Phylogenetic patterns of human respiratory picornavirus species, including the newly identified group C rhinoviruses, during a 1-year surveillance of a hospitalized patient population in Italy

Human rhinovirus species C (HRV-C) was the second most common HRV species detected in hospitalized patients in Italy with acute respiratory disease during a 1-year surveillance period. Sequencing of the picornavirus VP4/VP2 region allowed molecular typing of HRV-A and HRV-B and provisional typing of HRV-C.     

Detection of human rhinovirus C in children with acute lower respiratory tract infections in South Korea

Recently, HRV-C was identified as a new species of HRV, but its spectrum of clinical disease is still not clear. The purpose of this study was to investigate the molecular epidemiology of HRVs in children with acute lower respiratory tract infections (LRTIs). A total of 54 HRV-positive samples that were negative for other respiratory viruses were sequenced. HRV-A was detected in 33, HRV-B in 4, and HRV-C in 17 of these samples. All HRV-C-positive patients showed favorable clinical outcomes. We confirmed the presence of HRV-C in children with LRTIs, but its association with clinical severity is not clear.     

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 epidemiology and genetic diversity of human rhinovirus affecting hospitalized children in Rome

Human rhinoviruses (HRV) have been re-classified into three species (A–C), but the recently discovered HRV-C strains are not fully characterized yet. This study aimed to undertake a molecular and epidemiological characterization of HRV strains infecting children hospitalized over one year in two large research hospitals in Rome. Nasal washings from single HRV infections were retrospectively subjected to phylogenetic analysis on two genomic regions: the central part of the 5′Untranslated Region (5′UTR) and the Viral Protein (VP) 4 gene with the 5′ portion of the VP2 gene (VP4/2). Forty-five different strains were identified in 73 HRV-positive children: 55 % of the cases were HRV-A, 38 % HRV-C and only 7 % HRV-B. HRV-C cases were less frequent than HRV-A during summer months and more frequent in cases presenting wheezing with respect to HRV-A. Species distribution was similar with respect to patient age, and seasonality differed during summer months with fewer HRV-C than HRV-A cases. On admission, a significantly higher number of HRV-C cases presented with wheezing with respect to HRV-A. The inter- and intra-genotype variability in VP4/2 was higher than in 5′UTR; in particular, HRV-A patient VP4/2 sequences were highly divergent (8–14 %) at the nucleotide level from those of their reference strains, but VP4 amino acid sequence was highly conserved. In HRV-C isolates, the region preceding the initiator AUG, the amino acids involved in VP4 myristoylation, the VP4–VP2 cleavage site and the cis-acting replication element were highly conserved. Differently, VP4 amino acid conservation was significantly lower in HRV-C than in HRV-A strains, especially in the transiently exposed VP4 N-terminus. This study confirmed the high number of different HRV genotypes infecting hospitalized children over one year and reveals a greater than expected variability in HRV-C VP4 protein, potentially suggestive of differences in replication.     

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.     

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.     

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.     

Epidemiologic,clinical, and virologic characteristics of human rhinovirus infection among otherwise healthy children and adults: Rhinovirus among adults and children

Backgroundhuman rhinovirus (HRV) is a major cause of influenza-like illness (ILI) in adults and children. Differences in disease severity by HRV species have been described among hospitalized patients with underlying illness. Less is known about the clinical and virologic characteristics of HRV infection among otherwise healthy populations, particularly adults.Objectivesto characterize molecular epidemiology of HRV and association between HRV species and clinical presentation and viral shedding.Study designobservational, prospective, facility-based study of ILI was conducted from February 2010 to April 2012. Collection of nasopharyngeal specimens, patient symptoms, and clinical information occurred on days 0, 3, 7, and 28. Patients recorded symptom severity daily for the first 7 days of illness in a symptom diary. HRV was identified by RT-PCR and genotyped for species determination. Cases who were co-infected with other viral respiratory pathogens were excluded from the analysis. We evaluated the associations between HRV species, clinical severity, and patterns of viral shedding.Resultseighty-four HRV cases were identified and their isolates genotyped. Of these, 62 (74%) were >18 years. Fifty-four were HRV-A, 11HRV-B, and 19HRV-C. HRV-C infection was more common among children than adults (59% vs. 10%, P < 0.001). Among adults, HRV-A was associated with higher severity of upper respiratory symptoms compared to HRV-B (P = 0.02), but no such association was found in children. In addition, adults shed HRV-A significantly longer than HRV-C (P trend = 0.01).Conclusionsamong otherwise healthy adults with HRV infection, we observed species-specific differences in respiratory symptom severity and duration of viral shedding.     

