Genotyping of rhinoviruses in acute respiratory tract infections

Introduction: Rhinovirus (RV) is the most frequent cause of acute respiratory illness. RV is usually the causative agent of upper respiratory infections with mild symptoms and cold. However, it is related with asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD), pneumonia, sinusitis otitis and wheezing especially in children. RV was identified in three species by molecular methods which are RV-A, RV-B and RV-C. In most of the cases, it was reported that RV-A and RV-C were related to lower respiratory tract infections and asthma inflammation, while RV-B was rarely reported in lower respiratory tract infections. It was also reported that RV-C causes severe infections in children. The aim of the present study was detection of RV species by sequence analysis among the patients with respiratory tract infections and RV positive in nasopharyngeal samples. Additionally, the relation between species and clinical picture, viral load, age and gender was also in the purpose of study. Materials and Methods: The study group was consisted of 96 patients (50 children and 46 adults) who admitted to Adnan Menderes University Hospital with acute respiratory claims and were detected as RV positive previously with two commercial kits [(FTD Respiratory pathogens 21 (Fast-track diagnostics Ltd., Malta) and Anyplex™ II RV16 Detection V1.1 (Seegene Inc., Seoul, Korea)]. Viral loads of samples were determined with a quantitative real-time PCR (LightCycler® 480 RNA Master Hydrolysis Probes, USA). Following the real-time in order to represent the amplicons FastStart High Fidelity PCR System, dNTPack was used as a conventional PCR method. After purification of PCR products BigDye® Terminator v3.1 Cycle Sequencing Kit was used in sequence analysis PCR. Purified PCR products were also loaded to ABI Prism 3100/3100 and sequencing was performed in two directions. Data from study was evaluated with SPSS 17.0 (Inc, Chicago, IL, USA) pocket program for Windows. Chi-square test was used for analytical comparisons. Phylogenetic analysis was performed with Geneious 9.1.3 program. In the analysis a 400 bp region of VP4/VP2 rhinovirus capsid protein coding region was included. Rhinovirus species were determined by analysing the sequences with references for VP4/VP2 coding region by neighbour-joining method. References were downloaded from GenBank (http://www.ncbi.nlm.nih.gov). Test parameters were set as minimum identification 70%, Bootstrap value 1000 replication. Clustering of sequences with references was the approach that we used to determine species of isolates. Results: The age of children were changing from 22 days to 16 years and the mean was 24 months (6,19-72) and the age of adults were changing from 18 to 86 and the mean was 57,63±15,68. After the quantitative PCR analysis, 31 isolates were excluded from the study among 127 samples which were positive with commercial multiplex PCR. Of the 96 samples in which expected amplicons were observed in 32 of adults and 33 of children, totally 65 isolates. After sequence analysis, 28 were (43,07%) RV-A, 7 were (10,76%) RV-B and 28 were (43,07%) RV-C; moreover one sample was EV-D68 (1,53%) and one sample was EV-C (1,53%). The distribution of species in adults was: 15 (48,3%) RV-A, 5 (16,1%) RV-B and 11 (35,4%) RV-C. The distribution of species in children was 13 (%40,6) RV-A, 2 (%6,3) RV-B and 17 (%53,1) RV-C. There was statistically significant relations with RV species and clinical Picture, viral loads, age and gender in both of the age groups (p> 0,05). Discussion: We detected RV-A and RV-B species in same rates, RV-B in lower rate. In child patients RV-C, in adult patients RV-A was the most common species. No statistically significant relationship was found with RV species and clinical severity, viral loads, age and gender in both of the age groups. This was the first study from Turkey that reported the RV species. The findings will have a contribution to understanding of rhinovirus epidemiology and pathogenesis. There is a gap in the field of clinical picture and RV species and that indicates the need for further studies including larger patient populations.