Epidemiological Investigation Of Canine Coronavirus Infection In Northeast China,2019-2021

Canine coronavirus(CCoV)can cause gastroenteritis symptoms of varying degrees in dogs,which mainly causes acute diarrhoea in puppies and sometimes even death in dogs.CCoV has a high clinical detection rate,which seriously threatens the health of dogs.In recent years,with the continuous expansion of China’s pet industry,CCoV has spread widely in Chinese pet dogs,causing harm to the healthy development of China’s pet industry.The host range and epidemic range of CCoV continue to expand,and the epidemic strains also continue to change.It is of great significance to strengthen the epidemiological surveillance of CCoV and keep track of the epidemic and genetic variation of CCoV for comprehensive prevention and control of CCoV.In this study,325 dog feces and swab samples were collected from eastern Inner Mongolia,Heilongjiang province,Jilin province,and Liaoning province.Through RT-PCR detection,sequence determination and epidemiological analysis,the current epidemic canine coronavirus strains and evolution characteristics in northeast China were studied,and coinfection detection and virus isolation were carried out.Of 325 samples,17.5%(57/325)were positive for CCoV.The prevalence of CCoV in dogs varies significantly by age,season,immunization status,and region.CCoV infection occurred in both adult and young dogs,and 82.5% of CCoV infection mainly occurred in young dogs.The positive rate of CCoV was higher in winter and spring.CCoV was detected in 37 non-immunized dogs(64.9%),10 incomplete immunized dogs(17.5%)and 10 fully immunized dogs(17.5%).In terms of regional distribution,the positive rate was higher in Liaoning Province.In 57 positive samples,35.1%(20/57)of CCoV-Ⅰ 38.6%(22/57)of CCoV-Ⅱa and 0%(0/57)of CCoV-Ⅱb were infected alone.Mixed infection of CCoV-I and CCoV-IIa accounted 12.3%(7/57),CCoV-Ⅰ and CCoV-Ⅱb mixed infection was not detected(0/57),and CCoV-Ⅱa and CCoV-Ⅱb mixed infection accounted for 12.3%(7/57).CCoV-Ⅰ,CCoV-Ⅱa and CCoV-Ⅱb mixed infection accounted for 1.8%(1/57).Sequence alignment and phylogenetic analysis of the M and S gene sequences showed that the CCoV-I and CCoV-II strains of canine coronavirus currently circulating in northeast China had genetic diversity compared with the reference strains at home and abroad.Sequence analysis showed that the nucleotides of M gene of different genotypes were highly similar and conserved.Phylogenetic analysis of the non-recombination sequences of CCoV M gene showed that two main CCoV genotypes could be identified according to MCC and NJ trees: CCoV-I and CCoV-II.CCoV-Ⅰ of the M gene was more closely related to the Brazilian strain.The CCoV-II had a high similarity with the reference strain in Nanjing,but had a distant relationship with the reference strains in other provinces,suggesting that they were not homologous strains or had mutated during transmission.The S gene was highly variable.The nucleotide homologies and amino acid homologies of 11 strains of CCoV-I S gene were93.4%-100% and 98.9%-100%,respectively.The nucleotide and amino acid homologies of the 8 strains CCoV-IIa S genes were 93.5%-100% and 95.2%-100%,respectively.The S gene varied greatly among different genotypes,but the same genotypes had high similarity,and S gene had high viral diversity.In all identified CCoV-I isolates,only one amino acid in S protein was replaced.C→A and A→G mutations were found in all 8 CCoV-IIa strains in this study,which were located at position 457 and 505,respectively.The mutation frequency of CCoV-Ⅱa strain is high,and the effect of mutation is still unknown,which deserves attention.Both the NJ tree and the MCC tree of S gene showed had a close relationship with Brazil,and the CCoV-IIb showed high affinity with strains from European countries.The CCoV-IIa strain was closely related to the Chinese reference strain MT114554,and was significantly different from other domestic and foreign reference strains.The co-infection rate of CCoV,Ast V(25 cases,43.9%)and CDV(14 cases,24.6%)was higher,followed by CPV(11 cases,19.3%)and CNV(1 case,1.8%),CCoV was mainly infected with CDV and Ast V in Heilongjiang province.CCoV was mainly mixed with CPV and Ast V in Jilin Province.The mixed infection rate of CCoV,CDV,CPV and Ast V was high in Liaoning Province.One CDV was detected in the eastern part of Inner Mongolia Province,but no other pathogens were detected.Subsequently,fresh feces samples of CCoV dogs with single infection were isolated and identified.MDCK was used to isolate the virus.Three strains of the samples were passed on the cells by virulent passage and adding new cells to the sixth generation,and CPE appeared in each generation.All the results were CCoV-Ⅱa.Data from this study provide evidence that CCoV-Ⅰ,CCoV-Ⅱa,and CCoV-Ⅱb strains are cocirculating in diarrheal dogs in northeast China,with high co-infection rates observed with Ast V and CDV.Three strains of CCoV-Ⅱa were isolated successfully.In this study,the etiology and evolution of canine coronavirus in Northeast China were studied,but epidemiological surveillance should be continued in the future.This study provides a theoretical basis for understanding the epidemic situation of canine coronavirus in northeast China,provides a basis for the research and development of vaccines against domestic strains in the future,and provides ideas for clinical diagnosis and treatment in the future.