Preliminary Evaluation Of Immune Response To Porcine Deltacoronavirus And Proteomic Analysis Of Infected LLC-PK Cells

Porcine deltacoronavirus?PDCoV?is a newly emerging of enteric coronavirus in swine that can infect pigs of different ages,but neonatal piglets are more susceptible.After infection,pigs mainly shows severe diarrhea,vomiting,dehydration and other clinical symptoms.PDCoV was first reported in Hong Kong,China in 2012.However,the outbreak of PDCoV occured in Ohio,the United States in 2014.Subsequently,it spread quickly in other pig states in the United States,resulting in 30-40%mortality of neonatal piglets.In recent years,PDCoV has been prevalent in pig farms in many countries,including China,Canada,Japan,South Korea,Thailand,Mexico and other countries.At present,due to the unclear transmission and pathogenic mechanism of PDCoV,and the lack of effective vaccine and prevention and control measures,incurring enormous huge economic losses to many countries and still a severe challenge to the pig industry.Based on this,the research carried out the following research on PDCoV,in order to improve the rapid diagnosis of PDCoV,lay the foundation for clarify its pathogenesis and vaccine research and development.1.Establishment of PDCoV LFD-RPA rapid diagnosis methodSpecific primers and probes were designed according to the published conserved sequence of PDCoV N gene?GenBank accession No:KJ481931?,and a method for the detection of PDCoV was established by recombinase polymerase amplification combined with a lateral flow dipstick?LFD-RPA?.The results show that LFD-RPA can be performed under a broad range of temperature conditions from 10³7?,and the detection of PDCoV can be completed in 20 min at 37?.After verification,the detection limit of this method is 1×10² copies/?L,and PDCoV can be detected specifically,showing good sensitivity and specificity.Meantime,the results of inter-and intra batch repeatability test confirmed that LFD-RPA had good repeatability.In addition,the validity of LFD-RPA was evaluated by detecting 68 clinical diarrhea samples?48 fecal swab specimens and 20 intestinal specimens?.The results showed that the positive rates of PDCoV detected by LFD-RPA and RT-PCR were 26.47%?18/68?and 23.53%?16/68?,respectively,indicating that LFD-RPA detection method is effective and accurate.2.Isolation,identification and biological characteristics of PDCoV strainA cell-adapted strain CH/XJYN/2016?GenBank accession No:MN064712?was successfully isolated from pig diarrhea samples collected in Xinjiang Uygur Autonomous Region,China by using LLC porcine kidney?LLC-PK?cells.The typical cytopathic effect?CPE?began to appear when the strain was blinded to P5generation in LLC-PK cells.At present,CH/XJYN/2016 strain has been stably passed on to P140 generation,and the virus title can reach 10⁷.8.8 TCID₅₀/ml.By indirect immunofluorescence assay,PDCoV antigen showed green fluorescence.At the same time,it was observed that PDCoV particles were oval and had a typical"crown"like structure by transmission electron microscop.The whole genome sequence of CH/XJYN/2016 strain was obtained by RT-PCR,with a total length of 25 833 nt.Sequence analysis showed that the S gene of CH/XJYN/2016 strain had97.70-98.74%nucleotide homology with that of other PDCoV isolates,of which the genetic distance was the closest to HKU15-44?GenBank accession No:JQ065042?in Hong Kong,China,with 98.74%nucleotide homology.Compared with other reported PDCoV strains,37 single nucleotide substitutions and 3 base insertions were observed in S gene of CH/XJYN/2016 strain.In addition,phylogenetic tree analysis of S gene showed that CH/XJYN/2016 strain was closely related to PDCoV strains isolated from China,the United States,Japan and South Korea,but far from PDCoV strains isolated from Thailand,Vietnam and Laos,suggesting that PDCoV strains isolated from China,the United States,Japan and South Korea may all originate from the same ancestor.3.Pathogenicity analysis of PDCoV on piglets of different ages and preliminary evaluation of immune responseCH/XJYN/2016 strain P6(10⁴ TCID₅₀/mL)was inoculated orally to 4-day-old piglets,which could cause diarrhea,vomiting,depression and other clinical symptoms after 1² days post-infection?dpi?.After dissecting the infected piglets,It was found that the intestine of infected piglets was seriously damaged,especially the small intestine,which was congested,thin and filled with yellow liquid.Histopathological results showed that the intestinal villi were shortened,fused and shed.In addition,the results of immunohistochemistry confirmed that PDCoV antigen was mainly present in intestinal villi cells.In conclusion,PDCoV CH/XJYN/2016 strain showed high pathogenicity to newborn piglets.PDCoV CH/XJYN/2016 strain P6 virus culture was inoculated orally to45-day-old piglets.In G1-G4 infected group(10⁴-10¹ TCID₅₀/ml),diarrhea and virus shedding occurred in succession within 3-14 dpi.The serum levels of PDCoV specific IgG,IgA and virus-neutralization?VN?antibody and IFN-?were exhibited significantly increased after first challenge?P<0.05?.However,at 21 dpi,all piglets returned to normal,and the piglets were inoculated with CH/XJYN/2016 strain P6orally again.All pigs were completely protected against rechallenge at 14 dpi.The serum and saliva levels of PDCoV specific IgG,IgA,and VN antibody increased further after rechallenge.Notably,the serum IFN-?antibody levels in pigs increased significantly during the first 7 dpi of inoculation,but then decreased continuously.Meanwhile,the median pig diarrhea dose(PDD₅₀)of PDCoV in conventional weaned pigs was determined(2.0log₁₀PDD₅₀/3mL).In addition,an inactivated cell-adapted CH/XJYN/2016-based vaccine was developed with aluminum adjuvant and 206adjuvant,and pigs were inoculated.It was found that compared with nonvaccinated pigs,weaned pigs given the inactivated vaccine developed a potent humoral immune response and showed no clinical signs or viral shedding after challenge,indicating a potent protective effect of the vaccine against PDCoV infection.4.Proteomic analysis of LLC-PK cells infected by PDCoVUsing liquid chromatography tandem-mass spectrometry?LC-MS/MS?combined with tandem mass tag?TMT?labeling was performed to compare the expression of differential protein in the PDCoV-infected and mock-infected LLC-PK cell at 18 h postinfection?hpi?.A total of 275 differentially expressed proteins?DEPs?were identified,of which 128 were significantly up-regulated and 147 significantly down-regulated.Bioinformatics analysis revealed that those DEPs were mainly involved to defense response,apoptosis and immune system.Base on DEPs bioinformatics analysis,indicating that PDCoV infection may utilize the apoptosis pathway of host cells to achieve maximum viral replication.Meantime,the host may be able to stimulate the transcription of the interferon-stimulated genes?ISGs?though the JAK/STAT signaling pathway to resist the virus.Furthermore,parallel reaction monitoring?PRM?was used to quantitatively verify the candidate proteins.The results showed that it was consistent with the quantitative data of TMT,which further confirmed the validity and accuracy of proteomics data.