A 70-year-old man presented to our emergency hospital with dry cough and shortness of breath. He experienced gradual worsening of his symptoms for 10 days since their initiation. On physical examination, the patient's oxygen saturation was 95%. Computed tomography (CT) of the chest revealed bilateral and peripheral ground-glass opacities. The polymerase chain reaction (PCR) for SARS-CoV-2 sampling from his nasopharyngeal swab was positive. He was admitted to the intensive care unit for COVID-19 and was administered oxygen, methylprednisolone, remdesivir, and tocilizumab for 4 days, 5 days, 3 days, and 1 day, respectively, which improved his dyspnea and chest CT findings (Figure 1).
His medical history included diabetes mellitus and hypertension. Approximately 1 month before admission, he had experienced paroxysmal chest pain. It was transient; therefore, he did not go to the hospital. On the day of admission, an electrocardiogram demonstrated an inverted T wave in V1-3 and non-specific ST-segment depression. Serum levels of troponin I and creatine kinase MB were elevated, suggesting acute coronary syndrome (ACS). These levels decreased in 1 day. Cardiac echocardiography revealed no wall motion abnormalities and a normal ejection fraction. Although the patient complained of no chest symptoms after admission, ACS was suspected. Therefore, the patient was administered nitrates with continuous treatment for his respiratory symptoms, waiting for further cardiac investigation. The PCR test result of his nasopharyngeal swab 9 days after admission was negative. He underwent coronary angiography 14 days after admission. Coronary angiogram demonstrated three-vessel disease of the left main trunk, which required coronary artery bypass grafting (CABG) (Figure 1).
Coronary artery bypass grafting was scheduled using an off-pump procedure, 37 days after admission. The patient was confirmed to have a negative PCR test result with his nasopharyngeal swab 5 days before off-pump coronary artery bypass grafting (OPCAB). One hour before entering the operating room, intra-aortic balloon pump (IABP) was inserted through the femoral artery under local anesthesia. Residual blood at the time of IABP insertion was preserved for analysis. During the OPCAB procedure, the saphenous vein was harvested for grafting, and residual tissues after anastomosis were preserved for analysis. Surgery was performed uneventfully. He was transferred to the postoperative intensive care unit, intubated, and sedated. Suctioning from the endotracheal tube was performed to remove the secretions during intubation. The secretion was used for PCR testing for SARS-CoV-2. The result of the PCR test was positive.
The patient was discharged on the 12th day following the surgery without any complications (Figure 1). At present, the patient is in good physical condition and did not present with cardiovascular complications as well as post-COVID-19 syndrome, 18 months after OPCAB.
Total RNA was extracted from the saphenous vein using Isogen  (Nippon Gene, Japan), according to the manufacturer's instructions. Reverse transcription of one microgram of RNA was performed using RiverTraAce  (Toyobo, Japan) in the presence of random hexamers and oligo(dT). To investigate virus replication, quantitative PCR was performed using the Power SYBR Green RT-PCR master mix (Applied bioystems, United States) in the presence of a nucleocapsid phosphoprotein gene-specific primer set designed by the National Institute of Infectious Diseases, Japan. The primer sequences synthesized in the 5'-to-3' direction were CACATTGGCACCCGCAATC as the forward primer (N_Sarbeco_F1) and GAGGAACGAGAAGAGGCTTG as the reverse primer (N_Sarbeco_R1). The amplification plot is shown in Figure 2.
Redundant tissue from the saphenous vein was fixed with 4% para-formaldehyde (pH 7.0). The fixed tissue was soaked in 30% sucrose (wt/vol) in Tris-buffered saline containing 0.1% tween-20 (TBS-T) and cryosectioned. Tissue sections were incubated with a primary antibody, a 1:100 diluted mouse anti-spike antibody (GeneTex, Irvine, CA, United States 6H3) or a 1:100 diluted mouse anti-nucleocapsid antibody (GeneTex, Irvine, CA, United States 1A9) at 4 C overnight. The sections were washed with TBS-T four times prior to being incubated with the secondary antibody, 1:200 diluted donkey Alexa Fluor 488 anti-mouse IgG (H + L) (Thermo Fisher Scientific, Waltham, MA, United States A21202), at room temperature for 30 min. After nuclear staining with DAPI (Thermo Fisher Scientific, Waltham, MA, United States) was performed, fluorescence signals were observed using fluorescence microscopy (Eclipse Ti2; NIKON, Tokyo, Japan). Immunohistochemistry revealed small clusters of antigen-positive speckles distinguished by autofluorescence (Figures 3A,B).
The saphenous vein was fixed with the Karnovsky's formulation of 2% para-formaldehyde and 2.5% glutaraldehyde 1-mM CaCl2 in a 0.1-M HEPES buffer and then post-fixed with 1.0% osmium tetroxide in a 0.1-M HEPES buffer and 0.1-M sucrose. After additional buffer washes, the samples were dehydrated with a graded ethanol series and embedded in epoxy resin. Thin sections were cut using a Leica ultramicrotome, stained with 1% uranyl acetate for 30 min, and washed with distilled H2O. A Joel transmission electron microscope (JEM-1400A) was used for observation at 120 kV. Digital images were acquired using a Bioscan Camera Model 792 (Gatan, Inc., Japan) controlled by a digital micrograph 3.4 (Gatan, Inc.). Viral particles were observed in the saphenous vein (Figure 3C). An area devoid of viral particles was also observed (Supplementary Figure 1).
HepG2 cells were cultured in a 12 well plate. Cells that reached 60% confluence were stimulated with patient serum and healthy control serum from a volunteer with no history of SARS-CoV-2 infection at 10% concentration for 18 h. We performed three independent experiments. Total RNA was recovered from each well by Isogen (Nippon Gene, Japan). Of the total RNA, 500 ng of RNA was reverse-transcribed using the Versa cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, United States). One microliter of cDNA was amplified with primers (human ISG15 Fw: CACCTGAAGCAGCAAGTGAGCGGGCTGGAG, human ISG15 Rv: CCGCAGGCGCAGATTCATGAACACGGTGCT, human MxAFw: GCCAGCAGCTTCAGAAGGCCATGCTGC AGC, human MxARv: GGGCAAGCCGGCGCCGAGCCTGCG TCAGCC, human 18S Fw: TCAACTTTCGATGGTAGTC GCCGT, human 18S Rv: TCCTTGGATGTGGTAGCCGTTTCT) using the Power Cyber Green master mix (Thermo Fisher Scientific, Waltham, MA, United States) and quantitative PCR system, Rotor gene 2 (QIAGEN, Netherlands). Serial dilutions of one of the samples were amplified using each primer pair for quantification. The quantified expression levels were normalized to the result of 18S ribosomal RNA that was used as the internal control. The data obtained from three independent wells are expressed as mean +- standard deviation. Statistical analyses were performed using the Microsoft Excel Student's T-test. Statistical significance was set at p < 0.05. As shown in Figure 4, type-I IFN stimulated genes, interferon-stimulated gene 15 (ISG 15) and myxovirus resistance A (MxA) expression levels were significantly higher in the HepG2 cells stimulated by the patient's serum.