Assay for 5' noncoding region analysis of all human rhinovirus prototype strains

Increasing recognition of the association of rhinovirus with severe lower respiratory tract illnesses has clarified the need to understand the relationship between specific serotypes of rhinovirus and their clinical consequences. To accomplish this, a specific and sensitive assay to detect and serotype rhinovirus directly from clinical specimens is needed. Traditional methods of serotyping using culture and serum neutralization are time-consuming, limited to certain reference laboratories, and complicated by the existence of over 100 serotypes of human rhinoviruses (HRVs). Accordingly, we have developed a sequence-based assay that targets a 390-bp fragment accounting for approximately two-thirds of the 5′ noncoding region (NCR). Our goal was to develop an assay permitting amplification of target sequences directly from clinical specimens and distinction among all 101 prototype strains of rhinoviruses. We determined the sequences of all 101 prototype strains of HRV in this region to enable differentiation of virus genotypes in both viral isolates and clinical specimens. We evaluated this assay in a total of 101 clinical viral isolates and 24 clinical specimens and compared our findings to genotyping results using a different region of the HRV genome (the VP4-VP2 region). Five specimens associated with severe respiratory disease in children did not correlate with any known serotype of rhinovirus and were found to belong to a novel genogroup of rhinovirus, genogroup C. Isolates were also found that corresponded to the genogroup A2 variant identified in New York and Australia and two other novel group A clusters (GAC1 and GAC2).     

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.     

Rhinovirus infections in western Sweden: a four-year molecular epidemiology study comparing local and globally appearing types

Human rhinovirus (HRV) is a highly prevalent pathogen and a major cause of acute respiratory tract infection (ARTI). HRV express less seasonality than other viral ARTIs, which typically appear as seasonal epidemics lasting for 1–2 months. The aim of this study was to investigate the seasonal patterns of HRV types over four consecutive years in one geographic region. HRV identified in respiratory samples from 114 patients over a four-year period were analysed by VP4/VP2 sequencing. HRV-A was found in 64, HRV-B in 11 and HRV-C in 37 cases. Overall, 33 different HRV-A types, nine B types and 21 C types were found. As many as 21 of the HRV types appeared during several seasons, with a maximum time-span of four years. Some types appeared during successive seasons and, in some cases, phylogenetic analysis indicated extended periods of circulation locally. Most of the strains were closely related to HRV identified in other parts of the world during the same time period. HRV strains that circulate locally represent many types and seem to reflect that HRV infections are highly globalised. The existence of simultaneous or successive epidemics with different HRV types in combination with the ability of each type to remain in the local population over extended periods of time may contribute to explaining the high rate of HRV infections.     

Enhanced severity of virus associated lower respiratory tract disease in asthma patients may not be associated with delayed viral clearance and increased viral load in the upper respiratory tract.

BACKGROUND: Viral respiratory infections, particularly human rhinovirus (HRV) infections, are the most common cause of asthma exacerbation. HRV infections usually lead to more severe and longer duration of lower respiratory tract (LRT) symptoms in asthmatics than in otherwise healthy individuals. However, the exact mechanism by which viruses contribute to exacerbation of asthma is unknown. OBJECTIVES: The main objective of our study was to investigate the relationship of the enhanced severity of LRT symptoms to viral dynamics or cytokine responses in the upper respiratory tract (URT). STUDY DESIGN: Therefore, we conducted a longitudinal study in which asthmatics and healthy controls were followed during natural viral respiratory tract infections. RESULTS: Our study confirmed that viral respiratory tract infections caused more severe problems of the LRT in asthma patients as compared to healthy controls. However, for all subjects, the severity of LRT symptoms were not related to viral load or prolonged viral shedding in the URT. In addition, we did not detect differences in proinflammatory cytokines in the URT between asthmatics and controls. CONCLUSION: Persistence of the virus, as well as viral load in the URT, may not be associated with the induction and/or persistence of asthmatic symptoms.     

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.     

Molecular and biological characterization of the 5 human-bovine rotavirus (WC3)-based reassortant strains of the pentavalent rotavirus vaccine, RotaTeq®

