Pathology and Pathogenesis of SARS-CoV-2 Associated with Fatal Coronavirus Disease, United States Roosecelis B. Martines,1 Jana M. Ritter,1 Eduard Matkovic, Joy Gary, Brigid C. Bollweg, Hannah Bullock, Cynthia S. Goldsmith, Luciana Silva-Flannery, Josilene N. Seixas, Sarah Reagan-Steiner, Timothy Uyeki, Amy Denison, Julu Bhatnagar, Wun-Ju Shieh, Sherif R. Zaki; COVID-19 Pathology Working Group2 An ongoing pandemic of coronavirus disease (CO- United States; since then, all 50 US states, District of VID-19) is caused by infection with severe acute respi- Columbia, Guam, Puerto Rico, Northern Mariana Is- ratory syndrome coronavirus 2 (SARS-CoV-2). Charac- lands, and US Virgin Islands have confirmed cases of terization of the histopathology and cellular localization COVID-19 (2–4). of SARS-CoV-2 in the tissues of patients with fatal CO- Coronaviruses are enveloped, positive-strand- VID-19 is critical to further understand its pathogenesis ed RNA viruses that infect many animals; human- and transmission and for public health prevention mea- adapted viruses likely are introduced through zoo- sures. We report clinicopathologic, immunohistochemi- notic transmission from animal reservoirs (5,6). Most cal, and electron microscopic findings in tissues from known human coronaviruses are associated with 8 fatal laboratory-confirmed cases of SARS-CoV-2 in- mild upper respiratory illness. SARS-CoV-2 belongs fection in the United States. All cases except 1 were in to the group of betacoronaviruses that includes severe residents of long-term care facilities. In these patients, SARS-CoV-2 infected epithelium of the upper and lower acute respiratory syndrome coronavirus (SARS-CoV) airways with diffuse alveolar damage as the predominant and Middle East respiratory syndrome coronavirus pulmonary pathology. SARS-CoV-2 was detectable by (MERS-CoV), which can infect the lower respiratory immunohistochemistry and electron microscopy in con- tract and cause a severe and fatal respiratory syn- ducting airways, pneumocytes, alveolar macrophages, drome in humans (7). SARS-CoV-2 has >79.6% simi- and a hilar lymph node but was not identified in other larity in genetic sequence to SARS-CoV (5). extrapulmonary tissues. Respiratory viral co-infections SARS-CoV-2 is highly transmissible among hu- were identified in 3 cases; 3 cases had evidence of bac- mans; fatality rates for COVID-19 vary and are higher terial co-infection. among the elderly and persons with underlying condi- tions or immunosuppression (8,9). The current knowl- he ongoing global pandemic of coronavirus dis- edge about COVID-19 pathogenesis and pathology in Tease (COVID-19), caused by severe acute respi- fatalities is based on a small number of described cas- ratory syndrome coronavirus 2 (SARS-CoV-2), was es and extrapolations from what is known about other identified in Wuhan, Hubei Province, China, and has similar coronaviruses, such as SARS-CoV and MERS- spread rapidly around the world (1,2). As of May CoV (10–18). Pathologic evaluation and determination 18, 2020, World Health Organization official data re- of virus distribution and cellular localization within ported 4,628,903 confirmed cases and 312,009 deaths tissues is crucial to elucidating the pathogenesis of (2). On January 20, 2020, the Centers for Disease Con- these fatal infections and can help guide development trol and Prevention (CDC) confirmed a case in the of therapeutic and preventive countermeasures. We report on the histopathologic features and detection of Author affiliations: Centers for Disease Control and Prevention, virus in tissues by immunohistochemistry (IHC) and Atlanta, Georgia, USA (R.B. Martines, J.M. Ritter, E. Matkovic, electron microscopy (EM) from 8 confirmed fatal cases J. Gary, B.C. Bollweg, C.S. Goldsmith, L. Silva-Flannery, of COVID-19 in the United States. J.N. Seixas, S. Reagan-Steiner, T. Uyeki, A. Denison, J. Bhatnagar, W.-J. Shieh, S.R. Zaki); Synergy America Inc., 1These authors contributed equally to this article. Atlanta (H. Bullock). 2Members of the COVID-19 Pathology Working Group: Rhonda DOI: https://doi.org/10.3201/eid2609.202095 Cole, Amanda Lewis, Pamela Fair, Lindsey Estetter. Emerging Infectious Diseases • www.cdc.gov/eid •Vol. 26, No. 9, September 2020 2005 SYNOPSIS Materials and Methods For double-stained assays, we used Envision G/2 Double Stain System, Rabbit/Mouse (DAB Perma- Study Patients and Data Collection nent Red) from Agilent Technologies (https://www. As part of the public health response to COVID-19, agilent.com). We used antibodies against CD163 (Lei- the CDC Infectious Diseases Pathology Branch (Di- ca Biosystems, https://www.leicabiosystems.com) vision of High-Consequence Pathogens and Pathol- or surfactant apoprotein A (Dako, https://www. ogy, National Center for Emerging and Zoonotic agilent.com), followed by the anti–SARS-CoV nucleo- Infectious Diseases) was consulted on autopsies of capsid antibody (Novus Biologicals). We performed 8 patients with laboratory evidence of SARS-CoV-2 all assays according to the manufacturer’s