Proteome Analysis Of Pedv-infected Vero Cells And Attenuation Of Pedv By Serial Passage In Vero Cells

Porcine epidemic diarrhea is caused by porcine epidemic diarrhea virus(PEDV)with vomiting,dehydration,metabolic acidosis and high mortality rate in neonatal suckling pigs.The disease was reported as early as 1970s in the UK and Belgium.In the next 30 years,PED is mainly outbreak in Asia and Europe,result in great economic losses to the pig industry.In China,because the TGEV-PEDV bivalent inactivated vaccine and TGEV-PEDV bivalent live vaccine is widely used,which is very important to control the infection of PEDV.However,since the end of 2010,due to the emergence of PEDV variant strain caused diarrhea outbreak in China and presents new characteristics:the high morbidity,high mortality and with a widely range of disease outbreak.Even if the pig are not escape by immuned.In 2013,PEDV variant strain occured in the United States,Canada and so on which had never happeded it ago and it caused a large number of mortality when disease onsets.PED has caused significant economic losses to the pig industry.At present,understanding the pathogenic mechanism of PEDV variant strain is very limited,and the variant strain vaccine has become the top priority to control the infection of PEDV variants.Therefore,this study is attempt to analyze the protein profile of PEDV-infected cells by quantitative proteomics,and evaluation of PEDV in Vero cell continuous passage of virulence and immunogenicity.The specific contents are as follows:1.Proteomic analysis on PEDV-infected host cellsQuantitative proteomic methods are widely accepted tools with which to study virus-host cell interactions.In this study,isobaric tags for relative and absolute quantification(iTRAQ)approach was used to identify the di?erentially expressed proteins in Vero cells after PEDV infection.In total,49 di?erentially regulated proteins were identified,including 8 upregulated proteins and 41 downregulated proteins.A bioinformatic analysis showed that these di?erentially expressed cellular proteins were involved in apoptosis,signal transduction,and stress responses.Based on these differentially expressed proteins,we propose that PEDV might utilize apoptosis and extracellular signal regulated kinases pathways for maximum viral replication.We analyzed expression levels of HSDL2,14-3-3 gamma,and HSP27 in PEDV-infected Vero cells by Western blotting.The ratios of these three proteins between infected and uninfected cells were consistent with those determined using the iTRAQ labeled LC-MS/MS system.We believe our findings provide valuable information with respect to better understanding the host response to PEDV infection.2.Analysis of PEDV strains AJ1102 of different passages of the whole genome sequencePEDV strains AJ1102 was passed serially in Vero cell to the 120th generation,and the complete genome sequence analysis of P11,P40,P70,P100 and P120 found that their complete genome sequences length is 28042bp,28042bp,28054bp,28051 bp and 28051 bp respectively,sequence alignment revealed that PEDV non-coding regions,non-structural protein gene and S gene mutation rate was obviously higher than the other gene mutations in the process of passaging and mutations mainly occurred in P1 to P11.It is worth noting that two characteristic mutations occurred during the passage.One appeared in the generation P70:the inserted 12 base between 3087-3088 base of gene S resulted in the insertion of four amino acids between 1269-1270 amino acids(AIVD);Another characteristic mutation appeared in the generation P100:the defecting bases of 669-671(TTT)of gene M caused that the 223 histidine mutated to Glutamine and 224 lutamine is missing.The two characteristic mutations are stable until P120.3.The pathogenic analysis for different passages of the PEDV strains AJ11023-5 day-old and 24-26 days-old piglets were inoculated orally by P11,P40,P70,P100 and P120 PEDV strains AJ1102.Animal experiment show that:more than 80%of the 3-5 days old and 24-26 days old piglets diarrhea caused by P11 and P40,and P11 challenge group lead the death of one 3-5 days old piglet,while the three passages P70,P100 and p120 challenged groups does not cause 3-5 days-old and 24-26 days-old piglets diarrhea,indicating that the virulence in p70 has been greatly reduced.4.The immunogenic analysis for different passages of the PEDV strains AJ1102Piglets were inoculated by P40,P70,P100 and P120 of PEDV strains AJ1102 though muscle,we found that:the P70 immune group produce neutralizing antibody earliest,all immunized Piglets' neutralizing antibody reached above 1:19.0(1:19.0 to 1:27.0)in 21 days;P100 and P120 vaccinated piglets antibody were lower than 1:11.3 which were significantly lower than P70 immune group indicated that the immunogenicity of P100 and P120 were declined.Although the immunogenicity of P100 and P120 declined,the piglets also can obtain protection after virus challenged,this may due to the other immunity induced by viral immuned piglets played an important role during virus challenged.5.The effect on virulence of the insertion of PEDV strain AJ1102 S geneAs the virulence of PEDV strains AJ1102 declined significantly after passaged 70 generation.Compared the genome of P70 with those of P11 and P40,one characteristic variation is that S gene has four amino acids(AIVD)insertion between 1269-1270,and insertion maintains in the subsequent passages,we speculate that the four amino acid insertion may cause the virus virulence decline.In order to confirm this assumption,first of all,we found that4 amino acids insertion first appeared In P55 through sequencing the S gene of each generation virus.Then we cloned P55 without 4 amino acids insertion and P55-In with four amino acid insertion.Animal experiments conformed that,compared to the P11,P55 become less virulent but still be strongly pathogenic to 3-5 days-old piglets,and P55-In was non-pathogenic to 3-5 days-old piglets,which demonstrated that the insertion of four amino acid in S gene is an important for declining the virulence.6.The deletion of PEDV strain AJ1102 M gene effect on immunogenicityIn order to insure the deletion of 669-671 in gene M of PEDV strains AJ1102 whether effects the immunogenicity of the virus.We first determined the lack of three bases appear in the 82th generation by RT-PCR and sequencing,we cloned P82 without the deletion and P82-Del with the deletion of the three bases.Then we passed the two clones to 100 generations respectively.The immunogenicity comparating experiment confirmed that,several piglets in P70,P82,P100 immune groups produced low levels of neutralizing antibodies after immuned 7 days.The antibodies of all the immunized piglets were reached above 1:19(1:19 to 1:27)after immuned 21 days.However P82-Del and P100-Del immune group produce neutralizing antibody relatively late.The part of piglets produces low levels of neutralizing antibodies after immuned 14d and the piglets antibody were no significantly increased(all<1:13.5)after immuned 21 days.But all the immune groups can protect against PEDV strains AJ1102 challenged.It was indicatde that the deletion of the three bases in the M gene has a certain extent effects on immunogenicity of virus,and also illustrating that the other immune responses induced by virus plays a very important role in protecting immuned piglets.