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.     

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 Rhinoviruses

Human rhinoviruses (HRVs), first discovered in the 1950s, are responsible for more than one-half of cold-like illnesses and cost billions of dollars annually in medical visits and missed days of work. Advances in molecular methods have enhanced our understanding of the genomic structure of HRV and have led to the characterization of three genetically distinct HRV groups, designated groups A, B, and C, within the genus Enterovirus and the family Picornaviridae. HRVs are traditionally associated with upper respiratory tract infection, otitis media, and sinusitis. In recent years, the increasing implementation of PCR assays for respiratory virus detection in clinical laboratories has facilitated the recognition of HRV as a lower respiratory tract pathogen, particularly in patients with asthma, infants, elderly patients, and immunocompromised hosts. Cultured isolates of HRV remain important for studies of viral characteristics and disease pathogenesis. Indeed, whether the clinical manifestations of HRV are related directly to viral pathogenicity or secondary to the host immune response is the subject of ongoing research. There are currently no approved antiviral therapies for HRVs, and treatment remains primarily supportive. This review provides a comprehensive, up-to-date assessment of the basic virology, pathogenesis, clinical epidemiology, and laboratory features of and treatment and prevention strategies for HRVs.     

Clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy

BackgroundHuman rhinoviruses (HRVs) are common causes of upper respiratory tract infection (URTI) in hematologic malignancy (HM) patients. Predictors of lower respiratory tract infection (LRTI) including the impact of HRV species and types are poorly understood.ObjectivesThis study aims to describe the clinical and molecular epidemiology of HRV infections among HM patients.Study designFrom April 2012–March 2013, HRV-positive respiratory specimens from symptomatic HM patients were molecularly characterized by analysis of partial viral protein 1 (VP1) or VP4 gene sequence. HRV LRTI risk-factors and outcomes were analyzed using multivariable logistic regression.ResultsOne hundred and ten HM patients presented with HRV URTI (n = 78) and HRV LRTI (n = 32). Hypoalbuminemia (OR 3.0; 95% CI, 1.0–9.2; p = 0.05) was independently associated with LRTI, but other clinical and laboratory markers of host immunity did not differ between patients with URTI versus LRTI. Detection of bacterial co-pathogens was common in LRTI cases (25%). Among 92 typeable respiratory specimens, there were 58 (64%) HRV-As, 12 (13%) HRV-Bs, and 21 (23%) HRV-Cs, and one Enterovirus 68. LRTI rates among HRV-A (29%), HRV-B (17%), and HRV-C (29%) were similar. HRV-A infections occurred year-round while HRV-B and HRV-C infections clustered in the late fall and winter.ConclusionsHRVs are associated with LRTI in HM patients. Illness severity is not attributable to specific HRV species or types. The frequent detection of bacterial co-pathogens in HRV LRTIs further substantiates the hypothesis that HRVs predispose to bacterial superinfection of the lower airways, similar to that of other community-acquired respiratory viruses.     

Human enterovirus and rhinovirus infections are associated with otitis media in a prospective birth cohort study

BackgroundHuman enteroviruses (HEVs) and rhinoviruses (HRVs) have been linked to acute otitis media (AOM).ObjectivesThe present study evaluates the aforementioned association in a birth cohort setting.Study designThe cohort included 286 healthy infants (191 boys) followed from birth up to the age of 2 years in the Type 1 Diabetes Prediction and Prevention study in Finland. Stool samples were collected monthly and analyzed for the presence of HRV and HEV RNA using RT-PCR. Clinical symptoms were recorded by a questionnaire every 3–6 months.ResultsAltogether 610 AOM episodes were reported during the follow-up. 9.8% of the stool samples were positive for HRV and 6.8% for HEV. HRV positivity peaked at the age of 3–6 months declining gradually after this age, whereas HEV positivity peaked later, at the age of 12–24 months. The risk of AOM was increased in children who were HEV positive at least once at the age of 6–12 months (OR 2.2 [95%CI 1.1–4.2], P = 0.023) or who were HRV positive at least once at the age of 18–24 months (OR 2.3 [95%CI 1.0–5.2], P = 0.042). Having an older sibling, short breast-feeding and maternal smoking during pregnancy were also significantly associated with AOM.ConclusionsHRV and HEV infections are frequent during the first months of life. The observed trend for increased risk of AOM in HRV and HEV positive children is in line with the results from hospital series suggesting that these viruses may play an independent role in the pathogenesis of AOM.     

