Rhinovirus genome evolution during experimental human infection - PubMed (original) (raw)

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Rhinovirus genome evolution during experimental human infection

Samuel Cordey et al. PLoS One. 2010.

Abstract

Human rhinoviruses (HRVs) evolve rapidly due in part to their error-prone RNA polymerase. Knowledge of the diversity of HRV populations emerging during the course of a natural infection is essential and represents a basis for the design of future potential vaccines and antiviral drugs. To evaluate HRV evolution in humans, nasal wash samples were collected daily for five days from 15 immunocompetent volunteers experimentally infected with a reference stock of HRV-39. In parallel, HeLa-OH cells were inoculated to compare HRV evolution in vitro. Nasal wash in vivo assessed by real-time PCR showed a viral load that peaked at 48-72 h. Ultra-deep sequencing was used to compare the low-frequency mutation populations present in the HRV-39 inoculum in two human subjects and one HeLa-OH supernatant collected 5 days post-infection. The analysis revealed hypervariable mutation locations in VP2, VP3, VP1, 2C and 3C genes and conserved regions in VP4, 2A, 2B, 3A, 3B and 3D genes. These results were confirmed by classical sequencing of additional samples, both from inoculated volunteers and independent cell infections, and suggest that HRV inter-host transmission is not associated with a strong bottleneck effect. A specific analysis of the VP1 capsid gene of 15 human cases confirmed the high mutation incidence in this capsid region, but not in the antiviral drug-binding pocket. We could also estimate a mutation frequency in vivo of 3.4x10(-4) mutations/nucleotides and 3.1x10(-4) over the entire ORF and VP1 gene, respectively. In vivo, HRV generate new variants rapidly during the course of an acute infection due to mutations that accumulate in hot spot regions located at the capsid level, as well as in 2C and 3C genes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Kinetics of HRV-39 infection in inoculated patients.

The course of infection is shown from days 0 to 5 for each patient (P). The relative HRV RNA values are expressed as 1/CT and converted into % with 100% being arbitrarily set for each patient at day 1. The mean value is represented by the black line. Subjects analyzed further by ultra-deep sequencing are marked with an asterisk.

Figure 2

Figure 2. Representation of all specific or common minority mutations to be found in the same loci for the four samples analysed by ultra-deep sequencing.

(A) Venn plot showing the minority mutations present initially in the HRV-39 inoculum and those present in HeLa-A, P1073 and P1077 after 5 days of infection. (B) Minority mutations present in the initial inoculum and in subjects P1073 and P1077 after 5 days of viral replication in the nasopharyngeal epithelium are represented by black and blue at their respective positions along the HRV-39 ORF. Blue bars represent the 14 minority mutations present in the Venn plot in the four viral populations.

Figure 3

Figure 3. Change in mutation frequencies (minority and majority mutations, colored bars) and minority mutation densities (curves).

Colored bars represent the difference in proportions of each nucleotide between the inoculum and the final (5 days' post-infection) sample in HeLa (A), subjects P1073 (B) and P1077 (C). As each gain in proportion by one nucleotide must be a loss by another, the changes sum to zero. Crosses indicate non-synonymous mutations. Curves indicate minority mutation densities (estimated by a Gaussian kernel function), including mutations whose nucleotide proportions did not change between the inoculum and the final sample.

Figure 4

Figure 4. Comparison of ORF consensus sequences in HeLa-OH and human volunteers.

(A) Five HeLa-OH (A to E) and samples from five human volunteers (P1062, P1120, P1073, P1074 and P1077) collected 5 days' post-infection were analysed by classical sequencing method. All mutants present after 5 days of infection, as well as those already present as minority mutations in the initial HRV-39 inoculum, are shown at their respective genome position. Synonymous, non-synonymous and non-conservative mutations are represented by black, and blue and red symbols, respectively. (B) The conservation plot was calculated based on an alignment of 99 rhinovirus serotypes as previously published . The average sequence identity for the ORF was 69.9% (blue dashed line).

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