Molecular Epidemiology Investigation Of α-Cov And β-CoV In Animals Of Shandong Province

Coronaviruses belong to the genus Coronavirus of the Nidovirales Family(Coronaviridae).Since the 1990s,the cross-species transmission of multiple coronaviruses has caused major animal and human diseases.Coronaviruses have increasingly affected the health of humans and animal husbandry,causing immeasurable losses.In December 2019,the new coronavirus swept across China in less than a month,and then spread across the world.As of March 18,2021,more than 120 million people have been infected worldwide,and the cumulative death toll has exceeded 30 million.The coronavirus has caused such great harm and economic losses to human society more than once.The seriousness was first reported in Guangdong Province,China at the end of 2002.Serious Acute Respiratory Syndrome(SARS),before active public health intervention strategies were successfully contained,had infected more than 8,000 people and killed nearly 800 people.In September 2012,WHO discovered another virus MERS-CoV,which can cause acute respiratory syndrome and renal failure,and it has also seriously endangered the safety of human life and property.Animals play an important role in the spread of coronaviruses as intermediate hosts,enabling viruses to spread from natural hosts to humans.Understanding the diversity of coronaviruses in various animals is essential for better prevention of emergencies.Therefore,animals Coronavirus surveillance is an essential step.This experiment aims to monitor the distribution and prevalence of alpha and beta coronaviruses in animals,to provide early warning of infectious diseases that may be caused byɑ-CoV andβ-CoV in animals.In order to establish a real-time fluorescent quantitative PCR(RT-q PCR)method for the accurate detection and clinical use ofα-CoV andβ-CoV,this study refers to the published sequences of 27α-CoV strains and 36β-CoV strains,and designs merged primers,which are verified and optimized by experiments.The results show that the optimized specific primer ofα-CoV was 2μmol/L and the annealing temperature was 55℃;the optimized specific primer ofβ-CoV was 2μmol/L and the annealing temperature was 55℃.A real-time fluorescent quantitative PCR method that can quickly and accurately detectα-CoV andβ-CoV was established.This method has strong specificity,high sensitivity and good reproducibility.Among the 10 common viruses that harm the livestock industry and poultry production,only PEDV and TGEV of the genusαcan be detected.The corresponding standard curve of RT-q PCR for detectingα-CoV is:y=-3.9114x+43.779,the correlation coefficient is 0.998,the minimum detection limit ofα-CoV is 1.0×10~2copies/μL,and the sensitivity is 100 times that of ordinary PCR methods.The corresponding standard curve of RT-q PCR for detectingβ-CoV is:y=-3.8821x+44.73,correlation coefficient 0.993,minimum detection limit is1.0×10~3copies/μL,and the sensitivity is 1000 times that of ordinary PCR methods.The results of the repeatability test showed that the coefficient of variation ofα-CoV was not higher than2.0%,and the coefficient of variation ofβ-CoV was not higher than 2.5%.This study has collected 515 samples in autumn and 428 samples in winter.The samples include domestic animals(pigs,cattle,sheep,donkeys),fur animals(mink,raccoon,fox,rabbit),and experimental animals(mice,guinea pigs,rabbits).zoo wild animals(gorillas,pandas,lions,tigers,elephants,etc.),pets(dogs,cats).The established real-time fluorescent quantitative PCR method was used to detect 943 samples.Among the autumn samples,the positive rate of domestic animalɑ-CoV was 2.6%,and the positive rate ofβ-CoV was 5.2%;the positive rate of fur animalɑ-CoV was 4.2%,andβ-CoV positive rate was 2.8%;experimental animalɑ-CoV positive rate was 1.4%,andβ-CoV positive rate was 7.0%;wild animalɑ-CoV positive rate was 2.1%,andβ-CoV positive rate was 17.0%;The positive rate of petɑ-CoV was 14.3%,andβ-CoV was 4.3%.In the winter samples,the positive rate ofɑ-CoV in domestic animals was 3.8%,and the positive rate ofβ-CoV was 3.4%;the positive rate of experimental animals was 4.3%,and the positive rate ofβ-CoV was 4.3%;wild animals were positive forɑ-CoV The positive rate ofβ-CoV was 3.8%,and the positive rate ofβ-CoV was 23.1%;the positive rate of wild animalɑ-CoV was 3.8%,and the positive rate ofβ-CoV was 23.1%.From the comprehensive point of view of the test results,whether in autumn or winter,the detection rate ofβ-CoV in wild animals in zoos is relatively high,indicating that wild animals are more susceptible toβ-CoV and may be the host or intermediate host of multipleβ-CoV.The monitoring ofβ-CoV should be strengthened.Due to the relatively high detection rate ofβ-CoV in the feces of wild animals,three of the strong positive samples were selected and sent to Shanghai Probe Biological Co.,Ltd.for metagenomic sequencing.The samples are green monkey,camel,and male lion feces,but because there are fewer green monkey and male lion feces,only camel feces samples get good results.The whole genome sequence of a camel coronavirus belonging to theβ-CoV a subgroup was obtained by sequencing.The total length is 28702 bp,and the homology with bovine coronavirus is 91.1%.It has the lowest homology with cat coronavirus,only 41.3%.Metagenomic sequencing can be used for the detection of unknown coronaviruses in fecal samples of wild animals and the whole genome sequencing of new coronaviruses.In summary,this study established a real-time fluorescent quantitative PCR method forɑ-CoV andβ-CoV,and applied it to the molecular epidemiological investigation of two important coronavirus genera in various animals.This research provides a reliable and powerful technical stock for monitoring the epidemic situation of animal coronaviruses and early warning of cross-species transmission.