key: cord-345044-2fez1gu0 authors: Proenca‐Modena, José Luiz; de Souza Cardoso, Ricardo; Criado, Miriã Ferreira; Milanez, Guilherme Paier; de Souza, William Marciel; Parise, Pierina Lorencini; Bertol, Jéssica Wildgrube; de Jesus, Bruna Lais Santos; Prates, Mirela Cristina Moreira; Silva, Maria Lúcia; Buzatto, Guilherme Pietrucci; Demarco, Ricardo Cassiano; Valera, Fabiana Cardoso Pereira; Tamashiro, Edwin; Anselmo‐Lima, Wilma Terezinha; Arruda, Eurico title: Human adenovirus replication and persistence in hypertrophic adenoids and palatine tonsils in children date: 2019-03-18 journal: J Med Virol DOI: 10.1002/jmv.25441 sha: doc_id: 345044 cord_uid: 2fez1gu0 The role of human adenovirus (HAdV) infection in different acute diseases, such as febrile exudative tonsillitis, conjunctivitis, and pharyngoconjunctival fever is well established. However, the relationships, if any, of HAdV persistence and reactivation in the development of the chronic adenotonsillar disease is not fully understood. The present paper reports a 3‐year cross‐sectional hospital‐based study aimed at detecting and quantifying HAdV DNA and mRNA of the HAdV hexon gene in adenoid and palatine tonsil tissues and nasopharyngeal secretions (NPS) from patients with adenotonsillar hypertrophy or recurrent adenotonsillitis. HAdV C, B, and E were detectable in nearly 50% of the patients, with no association with the severity of airway obstruction, nor with the presence of recurrent tonsillitis, sleep apnea or otitis media with effusion (OME). Despite the higher rates of respiratory viral coinfections in patients with HAdV, the presence of other viruses, including DNA and RNA viruses, had no association with HAdV replication or shedding in secretions. Higher HAdV loads in adenoids showed a significant positive correlation with the presence of sleep apnea and the absence of OME. Although this study indicates that a significant proportion (~85%) of individuals with chronic adenotonsillar diseases have persistent nonproductive HAdV infection, including those by HAdV C, B, and E, epithelial and subepithelial cells in tonsils seem to be critical for HAdV C production and shedding in NPS in some patients, since viral antigen was detected in these regions by immunohistochemistry in four patients, all of which were also positive for HAdV mRNA detection. Human adenovirus (HAdV) is a nonenveloped icosahedral DNA virus that is highly prevalent in human populations. 1 Since its discovery in the early 1950s, 2,3 more than 84 HAdV genotypes, including all the 50 previously characterized serotypes were described. Currently, seven HAdV species (A-G) have been identified and are classified in the genus Mastadenovirus of the family Adenoviridae. 4 HAdV can infect a large variety of cell types and tissues in humans, leading to a broad array of diseases, including acute respiratory infections (ARI), 5 febrile exudative tonsillitis, 6 acute conjunctivitis, 7 cystitis, gastroenteritis, 4 and rare cases of encephalitis, 8 myocarditis, 9 and hepatitis. 10 Although HAdV infections are generally asymptomatic in immunocompetent individuals, acute HAdV diseases have a significant impact on children (especially under 4 years of age), elderly, immunosuppressed individuals, and military recruits. 4 While HAdV can replicate in several cells types in vitro and is associated with productive infections in different tissues in humans, several HAdV species present varied tissue specificities. For instance, HAdV C (serotypes 1, 2, 5, and 6) are commonly associated with acute tonsillitis and respiratory diseases, whereas HAdV-F (serotypes 40 and 41) and HAdV D (serotypes 8, 19 , and 37) are typically associated with gastrointestinal infections and a relatively severe and highly contagious form of epidemic keratoconjunctivitis, respectively. 1 Following the HAdV replication cycle, the viral genome can persist in the nucleus. 11, 12 Such a fact is best exemplified by the persistence of HAdV C after primary infections of the respiratory tract, with intermittent viral excretion in nasopharyngeal secretions (NPS) and feces. [13] [14] [15] Numerous studies have shown that lymphocytes of tonsils and adenoids are essential sites of HAdV persistence, namely of the species C. 16 Indeed, seminal studies indicated the ability of HAdV to persist in tonsils and adenoids, since it was possible to recover HAdV from these tissues weeks to months after the establishment of explant cultures. 2, 17 More recent studies using tissue cell separation and sorting have revealed that HAdV DNA is present in T lymphocytes of tonsils and adenoids. 18 In addition, several established human lymphocyte cell lines, including a lymphoblastoid cell line derived from a bone marrow transplant recipient with adenovirus pneumonia, may sustain prolonged and noncytopathic adenovirus infection. 