马立克氏病病毒可感染鸡大脑小胶质细胞并促进其Toll样受体15和1LB基因的转录

Marek's Disease Virus Can Infect Chicken Brain Microglia and Promote the Transcription of Toll-like Receptor 15 and 1LB Genes

体外培养雏鸡大脑神经胶质细胞并纯化得到小胶质细胞。分别用马立克氏病病毒(Marek's disease virus,MDV)广西超强毒力(vv)株YL040920和弱毒疫苗株CVI988/Rispens感染小胶质细胞,每日观察病毒感染后的细胞病变效应(CPE);用免疫组织化学法检测病毒感染后小胶质细胞中MDV MEQ蛋白的表达,并用荧光定量PCR(qPCR)以及qRT-PCR法分别检测MDVmeq基因(meq-DNA)的复制以及gB基因mRNA(gB-mRNA)的转录水平。同时用qRT-PCR法检测MDV感染对小胶质细胞Toll样受体家族(TLRs)基因转录的影响。结果发现,YL040920株和CVI988/Rispens株病毒均能感染小胶质细胞,病变细胞呈多灶性脱落并形成空斑,随病变进展迅速增大并融合。可在MDV感染的小胶质细胞核内检测到MEQ蛋白,并在感染细胞中检测到meq-DNA的复制和gB-mRNA的转录。YL040920株感染的小胶质细胞中病毒载量和gB-mRNA的转录水平显著低于以相同病毒量CVI988/Rispens株感染的小胶质细胞中的相应量(P≤0.05/0.001)。MDV感染可上调小胶质细胞TLR15和TLR1LB基因的转录水平。小胶质细胞中TLR15和TLR1LB mRNA的转录水平在YL040920感染1 d后升高,于3 dpi达到最高值,到5 dpi时有所降低。检测到小胶质细胞中TLR1LB mRNA转录水平在CVI988/Rispens感染1 d后升高,到3 dpi时达最高值;而TLR15 mRNA的转录水平在3 dpi后开始升高,二者在5 dpi时均降低。比较YL040920和CVI988/Rispens对TLRs mRNA转录的影响发现vvMDV YL040920感染小胶质细胞后主要促进其TLR15基因的转录(P≤0.01/0.001),而弱毒疫苗株CVI988/Rispens感染后主要促进TLR1LB基因的转录(P≤0.001)。

Microglial cells were purified from a mixed neuroglia culture prepared from the neonatal chicken brain in vitro, and were infected with the vvMDV YL040920 isolate and an attenuated MDV vaccine strain CVI988/Rispens, respectively. The presence of cytopathic effect （CPE） was examined daily, and the MEQ expression in MDV-infeeted microglia was detected by immunohistochemistry assay. DNA replication of the MDV meq gene and transcription of the gB gene were determined by real-time quantitative PCR （qPCR） and qRT-PCR, respectively. The transcripts of Toll-like receptor （TLR） mRNA in microglia post MDV infection were quantified by qRT-PCR. The results of this study showed that both vvMDV YL040920 and attenuated vaccine strain CVI988/Rispens could infect microglia and produce characteristic CPE with plaque formation. The plaques were formed due to cells shedding at multi-sites, then quickly expanded and integrated. Furthermore, the MEQ protein was detected in nuclei of YL040920 and CVI988/ Rispens-infected microglia, and MDV meq DNA replication and gB gene transcription in MDV-infected microglia were also confirmed. Although both MDV DNA copies and gB transcripts were increased in the vi- rus-infected microglia, the higher viral DNA load and gB transcript were observed for CVI988/Rispens than for YL040920 in vitro （P≤O. 05/0. 001）. The transcriptions of TLR15 and TLRILB gene were found to be up-regulated in microglia following MDV infection in vitro. Purified microglia infected with YL040920 was observed increased TLR15 and TLR1LB transcripts as early as 1 day post infection （dpi）, and reached its peak level at 3 dpi, then decreased mildly at 5 dpi. For CVI988/Rispens, it induced an increase of TLR15 transcript as early as 1 dpi, and rose rapidly at 3 dpi, and then decreased slightly at 5 dpi. At the same time, CVI988/Rispens induced the increase of chTLR1LB transcript at 3 dpi and decreased at 5 dpi. By comparing the TLRs transcription between YL040920 and CVI988/Rispens-infected microglia, it was suggested that vvMDV YL040920 might induce more TLR15 transcript than the attenuated vaccine strain CVI988/Rispens （P≤0.01/0. 001）, while CVI988/Rispens induced more TLR1LB transcript than YL040920 （P≤0. 001）.