A previously undescribed coronavirus associated with respiratory disease in humans

Abstract

The etiology of acute respiratory tract illnesses is sometimes unclear due to limitations of diagnostic tests or the existence of as-yet-unidentified pathogens. Here we describe the identification and characterization of a not previously recognized coronavirus obtained from an 8-mo-old boy suffering from pneumonia. This coronavirus replicated efficiently in tertiary monkey kidney cells and Vero cells, in contrast to human coronaviruses (HCoV) 229E and OC43. The entire cDNA genome sequence of the previously undescribed coronavirus was determined, revealing that it is most closely related to porcine epidemic diarrhea virus and HCoV 229E. The maximum amino acid sequence identity between ORFs of the newly discovered coronavirus and related group 1 coronaviruses ranged from 43% to 67%. Real-time RT-PCR assays were designed to test for the prevalence of the previously undescribed coronavirus in humans. Using these tests, the virus was detected in four of 139 individuals (3%) who were suffering from respiratory illness with unknown etiology. All four patients suffered from fever, runny nose, and dry cough, and all four had underlying or additional morbidity. Our data will enable the development of diagnostic tests to study the prevalence and clinical impact of this virus in humans in more detail. Moreover, it will be important to discriminate this previously undescribed coronavirus from HCoV 229E and OC43 and the severe acute respiratory syndrome coronavirus.

Figures

Fig. 1.

Characteristics of the previously undescribed Dutch HCoV. CPE caused by HCoV-NL at 7 days after inoculation of Vero cells are shown (B) with mock-infected cells as controls (A). The cell-free supernatant from these cells revealed the presence of coronavirus-like particles by negative contrast electron microscopy at ×140,000 (C).

Fig. 2.

Genome organization of HCoV-NL. All putative ORFs of 50 or more amino acid residues in the HCoV-NL genome are shown, with known homologues in HCoV-229E shown in black and the additional 24 small ORFs shown in gray.

Fig. 3.

Phylogenetic analysis of HCoV-NL and other coronaviruses. The full-length genome (A), replicase 1ab (B), spike (C), membrane glycoprotein E (D), matrix protein (E), and nucleocapsid protein (F) nucleotide sequences were aligned with those of known coronaviruses and maximum likelihood trees were constructed by using 100 bootstraps and three jumbles. Bars roughly represent 10% nucleotide sequence differences between closely related strains. PEDV, porcine epidemic diarrhea virus; TGEV, transmissible gastroenteritis virus; BCoV, bovine coronavirus; MHV, murine hepatitis virus; IBV, infectious bronchitis virus; SARS-CoV, SARS coronavirus; PRCoV, porcine respiratory coronavirus; PHEV, porcine hemagglutinating encephalomyelitis virus; RCoV, rat coronavirus; FCoV, feline coronavirus; CCoV, canine coronavirus.