Rapid identification of emerging pathogens: coronavirus

Abstract

We describe a new approach for infectious disease surveillance that facilitates rapid identification of known and emerging pathogens. The process uses broad-range polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry for accurate mass measurements of PCR products, and base composition signature analysis to identify organisms in a sample. We demonstrate this principle by using 14 isolates of 9 diverse Coronavirus spp., including the severe acute respiratory syndrome-associated coronavirus (SARS-CoV). We show that this method could identify and distinguish between SARS and other known CoV, including the human CoV 229E and OC43, individually and in a mixture of all 3 human viruses. The sensitivity of detection, measured by using titered SARS-CoV spiked into human serum, was approximate, equals1 PFU/mL. This approach, applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens, is capable of automated analysis of >900 PCR reactions per day.

Figure 1

Electrospray ionization Fourier transfer ion cyclotron resonance (ESI-FTICR) mass spectrum from the polymerase chain reaction (PCR) amplicons from the severe acute respiratory syndrome (SARS)-associated coronavirus obtained with the propynylated RNA-dependent RNA polymerase primer pairs. The electrospray ionization conditions separate the sense and antisense strands of the PCR products. Multiple charge states are observed across the m/z range shown. The inset shows an expanded view of the isotope envelope of the (M-27H+)27- species. As enumerated in Table 1, the derived molecular masses for the amplicon strands are 27298.518 (+ 0.03) Da and 27125.542 (+ 0.03) Da, corresponding to an unambiguous base composition of A27G19C14T28/ A28G14C19T27 for the double-stranded amplicon, the composition expected for the SARS isolate.

Figure 2

Detection of 3 human coronavirus (CoV) in a mixture. The deconvoluted (neutral mass) mass spectra obtained for the RNA-dependent RNA polymerase primer for the 3 human CoV, HCoV-229E, HCoV-OC43, and severe acute respiratory syndrome–associated CoV, which were tested individually and in a mixture are shown. Forward and reverse amplicons are shown with the measured monoisotopic masses for each strand. Colors of the monoisotopic masses for the mixed spectra correspond to the individual viral species.

Figure 3

Spatial representation of base compositions for the 3 coronavirus (CoV) species known to infect humans. Severe acute respiratory syndrome (SARS), HCoV-OC43, and HCoV-229E base compositions in the region amplified by RNA-dependent RNA polymerase primers (Table 1) are plotted on the A, G, and C axes. T counts are shown by the tilt of the symbol. Within a species, all known isolates of each virus (37 isolates for SARS, 4 for HCoV-229E, and 2 for OC43) had identical sequences in this region. Δ_bc represents the number of changes in the A, G, C, and T bases needed for 1 species to be misidentified as another in the direction of the arrow. Δ_m represents the pairwise mutation distance between 2 species, or the cumulative probability of Δ_bc occurring.

Figure 4

Representation of the mutational distances calculated for the 2 selected primer sets overlaid on the coronavirus phylogenetic tree. Each oval represents grouping of members contained within it; numbers next to the group indicate the maximum distance between any 2 members of the group. Distances are computed as the base 10 logarithm of the geometric average of the pair-wise probabilities for any given pair of base compositions.