key: cord-1040523-gs9d8y55 authors: Marin, J; Jeler-Kačar, D; Levstek, V; Maček, V title: Persistence of Viruses in Upper Respiratory Tract of Children with Asthma date: 2000-07-31 journal: Journal of Infection DOI: 10.1053/jinf.2000.0688 sha: 831aec32242ea393943b6d0d9f11796fcbad5329 doc_id: 1040523 cord_uid: gs9d8y55 Abstract Objectives: Nasopharyngeal swabs of 50 asthmatic children in the symptom-free period were examined for the presence of adenoviruses, rhinoviruses and coronaviruses. A control group of 20 healthy individuals was included in this study. Methods: A polymerase chain reaction was used to detect adenovirus DNA and rhinovirus and coronavirus complementary DNA. The fragments of amplified genetic material were visualized with the use of agarose gel electrophoresis. Results: Adenovirus DNA was found in 78.4% of asthmatic children, rhinovirus RNA in 32.4% and coronavirus RNA in 2.7%. Adenovirus DNA was detected in one of the 20 nasopharyngeal swabs of healthy controls; the rest of the control samples were negative. Conclusions: The persistent presence of viruses in the upper respiratory tract of asthmatic children shows a possible connection between viral infections and asthma. Asthma is the most frequent chronic respiratory disease, affecting up to 10% of children. 1,2 Asthma induced by allergens has been studied intensively and the pathogenesis is well defined. In addition to allergens, respiratory viruses have been proven to be important factors in provoking asthmatic attacks. 2-4 Virus-induced damage to the respiratory epithelium results in locally intensified activity of various cells involved in the immune response. 5, 6 The presence of viruses in the respiratory tract forms a permanent source of foreign proteins, which might be the cause of subsequent pathological changes. 7 Rhinoviruses, coronaviruses, adenoviruses, respiratory syncytial virus and other paramixoviruses are most frequently presumed to be the potential cause of virus-induced asthma exacerbation. [8] [9] [10] A vast number of virus and host characteristics dictate the type of infection, either persistent or latent, and determine host response and the resulting development of the disease. [11] [12] [13] Destruction of the infected cell or reorganization of viral antigens within the cell or on the cell membranes are commonly considered to be the conse-quence of virus infection. The host's immune system should finally destruct viruses. 4 In this study nasopharyngeal swabs were taken from asthmatic children whose asthma was well controlled, and when they were at least 3 weeks free of any respiratory infections. The samples were examined for the presence of adenovirus DNA and rhinovirus and coronavirus RNA. Fifty children, aged between 5 and 17 years with moderate asthma, were included in the study. Up to five subsequent nasopharyngeal swabs were taken from each child during the symptom-free period. For at least 3 weeks they displayed no symptoms of upper airway infections and with peak expiratory flow rates (PEFR), above 80% of their best PEFR. When the samples were taken, they had been receiving inhaled glucocorticoids (fluticasone 100 -250 g daily) for at least 6 months. A total of 88 samples from the asthmatic children and 20 samples from the control group (aged 5-24 years) were examined for the presence of the previously mentioned viruses. To ensure a positive control, the following viruses were included in the diagnostic procedure: adenovirus type 1 and type 5 (Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia); rhinovirus 1B, 632, 90-05 (ATCC, USA); and human coronavirus, strain 229E, lot 5W (ATCC, USA). The samples in transport medium were vigorously vortexed and subsequently centrifuged at 2000 rpm for 10 min. The pellet was resuspended in 400 l of PBS and kept in 200 l aliquots at [70°C until used. Isolation of DNA Two hundred microlitres of cell suspension were used for the isolation of DNA using the QIAamp Blood Kit (QIAGEN Gm bH, Germany). 14 One hundred microlitres of isolated DNA were kept in an elution buffer at [20°C, unless immediatly used for PCR. PCR reaction with outer primers was followed by nested PCR (n-PCR) with inner primers. Two control adenovirus types were included in each PCR reaction. PCR was carried out in a volume of 50 l. The PCR mixture contained 5 l of DNA preparation, 10 reaction buffer, 25 mM MgCl 2 , 20 mM dNTP, 50 M of each primer (TIB MOLBIOL, Berlin, Germany) (Table I) , 5 U/l of DNA Taq polymerase and DEPC water. All the reagents were provided by Perkin Elmer (New Jersey, USA). The reaction mixture was placed in a thermal cycler (Eppendorf Gradient) and heated to 93°C for 4 min. The samples were subjected to 40 cycles at 93°C (30 s) for denaturation, at 60°C (30 s) for annealing and at 72°C (60 s) for elongation. In the subsequent n-PCR the pair of inner primers was used ( Table I) . The original DNA samples were replaced by amplicons of the first PCR. 15 Isolation of RNA One hundred and forty microlitres of frozen clinical material were used for isolation of RNA following RNeasy Total RNA purification Protocol (QIAGEN GmbH, Germany). 16 Fifty microlitres of RNA was eluted in DEPC water and kept at [70°C until tested in PCR. 15 Detection of rhinovirus and coronavirus RNA RNA samples were reverse transcribed into complementary DNA copy (c-DNA) at 37°C for 1 h in the 50 µl reaction mixture containing 10 reaction buffer, 25 mM MgCl 2 , 20 mM dNTP, 20 U/l of RNase inhibitor, 21 U/l of AMV reverse transcriptase, 50 µM of each random primer and DEPC water. All reagents were provided by Perkin Elmer (New Jersey, USA). Five microlitres of c-DNA product were subsequently amplified in 50µl PCR reaction mixture consisting of 10 reaction buffer, 25 mM MgCl 2 , 20 mM dNTP, 5 U/l of DNA Taq polymerase (all reagents provided by Perkin Elmer, New Jersey, USA) and 50 µM of each specific primer for rhinoviruses and coronaviruses (TIB MOLBIOL, Berlin, Germany) (Table I) . 