Analysis Of The Effects Of Bovine MiR-29b Overexpression On BALB/c Mice Infected With Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus(BVDV)is the main cause of bovine viral diarrhea/mucosal disease(BVD/MD),causing bovine diarrhea,hyperthermia,immunosuppression,mucosal congestion.Other symptoms can also cause milk production in dairy cows,miscarriage of pregnant cows,stillbirth and persistent infection of cattle.BVDV spreads widely in cloven-hoofed animals and endangers the safety of some wild animals,causing huge economic losses to the livestock industry and related industries.Previous studies have found that miR-29b targets and regulates autophagy associated gene(ATG)14 and ATG9A,and inhibits autophagy;while high expression of miR-29b significantly inhibits BVDV in bovine kidney cell MDBK.The replication,but whether high expression of miR-29b affects the replication of BVDV in vivo has not been reported yet,and it is urgent to study and explore,in order to provide a new method for prevention and control of BVD.Objective:To explore the effect of miR-29b overexpression on BVDV-infected BALB/c mice.Methods:The pre-miR-29b gene fragment was amplified by MDBK genome and cloned into pLL3.7 lentiviral vector;recombinant plasmid pLL3.7-pre-miR-29b or pLL3.7 plasmid and three helper plasmids were constructed.Co-transfection into HEK-293T cells,packaging lentivirus,and measuring its titer;purchasing 4-5 week old BALB/c mice,randomly divided into 5 groups,6 in each group,two consecutive tail vein injections 2.5 ×107 IU pLL3.7-pre-miR-29b lentivirus or pLL3.7 lentivirus suspension(negative control),96 h after infection,the mice were sacrificed and organ tissues were harvested,total miRNA was extracted,and miR-29b was detected by TaqMan qPCR Expression:At 96 h after lentiviral infection,BVDV was infected by intranasal route;at different time points after infection(0,2,4,10,15 d),the mice were sacrificed and tissues and organs were collected to extract total RNA.Real-time quantitative RT-PCR was used to detect BVDV load.Pathological sections were also prepared.The overexpression of miR-29b was observed to affect the pathological changes of BALB/c mice caused by BVDV infection.The results showed that the pLL3.7-pre-miR-29b plasmid was successfully constructed;pLL3.7-pre-miR-29b and pLL3.7 lentivirus were successfully packaged,and the lentivirus titer after concentration was 1.34 × 108 IU/mL;After intravenous lentivirus,mice infected with pLL3.7-pre-miR-29b lentivirus had higher levels of miR-29b expression in different tissues compared with the control;BVDV challenge was detected by fluorescence quantitative RT-qPCR.After 4 days,the relative copy number of BVDV 5’ UTR of each tissue in the treatment group gradually formed with the control group;after the 10th day,pLL3.7-pre-miR-29b in the treatment group significantly inhibited the copy number of BVDV;Compared with the treatment group,the lesions in the tissues of the control group were more serious,and some tissues showed signs of degeneration and necrosis.Conclusion:pLL3.7-pre-miR-29b lentiviral infection can significantly reduce the copy number of BVDV in various tissues,inhibit the replication of BVDV in vivo,and alleviate the pathological changes of various tissues and organs of BALB/c mice caused by BVDV infection.BALB/c mice have a certain protective effect.This study explored the role of miR-29b in influencing BVDV-infected BALB/c mice,and provided a theoretical basis for the development of effective strategies and methods for anti-BVDV.