Identification of a Novel Coronavirus Causing Severe Pneumonia in Human: A Descriptive Study
Introduction
Coronaviruses (CoVs) are enveloped viruses with a single positive-stranded RNA genome, typically ranging from 26 to 32 kilobases in length. They belong to the subfamily Orthocoronavirinae within the family Coronaviridae and are classified into four genera: Alphacoronaviruses (α), Betacoronaviruses (β), Gammacoronaviruses (γ), and Deltacoronaviruses (δ). The viral genome encodes four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), along with several non-structural proteins and unique accessory proteins.
CoVs infect humans and a variety of avian and mammalian species worldwide. Six CoVs are known to infect humans, including two α-CoVs (229E and NL63) and four β-CoVs (OC43, HKU1, severe acute respiratory syndrome [SARS]-CoV, and Middle East respiratory syndrome [MERS]-CoV). All human CoVs are zoonotic, with bats considered a key reservoir. Since the beginning of the 21st century, two zoonotic CoVs, SARS-CoV and MERS-CoV, have caused severe human diseases. The SARS-CoV outbreak in 2003 resulted in 8,096 cases and 774 deaths worldwide, while MERS-CoV, identified in 2012, has infected 2,494 people with a case fatality rate of 34.4%.
Methods
Ethical Approval This study was conducted in accordance with the Declaration of Helsinki and approved by the National Health Commission of the People’s Republic of China and the Ethics Commission of Wuhan Jinyintan Hospital (No. KY-2020-01.01). Written informed consent was waived due to the context of emerging infectious diseases.
Clinical Specimen and Data Collection Bronchoalveolar lavage (BAL) specimens were collected from five patients hospitalized with pneumonia at Wuhan Jinyintan Hospital, Hubei province, China, from December 18 to 29, 2019. Clinical data, demographic characteristics, underlying medical conditions, clinical signs and symptoms, chest radiographic findings, clinical laboratory testing results, travel history, recent animal exposure, and outcomes were gathered.
Genome Sequencing Nucleic acids were extracted from 200 μL of BAL using the Direct-zol RNA Miniprep kit and Trizol LS. The sequencing library was constructed using a transposase-based methodology and sequenced on an Illumina NextSeq platform. Quality control processes included removal of low-complexity reads, adapter trimming, low-quality reads removal, short reads removal, host removal, and ribosomal reads removal. Taxonomic assignment was performed with Kraken 2 against reference databases.
Phylogenetic Analysis Multiple sequence alignment was performed using the ClustalW program in MEGA software. Phylogenetic trees were constructed using the maximum-likelihood method. Full-genome viral sequences were deposited in the Global Initiative on Sharing All Influenza Data (No. EPI_ISL_402123, EPI_ISL_403928-31) and the Genome Warehouse in the National Genomics Data Center.
Virus Isolation BAL specimens were inoculated onto Vero cells and observed daily for cytopathic effect (CPE). Viral particles were negatively stained with phosphotungstic acid and characterized using electron microscopy. Viral nucleic acids were confirmed by RT-PCR with specific primers.
Immunofluorescence Assay Spot slides were prepared by applying virus-infected or non-infected cell suspensions onto Teflon-coated slides. Cells were fixed with paraformaldehyde, blocked, and stained with serum from a convalescent patient or a healthy person. Goat anti-human immunoglobulin G conjugated with fluorescein isothiocyanate was used as the secondary antibody.
Results
General Information of Patients Five patients, aged 41 to 65, presented with fever, cough, and dyspnea. Three had exposure to the Huanan Seafood Market, while two did not. All patients had complications of acute respiratory distress syndrome, and one patient died. Chest radiography revealed diffuse opacities and consolidation.
Novel CoV Identification by Next-Generation Sequencing Sequence results revealed a previously unknown β-CoV strain in all five patients, with 99.8% to 99.9% nucleotide identities among the isolates. The virus showed 79.0% nucleotide identity with SARS-CoV and 51.8% with MERS-CoV. The virus is phylogenetically closest to a bat SARS-like CoV (SL-ZC45) with 87.6% to 87.7% nucleotide identity but is in a separate clade.
Phylogenetic Analysis The novel CoVs are most similar to bat SARS-like CoVs (SL-CoVs) ZXC21 and ZC45, with 87.6% to 87.7% nucleotide identity. The virus has a single intact open reading frame gene 8, characteristic of bat-origin CoVs. The amino acid sequence of the tentative receptor-binding domain resembles that of SARS-CoV, indicating potential use of the same receptor.
Viral Culture CPE was observed in 30% of Vero cells inoculated with the novel CoV after two passages. Electron microscopy revealed characteristic CoV particles with surface projections. Immunofluorescence assays confirmed the presence of viral particles in the cells.
Clinical Features and Outcomes Patients presented with fever, cough, and dyspnea. Chest radiography showed bilateral ground-glass opacities and consolidation. Complications included acute respiratory distress syndrome, septic shock, and acute kidney injury. One patient died, one was discharged, and three remained hospitalized.
Discussion
The identification of a novel bat-borne CoV associated with severe and fatal respiratory disease in humans highlights the potential threat to public health. The virus is distinct from known human CoVs, including SARS-CoV and MERS-CoV, and is most closely related to bat SARS-like CoVs. The presence of ORF3 and intact ORF8 gene regions indicates a bat origin. The receptor-binding domain of the novel CoV resembles that of SARS-CoV, suggesting potential use of the same receptor.
The rapid progression of the disease, with major lower respiratory pathology and no obvious upper respiratory symptoms, underscores the need for further exploration of the viral receptor distribution in organs. The possibility of unrecognized mild or subclinical infections should be clarified, as identification of such infections is critical for controlling disease spread. Development of serological assays would be beneficial for detecting such infections at the population level.
In conclusion, the identification of this novel bat-borne CoV poses a potential threat to public health. Clarification of the source and transmission mode of these infections is urgently needed to prevent a potential epidemic.
doi.org/10.1097/CM9.0000000000000722
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