Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections

Viruses are the major cause of pediatric acute respiratory tract infection (ARTI) and yet many suspected cases of infection remain uncharacterized. We employed 17 PCR assays and retrospectively screened 315 specimens selected by season from a predominantly pediatric hospital-based population. Before the Brisbane respiratory virus research study commenced, one or more predominantly viral pathogens had been detected in 15.2% (n = 48) of all specimens. The Brisbane study made an additional 206 viral detections, resulting in the identification of a microbe in 67.0% of specimens. After our study, the majority of microbes detected were RNA viruses (89.9%). Overall, human rhinoviruses (HRVs) were the most frequently identified target (n = 140) followed by human adenoviruses (HAdVs; n = 25), human metapneumovirus (HMPV; n = 18), human bocavirus (HBoV; n = 15), human respiratory syncytial virus (HRSV; n = 12), human coronaviruses (HCoVs; n = 11), and human herpesvirus-6 (n = 11). HRVs were the sole microbe detected in 37.8% (n = 31) of patients with suspected lower respiratory tract infection (LRTI). Genotyping of the HRV VP4/VP2 region resulted in a proposed subdivision of HRV type A into sublineages A1 and A2. Most of the genotyped HAdV strains were found to be type C. This study describes the high microbial burden imposed by HRVs, HMPV, HRSV, HCoVs, and the newly identified virus, HBoV on a predominantly paediatric hospital population with suspected acute respiratory tract infections and proposes a new formulation of viral targets for future diagnostic research studies.     

Newly identified human rhinoviruses: molecular methods heat up the cold viruses

Human rhinovirus (HRV) infections cause at least 70% of virus‐related wheezing exacerbations and cold and flu‐like illnesses. They are associated with otitis media, sinusitis and pneumonia. Annually, the economic impact of HRV infections costs billions in healthcare and lost productivity. Since 1987, 100 officially recognised HRV serotypes reside in two genetically distinct species; HRV A and HRV B, within the genus Enterovirus, family Picornaviridae. Sequencing of their ～7kb genomes was finalised in 2009. Since 1999, many globally circulating, molecularly‐defined ‘strains’, perhaps equivalent to novel serotypes, have been discovered but remain uncharacterised. Many of these currently unculturable strains have been assigned to a proposed new species, HRV C although confusion exists over the membership of the species. There has not been sufficient sampling to ensure the identification of all strains and no consensus criteria exist to define whether clinical HRV detections are best described as a distinct strain or a closely related variant of a previously identified strain (or serotype). We cannot yet robustly identify patterns in the circulation of newly identified HRVs (niHRVs) or the full range of associated illnesses and more data are required. Many questions arise from this new found diversity: what drives the development of so many distinct viruses compared to other species of RNA viruses? What role does recombination play in generating this diversity? Are there species‐ or strain‐specific circulation patterns and clinical outcomes? Are divergent strains sensitive to existing capsid‐binding antivirals? This update reviews the findings that trigger these and other questions arising during the current cycle of intense rhinovirus discovery. Copyright © 2010 John Wiley & Sons, Ltd.     

Epidemiological analysis and follow‐up of human rhinovirus infection in children with asthma exacerbation

To determine the prevalence of human rhinovirus (HRV) infection in children with acute asthma exacerbations, investigation of HRV viral load and severity of asthma exacerbations is also required. Nasopharyngeal aspirates and swabs were collected and assessed for respiratory viruses. HRV‐positive samples were sequenced to identify types and determine viral load. Outpatients with asthma exacerbations underwent follow‐up evaluations, their swabs were collected and clinical outcomes were recorded at their next clinic visit 4 weeks later. One hundred forty‐three inpatients and 131 outpatients, including 88 patients with asthma exacerbations and 43 controls with stable asthma were recruited. HRV‐A was mainly detected in September and February (45.5% and 33.3%, respectively), while HRV‐C was mainly detected in November and April (70.0% and 55.6%, respectively). HRV‐C was the primary type and was primarily found in inpatients with severe asthma exacerbations. HRV‐A viral load in the group of inpatients with severe exacerbations was higher than in the mild and moderate groups (P < 0.001 and P = 0.022). The HRV‐A viral load of both inpatients and outpatients was higher than that of HRV‐C (P < 0.001 and P = 0.036). The main genotypes were HRV‐C53 and HRV‐A20 among inpatients, and this genotype caused more severe clinical manifestations. HRV persisted for no more than 4 weeks, and their symptoms or signs of disease were well‐controlled well. HRV‐C was most frequently detected in asthma exacerbations. HRV‐A with high viral load led to severe asthma exacerbations.

