Genomic Characteristics And Pathogenicity Analysis Of Different Passages Of Porcine Deltacoronavirus NH Strain

Porcine deltacoronavirus(PDCoV)is a new coronavirus which can cause swine diarrhea in recent years.PDCoV was first reported in Hong Kong in 2012.It was detected in US herds in 2014 and quickly spread to more than 20 US states and Canada.Watery diarrhea,vomiting,dehydration and death are the main clinical symptoms.It has caused huge economic losses to the pig industry.Therefore,isolating the virus and analyzing its genetic evolution characteristics and pathogenicity differences during cell passaging will help to study the molecular mechanism of PDCoV attenuation and have great significance for the development of attenuated vaccines.In this study,the virus titer and plaque size of PDCoV NH strain passaged in ST cells were measured.The results showed that the virus titer increased with the increase of the passage.The plaques produced by low-generation passage were small,and the plaques produced by high-generation passage were large.There was no significant difference in plaque size after passaging 50 times in cell.Using 19 pairs of primers,complete genome sequence of NH-P10,NH-P20,NH-P30,NH-P40,NH-P50,NH-P60,NH-P70,NH-P80,NH-P90,NH-P100,NH-P110,NH-P120,NH-P130,NH-P140,NH-P150,and NH-P160 were sequenced.The results indicated that the NH strain did not have base deletion or insertion but nucleotide and amino acid mutations during serial passage in vitro.These nucleotide and amino acid mutations were scattered in different regions of the genome and mainly in S gene.The results showed that the virulence of the NH strain might be the result of multiple amino acid mutations rather than a single amino acid mutation.Based on the whole genome sequencing results,NH-P10,NH-P50,NH-P100 and NH-P150 were selected to conduct a challenge experiment on 3-day-old healthy piglets to verified whether NH had been attenuated during serial passage.The results showed that both the NH-P10 and NH-P50 challenged groups had severe diarrhea.The NH-P100 challenged group of 1 piglet had transient soft feces,and then returned to normal.The NH-P150 challenge group showed no abnormal performance.Post-mortem examination of piglets in each group revealed that the intestinal wall of the small intestine in the NH-P10 and NH-P50 challenge groups became thinner,permeable,and filled with yellow water-like feces.One piglet in the NH-P100 challenge group showed that the intestinal wall of the small intestine became thinner and filled with yellow liquid.The other had no visible change.The NH-P150 challenge group and the control group were normal.Histopathological examination results showed that the small intestinal villi of NH-P10 and NH-P50 challenged piglets shed.NH-P100 and NH-P150 showed no clear histologic lesions.IHC results showed that the viruses in the NH-P10 and NH-P50 challenge groups were mainly distributed in small intestinal villi of jejunum and ileum,and no viruses were detected in the NH-P100,NH-P150 and control groups.Fluorescence quantitative PCR was used to determine the viral load from the intestine to the end of each group.The results showed that NH-P10 and NH-P50 could detect viral nucleic acid in each intestinal segment and the viral RNA were high.NH-P100 and NH-P150 could also detect viral nucleic acid,but the viral RNA were low.Besides the amino acid positions of 182,396,616,707 and 797,the sequence of the S protein was consistent with the amino acids mutations in vitro and in vivo.Compared with the low-passaging strains(NH-P10 and NH-P50),the two high-passaging strains(NH-P100 and NH-P150)had two potential glycosylation sites disappear due to amino acid mutations at positions 162 and 168 and caused the antigen index mutation at the corresponding position according to the predicted results of N-glycosylation site and antigenicity of S protein.In conclusion,this study analyzed the evolutionary characteristics of the whole genome of the NH strain during the passaging on ST cells.Gene mutations increased with serial passaging.Mutations mainly occurred on S gene.Animal experiment indicated the virulence of high-passageing strains were attenuated or non-pathogenic to suckling piglets.The above research has laid the foundation for studying the molecular mechanism of PDCoV weakening and developing PDCoV attenuated vaccine.