Severe Acute Respiratory Syndrome Coronavirus 2 From Patient With Coronavirus Disease, United States

Jennifer Harcourt; Azaibi Tamin; Xiaoyan Lu; Shifaq Kamili; Senthil K. Sakthivel; Janna Murray; Krista Queen; Ying Tao; Clinton R. Paden; Jing Zhang; Yan Li; Anna Uehara; Haibin Wang; Cynthia Goldsmith; Hannah A. Bullock; Lijuan Wang; Brett Whitaker; Brian Lynch; Rashi Gautam; Craig Schindewolf; Kumari G. Lokugamage; Dionna Scharton; Jessica A. Plante; Divya Mirchandani; Steven G. Widen; Krishna Narayanan; Shinji Makino; Thomas G. Ksiazek; Kenneth S. Plante; Scott C. Weaver; Stephen Lindstrom; Suxiang Tong; Vineet D. Menachery; Natalie J. Thornburg

Disclosures

Emerging Infectious Diseases. 2020;26(6):1266-1273. 

In This Article

Results

A patient was identified with confirmed COVID-19 in Washington State on January 22, 2020. CPE was not observed in mock infected cells (Figure 1, panel A). Cycle threshold (Ct) values were 18–20 for NP specimens and 21–22 for OP specimens.[1] The positive clinical specimens were aliquoted and refrozen inoculated into cell culture on January 22, 2020. We observed CPE 2 days postinoculation and harvested viral lysate on day 3 postinoculation (Figure 1, panels B, C). We used 50 μL of passage 1 viral lysates for nucleic acid extraction to confirm the presence of SARS-CoV-2 by using the CDC molecular diagnostic assay.[1] The Ct values of 3 nucleic acid extractions were 16.0–17.1 for nucleocapsid portion 1, 15.9–17.1 for nucleocapsid portion 2, and 16.2–17.3 for nucleocapsid portion 3, which confirmed isolation of SARS-CoV-2 (Ct <40 is considered a positive result). We also tested extracts for 33 additional different respiratory pathogens by using the Fast Track 33 Assay. No other pathogens were detected. Identity was additionally supported by thin-section electron microscopy (Figure 1, panel D). We observed a morphology and morphogenesis characteristic of coronaviruses.

Figure 1.

Cytopathic effect caused by severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States, 2020. A–C) Phase-contrast microscopy of Vero cell monolayers at 3 days postinoculation: A) Mock, B) nasopharyngeal specimen, C) oropharyngeal specimen. Original magnifications ×10). D) Electron microscopy of virus isolate showing extracellular spherical particles with cross-sections through the nucleocapsids (black dots). Arrow indicates a coronavirus virion budding from a cell. Scale bar indicates 200 nm.

We used isolates from the first passage of an OP and an NP specimen for whole-genome sequencing. The genomes from the NP specimen (GenBank accession MT020880) and OP specimen (GenBank accession no. MT020881) showed 100% identity with each other. The isolates also showed 100% identity with the corresponding clinical specimen (GenBank accession no. MN985325).

After the second passage, we did not culture OP and NP specimens separately. We passaged virus isolate 2 more times in Vero CCL-81 cells and titrated by determining the 50% tissue culture infectious dose (TCID50). Titers were 8.65 × 106 TCID50/mL for the third passage and 7.65 × 106 TCID50/mL for the fourth passage.

We passaged this virus in the absence of trypsin. The spike protein sequence of SARS-CoV-2 has an RRAR insertion at the S1-S2 interface that might be cleaved by furin.[16] Highly pathogenic avian influenza viruses have highly basic furin cleavage sites at the hemagglutinin protein HA1-HA2 interface that permit intracellular maturation of virions and more efficient viral replication.[17] The RRAR insertion in SARS-CoV-2 might serve a similar function.

We subsequently generated a fourth passage stock of SARS-CoV-2 on VeroE6 cells, another fetal rhesus monkey kidney cell line. We sequenced viral RNA from SARS-CoV-2 passage 4 stock and confirmed it to have no nucleotide mutations compared with the original reference sequence (GenBank accession no. MN985325). SARS-CoV has been found to grow well on VeroE6 cells and MERS-CoV on Vero CCL81 cells.[18,19] To establish a plaque assay and determine the preferred Vero cell type for quantification, we titered our passage 4 stock on VeroE6 and VeroCCL81 cells. After infection with a dilution series, SARS-CoV-2 replicated in both Vero cell types; however, the viral titers were slightly higher in VeroE6 cells than in Vero CCL81 cells (Figure 2, panel A). In addition, plaques were more distinct and visible on Vero E6 cells (Figure 2, panel B). As early as 2 days postinoculation, VeroE6 cells produced distinct plaques visible by staining with neutral red. In contrast, Vero CCL81 cells produced less clear plaques and was most easily quantitated by staining with neutral red 3 days postinoculation. On the individual plaque monolayers, SARS-CoV-2 infection of Vero E6 cells produced CPE with areas of cell clearance (Figure 2, panel C). In contrast, Vero CCL81 cells had areas of dead cells that had fused to form plaques, but the cells did not clear. Together, these results suggest that VeroE6 cells might be the best choice for amplification and quantification, but both Vero cell types support amplification and replication of SARS-CoV-2.

Figure 2.

Viral propagation and quantitation of severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States, 2020. A) Two virus passage 4 stocks (black and gray circles) were quantified by using plaque assay at day 2 (solid circles) and day 3 (open circles) postinfection of Vero E6 and Vero CCL81 cells. B) Plaque morphology for virus on Vero E6 and Vero CCL81 at day 2 and day 3 postinoculation. C) Cell monolayers 2 days postinfection of Vero E6 (top) and Vero CCL81 (bottom) at 3 dilutions. Original magnifications ×40.

