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基孔肯雅热是由基孔肯雅病毒（CHIKV）导致的一种疾病，目前在非洲、亚洲和美洲由蚊子传播给人类; 基孔亚病毒的感染与严重的心脏症状有关，然而，CHIKV病毒如何导致心脏病还不得而知。

2023年8月3日，来自纽约大学格罗斯曼医学院和康奈尔医学院的一组研究人员采用分子表达，免疫荧光，免疫组化，细胞因子检测等实验方法，结合单细胞分析以及Visiopharm专业图像处理软件，通过自建算法识别凋亡信号，探讨了线粒体抗病毒信号蛋白（MAVS）在CHIKV感染中的作用，并发表于 Nature Communications杂志。

Chikungunya fever is a disease caused by the chikungunya virus (CHIKV), a disease currently transmitted to humans by mosquitoes in Africa, Asia, and the Americas; infection with the chikungunya virus has been associated with severe cardiac symptoms; however, how the CHIKV virus leads to heart disease is unknown.

On 3 August 2023, a group of researchers from New York University's Grossman School of Medicine and Cornell Medical College explored the role of the mitochondrial antiviral signalling protein (MAVS) in CHIKV infection by using molecular expression, immunofluorescence, immunohistochemistry, and cytokine assays in conjunction with single-cell analyses and the Visiopharm professional image-processing software, which identifies apoptotic signals through a self-constructed algorithm. CHIKV infection, and published in Nature Communications.

研究目的及结论

Objective and conclusion of the research

这篇文章探讨 CHIKV 感染与心脏病之间的关系；评估免疫系统对 CHIKV 感染的反应；探讨 CHIKV 感染引起的心肌炎和血管炎以及 CHIKV 心脏感染的模型和机制；强调监测 CHIKV 感染患者心脏功能的重要性。

通过利用小鼠模型和人类原代心脏细胞，研究人员发现 CHIKV 在心脏成纤维细胞内具有很强的复制能力；由于 I 型干扰素反应的激活，导致免疫功能正常的小鼠能够有效地消灭 CHIKV 而不会造成实质性伤害，而在缺乏 MAVS 的情况下，长期感染会导致心肌炎和血管炎的发生，进而引起长期的心血管并发症，强调MAVS 信号对于从心脏组织中清除病毒的必要性。

文章通过CASP3信号的表达，采用免疫组化的方法进行检测，图片通过Visiopharm软件进行定量处理分析。对整片扫描的幻灯片进行分析，以确定裂解的CASP3阳性细胞的数量。文章采用2021.09版本的Visiopharm图像分析软件。首先将图像导入到Visiopharm软件中，运行图像预处理步骤，手动勾画出感兴趣区域（ROI），借用Visopharm强大的数据库以及内置的标准算法，通过颜色去卷积和过滤来创建增强DAB信号和核信号的特征，使用可训练的线性贝叶斯算法完成分割，软件内置的后处理步骤应用于定义阳性或阴性的细胞核，计算每个ROI的总细胞核数量、阳性和阴性细胞核数量。完成DAB染色的小鼠心脏细胞的分析，与模拟感染对照组相比，WT 小鼠感染后显示出相似水平的 CASP3 信号，而 Ifnar1-/- 小鼠心肌内的 CASP3 水平显著增加（图 3），这表明无限制的 CHIKV 感染可导致心脏组织损伤。

This article explores the relationship between CHIKV infection and heart disease; assesses the immune system response to CHIKV infection; explores myocarditis and vasculitis caused by CHIKV infection as well as models and mechanisms of CHIKV cardiac infection; and highlights the importance of monitoring cardiac function in patients with CHIKV infection.

By using mouse models and human primary cardiac cells, the researchers found that CHIKV has a high replication capacity in cardiac fibroblasts; due to the activation of the type I interferon response, which results in immunocompetent mice being able to efficiently destroy CHIKV without substantial harm, while in the absence of MAVS, prolonged infection leads to myocarditis and vasculitis, which can in turn cause long-term cardiovascular complications. The necessity of MAVS signalling for virus clearance from cardiac tissue is emphasised.

In the article, the expression of CASP3 signalling was detected by immunohistochemistry and the images were analysed by Visiopharm software for quantitative processing. Whole scanned slides were analysed to determine the number of lysed CASP3 positive cells. The article used Visiopharm image analysis software version 2021.09. The images were first imported into Visiopharm software, the image preprocessing step was run to manually outline the region of interest (ROI), borrowing Visopharm's powerful database as well as the built-in standard algorithms to create features that enhance the DAB signal and the nuclear signal through colour de-convolution and filtration, and the segmentation was completed using trainable linear Bayesian algorithms, and the software's in-built post-processing step is applied to define positive or negative nuclei, calculating the total number of nuclei, positive and negative nuclei for each ROI. Completion of the analysis of DAB-stained mouse cardiac cells showed similar levels of cleaved CASP3 signals after infection in WT mice compared to mock-infected controls, whereas cleaved CASP3 levels were significantly increased in the myocardium of Ifnar1-/- mice (Fig. 3), suggesting that unrestricted CHIKV infection can lead to cardiac tissue damage.