19, 20 Although substantial knowledge has been obtained regarding mechanisms associated with viral persistence in human cell lines in vitro, 21 Quantification of the HadV genome and the detection of mRNA of the HAdV hexon gene was performed in human adenoids and tonsils, NPS, and peripheral blood (PB) from patients with tonsillar hypertrophy and were compared to those obtained in samples from control patients. The present study was conducted according to the principles expressed in the Helsinki Declaration and was approved by the local Research Ethics Committee (#10466/2008). All patients and caregivers signed informed consent and voluntarily agreed to participate in the survey. This was a cross-sectional study that evaluated the presence of HAdV in different samples of tissues and secretions from the upper respiratory tract of children with obstructive sleep apnea (OSA) or recurrent tonsillitis, comparing the results with control patients. Fragments of surgically removed adenoids and palatine tonsils (PTs), as well as samples of NPS and PB, were obtained from 180 patients (93 males) aged 1 to 18 years (median 5.0 years) who underwent adenotonsillectomy due to OSA or recurrent tonsillitis. Small punch biopsies from tonsillar tissues, NPS, and PB were also obtained from 12 control patients (7 males, median 3.0 years) undergoing cochlear implantation in the absence of chronic adenotonsillitis, without ARI symptoms and with normal nasofibroscopy. All patients enrolled in the study were undergoing treatment at the Otorhinolaryngology HAdV detection was performed by TaqMan real-time PCR (real-time PCR) following a previously published protocol. 23 for 2 hours before the PCR to ensure target-specific amplification. As a negative control, the same RNA extraction product was used for real-time PCR without previous RT. Samples were considered PCRpositive for HAdV hexon mRNA only when they were also simultaneously negative for the same target using the extracted RNA without previous RT. All samples, including all cDNAs and the RNAs pretreated with DNAse, were tested by real-time PCR for β-actin mRNA, following the previously described protocol. 24 A molecular typing assay based on conventional nested-PCR amplification and sequencing of a hypervariable region contained in the hexon gene was performed to determine which species of HAdV were present in the patients included in this study, following a previously published protocol. 25 Briefly, the first PCR reaction was conducted A maximum likelihood (ML) phylogenetic tree was inferred using nucleotide sequences from strains of adenoviruses described in this study and representative members of the Adenoviridae family. Multiple sequence alignment was generated using MAFFT v.7 26 with manual adjustments. The ML tree was constructed using the IQ-TREE version 1.6.8 software with 1000 ultrafast bootstraps and the best-fit nucleotides model determined by Bayesian Information Criterion, which considered 88 reversible DNA substitution models. 27, 28 Statistical support for individual nodes was estimated using the bootstrap value, and the phylogenetic tree was visualized with the In this study, the association between the replication of HAdV and the presence of other respiratory viruses in adenotonsillar tissue were analyzed. All samples were tested for the presence of the following respiratory viruses by real-time PCR, according to previously described procedures 23 The patient groups were compared using the Chi-square and Fisher's Exact tests; viral loads among patient groups were assessed using the Mann-Whitney or unpaired t test. Comparisons between three or more groups were conducted with one-way analysis of variance and the Bonferroni test. All assays were carried out using the GraphPad Prism software version 5.00 for Mac (GraphPad Software, San Diego, CA), and a P value of less than or equal to 0.05 was adopted for significance. (OME), and allergy (Tables 1 and 2 ). Among all the analyzed parameters, the only fact worth mentioning was that the viral codetections were significantly more frequent (P = 0.002) in tissues positive for HAdV than in HAdV-negative ones ( Table 2 ). The median HAdV load determined by qPCR in adenoids from patients with chronic adenotonsillar disease was 1.6 × 10 5 copies of genome/g (mean 9.4 × 10 6 ± 5.1 × 10 7 copies/g), while in the control patients, the median HAdV load was 2.6 × 10 6 copies/g (mean 3.9 × 10 7 ± 6.5 × 10 7 copies/g). In the PTs from patients with chronic adenotonsillar disease, the median HAdV load was 5.5 × 10 4 copies/g (mean 7.0 × 10 5 ± 1.8 × 10 6 copies/g), whereas, in the control group, the median was 1.8 × 10 5 copies/g (mean 1.8 × 10 5 ± 3.2 × 10 4 copies/g). Regarding the NPS samples, the median HAdV load was 1.4 × 10 4 copies/mL (mean 1.2 × 10 6 ± 7.2 × 10 6 copies/mL) and 3.4 × 10 4 copies/mL (mean 3.4 × 10 4 ± 4.7 × 10 4 copies/mL) in the patients with and without chronic adenotonsillar disease, respectively. The median HAdV load was almost three times higher in the adenoids than the other infection sites, although the difference was not significant ( Figure 1A) . However, HAdV loads in the adenoids were not uniformly high when compared to the other sampling sites of the same patients ( Figure 1B) . Differences in HAdV viral loads between patients with chronic adenotonsillar disease and the controls were not significant ( Figure 1C -E). In general, the HAdV loads in the adenoids, PTs, and NPS were not significantly different among the patients with and without any of the several clinical features analyzed in the present study ( Figure 1C -H). Of note, the HAdV viral loads were significantly higher in patients with sleep apnea (1.4 × 10 7 ± 6.7 × 10 7 copies/g) than in those without the condition (2.4 × 10 6 ± 7.3 × 10 6 copies/g; P = 0.03), although lower in patients with OME (1.6 × 10 6 ± 5.6 × 10 6 copies/ g) than in those without the disease (1.07 × 10 7 ± 5.6 × 10 7 copies/g; P = 0.006). The HAdV load was also significantly lower in PTs from patients with OME (1.6 × 10 5 ± 3.1 × 10 5 copies/g) than those without the condition (1.1 × 10 6 ± 2.4 × 10 6 copies/g; P = 0.02), suggesting The high frequency (27%) of patients with significant viral loads in the adenotonsillar tissues (>10 6 (Table 3) . Remarkably, mRNA for the HAdV hexon gene was detected in one of the 3 (33.3%) HAdV-positive adenoid biopsies obtained from control patients without adenotonsillar diseases ( To HAdV is among the leading causative agents of ARI in humans. 