Genetic Variation Regularity And Type? Interferon Escaping Mechanism Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea(PED)is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV).The clinical characteristics within infected-piglets show severe diarrhea,dehydration.And it leads to severe atrophic enteritis,and higher morbidity and mortality.Since it was first reported in 1971 in England,it had spread to Europe,Asia,America and Australia,causing significant economic losses to the global pig industry.In the process of clinical spread,PEDV genome underwent continual mutation and evolution to strengthen its pathogenicity,which caused that the current vaccine could not provide effective immune protection,thus showed a great difficulty of PEDV prevention and control in the swine industry.In addition,PEDV infection could significantly inhibit the transcription and expression of interferon,and then escaped the innate immune response.In this study,we mainly focus on the exploration of genetic variation and evolution regularity of PEDV,identification of the functional site involving the virus infection,and investigation of the immune escape mechanism,which will help to lay the basis for the effective prevention and control of this disease.1 Epidemiological and genetic characteristics of porcine epidemic diarrhea virusPorcine epidemic diarrhea has become pandemic in the Asian pig-breeding industry,causing significant economic loss.In the present study,11 complete genomes of porcine epidemic diarrhea virus(PEDV)field isolates from China were determined and analyzed.Frequently occurring mutations were observed,which suggested that full understanding of the genomic and epidemiological characteristics is critical in the fight against PEDV epidemics.Comparative analysis of 49 available genomes clustered the PEDV strains into pandemic(PX)and classical(CX)groups and identified four hypervariable regions(V1 to V4).Further study indicated key roles for the spike(S)gene and the V2 region in distinguishing between the PX and CX groups and for studying genetic evolution.Genotyping and phylogeny-based geographical dissection based on 219 S genes revealed the complexity and severity of PEDV epidemics in Asia.Many subgroups have formed,with a wide array of mutations in different countries,leading to the outbreak of PEDV in Asia.Spatiotemporal reconstruction based on the analysis suggested that the pandemic group strains originated from South Korea and then extended into Japan,Thailand,and China.However,the novel pandemic strains in South Korea that appeared after 2013 may have originated from a Chinese variant.Thus,the serious PED epidemics in China and South Korea in recent years were caused by the complex subgroups of PEDV.The data in this study have important implications for understanding the ongoing PEDV outbreaks in Asia and will guide future efforts to effectively prevent and control PEDV.2 Identification of two mutation sites in spike and envelope proteins mediating optimal cellular infection of porcine epidemic diarrhea virus from different pathwaysEntry of the ?-coronavirus porcine epidemic diarrhea virus(PEDV)requires specific proteases to activate spike(S)protein for the membrane fusion of the virion to the host cell following the receptor binding.Herein,PEDV isolate 85-7 could proliferate and induce cell-cell fusion in a trypsin independent manner on Vero cells,and eight homologous mutation strains were screened by continuous proliferation in the absence of trypsin on Vero cells.According to the whole genome sequence comparative analysis,we identified four major variations located in nonstructural protein 2(Nsp2),S,open reading frame 3(ORF3),and envelope(E)genes,respectively.Comparative analyses of their genomic variations and proliferation characteristics identified a single mutation within the S2'cleavage site between C30 and C40 mutants:the substitution of conserved arginine(R)by a glycine(G)(R895G).This change resulted in weaker cell-cell fusion,smaller plaque morphology,higher virus titer and serious microfilaments' condensation.Further analysis confirmed that this mutation was responsible for optimal cell-adaptation,but not the determinant for trypsin-dependent entry of PEDV.Otherwise,a novel variation(16-20 aa deletion and an L25P mutation)in the transmembrane domain of the E protein affected multiple infection processes,including up-regulating the production of the ER stress indicator GRP78,improving the expression of pro-inflammatory cytokines IL-6 and IL-8,and promoting apoptosis.The results of this study provide a better understanding of the potential mechanisms of viral functional proteins in PEDV replication,infection,and fitness.3 Down-regulating heat shock protein 27 is involved in porcine epidemic diarrhea virus escaping from host antiviral mechanismPorcine epidemic diarrhea virus(PEDV),is the etiological agent of porcine epidemic diarrhea virus(PED),causes high mortality with severe vomiting,diarrhea,and dehydration in swine farms.In this study,the PEDV strain 85-7 could be proliferated effectively in MARC-145 cells,and caused a distinct inhibition of the expression of interferon-?(IFN-?,encoded by IFNB1),which suggested that a full understanding how this virus manipulates the host immune responses is critical in the fight against the spread of PEDV We found that,the infection of PEDV strain 85-7 significantly downregulated HSP27 production in MARC-145 cells,and overexpression of HSP27(encoded by HSPB1)decreased the virus titer by about 28-fold.Further study revealed that HSP27 could significantly activate the NF-?B phosphorylation,and thus increase the mRNA level of IFNB1 and downstream interferon-stimulated genes(ISGs)in MARC-145 cells.Indeed,treatment with IFN-? and the ISGs,including murine myxovirus resistance 1 and 2(Mxl and Mx2),showed direct anti-PEDV activity.Notably,the antiviral activity and transcription of the antiviral effectors induced by overexpression of HSP27 could be counteracted by the knockdown of IFNB1 via RNA interference,indicating that HSP27 was an upstream regulator of the intracellular antiviral effect against PEDV infection.This study is the first to link HSP27 to PEDV replication via the innate immunity response,which contributed to further clarify the mechanism of PEDV infection and the development of novel antiviral therapies.4 The identification of interferon-inducing characteristics in a PEDV variant strainIn previous study,the PEDV 85-7 parent strain was isolated and its eight homologous mutation strains were screened by continuous proliferation on Vero cells.Of them,the S2'mutant(C40)induced weaker cell fusion ability,and caused significantly increasing of the virus titer in Vero cells.While compared with the parent strain,the virus titer of C40 strain was significantly decreased within MARC-145 cell model,suggesting that it may be closely related to the interferon secretion pathway and S2' site mutation.By mRNA and LncRNA sequencing,we received differential expression profiles of the C40 strain compared to 85-7 parent strain before and after infection within MARC-145 cells by transcriptomics analyses.In addition,the above RNA-seq results were verified by qPCR assay,which showed consistent expression levels with each other.From the results,we found these differential genes could be involved in the macromolecules transcriptional modification(such as nucleic acid),cell membrane,organelles and other cell components'expression.Compared with parent strain,C40 variant strain could specifically regulate the expression of HSPA12B,DNAJC1 and DNAJC24,and significantly up-regulate the transcription of IL6,CXCL9 and IL12A.It is also significant different with parent strain that C40 strain initiated the transcription of IFNB1,IFNL1 and IFNL3,and greatly increased the transcriptional expression of the corresponding downstream interferon-stimulating gene(ISGs),which in turn led to a significant decrease in the virus titer.Meanwhile,C40 could specifically regulate 16 LncRNA transcription compared with that of parent strain,further studies had shown that LncRNA-L6 and LncRNA-L7 potential targeted the anti-PEDV genes IF144 and IL12A,respectively,and thus negatively regulated PEDV proliferation.This study showed that the specific mutation of PEDV specific gene could regulate the replication of PEDV by transforming the cellular antiviral immune response and LncRNA,and then provided the basis for the screening of its antiviral molecules,and illustrating the infection mechanism.