Zhang X, Hinton D, Park S, Liao C L, Lai M M, Stohlman S
Department of Neurology, University of Southern California School of Medicine, Los Angeles 90033, USA.
Adv Exp Med Biol. 1998;440:521-8. doi: 10.1007/978-1-4615-5331-1_67.
We have developed a defective-interfering (DI) RNA of mouse hepatitis virus (MHV) as a vector for expressing a variety of cellular and viral genes including the chloramphenicol acetyltransferase (CAT), hemagglutinin' esterase (HE), and gamma interferon. Here, we used the HE-expressing DI RNA for examining the role of HE protein in viral pathogenesis. The pseudorecombinant virus containing an expressed HE protein was generated by infecting cells with MHV-A59, which does not express, HE, and transfecting the in vitro-transcribed DI RNA containing the HE gene. These pseudorecombinant viruses (DE-HE A59) were then inoculated intracerebrally into mice. Viruses recovered from cells infected with A59 and transfected with DI RNA expressing the CAT gene (DE-CAT A59) were used as a control. At various time points after inoculation, mice were observed for clinical symptoms. Tissues (brains and livers) were obtained for determining the replication of DI RNA by RT-PCR, virus replication by plaque assay, antigen expression by immunohistochemistry, and pathological changes. Results showed that all mice infected with DE-CAT A59 succumbed to infection by 9 days postinfection (d p.i). These data are identical to the pathogenesis of the parental A59 virus, demonstrating that inclusion of the DI RNA did not by itself alter pathogenesis. In contrast, only 40% of mice infected with DE-HE A59 succumbed to infection. The subgenomic mRNAs transcribed from the DI vector were detected at 1 and 2 d p.i. but not at subsequent time points, indicating that the genes in the DI vector were expressed only at an early stage of viral infection. No significant difference in virus replication in the brains was detected between these two groups of mice, suggesting that virus replication in brains was not affected by the expression of the HE. Histopathological examination showed only a small increase in the extent of inflammatory cell infiltration and reduced viral antigen in the mice infected with DE-HE A59. There was no difference in virus replication in the livers at 2 and 4 d p.i., but a 3 log10 reduction was detected in the livers of mice infected with DE-HE A59 at 6 d p.i. Histological examination showed a significant reduction in viral antigen, inflammation and necrosis in mice infected with DE-HE A59. These results indicate that the expression of HE from the DI vector altered the viral pathogenesis. This study thus demonstrates the usefulness of this system in studying the role of viral or cellular genes expressed locally at the sites of viral infection in viral pathogenesis.
我们已经构建了一种小鼠肝炎病毒（MHV）的缺陷干扰（DI）RNA，作为表达多种细胞和病毒基因的载体，这些基因包括氯霉素乙酰转移酶（CAT）、血凝素酯酶（HE）和γ干扰素。在此，我们使用表达HE的DI RNA来研究HE蛋白在病毒致病机制中的作用。通过用不表达HE的MHV - A59感染细胞，并转染含有HE基因的体外转录DI RNA，产生了含有表达HE蛋白的假重组病毒。然后将这些假重组病毒（DE - HE A59）脑内接种到小鼠体内。从感染A59并转染表达CAT基因的DI RNA（DE - CAT A59）的细胞中回收的病毒用作对照。在接种后的不同时间点，观察小鼠的临床症状。获取组织（脑和肝）用于通过RT - PCR确定DI RNA的复制、通过蚀斑测定确定病毒复制、通过免疫组织化学确定抗原表达以及观察病理变化。结果显示，所有感染DE - CAT A59的小鼠在感染后9天（dpi）内均死于感染。这些数据与亲本A59病毒的致病机制相同，表明包含DI RNA本身并未改变致病机制。相比之下，感染DE - HE A59的小鼠中只有40%死于感染。在感染后1天和2天检测到从DI载体转录的亚基因组mRNA，但在随后的时间点未检测到，这表明DI载体中的基因仅在病毒感染的早期阶段表达。在这两组小鼠的脑中未检测到病毒复制的显著差异，这表明脑中的病毒复制不受HE表达的影响。组织病理学检查显示，感染DE - HE A59的小鼠中炎症细胞浸润程度仅略有增加，病毒抗原减少。在感染后2天和[此处原文可能有误，推测为4天]4天，两组小鼠肝脏中的病毒复制没有差异，但在感染后6天，感染DE - HE A59的小鼠肝脏中检测到病毒复制减少了3个对数10。组织学检查显示，感染DE - HE A59的小鼠中病毒抗原、炎症和坏死显著减少。这些结果表明，来自DI载体的HE表达改变了病毒致病机制。因此，本研究证明了该系统在研究病毒感染部位局部表达的病毒或细胞基因在病毒致病机制中的作用方面的实用性。
