致急性呼吸道感染人腺病毒的体外感染特性初步比较

Preliminary Comparison of in vitro Infection Characteristics of Human Adenovirus Causing Acute Respiratory-tract Infection

致急性呼吸道感染人腺病毒(Human adenoviruses,HAdV)的型别特异性体外感染可能与细胞因子诱导的差异有关,并推测型别特异性体外感染可能与肺细胞中型别特异性细胞因子的诱导有关。为了比较致急性呼吸道感染人腺病毒3型、5型、7型和55型的体外感染特性,临床分离多株人腺病毒3型、7型和55型接种人肺腺癌细胞(A549细胞)或人胚肺成纤维细胞(MRC-5细胞),比较其感染滴度;将人腺病毒3型、5型、7型和55型毒株接种MRC-5细胞和A549细胞进行空斑试验,比较其空斑形成以及大小、形态;接种感染MRC-5细胞,分别于感染后10h和26h收集细胞培养上清,用人炎症细胞因子抗体芯片同步检测10个主要炎症相关细胞因子包括IL-1α、IL-1β、IL-4、IL-6、IL-8、IL-10、IL-13、MCP-1、IFN-γ以及TNF-α的表达水平并进行分析。结果显示,与MRC-5细胞相比,A549细胞对这几种人腺病毒更加敏感;人腺病毒55型毒株与其它型别相比产毒量较高。人腺病毒在MRC-5细胞上难以形成空斑,而用A549细胞进行的空斑试验显示人腺病毒5型和55型毒株有较高的空斑形成单位,并且形成的空斑为规则的圆形且边缘清晰,空斑直径较大;而人腺病毒7型和3型毒株感染滴度较低,人腺病毒7型形成的空斑大小不均匀,形状不规则,人腺病毒3型形成的空斑直径很小,呈点状。炎症细胞因子检测结果显示,与正常对照组相比,野生型人腺病毒55型表达差异的炎症细胞因子是IL-1α、IL-1β和TNF-α;改造的人腺病毒3型感染MRC-5细胞后,两个时间点的各炎症细胞因子基本低表达。与正常对照组相比,人腺病毒感染MRC-5细胞后表达有差异的炎症细胞因子主要为IL-6和IL-8。

Type-specific in vitro infection of human adenoviruses(HAdV) causing acute respiratory-tract infection may be related to differences in cytokine induction,and type-specific in vitro infection may be related to the induction of type-specific cytokines in lung cells. In order to compare the characteristics of acute respiratory-tract infection induced by human adenovirus type 3,type 5,type 7 and type 55 in vitro,several strains of human adenovirus type 3,type 5,type 7 and type 55 were isolated clinically and inoculated into human lung adenocarcinoma cells(A549 cells)or human embryonic lung fibroblasts(MRC-5 cells)to compare the titers of infection. They were also inoculated into MRC-5 cells and A549 cells for plaque assays,and the plaque formation,size and morphology were compared. After virus inoculation into MRC-5 cells,the supernatants of cell culture were collected at 10 hrs and 26 hrs,respectively. The expression levels of 10 major inflammatory cytokines including IL-1,IL-1β,IL-4,IL-6,IL-8,IL-10,IL-13,MCP-1,IFN-γ and TNF-α were detected synchronously and analyzed with human inflammatory cytokine antibody microarray. The results showed that,compared with MRC-5 cells,A549 cells were more sensitive to these types of human adenovirus. Human adenovirus type 55 strain had higher virulence than the other types of human adenovirus. It was difficult for human adenovirus to form plaques on MRC-5 cells. Human adenovirus types 5 and type 55 strains showed relatively high plaque forming units in A549 cells,and they were regular,rounded with well-defined margins and large diameters. However,the infection titer of human adenovirus types 7 and type 3 were low,and the plaques formed by human adenovirus type 7 were uneven in size and irregular in shape. And the plaques formed by human adenovirus type 3 were small in diameter and punctate. The results of inflammatory cytokines assay showed that,compared with normal control group,inflammatory cytokines expressed after human adenovirus wild type 55 infection were IL-1α、IL-1β and TNF-α. After the modified human adenovirus type 3 infection of MRC-5 cells,expression of inflammatory cytokines was basically low at two time points. Compared with normal control group,IL-6 and IL-8 were the main inflammatory cytokines with different expression after human adenovirus infection of MRC-5 cells.