     

Rhinovirus-Infected Epithelial Cells Produce More IL-8 and RANTES Compared With Other Respiratory Viruses

Purpose

The environmental factors human rhinoviruses (HRVs) and house dust mites (HDMs) are the most common causes of acute exacerbations of asthma. The aim of this study was to compare the chemokine production induced by HRVs in airway epithelial cells with that induced by other respiratory viruses, and to investigate synergistic interactions between HRVs and HDMs on the induction of inflammatory chemokines in vitro.

Methods

A549 human airway epithelial cells were infected with either rhinovirus serotype 7, respiratory syncytial virus (RSV)-A2 strain, or adenovirus serotype 3 and analyzed for interleukin (IL)-8 and regulated on activation, normal T-cell expressed and secreted (RANTES) release and mRNA expression. Additionally, activation of nuclear factor (NF)-κB and activator protein (AP)-1 were evaluated. The release of IL-8 and RANTES was also measured in cells stimulated simultaneously with a virus and the HDM allergen, Der f1.

Results

HRV caused greater IL-8 and RANTES release and mRNA expression compared with either RSV or adenovirus. NF-κB and AP-1 were activated in these processes. Cells incubated with a virus and Der f1 showed an increased IL-8 release. However, compared with cells incubated with virus alone as the stimulator, only HRV with Der f1 showed a statistically significant increase.

Conclusions

IL-8 and RANTES were induced to a greater extent by HRV compared with other viruses, and only HRV with Der f1 acted synergistically to induce bronchial epithelial IL-8 release. These findings may correspond with the fact that rhinoviruses are identified more frequently than other viruses in cases of acute exacerbation of asthma.     

Rhinovirus infection and house dust mite exposure synergize in inducing bronchial epithelial cell interleukin‐8 release

Background Human rhinoviruses (HRVs) and house dust mites (HDMs) are among the most common environmental factors able to induce airway inflammation in asthma. Although epidemiological studies suggest that they also synergize in inducing asthma exacerbations, there is no experimental evidence to support this, nor any information on the possible mechanisms involved. Objective To investigate their interaction on the induction of airway epithelial inflammatory responses in vitro. Methods BEAS‐2B cells were exposed to activated HDM Dermatophagoides pteronyssinus major allergen I (Der p I), HRVs (HRV1b or HRV16) or both in different sequences. IL‐8/CXCL8 release, intercellular adhesion molecule (ICAM)‐1 surface expression and nuclear factor κB (NF‐κB) translocation were evaluated. Complementary, primary human bronchial epithelial cells (HBECs) exposed to both Der p I and RVs and IL‐8, IL‐6, IFN‐γ‐induced protein (IP)‐10/CXCL10, IFN‐λ1/IL‐29, regulated upon activation normal T lymphocyte expressed and secreted (RANTES)/CCL5 release were measured. Results RV and Der p I up‐regulated IL‐8 release, ICAM‐1 expression and NF‐κB translocation in BEAS‐2B cells. Simultaneous exposure to both factors, as well as when cells were initially exposed to HRV and then to Der p I, resulted in further induction of IL‐8 in a synergistic manner. Synergism was not observed when cells were initially exposed to Der p I and then to HRV. This was the pattern in ICAM‐1 induction although the phenomenon was not synergistic. Concurrent exposure induced an early synergistic NF‐κB translocation induction, differentiating with time, partly explaining the above observation. In HBECs, both HRV and Der p I induced IL‐8, IL‐6, IL‐29 and IP‐10, while RANTES was induced only by HRV. Synergistic induction was observed only in IL‐8. Conclusion HRV and enzymatically active Der p I can act synergistically in the induction of bronchial epithelial IL‐8 release, when HRV infection precedes or is concurrent with Der p I exposure. Such a synergy may represent an important mechanism in virus‐induced asthma exacerbations.     

