Comparison of Subgenomic and Total RNA in SARS-CoV-2-Challenged Rhesus Macaques

Developing therapeutic and prophylactic countermeasures for the SARS-CoV-2 virus is a public health priority. During challenge studies, respiratory viruses are delivered and sampled from the same anatomical location. ABSTRACT Respiratory virus challenge studies involve administration of the challenge virus and sampling to assess for protection in the same anatomical locations. It can therefore be difficult to differentiate actively replicating virus from input challenge virus. For SARS-CoV-2, specific monitoring of actively replicating virus is critical for investigating the protective and therapeutic efficacy of vaccines, monoclonal antibodies, and antiviral drugs. We adapted a SARS-CoV-2 subgenomic RNA (sgRNA) RT-PCR assay to differentiate productive infection from inactivated or neutralized virus. Subgenomic RNAs are generated after cell entry and are poorly incorporated into mature virions, and thus may provide a marker for actively replicating virus. We show envelope (E) sgRNA was degraded by RNase in infected cell lysates, while genomic RNA (gRNA) was protected, presumably due to packaging into virions. To investigate the capacity of the sgRNA assay to distinguish input challenge virus from actively replicating virus in vivo, we compared the E sgRNA assay to a standard nucleoprotein (N) or E total (both gRNA and sgRNA) RNA in convalescent rhesus macaques and in antibody-treated rhesus macaques after experimental SARS-CoV-2 challenge. In both studies, the E sgRNA assay was negative, suggesting protective efficacy, whereas the N and E total RNA assays remained positive. These data suggest the potential utility of sgRNA to monitor actively replicating virus in prophylactic and therapeutic SARS-CoV-2 studies. IMPORTANCE Developing therapeutic and prophylactic countermeasures for the SARS-CoV-2 virus is a public health priority. During challenge studies, respiratory viruses are delivered and sampled from the same anatomical location. It is therefore important to distinguish actively replicating virus from input challenge virus. The most common assay for detecting SARS-CoV-2 virus, reverse transcription PCR (RT-PCR) targeting nucleocapsid total RNA, cannot distinguish neutralized input virus from replicating virus. In this study, we assess SARS-CoV-2 subgenomic RNA as a potential measure of replicating virus in rhesus macaques.

[1]  V. Munster,et al.  ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques , 2020, Nature.

[2]  Rebecca J. Loomis,et al.  Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates , 2020, The New England journal of medicine.

[3]  D. Lauffenburger,et al.  Single-Shot Ad26 Vaccine Protects Against SARS-CoV-2 in Rhesus Macaques , 2020, Nature.

[4]  Lisa E. Gralinski,et al.  Potently neutralizing and protective human antibodies against SARS-CoV-2 , 2020, Nature.

[5]  Lisa E. Gralinski,et al.  SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract , 2020, Cell.

[6]  P. Sorger,et al.  SARS-CoV-2 infection protects against rechallenge in rhesus macaques , 2020, Science.

[7]  R. Baric,et al.  DNA vaccine protection against SARS-CoV-2 in rhesus macaques , 2020, Science.

[8]  P. Vollmar,et al.  Virological assessment of hospitalized patients with COVID-2019 , 2020, Nature.

[9]  Hyeshik Chang,et al.  The Architecture of SARS-CoV-2 Transcriptome , 2020, Cell.

[10]  E. Holmes,et al.  Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding , 2020, The Lancet.

[11]  Jing Zhao,et al.  Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus–Infected Pneumonia , 2020, The New England journal of medicine.

[12]  S. Lo,et al.  A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster , 2020, The Lancet.

[13]  Chuan Qin,et al.  From SARS to MERS, Thrusting Coronaviruses into the Spotlight , 2019, Viruses.

[14]  Matteo Ricchi,et al.  A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything , 2017, Front. Microbiol..

[15]  Richard G. Jarman,et al.  Vaccine Protection Against Zika Virus from Brazil , 2016, Nature.

[16]  I. Sola,et al.  Continuous and Discontinuous RNA Synthesis in Coronaviruses. , 2015, Annual review of virology.

[17]  S. Perlman,et al.  Coronaviruses: An Overview of Their Replication and Pathogenesis , 2015, Methods in molecular biology.

[18]  H. Margolis,et al.  Analytical and Clinical Performance of the CDC Real Time RT-PCR Assay for Detection and Typing of Dengue Virus , 2013, PLoS neglected tropical diseases.

[19]  Oumar Faye,et al.  One-step RT-PCR for detection of Zika virus. , 2008, Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology.

[20]  Stuart G. Siddell,et al.  A Contemporary View of Coronavirus Transcription , 2006, Journal of Virology.

[21]  D. Brian,et al.  Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Lai,et al.  Defective-interfering particles of murine coronavirus: Mechanism of synthesis of defective viral RNAs , 1988, Virology.

[23]  S. Sawicki,et al.  Coronavirus Transcription: A Perspective , 2005, Current topics in microbiology and immunology.