Rhinovirus genome variation during chronic upper and lower respiratory tract infections

Abstract

Routine screening of lung transplant recipients and hospital patients for respiratory virus infections allowed to identify human rhinovirus (HRV) in the upper and lower respiratory tracts, including immunocompromised hosts chronically infected with the same strain over weeks or months. Phylogenetic analysis of 144 HRV-positive samples showed no apparent correlation between a given viral genotype or species and their ability to invade the lower respiratory tract or lead to protracted infection. By contrast, protracted infections were found almost exclusively in immunocompromised patients, thus suggesting that host factors rather than the virus genotype modulate disease outcome, in particular the immune response. Complete genome sequencing of five chronic cases to study rhinovirus genome adaptation showed that the calculated mutation frequency was in the range observed during acute human infections. Analysis of mutation hot spot regions between specimens collected at different times or in different body sites revealed that non-synonymous changes were mostly concentrated in the viral capsid genes VP1, VP2 and VP3, independent of the HRV type. In an immunosuppressed lung transplant recipient infected with the same HRV strain for more than two years, both classical and ultra-deep sequencing of samples collected at different time points in the upper and lower respiratory tracts showed that these virus populations were phylogenetically indistinguishable over the course of infection, except for the last month. Specific signatures were found in the last two lower respiratory tract populations, including changes in the 5'UTR polypyrimidine tract and the VP2 immunogenic site 2. These results highlight for the first time the ability of a given rhinovirus to evolve in the course of a natural infection in immunocompromised patients and complement data obtained from previous experimental inoculation studies in immunocompetent volunteers.

Figure 1. Repartition of upper respiratory tract, lower respiratory tract, and protracted infection episodes according to HRV species.

A. Percentage of each species associated with upper respiratory tract infection (URT), lower respiratory tract infection (LRT), and protracted infection (PI) among lung transplant recipients and hospital patients. B. Detailed repartition of URT, LRT and PI episodes in lung transplant recipients, and pediatric and adult hospital patients according to HRV species. Details of HRV-A, -B and -C types are shown in Figures S1A to C.

Figure 2. Synonymous and non-synonymous mutation hot spot regions.

A. The synonymous (upper panel), 5′UTR and non-synonymous (lower panel) changes observed between the first and last samples sequenced from patients P37, V38, R58 and F78, as well as between the URT and LRT samples from patient A46, were plotted together along the HRV-2 genome (GenBank accession number X02316) to map mutation hot and cold spot regions. B. The conservation plot was calculated based on an alignment of the amino acid sequence of 99 rhinovirus serotypes and clinical strains as previously published . The average sequence similarity measure for the ORF was 0.114 (red line). sd: standard deviation.

Figure 3. Intra-host genetic variation in patient F78 at the majority and minority population level.

A. Phylogenetic tree of the 11 complete genome sequences corresponding to samples collected over a 27.4-month infection period in the URT and LRT of patient F78, as well as HRV-A67 reference type (FJ445149) and 3 HRV-9 complete genome sequences (FJ445114,15,17). HRV-A 67 and FJ445117 were used as outgroups to root the tree and are not shown. B. Minority mutation repartition along the HRV genome as recorded for the last 3 URT and LRT samples collected from patient F78. Curves indicate minority mutation densities (estimated by a Gaussian kernel function) along the genome. C. Changes specific to the URT and LRT. The sequences of the last URT and LRT samples collected from patient F78 were compared to the first complete genome sequence (F78 URT 1.4 m) and changes specific to the URT (upper panel) and LRT (lower panel) are mapped along the HRV genome.