RotaTeq® is a pentavalent rotavirus vaccine that contains five human-bovine reassortant strains (designated G1, G2, G3, G4, and P1) on the backbone of the naturally attenuated tissue culture-adapted parental bovine rotavirus (BRV) strain WC3. The viral genomes of each of the reassortant strains were completely sequenced and compared pairwise and phylogenetically among each other and to human rotavirus (HRV) and BRV reference strains. Reassortants G1, G2, G3, and G4 contained the VP7 gene from their corresponding HRV parent strains, while reassortants G1 and G2 also contained the VP3 gene (genotype M1) from the HRV parent strain. The P1 reassortant contained the VP4 gene from the HRV parent strain and all the other gene segments from the BRV WC3 strain. The human VP7s had a high level of overall amino acid identity (G1: 95-99%, G2: 94-99% G3: 96-100%, G4: 93-99%) when compared to those of representative rotavirus strains of their corresponding G serotypes. The VP4 of the P1 reassortant had a high identity (92-97%) with those of serotype P1A[8] HRV reference strains, while the BRV VP7 showed identities ranging from 91% to 94% to those of serotype G6 HRV strains. Sequence analyses of the BRV or HRV genes confirmed that the fundamental structure of the proteins in the vaccine was similar to those of the HRV and BRV references strains. Sequences analyses showed that RotaTeq® exhibited a high degree of genetic stability as no mutations were identified in the material of each reassortant, which undergoes two rounds of replication cycles in cell culture during the manufacturing process, when compared to the final material used to fill the dosing tubes. The infectivity of each of the reassortant strains of RotaTeq®, like HRV strains, did not require the presence of sialic acid residues on the cell surface. The molecular and biologic characterization of RotaTeq® adds to the significant body of clinical data supporting the consistent efficacy, immunogenicity, and safety of RotaTeq®.     

Clinical features of patients with acute respiratory illness and rhinovirus in their bronchoalveolar lavages.

BACKGROUND: Several reports in selected populations suggest that human rhinovirus (HRV) may be responsible for lower respiratory tract infections or pneumonia. We describe clinical features of all patients with rhinovirus cultured from their bronchoalveolar lavage (BAL) during a 10-yr period in a tertiary care center. METHODS: Results for viral culture of all lower respiratory specimens performed during a 10-year period at the University of Virginia Health Sciences Center were reviewed. A case was defined as any patient with a positive culture for HRV in a BAL specimen. A comprehensive review of the patients' medical records was performed. In one case, in situ hybridization (ISH) was performed in order to identify whether rhinoviral RNA was present in bronchial biopsy specimens. RESULTS: During the 10-year study period viruses were identified in 431 lower respiratory tract specimens, and were most frequently cytomegalovirus or herpes simplex virus. Twenty patients (ages, 2.5-86 year) had a bronchoalveolar specimen culture positive for HRV. All had an abnormal chest radiograph, 60% were admitted to the intensive care unit, and 25% expired during their hospitalization. In 18 patients (90%) various severe underlying conditions were identified including solid organ transplants in seven, malignancies in four and AIDS in two. An immunosuppressive disease or condition requiring immunosuppressive therapy was present in all cases. In addition to HRV, one or more potential pathogens were identified in respiratory specimens from 14 patients (70%). Histopathological abnormalities, ranging from fibropurulent debris in alveoli to diffuse alveolar damage, were present in 6 of 13 bronchial biopsies. In two cases without any other significant pathogens than HRV, acute inflammations with fibropurulent debris in alveoli were observed. One lung transplant patient showed intermittent recovery of HRV in her respiratory specimens during a 15-week time period, but ISH did not show HRV RNA in bronchial epithelial cells. CONCLUSION: Our observations suggest that HRV recovery from BALs or lower respiratory tract samples in highly immunocompromised patients is associated with severe lower respiratory tract illness. Whether HRV directly causes viral pneumonia or predispose to pulmonary injury and/or superinfection remains uncertain.     

Phylogenetic analysis of rhinovirus isolates collected during successive epidemic seasons

Human rhinoviruses (HRV) have been shown to be the major causative agent for mild respiratory infections, but also associated with more serious diseases, such as acute otitis media and pneumonia in children, and asthma. Despite the economical and medical importance of HRV, little is known about the circulation and genetic diversity of HRV during a given season. The aim of this study was to genetically characterize HRV strains causing acute respiratory infections in a cohort of small children during a 2 years follow-up time. Genetic relationships between 61 HRV field isolates were studied using partial genomic sequencing in the VP4/VP2 region (420 nt) and phylogenetic analysis of these sequences. Sequences from the clinical isolates clustered in the two previously known phylogenetic clades, the designated genetic group 2 (including HRV 14) being more predominant. The maximum genetic variation within group 1 was 32.3% and within group 2 it was 32.7%. Several distinct clusters could be observed, some of which were strictly seasonal, whereas some other variants were detected during several seasons. The results of this study show striking genetic diversity of the HRV strains circulating in a given community during a short time.     

The significance of rhinovirus detection in hospitalized children: clinical,epidemiological and virological features