Molecular characterization and distinguishing features of a novel human rhinovirus (HRV) C,HRVC-QCE,detected in children with fever,cough and wheeze during 2003

BackgroundHuman rhinoviruses (HRVs) are associated with more acute respiratory tract infections than any other viral group yet we know little about viral diversity, epidemiology or clinical outcome resulting from infection by strains, in particular the recently identified HRVs.ObjectivesTo determine whether HRVC-QCE was a distinct HRV-C strain, by determining its genome and prevalence, by cataloguing genomic features for strain discrimination and by observing clinical features in positive patients.Study designNovel real-time RT-PCRs and retrospective chart reviews were used to investigate a well-defined population of 1247 specimen extracts to observe the prevalence and the clinical features of each HRV-QCE positive case from an in- and out-patient pediatric, hospital-based population during 2003. An objective illness severity score was determined for each HRVC-QCE positive patient.ResultsDifferences in overall polyprotein and VP1 binding pocket residues and the predicted presence of a cis-acting replication element in 1B defined HRVC-QCE as a novel HRV-C strain. Twelve additional HRVC-QCE detections (1.0% prevalence) occurred among infants and toddlers (1–24 months) suffering mild to moderate illness, including fever and cough, who were often hospitalized. HRVC-QCE was frequently detected in the absence of another virus and was the only virus detected in three (23% of HRVC-QCE positives) children with asthma exacerbation and in two (15%) toddlers with febrile convulsion.ConclusionsHRVC-QCE is a newly identified, genetically distinct HRV strain detected in hospitalized children with a range of clinical features. HRV strains should be independently considered to ensure we do not overestimate the HRVs in asymptomatic illness.     

Infections and coinfections by respiratory human bocavirus during eight seasons in hospitalized children

The human bocavirus (hBoV) has been identified in respiratory infections in children in a large number of studies. Despite this, the pathogenic role of the HBoV is under discussion. The main objectives of the study were: to determine the incidence of HBoV in hospitalized children; to describe the main clinical features of the positive children; and to compare the data with those from other viral infections in the same population. A prospective study was performed between 2005 and 2013 including children up to 14‐year old with respiratory infection admitted to the Severo Ochoa Hospital (Spain). Nasopharyngeal aspirates were taken from 3,275 patients and were tested for HBoV and other 15 respiratory viruses by RT‐nested PCR. HBoV was detected in 319 patients (9.9%); 80 cases as a single pathogen, and 239 cases (75%) as coinfections with other viruses. The HBoV was the fourth most common virus detected, behind respiratory syncytial virus (39.8%), rhinovirus (30.6%), and adenovirus (15%). The most common clinical diagnosis, in cases that HBoV was detected as a single pathogen was asthma exacerbation followed by pneumonia. A seasonal distribution was shown, with higher positivity rates in December and January. Children affected by HBoV were older than children infected by other viruses. Differences in terms of clinical diagnosis were found, bronchiolitis diagnosis was lower compared with the other viruses, and HBoV was associated with diagnosis of pneumonia, with increased use of antibiotics (41.8%), and radiographic infiltrates (47%). These findings could suggest a pathogenic role of HBoV in respiratory infections in children under 14 years of age. J. Med. Virol. 88:2052–2058, 2016. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

     

