Molecular detection of SARS-CoV-2 in formalin-fixed, paraffin-embedded specimens
Authors: Liu, Jun; Babka, April M.; Kearney, Brian J.; Radoshitzky, Sheli R.; Kuhn, Jens H.; Zeng, Xiankun
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. The virus rapidly spread globally, resulting in a public health crisis including almost 5 million cases and 323,256 deaths as of May 21, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected FFPE cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross-detection of the respective SARS-CoV-2 proteins by IHC and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex FISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. It is hoped that these reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.
Acute SARS-CoV-2 Infection Impairs Dendritic Cell and T Cell Responses
Authors: Zhou, Runhong; To, Kelvin Kai-Wang; Wong, Yik-Chun; Liu, Li; Zhou, Biao; Li, Xin; Huang, Haode; Mo, Yufei; Luk, Tsz-Yat; Lau, Thomas Tsz-Kan; Yeung, Pauline; Chan, Wai-Ming; Wu, Alan Ka-Lun; Lung, Kwok-Cheung; Tsang, Owen Tak-Yin; Leung, Wai-Shing; Hung, Ivan Fan-Ngai; Yuen, Kwok-Yung; Chen, Zhiwei
The SARS-CoV-2 pandemic has resulted in millions of infections, yet the role of host immune responses in early COVID-19 pathogenesis remains unclear, By investigating 17 acute and 24 convalescent patients, we found that acute SARS-CoV-2 infection resulted in broad immune cell reduction including T, natural killer, monocyte, and dendritic cells (DCs). DCs were significantly reduced with functional impairment, and ratios of conventional DCs to plasmacytoid DCs were increased among acute severe patients. Besides lymphocytopenia, although neutralizing antibodies were rapidly and abundantly generated in patients, there were delayed receptor binding domain (RBD)- and nucleocapsid protein (NP)-specific T cell responses during the first 3 weeks after symptoms onset. Moreover, acute RBD- and NP-specific T cell responses included relatively more CD4 T cells than CD8 T cells. Our findings provided evidence that impaired DCs, together with timely inverted strong antibody but weak CD8 T cell responses, could contribute to acute COVID-19 pathogenesis and have implications for vaccine development.