guide- by reverse transcription PCR (RT-PCR) on respira- lines. We used SARS-CoV-2–infected Vero cells as a tory swab specimens collected either before or after positive control and used non–COVID-19 cases and death. We reviewed available medical records and normal rabbit serum in place of primary antibody as preliminary autopsy reports for information regard- negative controls. ing demographics, symptom history, underlying conditions, infectious disease testing, imaging study Electron Microscopy findings, treatment and advanced supportive care We obtained upper airway and lung tissue specimens received, and date of death. This investigation was from formalin-fixed samples, cut them into cubes, reviewed in accordance with CDC’s human subjects rinsed them with 0.1 mmol/L phosphate buffer, post- review procedures and was determined to not meet fixed them with 2.5% glutaraldehyde, and rinsed them the definition of research. in phosphate buffer. In addition, we removed tissue samples from areas corresponding to positive SARS- Histopathology and Immunohistochemistry CoV-2 immunostaining from paraffin blocks with a We performed routine hematoxylin–eosin stains for 2-mm punch or from 4-µm sections on glass slides; we histopathologic evaluation. We conducted an IHC deparaffinized the samples in xylene and rehydrated. assay for SARS-CoV-2 using a rabbit polyclonal an- We processed tissues for transmission EM as described tibody raised against SARS-CoV nucleocapsid (No- previously (20). We immersed the sections embedded vus Biologicals, https://www.novusbio.com) (19) at in epoxy resin in boiling water, removed them from the 1:100 dilution and a Mach 4 Universal AP Polymer slides with a razor blade, and cut out the areas of inter- Kit (Biocare Medical, https://biocare.net) with Per- est and glued them onto a blank EM block. We stained manent Red Chromogen (Cell Marque/Millipore Sig- EM sections with uranyl acetate and lead citrate and ma, https://www.cellmarque.com). We pretreated examined them on a Thermo Fisher/FEI Tecnai Spirit the slides with heat-induced epitope retrieval with or Tecnai BioTwin electron microscope. a citrate-based buffer (Biocare Medical). We ran ap- propriate negative controls in parallel, using normal RT-PCR rabbit serum in place of the primary antibody. We We extracted nucleic acids from formalin-fixed par- validated cross reactivity of the anti–SARS-CoV an- affin-embedded (FFPE) tissues and assessed them by tibody with SARS-CoV-2 by testing controls created a conventional RT-PCR specifically targeting the nu- from SARS-CoV-2–infected Vero cells embedded cleocapsid gene of SARS-CoV-2 (J. Bhatnagar, unpub. with normal human tissues; we used this control as data) and real-time RT-PCR/PCR targeting other the positive control for subsequent IHC assays. The respiratory pathogens, including influenza viruses, SARS-CoV nucleocapsid antibody did not cross-react respiratory syncytial virus, human parainfluenza with influenza A(H1N1) virus, influenza B virus, re- viruses, and Streptococcus pneumoniae, as described spiratory syncytial virus, parainfluenza virus type 3, previously (21,22). The SARS-CoV-2 nucleocapsid as- human coronavirus (HCoV) 229E, or MERS-CoV in say detects SARS-CoV-2 and SARS-CoV but does not PCR-confirmed tissue samples. For cases with bron- amplify MERS-CoV and other common human coro- chopneumonia, we performed IHC testing for bacte- naviruses, including alphacoronavirus (HCoV-NL63) rial agents using a mouse monoclonal antibody raised and betacoronavirus (HCoV-HKU1). against Streptococcus pneumoniae but known to also detect other Streptococcus spp. and a rabbit polyclonal Results anti-Klebsiella pneumoniae antibody (both from Ther- mo Fisher, https://www.thermofisher.com) known Clinical Data to also detect other gram-negative bacteria (Escherich- Of the 8 case-patients, 7 were residents of a long- ia coli, Haemophilus influenzae, and Pseudomonas spp.). term care facility (LTCF) in Washington state (Table 2006 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 26, No. 9, September 2020 Pathology and Pathogenesis of SARS-CoV-2 1) (23,24). Seven (87.5%) were White, non-Hispanic. or myocardial necrosis and no notable histopatho- The median age of the 8 case-patients was 73.5 years; logic changes in the intestine were seen in any case. 2 were <65 years of age. The median number of days Brain tissues were not available for histopathologic from illness onset until death was 12.5 (range 6–15). evaluation or testing. Common signs and symptoms reported included We detected SARS-CoV-2 by IHC in the upper fever (75%), cough (62.5%), and shortness of breath airways in 4/8 (50%) case-patients and in the lungs in (62.5%). All patients had abnormal findings on chest 7/8 (92%) case-patients. We observed immunostain- radiographs. Case-patients were all hospitalized for ing of viral antigens in upper airway and bronchio- a median of 3.5 days. Six (75%) patients received lar epithelium, submucosal gland epithelium, and mechanical ventilation; 2 received comfort care.