Human bocavirus in children with acute respiratory infections in Vietnam

Acute respiratory infections are the major cause of morbidity and mortality globally. Human bocavirus (HBoV), a novel virus, is recognized to increasingly associate with previously unknown etiology respiratory infections in young children. In this study, the epidemiological, clinical, and molecular characteristics of HBoV infections were described in hospitalized Vietnamese pediatric patients. From April 2010 to May 2011, 1,082 nasopharyngeal swab samples were obtained from patients with acute respiratory infections at the Children's Hospital 2, Ho Chi Minh City, Vietnam. Samples were screened for HBoV by PCR and further molecularly characterized by sequencing. HBoV was found in 78 (7.2%) children. Co‐infection with other viruses was observed in 66.7% of patients infected with HBoV. Children 12–24 months old were the most affected age group. Infections with HBoV were found year‐round, though most cases occurred in the dry season (December–April). HBoV was possible to cause severe diseases as determined by higher rates of hypoxia, pneumonia, and longer hospitalization duration in patients with HBoV infection than in those without (P‐value <0.05). Co‐infection with HBoV did not affect the disease severity. The phylogenetic analysis of partial VP1 gene showed minor variations and all HBoV sequences belonged to species 1 (HBoV1). In conclusion, HBoV1 was circulating in Vietnam and detected frequently in young children during dry season. Acute respiratory infections caused by HBoV1 were severe enough for hospitalization, which implied that HBoV1 may have an important role in acute respiratory infections among children. J. Med. Virol. 86:988–994, 2014. © 2013 Wiley Periodicals, Inc.

     

The use of multiplex PCR for the diagnosis of viral severe acute respiratory infection in children: a high rate of co-detection during the winter season

Respiratory tract infection is a major cause of hospitalization in children. Although most such infections are viral in origin, it is difficult to differentiate bacterial and viral infections, as the clinical symptoms are similar. Multiplex polymerase chain reaction (PCR) methods allow testing for multiple pathogens simultaneously and are, therefore, gaining interest. This prospective case-control study was conducted from October 2013 to February 2014. Nasopharyngeal (NP) and oropharyngeal (throat) swabs were obtained from children admitted with severe acute respiratory infection (SARI) at a tertiary hospital. A control group of 40 asymptomatic children was included. Testing for 16 viruses was done by real-time multiplex PCR. Multiplex PCR detected a viral pathogen in 159/177 (89.9 %) patients admitted with SARI. There was a high rate of co-infection (46.9 %). Dual detections were observed in 64 (36.2 %), triple detections in 17 (9.6 %), and quadruple detections in 2 (1.1 %) of 177 samples. Seventy-eight patients required intensive care unit (ICU) admission, of whom 28 (35.8 %) had co-infection with multiple viruses. AdV, HBoV, HRV, HEV, and HCoV-OC43 were also detected among asymptomatic children. This study confirms the high rate of detection of viral nucleic acids by multiplex PCR among hospitalized children admitted with SARI, as well as the high rate of co-detection of multiple viruses. AdV, HBoV, HRV, HEV, and HCoV-OC43 were also detected in asymptomatic children, resulting in challenges in clinical interpretation. Studies are required to provide quantitative conclusions that will facilitate clinical interpretation and application of the results in the clinical setting.     

Human rhinovirus in bronchial epithelium of infants with recurrent respiratory symptoms

BACKGROUND: Human rhinoviruses (HRVs) are a common cause of upper respiratory tract infections. There is growing evidence that HRVs are also important in lower respiratory tract infections and often induce asthma exacerbations. OBJECTIVE: We evaluated the presence of HRV in the lower respiratory tract by obtaining bronchial biopsies from infants with recurrent asthmalike respiratory symptoms. METHODS: A total of 201 steroid-naive infants age 3 to 26 months with recurrent respiratory symptoms for at least 4 weeks within the preceding 2 months were studied for lung function using body plethysmography. Bronchoscopy was performed in 68 children, and bronchial biopsies were available from 59 infants for HRV detection with in situ hybridization. RESULTS: Human rhinovirus was detected in 21 of 47 (45%) specimens. Abnormal lung function (decreased airways conductance) was found in 18 of 21 (86%) HRV(+) infants and in 15 of 26 (58%) HRV(-) infants (P = .037). Occurrence of a respiratory infection in the 6 weeks preceding bronchoscopy correlated with HRV positivity (P = .036). CONCLUSION: Human rhinovirus is frequently found in the lower airways in infants with recurrent respiratory symptoms, and the majority of these HRV(+) infants also showed increased airway resistance. CLINICAL IMPLICATIONS: Human rhinovirus is a common pathogen causing upper and lower respiratory symptoms. Follow-up of these infants will reveal whether the presence of HRV in the bronchial biopsy and abnormal lung function with recurrent respiratory symptoms predicts subsequent asthma.     