Screening Respiratory Samples for Detection of Human Rhinoviruses (HRVs) and Enteroviruses: Comprehensive VP4-VP2 Typing Reveals High Incidence and Genetic Diversity of HRV Species C

Rhinovirus infections are the most common cause of viral illness in humans, and there is increasing evidence of their etiological role in severe acute respiratory tract infections (ARTIs). Human rhinoviruses (HRVs) are classified into two species, species A and B, which contain over 100 serotypes, and a recently discovered genetically heterogeneous third species (HRV species C). To investigate their diversity and population turnover, screening for the detection and the genetic characterization of HRV variants in diagnostic respiratory samples was performed by using nested primers for the efficient amplification of the VP4-VP2 region of HRV (and enterovirus) species and serotype identification. HRV species A, B, and C variants were detected in 14%, 1.8%, and 6.8%, respectively, of 456 diagnostic respiratory samples from 345 subjects (6 samples also contained enteroviruses), predominantly among children under age 10 years. HRV species A and B variants were remarkably heterogeneous, with 22 and 6 different serotypes, respectively, detected among 73 positive samples. Similarly, by using a pairwise distance threshold of 0.1, species C variants occurring worldwide were provisionally assigned to 47 different types, of which 15 were present among samples from Edinburgh, United Kingdom. There was a rapid turnover of variants, with only 5 of 43 serotypes detected during both sampling periods. By using divergence thresholds and phylogenetic analysis, several species A and C variants could provisionally be assigned to new types. An initial investigation of the clinical differences between rhinovirus species found HRV species C to be nearly twice as frequently associated with ARTIs than other rhinovirus species, which matches the frequencies of detection of respiratory syncytial virus. The study demonstrates the extraordinary genetic diversity of HRVs, their rapid population turnover, and their extensive involvement in childhood respiratory disease.Human rhinoviruses (HRVs) have recently been classified in the genus Enterovirus, family Picornaviridae (34). Typically for picornaviruses, they are small, nonenveloped, single-stranded positive-sense viruses with a 7,200-base mRNA genome. HRVs are highly heterogeneous genetically and antigenically. A total of 101 serotypes have been classified, and these fall into two species, HRV species A (HRV-A; 74 serotypes) and HRV-B (25 serotypes); HRV serotype 87 is genetically most similar to members of human enterovirus (HEV) species D (HEV-D). Recently, several reports have described the detection and characterization of a series of new divergent rhinovirus variants that have provisionally been assigned to a new species, HRV-A2 or HRV-C (1, 13, 15, 16, 18, 22, 28).HRVs are the most common etiological agents of upper respiratory tract infections and regularly cause a mild, self-limiting illness that is often referred to as the common cold. HRV infections are transmitted most commonly by the respiratory-salivary route, both by person-to-person contact and by airborne transmission. In temperate countries, infections occur primarily in two peaks, with the first being in April and May and the second being in September and October (24, 38). HRV infections occur in all age groups during all seasons, with a 90% past infection frequency by the age of 2 years (2). While infections are frequently associated with mild upper respiratory tract symptoms, HRV infections have been implicated in more serious illnesses, including pneumonia, otitis media, exacerbations of asthma, and chronic obstructive pulmonary disease (for a review, see reference 20). Whether HRV-C is more likely to be etiologically associated with the more severe end of the HRV-associated spectrum of respiratory disease is currently under active debate and investigation (23, 40).In the study described here, we have coupled a sensitive PCR-based method for screening for HRV using primers from the conserved 5′ untranslated region (UTR) with a VP4-VP2 amplification and typing method that can be applied directly to clinical specimens. The method effectively amplifies and allows the genetic characterization of all three species of HRV as well as HEVs. When the method was applied to respiratory specimens collected in Edinburgh, United Kingdom, we were able to document the diversity and rapid turnover of circulating HRV strains, including species C variants, in a predominantly pediatric population.     

