Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study

Abstract

Background: Human infections with zoonotic coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, have raised great public health concern globally. Here, we report a novel bat-origin CoV causing severe and fatal pneumonia in humans.

Methods: We collected clinical data and bronchoalveolar lavage (BAL) specimens from five patients with severe pneumonia from Wuhan Jinyintan Hospital, Hubei province, China. Nucleic acids of the BAL were extracted and subjected to next-generation sequencing. Virus isolation was carried out, and maximum-likelihood phylogenetic trees were constructed.

Results: Five patients hospitalized from December 18 to December 29, 2019 presented with fever, cough, and dyspnea accompanied by complications of acute respiratory distress syndrome. Chest radiography revealed diffuse opacities and consolidation. One of these patients died. Sequence results revealed the presence of a previously unknown β-CoV strain in all five patients, with 99.8% to 99.9% nucleotide identities among the isolates. These isolates showed 79.0% nucleotide identity with the sequence of SARS-CoV (GenBank NC_004718) and 51.8% identity with the sequence of MERS-CoV (GenBank NC_019843). The virus is phylogenetically closest to a bat SARS-like CoV (SL-ZC45, GenBank MG772933) with 87.6% to 87.7% nucleotide identity, but is in a separate clade. Moreover, these viruses have a single intact open reading frame gene 8, as a further indicator of bat-origin CoVs. However, the amino acid sequence of the tentative receptor-binding domain resembles that of SARS-CoV, indicating that these viruses might use the same receptor.

Conclusion: A novel bat-borne CoV was identified that is associated with severe and fatal respiratory disease in humans.

Figure 1

Microbial species profiles (proportions) in bronchoalveolar lavage fluid specimens from Patient 1 (A), Patient 2 (B), Patient 3 (C), Patient 4 (D), and Patient 5 (E) analyzed by deep sequencing.

Figure 2

Characteristics of viral genes. The schematic diagram of the novel coronavirus (CoV)'s genome (A). Phylogenic analysis of viral whole genome (B), spike (C), nucleocapsid (D) and RNA-dependent RNA polymerase (E) genes. The novel CoV identified and closely related viruses are in red in the phylogenetic trees. Other out-group viruses from public database are shown in blue. Evolutionary distances were calculated with the maximum likelihood method. Amino acid sequence of the putative receptor binding domain (RBD) of IPBCAMS-WH-01/2019 compared with strains of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and bat SARS-like CoV (F).

Figure 3

Viral isolation and identification. The untreated control (left) and cytopathic effect (right) on Vero cells visualized with an inverted microscope (A, original magnification ×20). Viral particles were negative stained with 1% solution of phosphotungstic acid and observed by using electron microscopy analysis (B, scale bar 200 nm). Immunofluorescent assays with convalescent serum and anti-human immunoglobulin G conjugated fluorescein isothiocyanate show signals (green) in cytoplasma in Vero cells. The nuclei and cytoplasma were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue) and Evans blue (red), respectively (C, scale bar 20 μm).

Figure 4

Computed tomographic chest radiographs of Patient 2, obtained on day 10 from illness onset at aortic arch (A) and pulmonary vein (B) scan demonstrating bilateral ground-glass opacity and consolidation, and Patient 5 on day 12 (C) and 13 (D) after illness onset demonstrating white lungs.