc）,we established a HTS method for the development of anti-PEDV drugs with an accuracy and efficiency.Briefly,the natural products with a virus inhibition rate>90%and a cell viability rate>80%were primarily selected by luciferase activity assay and the cytotoxicity assay.The IC₅₀ and CC₅₀ of the selected products were measured by a dose-dependent test,and the products with the selectivity index（SI）>10 was reconfirmed as anti-PEDV drugs.Using this HTS method,25 products that significantly inhibited PEDV replication with SI>10 were screened from803 compounds in the natural product library.The SI of protoporphyrin IX and 7-ethylcamptothecin was>80,exhibiting the excellent anti-PEDV activity.Additionally,it was further determined that the screened 25 products could significantly inhibit the replication of YN150 and YN15 by TCID₅₀ assay with inhibition rates>90%,providing candidate drugs for the prevention of PEDV.Interestingly,7 of the 25 identified products were natural antioxidants,including Betulonic acid,Ursonic acid,esculetin,lithocholic acid,nordihydroguaiaretic acid,caffeic acid phenethyl ester,and grape seed extract.It was further determined by IFA analysis and TCID₅₀ assay that these seven antioxidant drugs inhibited PEDV replication in a dose-dependent manner.Meanwhile,they showed inhibition rates>60%at the concentration of2.5μmol/L.To investigate how the antioxidant drugs affects PEDV replication,the level of ROS in PEDV-infected cells were assessed.We found that PEDV infection induced an increase level of intracellular ROS,resulting in an activation of autophagy to benefit its replication.Namely,antioxidants effectively decreased the level of ROS in PEDV-infected cells,resulting in the block of autophagy and the inhibition of PEDV replication.The study provides a new idea for the development of anti-PEDV drugs and the prevention and treatment of PEDV.