Identification and seasonality of rhinovirus and respiratory syncytial virus in asthmatic children in tropical climate

Abstract Introduction: Asthma is a disease that has been associated with the presence of different genetic and socio-environmental factors. Objective: To identify and evaluate the seasonality of respiratory syncytial virus (RSV) and human rhinovirus (RV) in asthmatic children and adolescents in tropical climate, as well as to assess the socioeconomic and environmental factors involved. Methods: The study was conducted in a referral hospital, where a total of 151 children were recruited with a respiratory infection. The International Study of Asthma and Allergies in Childhood (ISAAC) protocol and a questionnaire were applied, and a skin prick test was performed. The nasal swab was collected to detect RV and RSV through molecular assay. National Meteorological Institute (INMET) database was the source of climatic information. Results: The socio-environmental characterization of asthmatic children showed the family history of allergy, disturbed sleep at night, dry cough, allergic rhinitis, individuals sensitized to at least one mite. We identified RV in 75% of children with asthma and 66.7% of RSV in children with asthma. There was an association between the presence of RV and the dry season whereas the presence of the RSV was associated with the rainy season. Contributing to these results, a negative correlation was observed between the RSV and the wind speed and the maximum temperature (T. Max) and a positive correlation with precipitation. Conclusions: The results suggest a high prevalence of RV and RSV in asthmatic children and the seasonality of these viruses were present in different climatic periods. This has significant implications for understanding short- and long-term clinical complications in asthmatic patients.


Clinical Perspectives
The prevalence of asthma has increased worldwide in developed countries, leading to numerous hospitalizations. The association between viral infections, wheezing in infants and exacerbation of asthma is well established. Viruses are known to have different seasonality in each region. This is the first study that identifies and observes the seasonality of rhinovirus (RV) and respiratory syncytial virus (RSV) in a tropical country and associates this seasonality with the development of asthma.
Our results show a high prevalence of RV and RSV and an association of rhinovirus with asthma. In addition, a correlation was observed between the dry season and the presence of RV and the rainy season with the presence of the RSV.
These data contribute to define public health policies and management and intervention strategies for the control of asthma and medium-term improvement in the quality of life of patients with allergic manifestations.
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INTRODUCTION
Asthma is a chronic inflammatory disease, characterized by an abrupt hypersensitivity reaction mediated by Immunoglobulin E 1 . The prevalence of asthma has increased worldwide in developed countries, leading to numerous hospitalizations and a considerable rise in morbidity and mortality 2 . Around 300 million people worldwide are affected by asthma. In Brazil, asthma is the fourth cause of death caused by respiratory diseases, and according to data from the Pan American Health Organization, Brazil has more than 15 million people with asthma 3 .
This pathology is the result of the interaction of genetic, immune, and environmental factors. Environmental exposure to allergens, irritants, and other specific factors leads to the development and maintenance of asthma symptoms. Different studies previously showed the association between viral infections, wheezing in infants, and exacerbation of asthma [4][5][6] . Infections with respiratory syncytial virus (RSV) and/or human rhinovirus (RV) are a fundamental cause in children's and adolescents' respiratory tract diseases and a major cause of bronchitis in children 1,7 . Some authors have already demonstrated that viral infections are capable of exacerbating asthma. As well as the exposure to RSV and RV serves as a primer for the development of this disease 8,9 . The mechanism by which RSV exacerbates asthma is associated with the T cell response characterized mainly by Th2 cytokine production, the same response observed during asthma episodes. Viral infections incidence suffers influence from seasonal factors such as precipitation, temperature, humidity, and wind speed, associated with the prevalence of respiratory diseases 10 . In countries of temperate zones, upper respiratory tract infections being more frequent in autumn and spring, rising during winter, following wheatear changes 11 . A study conducted in Germany suggests that humidity and temperature are Downloaded from http://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20200634/893031/bsr-2020-0634-t.pdf by guest on 20 September 2020 associated with hospitalizations due to lower respiratory tract infections by the Influenza virus, RSV, and RV 12 . Another study carried out in the Colombia, showed that the occurrence of acute respiratory infection in children was associated with air temperature and relative humidity 13 . A study conducted in China, in 2016, showed that RV was the main viral pathogen in wheezing children, especially in the summer 14 .
Other study carried out in China in 2020, showed the seasonality of RSV infection in hospitalized children and correlated with temperature 15 .
However, few studies have reported the seasonal variation of respiratory virus in tropical countries. The relationship between presence of respiratory virus and development of asthma has been controversial. The present study aims to identify and evaluate the seasonality of RSV and human rhinovirus RV in asthmatic children and adolescents in tropical climate, as well as to assess the socio-economic and environmental factors involved.

Climatic characterization
In the state of Maranhão, Brazil, the climate presents two well-established periods, the rainy season (months from January to June) and the dry season (months from July to August), which were defined according to weather studies by Silva, 2019 16 .
The data of meteorological parameters, including maximum daily, average and minimum temperature (° C), wind speed (Km / h), relative humidity (%) and

Subjects
The study was performed at Dr. Odorico de Amaral Matos Children's Hospital in the City of São Luís -MA, Brazil, from April 2018 to March 2019. 151 children aged 2 to 12 years were included in this study. The sample size was calculated using PASS 15® software, with the following parameters: prevalence 21,1% of children with respiratory infection in ambulatory 18 , level of significance (α) of 5 %, 80% test power, and tolerable error of 8%.
Upon hospital admission due to infection symptoms, patients were characterized as asthmatic and non-asthmatic, biological samples were collected, and then an immediate hypersensitivity test was performed according to the descriptions and protocols below. The clinical diagnosis of asthma was determinate by physicians from the hospital according to GINA (Global Initiative for Asthma) criteria: dyspnea, chronic cough; wheezing; chest tightness or chest discomfort, particularly at night or in the early hours of the morning; spontaneous improvement with or use of specific medications for asthma (e.g., bronchodilators, steroid anti-inflammatories). Besides, individuals who had three or more wheezing episodes within six months were considered to be wheezing. The clinical presentation of viral infection was characterized by watery nasal secretion, moderate cough, low hyperthermia, and wheezing 19 .