Sensitive and rapid detection of viruses associated with hand foot and mouth disease using multiplexed MALDI-TOF analysis

BackgroundHuman enterovirus (HEV) is the major cause of hand foot and mouth disease (HFMD). A powerful method for detecting HEVs associated with HFMD can provide results in a clinically relevant time frame. However, the limitations of the current enterovirus test make it difficult to identify multiple genotypes on the first pass.ObjectiveTo develop a more sensitive and easy applicable assay for detecting 18 HFMD-associated HEV serotypes in multiplex PCR products.Study design: A total of 241 clinical specimens were collected from HFMD patients during the 2010 outbreak in China. These samples were tested by DNA sequencing and MassARRAY analysis, respectively.ResultsAnalysis of a dilution series of plasmids revealed the detection limit per PCR reaction for the MassARRAY method was one copy for the tested HEVs. We compared results from 241 samples to those of the sequence analysis of the VP1 gene. The MassARRAY method detected all samples found positive by consensus PCR and sequencing method. Comparison of the results of MassARRAY and the DNA sequencing method found concordant results for 225 (93.4%) of the 241 samples. In 14 (5.8%) samples, the MassARRAY method detected multiple types, whereas the DNA sequencing method detected a single type. In another 2 (0.8%) samples, the MassARRAY method detected single types, whereas the DNA sequencing method detected no HEV.ConclusionsThe MassARRAY assay is a highly sensitive and accurate method for the type-specific detection of 18 HEVs in HFMD and is a powerful complement to current detection methods.     

Newly identified respiratory viruses in children with asthma exacerbation not requiring admission to hospital

There are few data describing the comprehensive identification in and influence of newly identified respiratory viruses on asthma exacerbations. Most studies focus on inpatients. In this preliminary study, the point prevalence and the associations of picornavirus species described recently and human bocavirus (HBoV) with the recovery from exacerbations in non‐hospitalized asthmatic children (median age 5.1 years) were examined. Human rhinoviruses (HRVs) were present in 52.6% of specimens, HBoV‐1 was in 7.7%. Viral co‐detections occurred in 25.6% of children and were associated (P = 0.04) with lower asthma quality of life scores upon presentation than were single viral detections. The undifferentiated presence or absence of virus did not influence the severity of asthma or recovery however when virus species were examined individually, specific clinical associations emerged. HRV species C (HRV‐Cs) were the viruses most frequently detected as single virus detections. Among 41 genotyped HRVs, more HRV‐Cs (n = 23) were identified than HRV‐As (n = 16) however HRV‐A detection was associated (P = 0.01) with worse asthma symptoms and cough for longer than was HRV‐C detection. Larger, PCR‐based studies are required to elucidate further the true impact of HRV species in childhood asthma exacerbations of both hospitalized and non‐hospitalized cohorts. J. Med. Virol. 82:1458–1461, 2010. © 2010 Wiley‐Liss, Inc.     

Improved detection of rhinoviruses in nasal and throat swabs by seminested RT-PCR

A seminested RT-PCR (nRT-PCR) was used to detect picornavirus (PV) RNA in cell cultures inoculated with rhinoviruses (HRVs) and enteroviruses (EVs). PCR tests in which a primary "touchdown" PCR was followed by secondary reactions using PV or HRV specific primers were able to differentiate HRVs of 48 serotypes from EVs. PVnRT-PCR and HRVnRT-PCR were then used to test nasal and throat swabs from adult subjects with naturally acquired respiratory virus infections. The swabs were also analysed for respiratory viruses by cell culture techniques and the rates of PV identification by the two methods were compared. PVnRT-PCR was found to be at least five times more sensitive than cell culture for the detection of PVs in these clinical specimens. Paired acute and convalescent serum samples were tested for complement fixing antibodies to adenovirus, influenza A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, and 3, Myco plasma pneumoniae, and Chlamydia psittaci. An enzyme-linked immunosorbent assay (ELISA) was used to detect rises in antibody level to coronavirus types 229E and OC43. The overall rate of pathogen identification in 159 swabs from adult asthmatics increased from 28% when only cell culture and serology were used to 57% when these methods were supplemented by PVnRT-PCR. © 1993 Wiley-Liss, Inc.     

