Construction Of The Recombinant Porcine Epidemic Diarrhea Virus Strain With Partial Deletion In The E Protein And Evaluation Of Its Immunogenicity

A single positive strand RNA virus named porcine epidemic diarrhea virus（PEDV）infects piglets and results in vomiting,diarrhea,and othersymptoms as well as a 100%mortality rate.PEDV was first identified in 1971 in Europe.A novel variant PEDV strain was found in ourcountry in 2010,which has brought great loss to the pork industry in China and all overthe world.One of the reasons forthe PEDV epidemic is that wild strains are constantly mutating,making existing vaccines less protective.Hence,there is an urgent need to rapidly develop new vaccines to prevent the spread of PEDV.Finding and using reverse genetic operating system to modify virulence genes is an important way to obtain live attenuated vaccines.To quickly obtain new live attenuated PEDV vaccine,this paperinnovatively found that amino acids 23-29 of the PEDV E（Envelope,E）protein affected the host immune response through the pathogenicity evaluation and sequence alignment,and constructed and rescued the recombinant strain with amino acid 23-29 deletion of the E protein.The pathogenicity and immunogenicity of the recombinant strain were evaluated.The specific results are listed as follows:1.Evaluation of the pathogenicity of the recombinant PEDV strains rCH/SX/2015 and rCH/SX/2016-S_HNXPIn the previous experiment to study the molecularmechanism of PEDV cell tropism,the G I recombinant rCH/SX/2015 and G II recombinant rCH/SX/2016-S_HNXP strains were constructed.However,the pathogenicity of the two strains remains unkown.The pathogenicity of the rCH/SX/2015 and rCH/SX/2016-S_HNXP strains were firstly evaluated in this study.Piglets were inoculated with 5×10⁶ TCID₅₀ rCH/SX/2015 orrCH/SX/2016-S_HNXP at the age of 2 days,the control group was inoculated with the same dose of DMEM orally.According to the results,all piglets in the rCH/SX/2016-S_HNXPgroup had diarrhea on day 1 and all died on day 2（4/4,100%）,togetherwith weight loss.No piglets in the control group and the rCH/SX/2015 group exhibit mortality（0/3,0）,diarrhea,orweight loss.The postmortem examination revealed thin,transparent intestinal walls in the rCH/SX/2016-S_HNXP group.The intestinal walls of rCH/SX/2015-infected animals were normal,similarto the mock-infected animals.The above results indicated that rCH/SX/2016-S_HNXP was a virulent strain while rCH/SX/2015 was a less virulent strain.To study the molecularmechanism of the attenuation of the rCH/SX/2015 strain,the amino acid sequences of the rCH/SX/2015 and rCH/SX/2016-S_HNXP strains were compared.Sequence alignment showed that seven amino acid deletions at aa23 to aa29 of the E protein were only found in rCH/SX/2015 strain but not in rCH/SX/2016-S_HNXPstrain.The predicted TM domain position of E_Δaa23-aa29 was changed from I₁₅-L₃₇ to L₁₀-F₃₂.The above results showed that rCH/SX/2015 was a less virulent strain and rCH/SX/2016-S_HNXP was a virulent strain.The E protein of rCH/SX/2015 strain possess a 7 amino acid deletion,while the E protein of rCH/SX/2016-S_HNXP is intact,and this deletion changes the transmembrane region of E protein.2.Construction and evaluation of biological characterization of the recombinant strain rPEDV-E_Δaa23-aa29 in vitro.The transmembrane region of coronavirus E protein is involved in many biological processes.Therefore,the deletion of these 7 amino acids in E protein was hypothesized to affect some functions of the virus.In view of this,the recombinant strain rPEDV-E_Δaa23-aa29with a 7-amino-acid region deletion in the E