A new study accepted by Cell showed that SARS-CoV-2 infects the nasal cavity to the greatest degree and infects and replicates progressively less well lower in the respiratory tract. The SARS-CoV-2 genome is made up of 16 nonstructural proteins, four structural proteins and eight accessory proteins. The spike protein mediates the viral entry by binding to the human ACE-2 enzyme and is the main target of host neutralising antibodies.
To help understand the organ-level architecture of receptor expression, the researchers developed a combination of RNA in situ hybridisation techniques and novel SARS-CoV-2 reporter viruses by reverse genetics. Reverse genetics is a technique that has been made possible by recombinant DNA technology, where researchers start from a protein or DNA for which there is no genetic information and work backwards to make a mutant gene, ending up with a mutant phenotype.
Reverse genetics allowed the researchers to generate a green fluorescent protein (GFP) reporter virus to explore the pathogenesis of SARS-CoV-2 and a luciferase reporter virus to demonstrate that sera from SARS and COVID-19 patients exhibited limited cross-coronavirus neutralisation.
They then used northern blot analysis to differentate between the reporter viral samples, the clinical isolates and wildtype samples. Ralph Baric, one of the authors, states that “this is a landmark study that reveals new and unexpected insights into the mechanisms that regulate disease progression and severity following SARS-CoV-2 infection” and that they also “describe a new reverse genetic platform for SARS-CoV-2 allowing [researchers] to produce key indicator viruses that will support national vaccine efforts”.
The team found that serine proteases and furin enhanced the replication efficiency of SARS-CoV-2 in vitro. As part of the study, the researchers also characterised ACE2 expression in the upper airways and lungs using high-sensitivity RNA in situ hybridisation. The analysis revealed the highest ACE2 expression was in the news and decreased throughout the lower respiratory tract. The team suggested that nasal surfaces may be the dominant initial site for infection, as it is primarily composed of ciliated cells in the superficial epithelium. Breathing may contribute to the spread of the infection to the lower airways, which aligned with the results of COVID-19 autopsy studies that found that the patchiness of SARS-CoV-2 matches the pathogenesis of an infection that originated in the upper-airway.
The techniques developed from the study, including a version of SARS-CoV-2 re-engineered to carry a fluorescent beacon, will help future research into the virus.
James Kiley, director of the Division of Lung Diseases at the National Heart, Lung and Blood Institute said that the “results open new directions for future studies that may guide therapeutic development and practices for reducing transmission and severity of COVID-19”.
