key: cord-0725876-3fd0t23z
authors: Kuypers, Jane; Perchetti, Garrett A.; Chu, Helen Y.; Newman, Kira L.; Katz, Joanne; Khatry, Subarna K.; LeClerq, Steven C.; Jerome, Keith R.; Tielsch, James M.; Englund, Janet A.
title: Phylogenetic characterization of rhinoviruses from infants in Sarlahi, Nepal
date: 2019-08-21
journal: J Med Virol
DOI: 10.1002/jmv.25563
sha: 388280d5ebdb61d0a58fc34497aa361161f7c651
doc_id: 725876
cord_uid: 3fd0t23z

PROBLEM: Rhinoviruses (RVs), the most common causes of acute respiratory infections in young children and infants, are highly diverse genetically. OBJECTIVE: To characterize the RV types detected with respiratory illness episodes in infants in Nepal. STUDY METHODS: Infants born to women enrolled in a randomized trial of maternal influenza immunization in rural, southern Nepal were followed with household‐based weekly surveillance until 180 days of age. Infants with respiratory symptoms had nasal swabs tested for twelve respiratory viruses. A subset with RV alone was selected for sequencing of the VP4/2 gene to identify RV types. RESULTS: Among 547 RV‐only positive illnesses detected from December 2012 to April 2014, 285 samples (52%) were sequenced. RV‐A, B, and C species were detected in 193 (68%), 18 (6%), and 74 (26%) specimens, respectively. A total of 94 unique types were identified from the sequenced samples, including 52 RV‐A, 11 RV‐B, and 31 RV‐C. Multiple species and types circulated simultaneously throughout the study period. No seasonality was observed. The median ages at illness onset were 88, 104, and 88 days for RV‐A, B, and C, respectively. The median polymerase chain reaction cycle threshold values did not differ between RV species. No differences between RV species were observed for reported respiratory symptoms, including pneumonia, or for medical care‐seeking. CONCLUSIONS: Among very young, symptomatic infants in rural Nepal, all three species and many types of RV were identified; RV‐A was detected most frequently. There was no association between RV species and disease severity.

Rhinoviruses (RVs) are the most common cause of acute respiratory infections in young children and infants. 1 RV infections are highly prevalent and associated with both mild upper respiratory tract and more severe lower respiratory tract (LRT) illnesses, including bronchiolitis, wheezing, and pneumonia. 2 RV are highly diverse genetically, with over 160 types, classified into three species, RV-A, RV-B, and RV-C. 3 Multiple species and types have been shown to circulate simultaneously in different populations including childcare facilities, 4 pediatric emergency department, 5 immunocompromised persons, 6 and hospitalized patients. 7 Individuals can be infected multiple times with different RV types. 4, 5 Some individuals have been shown to shed RV for long periods of time and detection of RV in asymptomatic persons is common. 2, 8 The diversity of RV species and types and the wide range of clinical symptoms seen in RV illnesses have led to the suggestion that specific species or types may cause more serious illness than other types. Some investigators have reported that RV-C caused more serious illness, especially wheezing and exacerbation of asthma, in some populations 5, [9] [10] [11] [12] [13] compared with illnesses caused by RV-A and B. However, no differences in illness severity between RV species were found in other studies. [14] [15] [16] [17] [18] [19] The epidemiology and clinical significance of specific RV types have not been analyzed for very young infants in a rural community setting. The molecular characterization of RV infections is limited for RV circulating in developing countries, including countries in Asia, such as Nepal. The detection and characterization of RV in settings with limited electricity, running water, or laboratory facilities have been difficult in the past but newer methods of specimen storage, transport, and molecular detection have permitted enhanced detection of RV and enabled the study of RV types. The aim of our study is to identify the RV species and types causing respiratory symptoms in a unique population of young infants in rural, southern Nepal. A secondary aim is to determine if there are relationships between RV species and types in demographics and illness severity in these very young infants in Nepal.

Infants born to women enrolled in a randomized trial of maternal influenza immunization in the Sarlahi District of southern Nepal were followed with household-based weekly surveillance from birth until 180 days of age. Detailed methods for and results of the trial have been published. 20, 21 cycle threshold (C t ) value was less than 30. Only one sample from each RV illness episode was sequenced. An RV illness episode was defined as a respiratory illness with an RV-positive swab. Unique RV illness episodes were defined as RV-positive swabs separated by greater than 21 days without an RV-positive swab.

A 543 base pair fragment of the RV VP4/2 gene was amplified in a semi-nested RT-PCR reaction. 26 PCR amplicons were purified after gel electrophoresis using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and sequenced with the PCR inner primers in both directions in an ABI 3730xI DNA Analyzer using the BigDye Terminator Kit (Applied Biosystems, NJ). Sanger sequencing was performed by GENEWIZ (Seattle, WA), which uses a Quality Score to assess sequence quality, similar to a Phred score, that is the average of the quality values (QVs) for each base in the sequence. To analyze the sequence, we used a QV cutoff of 30 when manually assessing the chromatogram quality, which equals a 0.001 error (or 1/1000) of probability in base calls. Sequences were compared with RV sequences in the NCBI GenBank database using BLAST to identify the RV type with greater than 90% identity. Sequences that did not match a known RV type in GenBank with at least 90% identity were identified as the RV species and the designation U (unknown type).

