Understanding the molecular basis leading the virulence of vesicular stomatitis virus in pigs

Comparison of the transcriptiomic profile using microarray analysis betwween primary porcine macrophages infected with recombinant vesicular stomatitis viruses Overall design: Infections of recombinant vesicular stomatitis viruses (VSV) were carried out on primary swine porcine macrophages. Six well plates containing 1x10^7 cells per well were infected at MOI of 10 TCID50 with each virus and incubated for five hours at 37 °C under 5% of CO2. Infections were conducted on triplicates. As a mock-infected samples 3 wells were incubated in the same conditions with macrophage media. After incubation, total RNA was extracted and used in microarray analysis using DNA microarrays (Array design #:Agilent-069270; 4 X 44k per slide) manufactured by Agilent Technologies. Low input RNA two color labeling kit (Agilent Technologies) was used to lable cRNA. Total RNA isolated from mock-infection was labeled with Cy5 as universal control. All samples including mock-infection were labeled with Cy3. One Cy3 labled sample was cohybrided with the Cy5 labled control in one hybridization chamber. After hybridization and washing, the arrays were scanned with a GenPix 4000B and the images were saved as tiff files. The expression data were extracted with Genpix 7.0 software for statisitcal analysis using R with librarys(limma, MASS, statmod, splines). LOESS method was applied to normalize interarray variation. The pig gene sequences were mapped to human genes with BLAST and manual annotation. Annotated probes were mapped to human genes, and human gene Entrezid were used in bioinformatic analysis with The Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool.

重组水疱性口炎病毒感染原代猪巨噬细胞的转录组谱基因芯片比较分析

实验设计：将重组水疱性口炎病毒（recombinant vesicular stomatitis viruses, VSV）感染原代猪巨噬细胞。使用每孔接种1×10^7个细胞的6孔板，以感染复数（multiplicity of infection, MOI）10 TCID50分别感染各病毒株，于37℃、5% CO₂条件下孵育5小时，实验设置3次生物学重复。设置空白感染对照：3孔细胞仅用巨噬细胞培养基孵育，保持相同培养条件。
孵育结束后，提取总RNA，采用安捷伦科技（Agilent Technologies）生产的DNA基因芯片（阵列设计编号：Agilent-069270；每张芯片含4×44k个探针）开展基因芯片分析。使用安捷伦低起始量RNA双色标记试剂盒（Low input RNA two color labeling kit）标记互补RNA（complementary RNA, cRNA）：以空白感染组提取的总RNA作为通用对照，采用Cy5荧光标记；所有样品（包括空白感染组）均采用Cy3荧光标记。将1份Cy3标记的样品与Cy5标记的对照共置于一个杂交舱中进行杂交。杂交及洗涤完成后，使用GenPix 4000B扫描仪扫描芯片，扫描图像以TIFF文件格式保存。使用GenPix 7.0软件提取表达数据，随后通过R语言结合limma、MASS、statmod、splines等程序包进行统计学分析。采用局部加权回归散点平滑法（LOESS）校正芯片间的杂交差异。
将猪基因序列通过BLAST比对并辅以人工注释定位至人类基因，将已注释的探针映射至人类基因，以人类基因的Entrez ID作为标识，通过在线工具注释、可视化和整合发现数据库（Database for Annotation, Visualization and Integrated Discovery, DAVID）进行生物信息学分析。