15 After heating the samples at 95°C for 5 min, 35 cycles were carried out at 94°C (60 s), 55°C (60 s) and at 72°C (90 s). For the detection of coronavirus RNA, n-PCR was performed using the inner pair of primers (Table I) according to the protocol used for adenovirus n-PCR. 15 In each PCR run, c-DNA of control viruses RNA was included. Detection of amplified products: amplified products were visualized on 1.5% gel with incorporated ethidium bromide after electrophoresis. The amplified fragments of expected lengths (Table I) PCR with the use of the outer pair of primers yielded negative results in all samples except in positive controls. However, with n-PCR, 142 bp fragment was observed in 69 out of 88 samples from asthmatic children. The 380 bp fragment signifies the amplification of rhinovirus RNA. Of the 74 samples examined, 24 were positive. The 116 bp fragment, observed after n-PCR, indicates amplified coronavirus RNA. Positive results were obtained only in two out of 74 examined samples. A few borderline PCR results were documented for all the three viruses searched (Table II) . Coexistence of nucleic acids (NAs) of two or all three viruses searched was occasionally noticed. The follow-up of these particular samples showed that half of them harboured two or three different virus NAs persistently, while in half of them coexistence of two or three different NAs changed occasionally. Only four samples examined were truly negative for the tested viruses. Adenovirus DNA was detected in one of the 20 samples of healthy individuals, while the rest of them were negative. The search for the presence of adenovirus, rhinovirus and coronavirus NAs in the upper respiratory tract of asthmatic children during the symptom-free period resulted in a high number of positive results: 78.4% of cases were positive for adenovirus DNA, 32.4% for rhinovirus RNA and 2.7% for coronavirus RNA. Johnston et al. found that upper respiratory tract viruses were associated with over 80% of asthma exacerbations in children. 3 However, our results relate to asymptomatic children, who were at least 3 weeks without any sign of respiratory infections. Nicholson and Beasley wanted to define the rate of virus participation in asthma exacerbation in adults. With the use of cell culture and serology, surprisingly low results were obtained. 4, 17 The reason seems to be that only active infections were demonstrated. Comparing virus identification rate in children with those in adult subjects demonstrates a 50% fall in isolation rate in adults. 18 Johnston maintains that the connection between upper respiratory infections and asthma exacerbation has long been recognized, but that its importance had been underestimated in most previous studies because of deficiencies in virusdetection methods used. 19 In view of the possible presence of either active or latent infection, we think that it was well advised to use PCR in our study. Another reason for the use of this method was that coronaviruses and rhinoviruses are rather difficult to grow in cultured cells. 20, 21 We have demonstrated viral DNA and RNA to be persistently present in most of asthmatic children even in the symptom-free periods. If the respiratory viruses infect only the upper respiratory tract, they affect the lower airway indirectly. 19 Gern and Fraenkel, however, confirmed that all respiratory viruses (with the exception of coronaviruses, for which no data exists) are able to infect the lower respiratory tract too. 22, 23 This raises the question as to what effects, relevant to asthma, they exhibit once they get there. Due to the high viral NAs detection rate in asthmatic children compared to the lower detection rate in the control group, we can speculate that the results could be related to the disease itself or to the glucocorticoid treatment, since there is data which suggests that glucocorticoid therapy increases the titre of respiratory viruses and prolongs viral shedding. 24, 25 Smernice za obravnavo bolnika z astmo Respiratory viruses and asthma Role of virus infection in excerbations in children with recurrent wheeze or cough Respiratory viruses and exacerbations of asthma in adults Persistent adenoviral infection and chronic airway obstruction in children Persistent and latent viral infections in pathology of asthma Latent adenoviral infection in the pathogenesis of chronic airways obstruction Viruses as precipitants of asthma symptoms The effects of rhinovirus infections on allergic airway responses New concepts in the pathogenesis of bronchial hyperrespondsivness and asthma Mediatorji takoja snje preoba cutljivosti pri astmi Asthma: past, present and future PCR: Protocols for Diagnosis of Human and Animal Virus Diseases Viral respiratory tract infection and excerbations of asthma in adult patients Acute respiratory illness in the community. Frequency of illness and the agents involved Viruses and asthma The biology and pathogenesis of coronaviruses Mechanism of transmission of rhinovirus infections Detection of rhinovirus colds in normal and asthmatic subjects Lower airways inflammation during rhinovirus colds in normal and asthmatic subjects Treatment of parainfluenza type 3 bronchiolitis and pneumonia in a cotton rat model using topical antibody and glucocorticoid Respiratory syncytial viral infection in children with compromised immune function This work is supported by grants from the Ministry of Science and Technology, Republic of Slovenia. We wish to thank to Mrs. Katja Sluga, Mrs. Snea zana Kramar and Ms. Katarina Marin for their excellent technical assistence.