Sequences were submitted to GenBank (accession numbers: Sequences from GenBank were included in the tree construction to identify RV types. Trees were visualized in FigTree (tree.bio.ed.ac.uk).

At each illness episode, the presence and duration (number of days) of signs and symptoms were recorded, including cough, fever, wheezing, difficulty breathing, and draining the ear. For each illness episode, the signs and symptoms were combined into a symptom score (each symptom was multiplied by the number of days for that symptom and the sum of all symptoms for all days was determined) and an episode duration (the total duration of any symptom). Care seeking was recorded and combined into a care rank score (no care = 0, nonmedical care = 1, medical care = 2, and doctor or hospital care = 3). Clinical characteristics were compared between infants infected with RV-A and B and those infected with RV-C using Wilcoxon rank-sum testing for medians and the χ 2 test of categorical variables. Associations between specific RV types that were detected KUYPERS ET AL. (Table 1) . A large number of types was detected for each species. Many RV-A and C types were detected four or more times. Total 51% of RV-A and C illnesses were in infants less than 90 days old compared with 39% of RV-B illnesses. However, no differences were seen between RV species in median infant ages at detection or in median PCR C t values ( Table 1) .

The phylogenetic trees generated from the RV-A, B, and C study sequences and reference sequences from GenBank are shown in Figure 2 . One RV-A sequence (detected in four illness episodes) and

three RV-C sequences (detected in five, one, and one illness episodes, respectively) did not match any known RV types in GenBank within 90% identity. Figure 3 shows the 94 unique RV types identified in the F I G U R E 

An LRT symptom was reported in 84 (72%) of 116 RV illnesses in infants less than 90 days old compared with 78 (56%) of 139 RV F I G U R E 2 Phylogenetic trees of RV partial VP4/2 gene sequences by RV species. A, RV-A types. B, RV-B types, C, RV-C types. Phylogenetic trees were generated from manually trimmed 420 base fragments using the maximum likelihood method and branch supported with 1000 bootstrap iterations using PhyML software. Bootstrap values are shown on tree nodes. Study sequences are identified by accession number and RV type. Reference sequences from GenBank are identified by accession number_Ref_RV type. RV, rhinovirus illnesses in infants 90 days or older (P < .01). We did not find any differences between illnesses caused by RV species A and B and those caused by RV-C for any reported respiratory symptom, including pneumonia, or for medical care-seeking (Table 2) .

We also evaluated whether illnesses with some RV types were associated with LRT symptoms compared with other types, regardless of species. Symptoms reported during illnesses with 20 RV types (17 RV-A and 3 RV-C) that were detected at least four times (range:

4-13) were differentiated into upper and lower tract symptoms. 4, 5, 7, 9, 12, [14] [15] [16] [17] [18] [19] 26, 27, 30, [32] [33] [34] [35] Our study is unique to many other studies in that we assessed viral types in a communitybased rural setting, using prospective surveillance, compared with the clinic or hospital-based surveillance in more urban settings.

Nonetheless, this study, in agreement with previous studies, has found high genetic diversity and rapid turnover of types, with few types being detected in consecutive seasons or over long periods of time. This simultaneous and successive circulation of different RV types may be one reason for the high incidence of RV infections throughout the year. 28 A previous report has described the risk factors for RV infections in infants in this Nepal study and provided more details on illness severity in the RV-positive infants. 36 In this report, analyses were limited to illnesses in which the only RV was detected to avoid confounding caused by coinfections with other respiratory viruses. LRT symptoms were more frequently reported in younger infants (<90 days old) compared with older infants (90-180 days old). We examined the association of RV species and types with age at infection, PCR C t value, and reported respiratory symptoms. RV species was not associated with age or PCR C t value, a surrogate for viral load. However, we tested infants only from birth to 6 months of age and were only able to sequence RV-positive samples with C t values less than 30. We did not observe any associations between RV species A and C and clinical signs or symptoms, pneumonia, or care-seeking in these infants. We previously did not observe an association between RV species in RV-positive febrile pregnant women in Care rank score the Nepal study and low birth weight in their infants. 37 No associations between RV species and illness severity have been reported by others. 4, [14] [15] [16] 18, 19, 30, 38 However, some studies have shown that RV-C species are more likely to cause LRT illnesses than RV-A 5,9 including specific clinical findings, such as wheezing, 12,39 asthma, 31 rhonchi, vomiting, 13 and viremia. 26 The discrepancies between these studies may be due to the patients that were tested. The majority of studies that showed an association between RV-C and more severe illnesses were conducted in children with LRT symptoms.

In addition to a lack of association between symptoms and RV species, we also did not see an association between specific RV-A and Abbreviations: CI, confidence interval; LRT, lower respiratory tract; RV, rhinovirus.

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