Because research has been initiated to study and respond to SARS-CoV-2, information about cell lines and types susceptible to infection is needed. Therefore, we examined the capacity of SARS-CoV-2 to infect and replicate in several common primate and human cell lines, including human adenocarcinoma cells (A549), human liver cells (HUH7.0), and human embryonic kidney cells (HEK-293T), in addition to Vero E6 and Vero CCL81 cells. We also examined an available big brown bat kidney cell line (EFK3B) for SARS-CoV-2 replication capacity. Each cell line was inoculated at high multiplicity of infection and examined 24 h postinfection (Figure 3, panel A). No CPE was observed in any of the cell lines except in Vero cells, which grew to >107 PFU at 24 h postinfection. In contrast, HUH7.0 and 293T cells showed only modest viral replication, and A549 cells were incompatible with SARS-CoV-2 infection. These results are consistent with previous susceptibility findings for SARS-CoV and suggest other common culture systems, including MDCK, HeLa, HEP-2, MRC-5 cells, and embryonated eggs, are unlikely to support SARS-CoV-2 replication.[20–22] In addition, SARS-CoV-2 did not replicate in bat EFK3B cells, which are susceptible to MERS-CoV. Together, the results indicate that SARS-CoV-2 maintains a similar profile to SARS-CoV in terms of susceptible cell lines.

Figure 3.

Cell lines from patient with coronavirus disease, United States, 2020, susceptible to SARS coronavirus 2 (SARS-CoV-2). Cell lines were infected with a high multiplicity of infection (>5), washed after adsorption, and subsequently harvested 24 h postinfection for viral titer and protein lysates. A) Viral titer for SARS-CoV-2 quantitated by plaque assay on Vero E6 cells 2 days postinoculation. Infected cell protein lysates were probed by using Western blotting with B) rabbit polyclonal anti-SARS N antibody or C) anti–SARS-CoV S protein antibody. Full-length spike protein (SFL) and spike protein S1 (S1) are indicated. N, nucleocapsid; S, spike protein; SARS, severe acute respiratory syndrome.

Having established robust infection with SARS-CoV-2 in several cell types, we next evaluated the cross-reactivity of SARS-CoV antibodies against the SARS-CoV-2. Cell lysates from infected cell lines were probed for protein analysis; we found that polyclonal serum against the SARS-CoV spike protein and nucleocapsid proteins recognize SARS-CoV-2 (Figure 3, panels B, C). The nucleocapsid protein, which is highly conserved across the group 2B family, retains >90% amino acid identity between SARS-CoV and SARS-CoV-2. Consistent with the replication results (Figure 3, panel A), SARS-CoV-2 showed robust nucleocapsid protein in both Vero cell types, less protein in HUH7.0 and 293T cells, and minimal protein in A549 and EFK3B cells (Figure 3, panel B). The SARS-CoV spike protein antibody also recognized SARS-CoV-2 spike protein, indicating cross-reactivity (Figure 3, panel C). Consistent with SARS CoV, several cleaved and uncleaved forms of the SARS-CoV-2 spike protein were observed. The cleavage pattern of the SARS spike positive control from Calu3 cells, a respiratory cell line, varies slightly and could indicate differences between proteolytic cleavage of the spike proteins between the 2 viruses because of a predicted insertion of a furin cleavage site in SARS-CoV-2.[16] However, differences in cell type and conditions complicate this interpretation and indicate the need for further study in equivalent systems. Overall, the protein expression data from SARS-CoV nucleocapsid and spike protein antibodies recapitulate replication findings and indicate that SARS-CoV reagents can be used to characterize SARS-CoV-2 infection.

Finally, we evaluated the replication kinetics of SARS-CoV-2 in a multistep growth curve. In brief, we infected Vero CCL-81 and HUH7.0 cells with SARS-CoV-2 at a low multiplicity of infection (0.1) and evaluated viral replication every 6 h for 72 h postinoculation, with separate harvests in the cell-associated and supernatant compartments (Figure 4). Similar to SARS-CoV, SARS-CoV-2 replicated rapidly in Vero cells after an initial eclipse phase, achieving 105 TCID50/mL by 24 h postinfection and peaking at >106 TCID50/mL. We observed similar titers in cell-associated and supernatant compartments, which indicated efficient egress. Despite peak viral titers by 48 h postinoculation, major CPE was not observed until 60 h postinoculation and peaked at 72 h postinoculation, indicating that infected monolayers should be harvested before peak CPE is observed. Replication in HUH7.0 cells also increased quickly after an initial eclipse phase but plateaued by 24 h postinoculation in the intracellular compartment at 2 × 103 TCID50/mL and decreased after 66 h postinoculation. Virus was not detected in the supernatant of infected HUH7 cells until 36 h postinoculation and exhibited lower titers at all timepoints (Figure 4). Major CPE was never observed in HUH7.0 cells. These results are consistent with previous reports for SARS-CoV and MERS-CoV, which suggested similar replication dynamics between the zoonotic CoV strains.[23,24]

Figure 4.

Multistep growth curve for severe acute respiratory syndrome coronavirus 2 from patient with coronavirus disease, United States, 2020. Vero CCL81 (black) and HUH7.0 cells (green) were infected at a multiplicity of infection of 0.1, and cells (solid line) and supernatants (dashed line) were harvested and assayed for viral replication by using TCID50. Circles, Vero CCL81 cells; squares, Vero CCL81 supernatants; triangles, HUH7.0 cells; inverted triangles, HUH7.0 supernatants. Error bars indicate SEM. TCID50, 50% tissue culture infectious dose.

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