Etiologies of Acute Bronchiolitis in Children at Risk for Asthma, with Emphasis on the Human Rhinovirus Genotyping Protocol

Abstract

This research aims to determine acute bronchiolitis' causative virus(es) and establish a viable protocol to classify the Human Rhinovirus (HRV) species. During 2021-2022, we included children 1-24 months of age with acute bronchiolitis at risk for asthma. The nasopharyngeal samples were taken and subjected to a quantitative polymerase chain reaction (qPCR) in a viral panel. For HRV-positive samples, a high-throughput assay was applied, directing the VP4/VP2 and VP3/VP1 regions to confirm species. BLAST searching, phylogenetic analysis, and sequence divergence took place to identify the degree to which these regions were appropriate for identifying and differentiating HRV. HRV ranked second, following RSV, as the etiology of acute bronchiolitis in children. The conclusion of the investigation of all available data in this study distributed sequences into 7 HRV-A, 1 HRV-B, and 7 HRV-C types based on the VP4/VP2 and VP3/VP1 sequences. The nucleotide divergence between the clinical samples and the corresponding reference strains was lower in the VP4/VP2 region than in the VP3/VP1 region. The results demonstrated the potential utility of the VP4/VP2 region and the VP3/VP1 region for differentiating HRV genotypes. Confirmatory outcomes were yielded, indicating how nested and semi-nested PCR can establish practical ways to facilitate HRV sequencing and genotyping.

Keywords: acute bronchiolitis; asthma; bioinformatics; genotyping; human rhinovirus; sequencing; virus.

Figure 1

Illustration of polymerase chain reaction (PCR) products for the six optimal assays (with HRV genome targets’ regions). Above are given the first- and second-round PCR products (i.e., the horizontal lines) for the six optimal assays in the research, including the lengths, placements (in relation to one another), and the VP target regions in the HRV genome. Product length (bp) is indicated, and the PCR product numbers give the positional number of the nucleotides inside the HRV genome (i.e., the 5′ and the 3′, on the left and right, respectively). Abbreviations: viral-capsid protein (VP).

Figure 2

The workflow of HRV genotype analysis. A, B, W, and Y represent the first-round PCR assays (master mixes); A:D, B:E, W:X, and Y:Z are the second-round PCR assays (master mixes). Abbreviations: PCR: polymerase chain reaction; RNA: ribonucleic acid.

Figure 3

Sequencing primers’ selection guideline.

Figure 4

Summary of the analysis steps of HRV genotyping. The boxes represent the main steps, while the points beside the boxes summarize the output. MSA: multiple sequence alignment.

Figure 5

Phylogenetic analysis of HRVs based on the VP4/VP2 region nucleotide sequences (MSA 2). By employing the VP4/VP2 MSA 2 (545 nucleotides), it was possible to complete the neighbor-joining analysis by employing the maximum composite likelihood model. In addition, by employing bootstrapping computed using 500 replicates, it was possible to evaluate the confidence of the sequence clustering. In addition, the branches and those bootstrap values that exceeded 40% are given, and phylogenetic analyses were completed by employing MEGA7. HRV strains in this research are circled in red. The ideal tree (with the sum of the branch lengths = 7.964) is given. Seventy nucleotide sequences were incorporated into the analysis, and positions containing insufficient information, such as gaps, were removed. Therefore, the data set included 417 positions in total.