Replication-Competent Virus Detected in Blood of a Fatal COVID-19 Case
Authors: Andrew Platt, Manmeet Singh, Sydney Stein, Shahabuddin Soherwardi, Emmie de Wit, Daniel S. Chertow
Journal: Annals of Internal Medicine (2023)

Letters12 December 2023Replication-Competent Virus Detected in Blood of a Fatal COVID-19 CaseFREEAndrew Platt, MD, PhD, Manmeet Singh, PhD, Sydney Stein, DVM, MPH, Shahabuddin Soherwardi, MD, Emmie de Wit, PhD, Daniel S. Chertow, MD, MPHAndrew Platt, MD, PhDLaboratory of Virology, National Institute of Allergy and Infectious Diseases, Emerging Pathogens Section, Critical Care Medicine Department, Clinical Center, and Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, Manmeet Singh, PhDLaboratory of Virology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, Sydney Stein, DVM, MPHLaboratory of Virology, National Institute of Allergy and Infectious Diseases, Emerging Pathogens Section, Critical Care Medicine Department, Clinical Center, and Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, Shahabuddin Soherwardi, MDHospitalist Department, TidalHealth Peninsula Regional, Salisbury, Maryland, Emmie de Wit, PhDLaboratory of Virology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, Daniel S. Chertow, MD, MPHLaboratory of Virology, National Institute of Allergy and Infectious Diseases, Emerging Pathogens Section, Critical Care Medicine Department, Clinical Center, and Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MarylandAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/L23-0253 SectionsAboutVisual AbstractPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail Background: SARS-CoV-2 infects respiratory tissues, and in some cases, replication-competent virus has been detected in extrapulmonary tissues, including brain (1). Viremic spread is suspected, and detection of viral RNA in blood is frequently reported (2). However, recovery of replication-competent virus from blood has not been previously demonstrated (3, 4).Objective: To confirm viremia in a fatal COVID-19 case where viral sequence in blood and tissues match.Case Report: A 76-year-old woman with a history of Turner syndrome, advanced dementia, and severe aortic stenosis was admitted to a community hospital in fall 2020 with new shortness of breath, hypoxia, and tachycardia per the patient's home nursing facility. Her heart rate was 120 beats/min, respiratory rate was 30 breaths/min, and peripheral oxygen saturation was 84% on bilevel positive airway pressure. Chest radiograph revealed bilateral infiltrates, and a nasopharyngeal swab was positive for SARS-CoV-2 by reverse transcriptase polymerase chain reaction. She required invasive mechanical ventilation and was treated with dexamethasone, remdesivir, empirical antibiotics, and vasopressors, developing oliguric renal failure and lactic acidosis. Considering the patient's critical illness and her end-of-life wishes, the family elected for comfort measures. She was compassionately extubated and died 1 day after admission.The next of kin consented for autopsy and were referred to the National Institutes of Health (NIH) Clinical Center in Bethesda, Maryland, for an autopsy study in COVID-19 (1). Perimortem blood drawn at time of death was collected in heparin EDTA tubes. Plasma was isolated and stored at −80°C. SARS-CoV-2 RNA and subgenomic RNA, suggestive of recent virus replication, were detected in plasma, lung, heart, and several additional tissues, and SARS-CoV-2 was detected by Vero E6 cell culture in lung and heart, but not plasma as previously reported (1). A second effort to isolate virus from plasma using a modified Vero cell line stably expressing human transmembrane protease serine 2 and angiotensin-converting enzyme 2 was successful. Subgenomic RNA polymerase chain reaction cycle threshold values decreased from 32.51 (3.48 × 104 RNA copies/mL) in the original plasma sample to 21.93 (1.14 × 109 RNA copies/mL) in the cell culture supernatant after 6 days. Cytopathic effect was seen in 2 of 2 replicates cultured with undiluted plasma and 1 of 2 replicates cultured with a 1:10 plasma dilution.Full-length genomic sequencing of pre- and postviral culture plasma, lung, and cardiac samples demonstrated more than 99% sequence similarity with multiple conserved mutations compared with the WA1 reference sequence, except for 3 synonymous and 1 nonsynonymous mutation present in 45% to 62% of reads in the postviral culture plasma sample (Figure).Figure. Sequencing results from primary samples and cell culture supernatants.NSP = nonstructural protein; UTR = untranslated region. Top. Representative schematic of the SARS-CoV-2 genome. Due to low input, a full-length contig could not be generated for the original plasma and lung samples. The location of the gaps in sequences is represented by green bars with diagonal lines for the plasma sample and by solid purple bars for the lung sample. Bottom. Synonymous and nonsynonymous mutations present in the SARS-CoV-2 sequence from the plasma, cell culture isolate from plasma, lung, cell culture isolate from the lung, and cell culture isolate from the heart as obtained by short-read sequencing and in reference to the SARS-CoV-2 2019-nCoV/USA-WA1/2020 genome (MN985325.1). Libraries were generated using the Illumina Ligation Prep kit sequenced on a MiSeq instrument (Illumina). The autopsy plasma sample had hybrid capture virus enrichment using myBaits Expert Virus SARS-CoV-2 panel (Arbor Biosciences) before sequencing. Values represent alternative variant percentage reads.