Human parainfluenza virus types 1–4 in hospitalized children with acute lower respiratory infections in China

Human parainfluenza viruses (HPIVs) are an important cause of acute lower respiratory tract infections (ALRTIs). HPIV‐4, a newly identified virus, has been associated with severe ALRTIs recently. A total of 771 nasopharyngeal aspirate samples were collected from hospitalized children between March 2010 and February 2011. HPIVs were detected by Nest‐PCR, and other known respiratory viruses were detected by RT‐PCR and PCR. All amplification products were sequenced. HPIVs were detected in 151 (19.58%) patients, of whom 28 (3.63%) were positive for HPIV‐4, 12(1.55%) for HPIV‐1, 4 (0.51%) for HPIV‐2, and 107 (13.87%) for HPIV‐3. Only three were found to be co‐infected with different types of HPIVs. All HPIV‐positive children were under 5 years of age, with the majority being less than 1 year. Only the detection rate of HPIV‐3 had a significant statistical difference (χ² = 29.648, P = 0.000) between ages. HPIV‐3 and HPIV‐4 were detected during the summer. Sixty (39.74%) were co‐infected with other respiratory viruses, and human rhinovirus (HRV) was the most common co‐infecting virus. The most frequent clinical diagnosis was bronchopneumonia, and all patients had cough; some patients who were infected with HPIV‐3 and HPIV‐4 had polypnea and cyanosis. No significant difference was found in clinical manifestations between those who were infected with HPIV‐4 and HPIV‐3. Two genotypes for HPIV‐4 were prevalent, although HPIV‐4a dominated. HPIV‐4 is an important virus for children hospitalized with ALRTIs in China. HRV was the most common co‐infecting virus. Two genotypes for HPIV‐4 are prevalent, HPIV‐4a dominated. J. Med. Virol. 88:2085–2091, 2016. © 2016 Wiley Periodicals, Inc.

     

Rhinovirus and the lower respiratory tract

Human rhinoviruses (HRVs) are well-recognised causes of common colds and associated upper respiratory tract complications such as sinusitis and otitis media. This article reviews information linking HRV infection to illness in the lower respiratory tract. HRVs are capable of efficient replication in vitro at temperatures present in the tracheobronchial tree and have been shown to cause productive infection, elaboration of cytokines and chemokines, and up-regulation of cell surface markers in human bronchial epithelial cells. In situ hybridisation studies have proven that HRV infection occurs in the tracheobronchial tree following experimental infection. Clinical studies report that HRV infection is the second most frequently recognised agent associated with pneumonia and bronchiolitis in infants and young children and commonly causes exacerbations of pre-existing airways disease in those with asthma, chronic obstructive pulmonary disease or cystic fibrosis. HRV infection is associated with one-third to one-half of asthma exacerbations depending on age and is linked to asthma hospitalisations in both adults and children. Limited information implicates HRV infection as a cause of severe lower respiratory tract illness in older adults and in highly immunocompromised hosts, particularly bone marrow transplant recipients. More information is needed about the pathogenesis of HRV infection with regard to lower respiratory tract complications in these diverse patient groups. Given the large unmet medical need associated with HRV infections, safe and effective antiviral agents are needed for both prevention and treatment of these infections.     

