| Porcine deltacoronavirus(PDCo V)is an emerging animal coronavirus in pigs causing diarrhea,vomiting,dehydration and have a higher incidence of infection in newborn piglets.PDCo V belongs to the newly members of deltacoronavirus genus in the Coronavirus family.PDCo V was initially reported in pigs in Hong Kong in 2012,and an outbreak of PDCo V was announced in some pig farms in the United States in 2014,the detection of PDCo V was reported subsequently in others states in the United States,Canada,Korea,and mainland China,Which caused higher economic losses to the pigs farms.There are currently no treatments or vaccines available for PDCo V.A local popular strain PDCo V CH-01 was isolated from pigs feces with diarrhea in Henan in this study,and the biological characteristics of this virus was investigated,and the genetic variation of the complete sequence of PDCo V CH-01 strain was analyzed.There are reported as following,1.Isolation and identification of PDCo V CH-01 strainThe feces samples were collected from pigs with diarrhea in Henan,and tested by conventional RT-PCR.Of the tested samples,all were negative for porcine epidemic diarrhea(PEDV),transmissible gastroenteritis virus(TGEV),reproductive and respiratory syndrome virus(PRRSV),pseudorabies virus(PRV),porcine circovirusvirus(PCV)and other swine enteric viruses,but only positive for PDCo V.The positive samples were filtered and inoculated on the LLC-PK and ST cells for virus isolation.A typical cypathic effect(CPE)was observed on ST and LLC-PK cells at 80 h post-infection(hpi),the cells and supernatants were harvested together(named as P0).The isolated strain is negative for other swine enteric viruses by RT-PCR assay.For serial passage on ST and LLC-PK cells,a typical CPE were characterized by rounded,clustered,and eventually detached from the monolayer on ST cells on P0,while it were showing rounded and clustered cells on LLC-PK cells on P0.After four passages the CPE is stably in the serial passage.The indirect immunofluorescence assay veified that the infectious-cells were PDCo V positive by using hypermmune pig antiserum against PDCo V,showing large numbers of IF-stained cells.All the above conformed the PDCo V CH-01 is successfully isolated in LLC-PK and ST cells.Plaque assay on the ST cells idendified the titer of CH-01-P5 is 1.23×107PFU·m L-1.The positive clones were picked for further passage,and infectious-virus titrations of P7,P15,P17,P30(TCID50)were measured,and ranged from 7.0-8.25 lgTCID50/0.1 m L.2.The biolgical characteristics of CH-01 strainThere are few reported about the biolgical characteristics about PDCo V.In order to study the virus growth curve,different MOI(10-2,10-4,10-6)of PDCo V CH-01 P30 strain were inoculated on the LLC-PK and the cells and supernatants were harvested together at 6,12,24 and 36 h for real time fluorogenic quantitative PCR(q RT-PCR),the results showed that the viral RNA level is the highest at 24 hpi(11.27 log10GE/m L)after different MOI infected cells,and then began to decline;The TCID50 results showed that the infectious-virus titrations is also the highest at 24 hpi(12.50 lg TCID50/0.1m L)after different MOI infected cells,and then began to decline.A series of physicochemical properties of CH-01 were explored,the results showed that the infectious-virus titrations of CH-01 with treatment at 37,42,50 for 30 min?,all decreased for 1-2 TCID50 and the virus could be totally inactivated with treatment at 65 and the ultraviolet for 30 min.The virus is ?sensitive to both acidic(p H3.0)and basic(p H9.0)environments at 37 ?,and which was thoroughly inactivated at p H9.0.The virus also exhibited sensitivity to some organic reagents such as diethyl ether and chloroform,the virus could be inactivated with 4.8% chloroform treatment.The hemagglutination test(HA)showed that the CH-01 did not induce appreciable HA activity against erythrocytes from chicken at 4 ?,37 and indoor environment,even trypsin treatment also did not ?induce appreciable HA activity.3.The complete sequence sequencing and phylogenetic analysis of CH-01 strainIn order to understand examine if genetic changes occurred in the PDCo V CH-01 during serial passages in cells cultures,refer to complete genome sequence of PDCo V HKU15-155(JQ065043)from Genbank,13 pairs of primers were designed to determinate the complete suquence of CH-01-P5 strains by RT-PCR segmented amplifying.The results showed that CH-01-P5 had the highest amino acid similarity with the field strain CH-01(100%),there were no nucleotide and amino acid changing between them,the cell-adapted passage CH-01 did not mutated,which was related to low cell passage.The phylogenetic analysis of CH-01-P5 strain complete sequence showed that 19 reference strains of PDCo V divided into two classes: Group 1 and Group 2.PDCo V strain CH-01-P5 and CH-01 shared 100% nucleotide identities with each other,they were in the Group 1 with PDCo V strains of China,United States and Korea.The amino acid homology of the complete sequence between CH-01-P5 strain and other PDCo V strains is 98.6%-100%.The PDCo V CH-01-P5 strain had a closer relationship with Hong Kong strain and other strains in china,and was less close to the PDCo V strains of United States and Korea,but is far to the PDCo V strains of Thailand?Vietnam and Lao.The analysis of CH-01-P5 strain structural gene sequence revealed that the amino acid homology of the M gene is 98.9%-100%,the N gene is 97.1%-100%,the S gene is 95.9-99.9%;and there were no amino acid in M,N and E gene,but S gene of CH-01-P5 strain had 3 bases deletion,and 12 bases mutation,of which had 5 amino acid mutation,it need to be further studied if the PDCo V CH-01-P5 strain in Henan has been changed.In the all,a PDCo V strain named CH-01 has been isolated successfully in this study,and the biolgical characteristics of PDCo V CH-01 strain was analyzed including the viral growth kinetics,physicochemical properties and phylogenetic,this study provide theoretical basis for the prevention and development of vaccines against PDCo V strain. |
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