Comparison of results of detection of rhinovirus by PCR and viral culture in human nasal wash specimens from subjects with and without clinical symptoms of respiratory illness

Human rhinoviruses (HRV) cause acute upper respiratory illness. The frequency of HRV-associated illnesses appears greater when PCR assays are used to detect rhinoviruses. The present study performed PCR-based detection of HRV upon entry of subjects into respiratory syncytial virus and parainfluenza type 3 vaccine trials when subjects were symptom-free and upon subsequent development of clinical symptoms of respiratory illness during the trial. The background of HRV PCR positivity in symptom-free individuals (30/139 [22%]) was only slightly lower than in those with respiratory illness (28/77 [36%]). For subjects with multiple samples, it was estimated that HRV was detectable by PCR for approximately 100 days before, during, and after clinical symptoms were documented. PCR is a remarkably more sensitive method of detecting HRV than is tissue culture. The presence of HRV RNA may not always reflect an association with infectious virus production. The limited association of HRV RNA with illness suggests caution in assigning causality of HRV PCR positivity to clinical symptoms of respiratory illness.     

Human rhinoviruses including group C are common in stool samples of young Finnish children

BackgroundHuman rhinoviruses (HRVs) are common causes of viral respiratory infections. They have been widely studied in respiratory samples in hospital patient series but only a few studies have been performed to assess their occurrence in other sample types and their circulation in healthy children background population.ObjectivesTo analyze the frequency of HRVs in the background population in Finland by screening HRV RNA from stool samples longitudinally collected in a cohort of young children.Study designAltogether 4184 stool samples were collected regularly from a cohort of children who were observed from birth. Samples were screened for the presence of RNA of HRVs using RT-PCR. HRV specific sequences were identified by sequencing the VP1 or VP4/VP2 coding region. Virus isolation was performed using four different cell lines and the result was confirmed by real time PCR.ResultsA total of 9% of the stool samples were positive for HRV RNA. Sequence analysis indicated that the most prevalent species was HRV-A, and the most prevalent serotype was HRV61. HRV-B and HRV-C species were also detected. One of the six tested rhinovirus positive samples retained its infectivity and was able to grow in RD and GMK cells.ConclusionsOur study shows that HRVs are frequently detected in the stool samples from the population of young children. We also show that HRV-C, which can cause severe illnesses in children, is commonly circulating in young children in Finland.     

Prevalence of human respiratory viruses in adults with acute respiratory tract infections in Beijing, 2005–2007

To determine the aetiological role and epidemiological profile of common respiratory viruses in adults with acute respiratory tract infections (ARTIs), a 2-year study was conducted in Beijing, China, from May 2005 to July 2007. Nose and throat swab samples from 5808 ARTI patients were analysed by PCR methods for common respiratory viruses, including influenza viruses (IFVs) A, B, and C, parainfluenza viruses (PIVs) 1–4, enteroviruses (EVs), human rhinoviruses (HRVs), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), human coronaviruses (HCoVs) OC43, 229E, NL63, and HKU1, and adenoviruses (ADVs). Viral pathogens were detected in 34.6% of patient samples, and 1.6% of the patients tested positive for more than one virus. IFVs (19.3%) were the dominant agents detected, followed by HRVs (6.5%), PIVs (4.3%), EVs (3.2%), and HCoVs (1.1%). ADVs, RSV and HMPV were also detected (<1%). The viral detection rates differed significantly between infections of the lower and upper respiratory tracts in the sample population: PIVs, the second most commonly detected viral agents in lower acute respiratory tract infections (LRTIs), were more prevalent than in upper acute respiratory tract infections, indicating that the pathogenic role of PIVs in LRTIs should be investigated. Currently, this study is the largest-scale investigation of respiratory virus infections in China with multiple agent detection, providing baseline data for further studies of respiratory virus infections in adults with ARTIs.     

Analysis of epitopes in the capsid protein of avian hepatitis E virus by using monoclonal antibodies

Avian hepatitis E virus (HEV) is related genetically and antigenically to human and swine HEVs and capsid protein of avian HEV shares approximately 48-49% amino acid sequence identities with those of human and swine HEVs. Six monoclonal antibodies (MAbs) were produced and used to locate different epitopes in the ORF2 region of aa 339-570 of avian HEV Chinese isolate. The results showed that five epitopes were located in the aa 339-414 region and one in the aa 510-515 region. Two epitopes located in aa 339-355 and aa 384-414 regions are the immunodominant epitopes on the surface of the avian HEV particles as demonstrated by immune capture of viral particles and immunohistochemical detection of the ORF2 antigens with two MAbs.