Kumar Ranjeet, Kolloli Afsal, Subbian Selvakumar
Public Health Research Institute, New Jersey Medical School, Rutgers University, 225 Warren Street, Newark, NJ 07103, USA.
Methods Protoc. 2021 Feb 27;4(1):18. doi: 10.3390/mps4010018.
The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) causes Coronavirus disease-2019 (COVID-19), which is an ongoing pandemic that has significantly affected the health, economy, and socio-economic status of individuals worldwide. Laboratory research using in vitro, ex vivo and in vivo models has been accelerated to understand the pathogenesis of SARS-CoV-2 infection. However, such experimental research involving SARS-CoV-2 is restricted to biocontainment/safety level-3 (BSL-3) settings, due to the high pathogenicity of this virus. Since many of the downstream analyses of SARS-CoV-2-infected biological samples need to be conducted in a non-BSL3 setting, it is important to ensure that the samples are fully decontaminated and safe for subsequent analysis. Here, we report the effectiveness of standard procedures used to fix cells and tissues for pathological analysis, including 2% or 4% paraformaldehyde, 50%-70% ethanol, 10% neutral buffered formalin and ultrafiltration using membranes with a molecular weight cut-off (MWCO) ranging from 3 to 30 kDa, for inactivating or eliminating SARS-CoV-2. We validated these methods in experimental laboratory samples, such as viral inoculum in cell culture media, SARS-CoV-2 infected host cells and animal tissue lysates. We found that 15 minutes' treatment of viral inoculum (10 plaque-forming units; PFU) or SARS-CoV-2 infected cells with paraformaldehyde or 70% ethanol resulted in complete inactivation of the virus. The treatment of infected hamster lung tissues with 10% neutral buffered formalin also fully inactivated the virus. However, only 3 kDa ultracentrifuge filter was effective in eliminating the virus to an undetectable limit in the filtrate. Our validated methods are useful for decontaminating biological samples to reduce infection risk and safe handling in BSL2 facilities.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）引发了2019冠状病毒病（COVID-19），这是一场仍在持续的大流行疾病，已对全球个人的健康、经济和社会经济状况产生了重大影响。为了解SARS-CoV-2感染的发病机制，利用体外、离体和体内模型的实验室研究已加速进行。然而，由于该病毒的高致病性，此类涉及SARS-CoV-2的实验研究仅限于生物安全/安全等级3（BSL-3）环境。由于SARS-CoV-2感染生物样本的许多下游分析需要在非BSL3环境中进行，因此确保样本得到充分净化并对后续分析安全至关重要。在此，我们报告了用于固定细胞和组织以进行病理分析的标准程序的有效性，包括2%或4%多聚甲醛、50%-70%乙醇、10%中性缓冲福尔马林以及使用截留分子量（MWCO）范围为3至30 kDa的膜进行超滤，用于灭活或消除SARS-CoV-2。我们在实验实验室样本中验证了这些方法，如细胞培养基中的病毒接种物、SARS-CoV-2感染的宿主细胞和动物组织裂解物。我们发现，用多聚甲醛或70%乙醇对病毒接种物（10个噬斑形成单位；PFU）或SARS-CoV-2感染的细胞进行15分钟处理可导致病毒完全失活。用10%中性缓冲福尔马林处理感染的仓鼠肺组织也可使病毒完全失活。然而，只有3 kDa的超速离心过滤器能有效将病毒消除至滤液中检测不到的水平。我们验证的方法有助于净化生物样本，以降低感染风险并在BSL2设施中安全处理。