Human bocavirus infection in young children with acute respiratory tract infection in Lanzhou,China

Human bocavirus (HBoV) is a recognized human parvovirus associated with acute respiratory tract infection. However, HBoV has yet to be established as a causative agent of respiratory disease. In this study, the epidemiological and virological characteristics of HBoV infection were studied in children with acute respiratory tract infection in China. In total, 406 children younger than 14 years of age with acute respiratory tract infection were included in this prospective 1‐year study. HBoV was detected in 29 (7.1%) of the 406 children. No clear seasonal fluctuation was observed in infection rates of HBoV. Of the 29 children infected with HBoV, 16 (55.2%) were coinfected with other respiratory viruses, most commonly respiratory syncytial virus (RSV). Viral coinfection with HBoV did not affect the severity of the respiratory disease (P = 0.291). The number of HBoV genome copies ranged from 5.80 × 10² to 9.72 × 10⁸ copies/ml in nasopharyngeal aspirates among HBoV‐positive specimens by real‐time PCR, and neither coinfection nor the severity of disease correlated with the viral load (P = 0.148, P = 0.354, respectively). The most common clinical features were cough and acute upper respiratory infection, and acute bronchopneumonia. Additionally, the NP‐1 gene of HBoV showed minimal sequence variation. These data suggest that HBoV is frequent in young children with acute respiratory tract infection in Lanzhou, China, and RSV is the most common coinfecting virus. There was no apparent association between the viral load of HBoV and coinfection or disease severity. The NP‐1 gene was highly conserved in HBoV. J. Med. Virol. 82:282–288, 2010. © 2009 Wiley‐Liss, Inc.     

Uncoating of human rhinoviruses

Human rhinoviruses (HRVs) are a major cause of the common cold. The more than one hundred serotypes, divided into species HRV‐A and HRV‐B, either bind intercellular adhesion molecule 1 (major group viruses) or members of the low‐density lipoprotein receptor (minor group viruses) for cell entry. Some major group HRVs can also access the host cell via heparan sulphate proteoglycans. The cell attachment protein(s) of the recently discovered phylogenetic clade HRV‐C is unknown. The respective receptors direct virus uptake via clathrin‐dependent or independent endocytosis or via macropinocytosis. Triggered by ICAM‐1 and/or the low pH environment in endosomes the virions undergo conformational alterations giving rise to hydrophobic subviral particles. These are handed over from the receptors to the endosomal membrane. According to the current view, the RNA genome is released through an opening at one of the fivefold axes of the icosahedral capsid and crosses the membrane through a pore presumably formed by viral proteins. Alternatively, the membrane may be ruptured allowing subviral particles and RNA to enter the cytosol. Whether a channel is formed or the membrane is disrupted most probably depends on the respective HRV receptor. Copyright © 2010 John Wiley & Sons, Ltd.     

Prevalence of viral respiratory tract infections in children with asthma

BACKGROUND: Previous studies support a strong association between viral respiratory tract infections and asthma exacerbations. The effect of newly discovered viruses on asthma control is less well defined. OBJECTIVE: We sought to determine the contribution of respiratory viruses to asthma exacerbations in children with a panel of PCR assays for common and newly discovered respiratory viruses. METHODS: Respiratory specimens from children aged 2 to 17 years with asthma exacerbations (case patients, n = 65) and with well-controlled asthma (control subjects, n = 77), frequency matched by age and season of enrollment, were tested for rhinoviruses, enteroviruses, respiratory syncytial virus, human metapneumovirus, coronaviruses 229E and OC43, parainfluenza viruses 1 to 3, influenza viruses, adenoviruses, and human bocavirus. RESULTS: Infection with respiratory viruses was associated with asthma exacerbations (63.1% in case patients vs 23.4% in control subjects; odds ratio, 5.6; 95% CI, 2.7- 11.6). Rhinovirus was by far the most prevalent virus (60% among case patients vs 18.2% among control subjects) and the only virus significantly associated with exacerbations (odds ratio, 6.8; 95% CI, 3.2-14.5). However, in children without clinically manifested viral respiratory tract illness, the prevalence of rhinovirus infection was similar in case patients (29.2%) versus control subjects (23.4%, P > .05). Other viruses detected included human metapneumovirus (4.6% in patients with acute asthma vs 2.6% in control subjects), enteroviruses (4.6% vs 0%), coronavirus 229E (0% vs 1.3%), and respiratory syncytial virus (1.5% vs 0%). CONCLUSION: Symptomatic rhinovirus infections are an important contributor to asthma exacerbations in children. CLINICAL IMPLICATIONS: These results support the need for therapies effective against rhinovirus as a means to decrease asthma exacerbations.