Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity

Abstract

Background: Between 50% and 80% of asthma exacerbations are associated with viral respiratory tract infections (RTIs), yet the influence of viral pathogen diversity on asthma outcomes is poorly understood because of the limited scope and throughput of conventional viral detection methods.

Methods: We investigated the capability of the Virochip, a DNA microarray-based viral detection platform, to characterize viral diversity in RTIs in adults with and without asthma.

Results: The Virochip detected viruses in a higher proportion of samples (65%) than did culture isolation (17%) while exhibiting high concordance (98%) with and comparable sensitivity (97%) and specificity (98%) to pathogen-specific polymerase chain reaction. A similar spectrum of viruses was identified in the RTIs of each patient subgroup; however, unexpected diversity among human coronaviruses (HCoVs) and human rhinoviruses (HRVs) was revealed. All but one of the HCoVs corresponded to the newly recognized HCoV-NL63 and HCoV-HKU1 viruses, and >20 different serotypes of HRVs were detected, including a set of 5 divergent isolates that formed a distinct genetic subgroup.

Conclusions: The Virochip can detect both known and novel variants of viral pathogens present in RTIs. Given the diversity detected here, larger-scale studies will be necessary to determine whether particular substrains of viruses confer an elevated risk of asthma exacerbation.

Figure 1

Summary of study participant distribution and Virochip results. Asterisks indicate viruses detected in specimens containing double infections. HRV′X′ is a divergent human rhinovirus (HRV) subgroup identified by array and sequence analysis. HCoV, human coronavirus; HMPV, human metapneumovirus; HPIV, human parainfluenza virus; IF, influenza virus; RSV, respiratory syncytial virus.

Figure 2

Clustogram of Virochip hybridization signatures. A, Nasal lavage specimens (X-axis) clustered according to the sum-normalized fluorescent intensity of array signal, with major viral oligonucleotide clusters (Y-axis) identified at right. B, Zoomed-in view of the 3 human coronavirus (HCoV) signatures detected by the Virochip. Each hybridization signature is boxed in white, with its corresponding HCoV type (OC43, NL63, and HKU1) indicated at the top of the clustogram; the identity of oligos lighting up within the clusters are indicated at right. HPIV, human parainfluenza virus; IF, influenza virus; RSV, respiratory syncytial virus.

Table 1

Detection rates for and viruses detected by culture and the Virochip microarray.

Table 2

Agreement between polymerase chain reaction (PCR) and the Virochip microarray for detection of rhinovirus.

Figure 3

Phylogenetic grouping of human rhinovirus (HRV) VP4/VP2 sequences. Red indicates HRVA subgroup members, blue indicates HRVB subgroup members, green indicates an HRV87 rhino/entero outlier, and the dashed circle highlights a branch of divergent HRV isolates (HRV′X′). The nos. at the ends of the branches indicate HRV reference serotype identifiers, and the circles at the ends of branches indicate the participant type and clinical outcome accompanying the cold event for clinical isolates.

Figure 4

Comparison of genomewide pairwise nucleotide sequence identity of human rhinovirus (HRV) A, HRVB, and HRV′X′ genomes. Top, HRV genome organization. Black bars above the genome schematic indicate classes of gene products and gene product identities, where known (ATPase, DEXH-box ATPase protein; NCR, noncoding region; POL, RNA-dependent RNA polymerase; PRO, viral protease; VP, viral protein; VPg, viral protein genomic encoding the 5′ protein that caps the viral genome). Gray shading of every other gene in the genome is provided for orientation in the lower panels. Coordinates for gene boundaries derived from alignment of 34 HRV reference genomes are shown below the genome schematic. The average percentage pairwise nucleotide identity scans were performed using a window of 100 nt, advanced in single-nucleotide steps across the genome. The red plot shows a representative subset of HRVA genome sequences (n = 27), the black plots show fully sequenced HRV′X′ genomes compared with HRVA genomes, and the blue plot shows fully sequenced HRVB (n = 7) and HRVA (n = 27) genomes. Dashed lines and the percentages shown at right indicate the overall genomewide average pairwise nucleotide identity for each comparison shown.