Inclusion and exclusion criteria
The inclusion criteria considered were age between 2 to 12 years (until the collection of biological material) and presenting respiratory infection. Children with pre-existing chronic lung disease, such as pneumonia, tuberculosis, and whooping cough, or under nebulizer therapy, were excluded.

Application of ISAAC and complementary questionnaires
The ISAAC (The International Study of Asthma and Allergies in Childhood) questionnaire was used to define asthma, rhinitis, and atopic eczema, with objective questions about the signs and symptoms of respiratory tract diseases 20 . A complementary questionnaire was applied to investigate the socio-environmental factors and family history of volunteers.

Immediate hypersensitivity skin test (prick test)
The skin prick test (Immunotech, FDA Allergenic Ltda, Rio de Janeiro, Brazil) was performed using extracts from domestic dust mite (Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Blomia tropicalis), cat, dog, grass, egg and milk, buffered saline (negative control), and histamine (positive control). The test was performed on the front of the forearm following the manufacturer's instructions.
Skin prick test responses were considered positive if the allergen caused a wheal with a diameter of at least 3 mm, after thirty minutes.

Viral samples
Respiratory samples were collected through a nasopharyngeal swab. After obtaining samples, those were disposed of in a 15 ml conical tube containing 2 ml of PBS (phosphate-buffered saline). Biological materials were centrifuged (3500 rpm for 10 minutes), and the supernatant was collected and stored at -80 ° C.

Extraction of total RNA
The total RNA was obtained using a set of QIAamp Viral RNA Mini Kit® reagents (QIAGEN, GmbH, Germany), following the manufacturer's instructions 21 .

Synthesis of the cDNA
The cDNA synthesis from the viral RNA extracted was performed using the Reverse Transcriptase SuperScript ™ II reagent set (Invitrogen, Gaithersburg, USA), following the instructions of the manufacturer's material.

Quantitative polymerase chain reaction (qPCR)
The identification of respiratory viruses was performed by qPCR in real-time

Statistical analysis
To compare the proportions of the classificatory variables, the chi independence square at the level of 5% and 10% (p <0.05 and p <0.10) was used. In some situations, the Yates' correction was applied. The binary logistic regression model was also used to analyze the effect between categorical and independent variables; a reference category odds ratio (OR = 1) was established, considering risk factors greater than one and protective factors less than 1. First, a univariable regression was performed, and then the multivariable, considering a significance level of 10% (p <0.10).
Pearson correlation coefficient (r) was used to analyze the correlation between RSV and RV with mean precipitation, mean temperature, and wind speed.  1).

Hypersensitivity test
The hypersensitivity test showed that 78.9% of asthmatic children with infection were sensitized to at least one allergen (

Identification and seasonality of RV and RSV in asthmatic or nonasthmatic children
It was observed that 75% of rhinovirus infected children were asthmatic patients, which resulted in a statistically significant difference. The rhinovirus was associated with the dry climate period of the region (

Correlation between the presence of RSV and RV and the climatic variables
Since it observed a difference in the seasonality of the virus, it was evaluated the climatic variables as wind speed, precipitation, maximum temperature (T. Max) and minimum temperature (T. Min). The data show a positive correlation with the precipitation variable (Table 5, p <0.10).
Also, we performed a temporal analysis of the data. Figure 1A shows the peak of RSV and RV infections. In the case of RSV infection, the peak occurs between February and March in the months with the highest precipitation. The peak of RV infection occurred between November and December, at the end of the dry period.
These observations show the influence of climatic variations on the distribution of these infections. In the temporal analysis of the wind velocity, it is observed that the peak incidence of the respiratory syncytial virus is in March, which is associated with low wind speed ( Figure 1B). The time series of maximum and minimum temperature and the respiratory syncytial virus is shown in Figure 1C, and the rhinovirus is shown in Figure 1D. Our results confirm the known relationship between a family history of allergies. Regarding the presence and seasonality of RV and RSV, our data showed an association between RV and dry climate, and an association between RSV and rainy climate Regarding the presence and seasonality of RV and RSV, our data showed an association between RV and dry climate, and an association between RSV and rainy climate. In Maranhão, the equatorial climate is dominant in the western part of the state, providing rainfall and high temperatures; the rest of the region is influenced by the tropical climate, with higher rainfall rates in the first months of the year, promoting respiratory viral infections. Once this difference between the rainy and dry climate was

AUTHOR CONTRIBUTIONS
Lopes and Falcai contributed equally to the study and had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Lopes and Falcai
Acquisition, analysis, or interpretation of data: All authors.  Table 1. Socio-environmental and clinical characteristics of asthmatic and nonasthmatic children with respiratory infection. Table 2. Allergic sensitization of the asthmatic and non-asthmatic children with respiratory infection. Table 3. Identification and seasonality of RV and RSV in children and adolescents with and without asthma.