* Additional mutations present in cell culture isolate from plasma samples. Download figure Download PowerPoint Several groups have reported detection of viral RNA in blood early during COVID-19 that typically becomes undetectable within 2 weeks of illness onset (2); however, previous efforts to isolate virus from blood have been unsuccessful (3, 4). The paradox of detecting replication-competent virus in extrapulmonary tissues but not blood from patients with COVID-19 has yet to be explained. The patient reported here was 1 of 44 autopsies done at the NIH during the first year of the pandemic, previously described in aggregate (1); we detected SARS-CoV-2 RNA in plasma from 12 patients. This patient had the highest level of SARS-CoV-2 RNA in plasma at 1.2 × 105 gene copies/mL, among the highest SARS-CoV-2 RNA and subgenomic RNA levels in other tissues, and was 1 of only 3 patients without an IgM or IgG antibody response to SARS-CoV-2 spike or nucleocapsid proteins (1, 5). We attempted viral culture of plasma using our modified Vero cell line in 7 additional patients (ages 30 to 53 years, samples 2 to 25 days from symptom onset) with SARS-CoV-2 RNAemia ranging from 7.8 × 103 to 9.5 × 104 gene copies/mL; all were negative.Discussion: Our case proves that replication-competent SARS-CoV-2 can traffic in blood during COVID-19 and seed tissues throughout the body. Use of a permissive Vero cell line increased sensitivity of detecting SARS-CoV-2 in plasma from our patient, as did using plasma from a critically ill but seronegative patient. Cell culture induced minor SARS-CoV-2 sequence variability, consistent with known Vero E6 cell culture effects, but overall, the same isolate was recovered before and after culture from the lungs, plasma, and heart of this patient. Further studies are needed to determine the implications of our findings for persons infected with SARS-CoV-2 variants, those with mild illness, persons recently vaccinated, or persons with waning immunity after natural infection or vaccination.References1. Stein SR, Ramelli SC, Grazioli A, et al; NIH COVID-19 Autopsy Consortium. SARS-CoV-2 infection and persistence in the human body and brain at autopsy. Nature. 2022;612:758-763. [PMID: 36517603] doi:10.1038/s41586-022-05542-y CrossrefMedlineGoogle Scholar2. Bwire GM, Majigo MV, Njiro BJ, et al. Detection profile of SARS-CoV-2 using RT-PCR in different types of clinical specimens: a systematic review and meta-analysis. J Med Virol. 2021;93:719-725. [PMID: 32706393] doi:10.1002/jmv.26349 CrossrefMedlineGoogle Scholar3. Andersson MI, Arancibia-Carcamo CV, Auckland K, et al. SARS-CoV-2 RNA detected in blood products from patients with COVID-19 is not associated with infectious virus. Wellcome Open Res. 2020;5:181. [PMID: 33283055] doi:10.12688/wellcomeopenres.16002.2 CrossrefMedlineGoogle Scholar4. Saá P, Fink RV, Bakkour S, et al; NHLBI Recipient Epidemiology and Donor Evaluation Study-IV-Pediatric (REDS-IV-P). Frequent detection but lack of infectivity of SARS-CoV-2 RNA in presymptomatic, infected blood donor plasma. J Clin Invest. 2022;132:e159876. [PMID: 35834347] doi:10.1172/JCI159876 CrossrefMedlineGoogle Scholar5. Huang N, Pérez P, Kato T, et al; NIH COVID-19 Autopsy Consortium, HCA Oral and Craniofacial Biological Network. SARS-CoV-2 infection of the oral cavity and saliva. Nat Med. 2021;27:892-903. [PMID: 33767405] doi:10.1038/s41591-021-01296-8 CrossrefMedlineGoogle Scholar Comments0 CommentsSign In to Submit A Comment Author, Article, and Disclosure InformationAuthors: Andrew Platt, MD, PhD; Manmeet Singh, PhD; Sydney Stein, DVM, MPH; Shahabuddin Soherwardi, MD; Emmie de Wit, PhD; Daniel S. Chertow, MD, MPHAffiliations: Laboratory of Virology, National Institute of Allergy and Infectious Diseases, Emerging Pathogens Section, Critical Care Medicine Department, Clinical Center, and Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MarylandLaboratory of Virology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MarylandHospitalist Department, TidalHealth Peninsula Regional, Salisbury, MarylandAcknowledgment: The authors thank Kent Barbian and Paul Beare for help with SARS-CoV-2 genome sequencing and related analysis. The following reagent was obtained through BEI Resources, the National Institute of Allergy and Infectious Diseases, and the NIH: Cercopithecus aethiops Kidney Epithelial Cells Expressing Transmembrane Protease, Serine 2 and Human Angiotensin-Converting Enzyme 2 (Vero E6-TMPRSS2-T2A-ACE2), NR-54970.Financial Support: By the Intramural Research Programs of the National Institute of Allergy and Infectious Diseases and the NIH Clinical Center.Disclosures: Disclosures can be viewed at www.acponline.org/authors/icmje/ConflictOfInterestForms.do?msNum=L23-0253.Corresponding Author: Andrew Platt, MD, PhD, National Institute of Allergy and Infectious Diseases, 10 Center Drive, Building 10, Room 141, Bethesda, MD 20892; e-mail, andrew.platt@nih.gov.This article was published at Annals.org on 12 December 2023. PreviousarticleNextarticle Advertisement FiguresReferencesRelatedDetails Metrics Current IssueJanuary 2024Volume 177, Issue 1Page: 113-115KeywordsAutopsyBloodCell culturesCOVID-19Infectious diseasesMutationRNA ePublished: 12 December 2023 Issue Published: January 2024 Copyright & PermissionsCopyright © 2023 by American College of Physicians. All Rights Reserved.PDF downloadLoading ...