| PDCo V is a member of the genus Deltacoronavirus of Coronaviridae,which belongs to the order Nidovirales.It is a novel porcine coronavirus,which mainly infects newborn piglets and causes diarrhea,vomiting,dehydration and even death.Clinically,PDCo V(Porcine deltacoronavirus,PDCo V)usually co-infect with PEDV(Porcine epidemic diarrhea,PEDV),TGEV(Transmissible gastroenteritis virus,TGEV)and PRo V(porcine rotavirus,PRo V).Since the first outbreak in the United States in 2014,PDCo V has rapidly spread to other countries around the world,causing massive economic losses to the global pig industry.Coronavirus not only encodes structural proteins and non-structural proteins involved in genome transcription and replication,but also encodes a series of special accessory proteins.Current studies showed that most of the accessory proteins are not necessary for virus replication,but may affect the pathogenicity of viruses.PDCo V,as the only deltacoronavirus successfully isolated in vitro,has been identified to encode three accessory proteins NS6,NS7 and NS7 a.Existing studies have shown that these three accessory proteins are involved in the regulation of the host's natural immune response,but their impact on virus replication and pathogenicity are still unclear.In view of this,this study successfully isolated the epidemic strain CHN-HG-2017 of PDCo V,established the first reverse genetic system of PDCo V in the world,and revealed the role of accessory protein of PDCo V in virus replication and pathogenicity for the first time.The main contents are as follows:1.Genomic characterization and pathogenicity of porcine deltacoronavirus strain CHN?HG?2017 from ChinaA PDCo V strain,named CHN-HG-2017,was successfully isolated from PDCo Vpositive feces of diarrhea piglets.The isolated virus was identified by indirect immunofluorescence and transmission electron microscopy.The alignment of nucleotide sequences showed that the whole genome of CHN-HG-2017 shared 97.6%-99.1%homology with other PDCo V strains.Recombination detection by RDP4 for whole genome sequences showed that that CHN-HG-2017 may be a recombinant strain of Chinese strain CH/SXD1/2015 and Vietnamese strain Vietnam/Ha Noi6/2015.Furthermore,animal experiments confirmed that PDCo V CHN-HG-2017 strain had strong pathogenicity to 5-day-old piglets,and typical symptoms such as diarrhea,vomiting,anorexia and lethargy appeared in piglets from 1-7 days post inoculation(DPI).Viral shedding was detected in rectal swabs until 14 DPI in the challenged piglets.Interestingly,high titers of virusneutralizing antibodies in sera were detected until 21 DPI.2.Generation of a full-length infectious c DNA clone of PDCo V strain CHN-HG-2017The full-length genome of PDCo V CHN-HG-2017 strain was divided into six constinuous fragments,and the full-length c DNA clone of the virus was obtained by in vitro ligation.After in vitro transcription and electroporation,the virus was successfully rescued.Comparing the rescued PDCo V with the wild-type PDCo V,the whole genome sequences of the two viruses were identical except for molecular markers,and they could produce the same cytopathic effect on LLC-PK1 cells.Further IFA and western blot assay showed the protein expression level of rescued PDCo V and wild-type PDCo V was equivalent.In addition,the two viruses generated nearly alike size plaques in LLC-PK1 cells and similar growth pattern were observed both in LLC-PK1 cells and IPI-2I cells.These results indicated that we successfully established the reverse genetic system of PDCo V,which laid a foundation for studying the role of virus-encoded protein in virus replication and pathogenesis.3.The role of PDCo V accessory protein in virus replication and pathogenicityThe recombinant PDCo V lacking NS6 or NS7 genes was successfully rescued by the established PDCo V reverse genetic system.The PDCo V with NS6 or NS7 deletion could still replicate in LLC-PK1 cells,which indicated that NS6 and NS7 were not necessary for the replication of PDCo V.Growth kinetics studies suggest that r PDCo V-?NS7 could replicate similarly to that of the wild-type PDCo V,whereas r PDCo V-?NS6-GFP exhibited a substantial reduction of viral titer,which indicated that the PDCo V accessory protein NS6 might play a key role in promoting virus replication.Meanwhile,animal studies demonstrated that piglets inoculated with r PDCo V-?NS7 or wild-type PDCo V showed similar diarrhea score and pathological damage.However,piglets infected with r PDCo V-?NS6-GFP did not show any clinical symptoms,indicating that NS6 protein is an important virulence factor of PDCo V and that the NS6-deficient mutant virus might be a promising live-attenuated vaccine candidate.In addition,we engineered a recombinant PDCo V with high expression of green fluorescent protein,which can be used as a tool for high-throughput drug screening and neutralizing antibody detection.4.Exploring the mechanism of PDCo V accessory protein NS6 affecting virus replication and pathogenicityTo further analyze the mechanism of PDCo V accessory protein NS6 affecting virus replication and pathogenicity,PDCo V NS6 was co-transfected into HEK-293 T cells with other structural proteins and accessory proteins NS7 and NS7 a.The results of immunoprecipitation showed that PDCo V accessory protein NS6 interacted with accessory proteins NS7 and NS7 a.The Flag tag was successfully inserted into the c-terminal of virus accessory protein NS6 by reverse genetic system,and the PDCo V recombinant virus expressing NS6-Flag was obtained.After infecting LLC-PK1 cells with recombinant virus,126 host proteins which may interact with accessory protein NS6 were identified by immunoprecipitation and mass spectrometry.GO annotation showed that most of the screened proteins were cytoskeletal proteins,and it was speculated that NS6,an accessory protein,might regulate the combination of virus and host cells,invasion and release by interacting with cytoskeletal related proteins,thus affecting the replication ability and pathogenicity of virus. |
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