Effects of Lentivirus-mediated Occludin Overexpression on BVDV Infection in BALB/c Mice

doi:10.13560/j.cnki.biotech.bull.1985.2021-1156

Abstract

Abstract:

This work aims to study whether the overexpression of tight junction protein（occludin,OCLN）affects the replication of bovine viral diarrhea virus（BVDV）in BALB/c mice. After lentivirus expression vector pLVML-Myc-bOCLN-linker-GFP-IRES-Puro was constructed,it was co-transfected into HEK-293T cells together with the helper plasmids pSPAX2 and pMD2.G. At 48 h post-transfection,OCLN-GFP lentivirus suspension was collected and its titer was measured. Meanwhile,the lentivirus generated with pLVML-Myc-Mcs-linker-GFP-IRES-Puro vector（GFP）was served as negative control. BALB/c mice aged 4-5 weeks were randomly divided into five groups：A（0 d）,B（4 d）,C（8 d）,D（10 d）and E（15 d）,with 6 mice in each group. Then 5× 10⁷ IU OCLN-GFP and GFP lentivirus suspension were injected twice continuously at an interval of 48 h. At 96 h after the second injection,BALB/c mice were challenged with 1.68 × 10⁵ TCID₅₀ BVDV. At 0,4,8,10 and 15 d after the challenge,mice were dissected and tissues including liver,spleen,lung,kidney and small intestine were collected. The overexpression of OCLN was detected by real-time fluorescent quantitative PCR（qRT-PCR）and Western blot,and the viral loads of BVDV in different tissues were detected by qRT-PCR. Histopathological sections were made to observe the pathological changes of each tissue. As results,OCLN-GFP and GFP lentivirus were successfully generated. After 96 h of lentivirus infection in BALB/c mice,the expressions of OCLN mRNA and protein increased significantly after OCLN-GFP lentivirus infection. Compared with the control group infected with GFP lentivirus,the BVDV viral load in tissues and organs of OCLN-GFP lentivirus infection experimental group increased significantly from 4 d after challenge. Meanwhile,compared with the control group,at 4 d after BVDV challenge,the lung and liver organs of the experimental group appeared obvious lesions at the earliest time;after 8 d of BVDV challenge,the pathological changes of liver,spleen and lung were more serious than those in the control group. In conclusion,the results shows that OCLN overexpression maysignificantly increase the replication of BVDV in BALB/c mice,and the pathological changes caused by BVDV infection are more serious,which provides an important basis for clarifying the molecular mechanism of OCLN mediated BVDV replication.

Key words: tight junction protein Occludin, lentivirus, bovine viral diarrhea virus, pathological section

WANG Wan-shun, FU Qiang, WEI Yu-rong, HU Xin-yan, CHEN Jun-zhen, LI Ze-yu, SHI Hui-jun. Effects of Lentivirus-mediated Occludin Overexpression on BVDV Infection in BALB/c Mice[J]. Biotechnology Bulletin, 2022, 38(6): 291-298.

Figures/Tables 6

Fig. 1 Lentivirus plasmid map and vector identification A：pLVML-Myc-bOCLN-linker-GFP-IRES-Puro lentiviral plasmid map. B：pLVML-Myc-Mcs-linker-GFP-IRES-Puro lentiviral plasmid map. C：PCR amplification（M：DNA marker 2000;1：negative control;2-4：monoclonal colonies）. D：OCLN gene sequence

Fig. 2 Detection of green fluorescence expression in the HEK-293T cells infected with lentivirus

Fig. 3 Detection of OCLN mRNA expressions in various tissues by qRT-PCR *P<0.05,**P<0.01

Fig. 4 Expressions of OCLN protein in various tissues by Western blot

Fig. 5 BVDV loading in various tissue by qRT-PCR ***P<0.001

Fig. 6 Detection of pathological changes of various tissues in BALB/c mice by pathological section

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