4 In addition to causing ARI, a previous study by our group showed that HAdV is one of the most frequent respiratory viruses detected in children with chronic adenotonsillar disease in the absence of ARI symptoms. 23 The near 50% detection rate of the HAdV genome reported herein confirms previous findings and agrees with adenoids being preferred sites of HAdV infection when compared with PTs. 16, 18 HAdV has been detected in PB from patients with HAdV-related tonsillitis in the presence of interleukin-6 production by endothelial cells, fibroblasts or activated T lymphocytes, an essential mechanism for the persistence of fever. 6 In the present study of patients without symptoms of ARI or acute tonsillitis, HAdV was undetectable in PB, suggesting that asymptomatic viremia is not frequent in asymptomatic HAdV carriers. As previously published by our group in a small cohort of patients, 23 HAdV was detected more frequently in tonsil tissues where codetection of other respiratory viruses was present. However, we demonstrated herein that such codetection is not linked to higher HAdV loads (>10 6 copies/g), nor with the detection of mRNA of the HAdV hexon gene. These findings indicate that the Although adenovirus DNA is frequently found in tonsils, adenoids, and intestinal tissues (varying from 30%-80% of cases), infectious viruses are rarely detected in these tissues, as measured by in situ hybridization or coculture with permissive cells. 16, 29 Corroborating these findings, we were able to detect HAdV-specific Persistent infection by HAdV has been associated with chronic airway obstructive diseases in children, such as asthma. 32, 33 In those studies, HAdV antigen or genome was found in bronchoalveolar lavage from more than 75% of children with asthma, respectively, by immunohistochemistry 32 or PCR. 33 In the present study, the detection of HAdV was not significantly correlated with chronic adenotonsillar disease, respiratory symptoms or OME, nor with any other detectable disease phenotype. Some clinical studies have associated the detection of respiratory viruses with OME. Viral infections caused by respiratory syncytial virus, influenza virus (types A and B), and adenovirus have been shown to increase the risk of OME, which can be in part attributed to these viral infections facilitating colonization of the nasopharynx by Streptococcus pneumoniae, Haemophilus influenzae, and M. catarrhalis, 34, 35 and the adhesion of S. pneumoniae to epithelial cells of the respiratory tract. 36 In contrast, the development of sleep apnea is partially associated with upper airway obstruction due to enlargement of the PTs and adenoids, seen significantly more often in obese patients with asymptomatic viral infections, such as those caused by adenoviruses. 37, 38 Proinflammatory cytokines released by visceral adipocytes seem to contribute to tonsillar inflammation and the development of sleep apnea. [39] [40] [41] Although no substantial association of HAdV and the severity of adenotonsillar enlargement was found in this study, a significant correlation was observed regarding HAdV quantities and the presence of sleep apnea or OME. The present study has shown that patients without OME had significantly higher viral loads than individuals with the condition. Some viruses, such as human cytomegalovirus, are known to target dendritic cells, subverting and compromising the host's adaptive immunity by interfering with the cellular transport of major histocompatibility complex molecules. 42, 43 Dendritic cells infected with HAdV strongly stimulate T cell proliferation, 44 which may result in increased cellular response to other infectious agents, protecting the host from the development of OME. In addition, persistent adenoviral infection, with small HAdV loads, could function as a chronic stimulus for the development of OME. In contrast, patients with sleep apnea exhibited significantly higher HAdV loads than individuals lacking the condition. Cellular and humoral responses are critical for the control of HAdV infection. The recruitment of macrophages and natural killer cells leads to the release of a range of proinflammatory cytokines, stimulating both CD4 + and CD8 + T cells, and, consequently, B cell proliferation with humoral antibody response. 45 Thus, in keeping with this idea, it is reasonable to consider that high levels of HAdV may induce the production of proinflammatory and vasoactive cytokines, which increase chances of developing apnea. In conclusion, the present study demonstrated that a high proportion of patients with the chronic adenotonsillar disease had persistent HAdV infection in the adenoids and tonsils. However, the presence of productive HAdV infection was not associated with the severity of nasal obstruction, recurrent tonsillitis or viral coinfections. The presence of higher HAdV loads in patients with apnea, in parallel with a protective effect against secretory otitis media, indicates that additional studies are required to provide a definitive role for HAdV during chronic adenotonsillar diseases. 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