Detection of the SARS-CoV-2 Nucleocaspid Protein (NP) Using Immunohistochemistry

Background

In December 2019, clusters of patients with pneumonia of unknown etiology were reported by the local health facilities in Wuhan, Hubei Province, China (Li et al., 2020; Zhu et al., 2020). In January 2020, the causative agent of mysterious pneumonia was identified as a novel coronavirus named as 2019 novel coronavirus (2019-nCoV) by the World Health Organization (WHO). This novel coronavirus was renamed as the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the consequent disease was designated as coronavirus disease 2019 (COVID-19) (Lu et al., 2020; Xu et al., 2020). According to the daily report of the WHO, the epidemic of SARS-CoV-2 has so far caused 81961 laboratory confirmed cases and 3293 death in China. Meanwhile, the number of confirmed COVID-19 and fatalities around the world are 465315 and 21031, respectively, by 26^th March 2020 (World Health Organization, 2020).

The importance of the secondary lymphoid organs, including spleen and lymph nodes (LNs), for resistance against infection is well established. Tissue-resident macrophages positioned in the splenic marginal zone (MZ) that are among the first cell types to encounter invading pathogens (Aichele et al., 2003; Mebius and Kraal, 2005). Similarly, the resident macrophages of the subcapsular sinus and hilar lymph nodes have been shown to play a protective role against viral infections by capturing viral particles (Junt et al., 2007; Tamburini et al., 2014). These macrophages present viral antigens to activate specific T cells, to induce activation, clonal proliferation, and subsequent killing of infected target cells through secretion of perforin, granzyme B, and interferon-γ etc (Backer et al., 2010; Bernhard et al., 2015; van Dinther et al., 2018). Moreover, these activated T cells also egress from spleen and LNs into blood circulation and play immune surveillance roles (Gebhardt et al., 2013). Unfortunately, our previous work has shown that lymphocytopenia is prevalent in COVID-19 patients, especially in aged and critically ill cases, suggesting that the immune system from COVID-19 patients might be neutralized by infection (Diao et al., 2020).

Therefore, understanding the destinies of lymphocytes in secondary lymphoid organs is critical for understanding SARS-CoV-2 viral infection, and the identification of methods to boost lymphocytes and enhance the host immunity in vivo.

In this study, the tissue samples of spleens and LNs are from six COVID-19 patients. Viral nucleocapsid protein (NP) antigen, cell apoptosis and proinflammatory cytokine expression were measured by immunohistochemistry. And the protocol is detailed below.