protein was constructed using rCH/SX/2016-S_HNXP as the backbone.Indirect Immunofluorescence Assay（IFA）,RT-PCR and sequencing results indicated that rPEDV-E_Δaa23-aa29 was successfully rescued.Next,the growth characteristics of the rPEDV-E_Δaa23-aa29 recombinant strains was evaluated in vitro.Viral plaque assay and Cytopathic effect（CPE）showed that rPEDV-E_Δaa23-aa29 produced smallerplaques and cytopathic effect than the parental strain.The results of growth curves showed that the titers of the rPEDV-E_Δaa23-aa29 on MARC145 and LLC-PK1 cells were lowerthan those of the rPEDV-E_wt.RT-q PCR results showed that E_Δaa23-aa29 mutations did not affect viral genome RNA synthesis.At 24 h and 48 h,compared with rPEDV-E_wt strain,the viral copies in the supernatant of the rPEDV-E_Δaa23-aa29 infected cells decreased by 2 log10 and 3 log 10,respectively.Moreover,the ratio of supernatant titers to cell titers in rPEDV-E_Δaa23-aa29 infected cells was significantly lowerthan that in rPEDV-E_wt infected cells.These results suggested that E_Δaa23-aa29 mutation reduced virus release.The ratio of viral copies to viral infectivity titers in the rPEDV-E_Δaa23-aa29 group was significantly higherthan the rPEDV-E_wt group,which indicated that the E_Δaa23-aa29 mutation reduced viral infectivity in vitro.The results of RT-q PCR indicated that the E_Δaa23-aa29 mutation induced higherexpression levels of type I and III interferon（IFN）and interferon stimulating genes（ISGs）in MARC145 and LLC-PK1 cells.These results suggested that the E_Δaa23-aa29mutation does not affect synthesis of viral genomic RNA,but reduced viral release and infectivity and induced higherexpression of type I and type III IFN in vitro.3.Evaluation of pathogenicity and immunogenicity of the recombinant strain rPEDV-EΔaa23-aa29.Attenuating the interferon antagonism of the viruses is a common way to attenuate the viruses.Therefore,the virulence of the rPEDV-E_Δaa23-aa29 strain may be attenuated.Piglets at 2 days of age were inoculated 10⁶ TCID₅₀ rPEDV-E_wt and rPEDV-E_Δaa23-aa29,and all the piglets infected with rPEDV-E_wt had severe diarrhea（7/7,100%）and died（7/7,100%）within 9 days.Piglets infected with rPEDV-E_Δaa23-aa29 exhibited milderdiarrhea symptoms,lowerfecal viral load,and lowermortality（3/6,100%）.In addition,rPEDV-E_Δaa23-aa29induced less severe pathological damage to the small intestine.The results of fecal IFN-βdetected assay showed that piglets in the rPEDV-E_Δaa23-aa29 challenge group induced higherexpression levels of IFN-βon day 1,which suggested that the E_Δaa23-aa29 mutation induces a strongerimmune response in vivo.To evaluate the immunogenicity of the the rPEDV-E_Δaa23-aa29 strain,survival piglets were inoculated with 5×10⁶ TCID₅₀ of rPEDV-E_wtvirus at 21 days,and the results showed that rPEDV-E_Δaa23-aa29 can effectively induce the production of Ig G,Ig A and neutralizing antibody.Piglets in the rPEDV-E_Δaa23-aa29challenged group showed delayed and milderdiarrhea,and lowerfecal viral load than those in the Mock-challenged group,but no pigs in the two groups died.RT-PCR and sequencing results showed that E_Δaa23-aa29 mutations were genetically stable both in vitro and in vivo.These results indicated that rPEDV-E_Δaa23-aa29 is attenuated but retains immunogenicity,and the E_Δaa23-aa29 mutation is genetically stable both in vitro and in vivo.In summary,PEDV E protein is identified as a virulence factorand a type I and III IFN antagonist from the perspective of the whole virus,which enriches the pathogenic mechanism of PEDV and also provides a new target forthe development of PED live-attenuated vaccines.