背景：基孔肯雅病毒（Chikungunya virus，CHIKV）是一种具有高度传染性，能够快速传播的蚊媒病毒，属于二类病原体。目前尚无获批上市的针对CHIKV的疫苗或药物。因此，开发针对这种病毒的控制和预防新举措就显得尤为迫切。然而，由于CHIKV的操作只能在Biosafety Level-3（BSL-3）及以上防护等级的实验室中进行，这极大地阻碍了CHIKV相关研究的进展。本文基于这一问题，制备了一种能够在Biosafety Level-2（BSL-2）实验室操作及应用的CHIKV假病毒，降低高致病性真病毒对实验环境的局限要求，促进对CHIKV更广泛的研究。

方法：本研究以HIV-1慢病毒载体系统为基础，利用实验室前期获得的CHIKV基因，构建含有双报告基因和表达CHIKV包膜蛋白的假病毒，并将其应用到中和抗体的检测当中。整个研究主要分为以下四个部分：第一部分，构建CHIKV假病毒包装系统，对该系统中的一系列包装条件进行优化; 第二部分，对优化后得到的病毒粒子进行超离浓缩，并对浓缩后得到的病毒粒子进行免疫印迹检测及电镜观察；第三部分，以该CHIKV假病毒为基础，建立96孔板微中和抗体检测系统，并对该系统中的一系列参数进行优化；第四部分，以所建立的CHIKV假病毒微中和抗体检测系统为基础，对CHIKV灭活病毒免疫兔血清、临床CHIKV康复患者血清进行中和效价检测。

结果：首先，我们将CHIKV包膜蛋白基因插入真核表达载体中，利用慢病毒包装系统进行CHIKV假病毒的构建，并对系统中影响病毒滴度的各种包装条件进行优化，最终确定的包装条件为：CHIKV病毒毒株选择1151D4f，去除Capsid表达基因，使用转移质粒pLVX-IRES-ZsGreen1-Luc，以及转染试剂Lipo3000，以质粒摩尔比例1:1:1的条件进行转染，得到的假病毒滴度为1.61×10⁵ TU/ml。然后，利用第一步成功构建的CHIKV假病毒，建立了假病毒微中和抗体检测系统，并对各种检测参数进行优化，最终确定的中和实验检测参数为：HEK-293T细胞50000个/孔，病毒接种量为500 TU/孔，于感染后96 h检测发光信号强度并计算中和抗体效价，能够得到最准确、最稳定的实验结果。接下来，利用上一步建立的CHIKV假病毒微中和抗体检测系统，对CHIKV灭活病毒免疫兔血清进行中和效价检测，通过与VSV-G假病毒中和实验结果进行对比，证明本研究所构建的CHIKV假病毒在中和抗体检测中具有良好的特异性。最后，我们也利用建立的CHIKV假病毒微中和抗体检测系统，对临床CHIKV康复患者血清进行了中和效价检测，并将检测结果与ELISA检测结果进行了对比，发现二者具有良好的相关性与一致性。由此可见，本研究所建立的CHIKV假病毒微中和抗体检测系统，具有良好的可靠性与灵敏度，可以替代传统的中和实验对血清等样品进行中和效价检测，且操作更加方便，结果更加客观。

结论：本研究成功构建了双报告基因CHIKV假病毒，并基于该假病毒建立了安全、可靠的微中和抗体检测系统，为在BSL-2实验室中，开展CHIKV相关研究，例如抗病毒药物筛选、单克隆抗体筛选、疫苗评价等提供了有效的手段。

Background Chikungunya virus (CHIKV) is a highly contagious, rapidly spreading mosquito-borne virus and is classified as a class II pathogen. There are currently no approved vaccines or drugs against CHIKV, so it is particularly urgent to develop new control and prevention measures against this virus. However, since the operation of CHIKV can only be carried out in laboratories with Biosafety Level-3 (BSL-3) and above, which greatly hinders the progress of CHIKV-related research. Based on this problem, this research strived to establish a CHIKV pseudovirus (PsV) that can be operated and applied in a Biosafety Level-2 (BSL-2) laboratory, reducing its limited requirements for the experimental environment and promoting more extensive research on CHIKV.

Methods In this study, based on the HIV-1 lentiviral vector system, the CHIKV gene obtained in the early stage of the laboratory was used to construct a pseudovirus containing dual reporter genes and expressing the CHIKV envelope glycoproteins, and it was applied to the detection of neutralizing antibodies. The whole study is mainly divided into the following four parts: the first part, the construction of the CHIKV pseudovirus packaging system, and the optimization of a series of packaging conditions in the system; the second part, the obtained PsV particles were concentrated by ultra-centrifuging, and further were subjected to WB detection and electron microscope observation; the third part, based on the CHIKV PsV, a 96-well plate-micro-neutralization assay system was established, and a series of parameters in the system were optimized; the fourth part, based on the established CHIKV PsV micro-neutralization assay system, the neutralizing titer of inactivated virus-immunized rabbit serum and clinical CHIKV recovered patients sera were determined.

Results Firstly, we inserted the CHIKV envelope glycoproteins gene into the eukaryotic expression vector, constructed the CHIKV PsV using the lentiviral packaging system, and optimized various factors affecting the virus titer in the system. The packaging conditions were determined as follows: CHIKV virus strain selected 1151D4f, removed the Capsid protein gene, co-transfected with the transfer plasmid pLVX-IRES-ZsGreen1-Luc with the plasmid molar ratio of 1:1:1 using transfection reagent Lipo3000. With the optimization, the virus titer is 1.61×10⁵ TU/ml. Then, using the CHIKV PsV successfully constructed in the first step, a PsV micro-neutralization assay system was established, and various parameters were optimized. The most accurate and stable results can be obtained by incubated HEK-293T cells 50000/well, virus 500 TU/well, and determining the luciferase activity after 96 h of infection. Next, using the CHIKV PsV micro-neutralization assay system established previously, the neutralization titer of the inactivated CHIKV-immunized rabbit serum was determined. And through comparing with VSV-G PsV, it is proved that the CHIKV PsV constructed in this study has good specificity in the detection of neutralization antibodies. Finally, we also used the established CHIKV PsV micro-neutralization assay to determine the neutralization titer of clinical CHIKV recovered patients-sera. And compared the results with the ELISA OD₄₅₀, it is found that CHIKV PsV neutralization titers have positive correlation and consistency with ELISA OD₄₅₀. It demonstrating that the CHIKV PsV micro-neutralization assay system established in this study has good reliability and sensitivity, and could replace the traditional neutralization assay to determine the neutralization titer of serum and other samples, with the operation is more convenient, the results are more objective.

Conclusion This study successfully established a double-reporter CHIKV PsV system, and established a safe and reliable micro-neutralization assay based on the CHKV PsV, which provides a safe and effective platform for screening anti-CHIKV drugs and evaluating vaccines against CHIKV.