Abstract
Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 viral proteins in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identified commercially available affinity-purified polyclonal antibodies raised against nucleocapsid and spike proteins of SARS-CoV-2 that provide sensitive and specific detection of the virus by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Two immunohistochemistry protocols are presented that are mutually validated by the matched detection patterns of the two distinct viral antigens in virus-infected cells within autopsy lung tissue of COVID-19 deceased patients. Levels of nucleocapsid protein in the lungs of COVID-19 decedents, as measured by quantitative histo-cytometry of immunohistochemistry images, showed an excellent log–linear relationship with levels of viral nucleocapsid RNA levels, as measured by qRT-PCR. Importantly, since the nucleocapsid protein sequence is conserved across all known viral strains, the nucleocapsid immunohistochemistry protocol is expected to recognize all common variants of SARS-CoV-2. Negative controls include autopsy lung tissues from patients who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. Sensitive detection of SARS-CoV-2 in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection, as well as provide insight into the clearance rate of virus in later COVID-19 disease stages. The protocols are also expected to be readily transferable to detect SARS-CoV-2 proteins in tissues of experimental animal models or animals suspected to serve as viral reservoirs.
Highlights
In December of 2019, an outbreak of pneumonia cases of unknown etiology occurred inWuhan, China, leading to the identification of a novel beta-coronavirus, named severe acute respiratory syndrome (SARS)-CoV2, as the causative agent [1,2,3]
COVID-19 patients with lower respiratory tract infection often develop acute respiratory disease syndrome (ARDS) that is observed in patients with severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), other corona-viral pneumonias
Since there was no positive control tissue available with known expression of viral proteins, we used a strategy of reciprocal cross-validation of the immunohistochemistry protocols by documenting corresponding staining patterns of the antibody directed to the nucleocapsid protein and the antibody directed to the spike protein in lungs from patients who died with known acute phase COVID-19 (Figure 1)
Summary
In December of 2019, an outbreak of pneumonia cases of unknown etiology occurred in. Wuhan, China, leading to the identification of a novel beta-coronavirus, named SARS-CoV2, as the causative agent [1,2,3]. COVID-19 patients with lower respiratory tract infection often develop acute respiratory disease syndrome (ARDS) that is observed in patients with severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), other corona-viral pneumonias. For rational development of treatments for COVID-19 there is a great need to understand the pathogenesis and pathology of COVID-19, in the respiratory tract, and in numerous other organs that become directly infected through vascular or neuronal spread. Numerous pathological changes appear secondary to, or indirectly from, viral infection, including aberrant immune cell activation, vascular changes, and coagulopathies, in turn affecting numerous organs [9,10,11]. One autopsy study reported IHC-detectable viral spike protein in lungs of 57% of COVID-19 fatalities (13/23) and concluded that “viral infection in areas of ongoing active injury contributes to persistent and temporally heterogeneous lung damage” [18], while another autopsy study used nucleocapsid protein immunohistochemistry and detected virus in only 7% (1/14) of COVID-19 decedents, and concluded that “direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression” [19]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.