Recent developments in molecular diagnostic tools have led to the easy and rapid detection of a large number of rhinovirus (HRV) strains. However, the lack of clinical and epidemiological data hampers the interpretation of these diagnostic findings. From October 2009 to January 2011, we conducted a prospective study in hospitalized children from whom samples were taken for the detection of respiratory viruses. Clinical, epidemiological and microbiological data from 644 patients with 904 disease episodes were collected. When HRV tested positive, strains were further characterized by sequencing the VP4/VP2 region of the HRV genome. HRV was the single respiratory virus detected in 254 disease episodes (28%). Overall, 99 different serotypes were detected (47% HRV-A, 12% HRV-B, 39% HRV-C). Patients with HRV had more underlying pulmonary illness compared with patients with no virus (p 0.01), or patients with another respiratory virus besides HRV (p 0.007). Furthermore, cough, shortness of breath and a need for oxygen were significantly more present in patients with HRV infection. Particularly, patients with HRV-B required extra oxygen. No respiratory symptom, except for oxygen need, was predictive of the presence of HRV. In 22% of HRV-positive disease episodes, HRV infection was hospital acquired. Phylogenetic analysis revealed several clusters of HRV; in more than 25% of these clusters epidemiological information was suggestive of transmission within specific wards. In conclusion, the detection of HRV may help in explaining respiratory illness, particular in patients with pulmonary co-morbidities. Identifying HRV provides opportunities for timely implementation of infection control measures to prevent intra-hospital transmission.     

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.     

Characterization of naturally occurring parainfluenza virus type 2 (hPIV-2) variants

BACKGROUND: Human parainfluenza viruses (hPIV) are respiratory pathogens responsible for upper and lower respiratory tract infections. In most labs, the clinical diagnosis of hPIV is routinely done using techniques based on the detection of viral antigens such as immunofluorescence assay or/and viral isolation. STUDY DESIGN: Five hPIV-2 isolated from respiratory samples exhibited unusual phenotypic and antigenic characteristics. These isolates showed important syncytial cytopathic effect and failed to react with one specific monoclonal antibody. These variant strains were subsequently compared with hPIV-2 prototype strain by cellular and molecular techniques. RESULTS: Both variant and prototype strains showed similar growth kinetics. Observation of plaque formation and syncytia assay indicated a more important fusogenic activity for the variant strains. Sequencing of fusion (F) and hemagglutinin-neuraminidase (HN) genes showed differences between the "atypical" hPIV-2 isolates and the Greer hPIV-2 prototype strain. These differences were analyzed with molecular modelling and structure prediction soft wares. A potential new glycosylation site in HN, in addition to minor changes observed in the predicted structure for the variant strains could explain their antigenic variation. Genetic changes in the fusion peptide and the cleavage site of F could also explain the difference observed in the fusion activity. CONCLUSIONS: Continuous global viral surveillance is essential to monitor antigenic changes that may occur in nature particularly with regards to the implementation of diagnostic assays. The differences observed in F and HN between the prototype strain and clinical hPIV-2 variants could also provide new data for the analysis of Paramyxovirus fusion mechanisms and their pathogenesis.     

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.     

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.     

Trypsin sensitivity of several human rhinovirus serotypes in their low pH-induced conformation

Five serotypes of human rhinovirus (HRV) were examined for sensitivity to trypsin at physiological pH, HRV1A, HRV2, and HRV14 were found to be resistant whereas in serotypes HRV49 and HRV89 degradation of VP2 was observed. However, exposure to low pH followed by neutralization, a treatment which causes irreversible conformational changes in the capsid, led to rapid cleavage by trypsin of VP1 in HRV1A, HRV2, and HRV49 at defined sites followed by degradation of VP2. In the case of HRV2, the cleavage site in VP1 was determined by direct protein sequencing and was shown to occur between Arg260 and Thr261, close to the C-terminus. HRV49 behaves similarly to HRV2 as expected from extensive sequence similarity in this region, whereas VP1 in HRV1A is most probably cleaved at a site closer to the C-terminus than that in HRV2. Although HRV14 contains the same amino acid pair present in HRV2 and HRV49, it was not cleaved under these conditions. HRV89, which lacks a basic residue at the corresponding position, was also insensitive. Examination of the cleavage site on the three-dimensional structural map of native HRV2 reveals that it is most probably buried inside the capsid and thus not accessible. Structural rearrangements of the viral capsid are thus necessary to account for the cleavage observed after low pH treatment.     

Detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients

The occurrence of human rhinoviruses (HRV) and its relationship to clinical and histopathological findings were investigated in 127 bronchoalveolar lavage specimens from 36 lung transplant recipients by real-time RT-PCR. In addition, 286 samples from 235 other immunocompromised and immunocompetent patients were also studied. HRV was detected in 41.7% of lung transplant recipients vs 14.5% of other patients (p < 0.0001), and no differences in viral load were observed. Acute respiratory insufficiency was found in 15 cases, three of which were HRV positive (viral load, 6.3x10⁶ RNA copies/ml in one patient with chronic graft dysfunction). A diagnosis of pneumonia was made in 10 out of 127 cases, two of which were HRV positive (viral load, 10³-10⁴ in cases of co-infection). Acute rejection was diagnosed in 12 cases, three of which were HRV positive (viral load, 10³ in two cases of co-infection and 10⁵ in a single infection). HRV infection may involve the lower respiratory tract, particularly in the presence of an impaired pulmonary background, such as a transplanted lung. Clinical evaluation should take into account the viral load, with a load of >10⁵ possibly being associated with clinical symptoms, although lower loads can be detected in both symptomatic and asymptomatic patients.