Tissue-based SARS-CoV-2 detection in fatal COVID-19 infections: Sustained direct viral-induced damage is not necessary to drive disease progression

Abstract

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although viral infection is known to trigger inflammatory processes contributing to tissue injury and organ failure, it is unclear whether direct viral damage is needed to sustain cellular injury. An understanding of pathogenic mechanisms has been handicapped by the absence of optimized methods to visualize the presence and distribution of SARS-CoV-2 in damaged tissues. We first developed a positive control cell line (Vero E6) to validate SARS-CoV-2 detection assays. We then evaluated multiple organs (lungs, kidneys, heart, liver, brain, intestines, lymph nodes, and spleen) from fourteen COVID-19 autopsy cases using immunohistochemistry (IHC) for the spike and the nucleoprotein proteins, and RNA in situ hybridization (RNA ISH) for the spike protein mRNA. Tissue detection assays were compared with quantitative polymerase chain reaction (qPCR)-based detection. SARS-CoV-2 was histologically detected in the Vero E6 positive cell line control, 1 of 14 (7%) lungs, and none (0%) of the other 59 organs. There was perfect concordance between the IHC and RNA ISH results. qPCR confirmed high viral load in the SARS-CoV-2 ISH-positive lung tissue, and absent or low viral load in all ISH-negative tissues. In patients who die of COVID-19-related organ failure, SARS-CoV-2 is largely not detectable using tissue-based assays. Even in lungs showing widespread injury, SARS-CoV-2 viral RNA or proteins were detected in only a small minority of cases. This observation supports the concept that viral infection is primarily a trigger for multiple-organ pathogenic proinflammatory responses. Direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression.

Keywords: COVID-19; Coronavirus; Diffuse alveolar damage; Nucleoprotein; RNA in situ hybridization; SARS-CoV-2; Spike protein.

Fig. 1

SARS-CoV-2 detection in infected Vero cell line. Cell pellet stained with hematoxylin and eosin (A). Immunohistochemical stain for the SARS-COV-2 nucleoprotein protein (B) and spike (C). RNA in situ hybridization for the sense RNA strand –S (D) and antisense RNA strand –SS (E) show cytoplasmic singles indicative of infected cells with active viral replication, although the density and confluence of signals was prone to obscure the nucleus in some cells. Immunofluorescence shows strong cytoplasmic staining within the majority of SARS-CoV-2–infected Vero cells (red) (F, inset of electron microscopy showing vesicles containing multiple viral particles).

Fig. 2

SARS-CoV-2 detection postmortem lung in case 7. The injured lung shows changes of diffuse alveolar damage including interstitial inflammation and hyaline membrane formation (A, hematoxylin and eosin stain). Virus is present in mostly within the alveolar spaces as detected by immunohistochemistry for the spike protein (B) and nucleoprotein (C), and in situ hybridization for the sense RNA strand (D). The viral infected cells show the same intra-alveolar distribution as the CD68 positive mononuclear phagocytic cells (E). The presence of SARS-CoV-2 was further confirmed by immunofluorescence for the nucleoprotein (F, red labeling).