Evaluation of real-time RT-PCR Rhino&EV/Cc r-gene® (bioMérieux) kit versions 1 and 2 for rhinovirus detection

BackgroundHuman rhinoviruses (HRVs) are frequent etiologic agents of tract infections, ranging from benign upper to potentially life-threatening lower respiratory tract infections. Diagnosis is based on molecular methods. 169 HRV types, belonging to species A, B and C, have been identified. This high genetic diversity makes it difficult to accurately detect circulating HRVs and to diagnose severe infection.ObjectivesTo comparatively assess the ability to detect HRV clinical isolates of the first version (V1) of the commercial real-time RT-PCR Rhino&EV/Cc r-gene^® (bioMérieux) kit, of an in-house RT-PCR followed by genotyping, considered as the reference method, and of the second version of this commercial test (V2).Study designFrom September 2011 to April 2013, HRVs were prospectively detected in 2525 respiratory specimens, using V1 in combination with the in-house reference RT-PCR. In November 2013, 85 specimens that had given initially false negative results with V1 were retested simultaneously with V1 and V2 and the in-house RT-PCR. In addition, 421 negative specimens with the in-house assay were prospectively tested with V2.ResultsAmong the 2525 specimens, V1 detected 80.7% (502/622) of in-house RT-PCR positive isolates: 85.3% (220/258) of HRV-A, 84.4% (27/32) of HRV-B and 74.9% (176/235) of HRV-C. Among the 85 respiratory samples tested with V1, V2 and the in-house RT-PCR, V2 was more efficient than V1 in detecting 16 HRV isolates: 11/33 (33.3%) of HRV-A and 5/47 (10.6%) of HRV-C tested. The analytical sensitivity of V2 was greater for 8/18 HRV-A genotypes and 2/22 HRV-C genotypes. Relative to the in-house assay, the specificity of V2 was 100% (421/421).ConclusionsThis study showed a slightly higher sensitivity of V2. However, diverse genotypes, especially HRV-C, were undetected.     

Human rhinovirus C in adult haematopoietic stem cell transplant recipients with respiratory illness

BackgroundA previously unidentified species of human rhinovirus, HRV-C, was described in 2006 in association with lower respiratory tract infection (LRTI). Features of infection in immunosuppressed adults are poorly characterised.ObjectivesThis study aims to determine the epidemiology of HRV-C in haematopoietic stem cell transplant (HSCT) recipients in a single centre.Study designA prospective cohort study of all HSCT recipients admitted to Westmead Hospital, Westmead, Australia from 1 July 2005 to 30 September 2007 was undertaken. Nose/throat samples were collected from all patients at the time of admission and patients developing pre-defined symptoms and/or signs of respiratory infection during the admission. Samples were processed and tested for rhinoviruses and 14 other respiratory viruses using nucleic acid-based methods, immunofluorescence and culture. HRV genotyping was performed by sequencing a region of the rhinovirus 5′ untranslated region (UTR). Clinical data on each episode were collected prospectively.ResultsHRVs were identified in 24 episodes: 8% of 299 episodes of clinically- defined respiratory infections and 39% of 61 episodes in which respiratory viruses were detected. HRV-C was most frequent (HRV-C: nine, HRV-A: eight and HRV-B: two). Seven episodes of HRV-C, five with pneumonia, occurred within 100 days of HSCT. Co-pathogens were frequent.ConclusionsThe newly described HRV-C was the most common rhinovirus group detected in HSCT recipients with respiratory infection, with co-pathogens being frequent. Further research is required to understand the activity and pathogenicity of this virus in HSCT recipients.     

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.     

Recombination in the evolution of human rhinovirus genomes

Human rhinoviruses (HRV) are highly prevalent human respiratory pathogens that belong to the genus Enterovirus. Although recombination within the coding region is frequent in other picornavirus groups, most evidence of recombination in HRV has been restricted to the 5’ untranslated region. We analysed the occurrence of recombination within published complete genome sequences of members of all three HRV species and additionally compared sequences from HRV strains spanning 14 years. HRV-B and HRV-C showed very little evidence of recombination within the coding region. In contrast, HRV-A sequences appeared to have undergone a large number of recombination events, typically involving whole type groups. This suggests that HRV-A may have been subject to extensive recombination during the period of diversification into types. This study demonstrates the rare and sporadic nature of contemporary recombination of HRV strains and contrasts with evidence of extensive recombination within HRV-A and between members of different species during earlier stages in its evolutionary diversification.     

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.     

Prospective evaluation of rhinovirus infection in healthy young children

BackgroundAlthough the incidence of human rhinovirus (HRV) infection is highest in young, no study has yet been published concerning the types of HRV circulating in this population, the incidence of symptomatic infections due to the different types, or duration of sheddingObjectivesThis prospective study evaluated the circulation of HRV species and types, and established the incidence of asymptomatic and symptomatic infections in young children.Study designThe study enrolled 93 healthy children aged <2 years, 88 of whom completed the follow-up of weekly household visits from November 2013 to February 2014. At each visit, a record was made of any signs and symptoms of acute infection, and a nasopharyngeal (NP) swab was taken in order to identify the HRVs by means of RT-polymerase chain reaction and to construct the phylogenetic tree of the HRV-positive cases.ResultsA total of 1408 NP samples were obtained and 326HRV infections were diagnosed (23.1%), leading to a mean number of 3.7 ± 2.3 infections per child: HRV-A in 72 cases (22.1%), HRV-B in 29 (8.9%), HRV-C in 122 (37.4%), and non-typeable HRV in 103 (31.6%). Shedding was significantly longer for HRV-A (14 days) and HRV-B (14 days) than HRV-C (7 days; p = 0.002 and p = 0.012). Most of the HRV infections (209/326, 64.1%) remained asymptomatic and, when symptomatic, were of marginal clinical relevance.ConclusionsIn healthy young children, HRV infection is extremely frequent, generally asymptomatic or with a mild clinical presentation, and viral shedding is limited in time.