如下图所示挑选了三张使用visiopharm软件的图像：

Three images using visiopharm software were selected, as shown below:

图1：CHIKV在心脏组织中积极复制并靶向心脏成纤维细胞。

c 来自2个独立实验的代表性图像，显示感染的心肌（刻度 =20μm）和主动脉瓣、左心房和血管（刻度 =30μm）。白色箭头：CHIKV感染的细胞。

e 来自三个独立实验的代表性荧光显微镜图像，这些实验对用不同心脏细胞类型标志物染色的 CHIKV 感染心脏的心室切片 Scale=15μm。CHIKV感染细胞与不同细胞类型标志物之间的共定位分析数据表示为与指示标记物重叠的总绿色信号（感染细胞）的百分比。在3-4个独立视野中对 n=3只小鼠进行共定位分析。

Fig. 1: CHIKV actively replicates in cardiac tissue and targets cardiac fibroblasts.

c Representative images from 2 independent experiments showing infected myocardium (scale = 20 μm) and aortic valve, left atria, and vessels (scale = 30 μm). White arrows: CHIKV-infected cells.

e Representative fluorescence microscopy images from three independent experiments of ventricular sections of CHIKV-infected hearts stained with different cardiac cell type markers Scale= 15 μm. Colocalization analysis between CHIKV-infected cells and different cell type markers Data are represented as the percentage of the total green signal (infected cells) overlapping with the indicated markers. Colocalization analysis was done in 3–4 independent fields for n = 3 mice.

图2：MAVS - / - 小鼠在CHIKV感染后发生心肌炎和大血管炎。

a 上图：实验设计的示意图。下图：来自至少四只独立小鼠的代表性 H&E，显示 CHIKV 感染的WT、Mavs+/-和 Mavs-/-心脏，分辨率为10dpi。刻度 = 200 μm。黑色方块表示所选部分（插图 i 和 ii）。插图 i：心肌区域插图 ii：附着在心脏底部的大血管量表=20μm。

b 具有心肌炎阳性体征的小鼠数量的量化该数据表示具有任何心肌炎体征的小鼠占所分析小鼠总数的百分比。WT（n=4）、Mavs+/−（n=6）和 Mavs−/−（n=13）。

c 大血管炎小鼠数量的量化。该数据表示具有任何血管炎迹象的小鼠占所分析小鼠总数的百分比。WT（n=4）、Mavs+/−（n=6）和Mavs−/−（n=13）。

Fig. 2: Mavs−/− mice develop myocarditis and large vessel vasculitis upon CHIKV infection.

a Upper panel: schematic representation of the experimental design. Bottom panel: Representative H&E from at least four independent mice showing CHIKV-infected WT, Mavs+/−, and Mavs−/− hearts at 10 dpi. Scale = 200 μm. The black square indicates the section selected (insets i and ii). Inset i: myocardial region Inset ii: large vessels attached to the base of the heart Scale = 20 μm.

b Quantification of the number of mice with positive signs of myocarditis the data represent the percentage of mice with any sign of myocarditis over the total number of mice analyzed. WT (n = 4), Mavs+/− (n = 6), and Mavs−/− (n = 13).

c Quantification of the number of mice with large vessel vasculitis. The data represent the percentage of mice with any sign of vasculitis over the total number of mice analyzed. WT (n = 4), Mavs+/− (n = 6), and Mavs−/− (n = 13).

图3：心脏组织中的局部IFN-I反应控制CHIKV感染并防止组织损伤。

g 左图：通过 IHC 对裂解的 CASP3 测量细胞凋亡染色。刻度=50μm。箭头突出显示凋亡病灶。右图：使用 Visiopharm 在整个心室切片（左心室、右心室和室中隔）中对裂解的 CASP3 细胞核与总细胞核进行定量。每个点代表一只鼠标。PBS对照（n=5），CHIKV感染的WT（n=5）和CHIKV感染的Ifnar1-/-小鼠（n=6）。

Fig. 3: Local IFN-I responses in cardiac tissue control CHIKV infection and prevent tissue damage.

g Left panel: Apoptosis staining was measured by IHC against cleaved CASP3. Scale= 50 μm. Arrows highlight apoptotic foci. Right panel: Quantifications of cleaved CASP3 nuclei versus total nuclei were performed in whole ventricular sections (LV, RV, and septum) using Visiopharm. Each dot represents one individual mouse. PBS control (n = 5), CHIKV- infected WT (n = 5), and CHIKV-infected Ifnar1−/− mice (n = 6).

Visiopharm软件在未来很多研究中都是必不可少的，其对选定的ROI进行定量分析，如细胞计数、蛋白表达分析和组织结构测量的优势，都可以更好更快地帮助我们获得研究所需的数据和内容，这是对基于分子细胞学的研究和发现无可比拟的一大助力。

Visiopharm software will be essential in many future studies, and its advantages of quantitative analysis of selected ROIs, such as cell counting, protein expression analysis, and tissue structure measurements, all help us to get the data and content we need for research better and faster. This is an unrivaled major aid to molecular cytology-based research and discovery.

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