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.     

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.     

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.     

Prospective genotyping of human rhinoviruses in children and adults during the winter of 2009-2010

BackgroundAbout 100 serotypes of human rhinovirus (HRV), classified into two species, have been identified by 1990. Uncultivable HRV variants have recently been identified and designated a new species. Recent improved diagnosis has led to a re-appraisal of the clinical impact of HRV infections in lower respiratory diseases.ObjectivesTo characterise clinical features in hospitalised patients with positive HRV RNA detection and to determine the distribution of HRV species in respiratory infections diagnosed during the winter of 2009–2010.Study designProspective virus typing was conducted by sequencing the VP4/VP2 genomic regions, and clinical data were collected.ResultsFifty-eight patients (for 63 respiratory specimens) were included. Phylogenetic analysis identified 52% of HRV species A, 6% of species B and 40% of species C, and revealed the co-circulation of 34 different HRV types during the study period. Three infants had successive infections with two or three different types. Five patients were admitted to an intensive care unit, four of them on arrival. Bronchiolitis, pneumonia and exacerbation of asthma were observed in 34/45 children. Pneumonia and severe exacerbation of chronic lung disease were observed in 8/13 adults, of whom 1, with immunocompromised status, died of multivisceral failure.ConclusionsThis study underlines the diversity of co-circulating strains and the potential severity of clinical presentations associated with HRV infections.     

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.     

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.     

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.     

Characterisation of a newly identified human rhinovirus, HRV-QPM, discovered in infants with bronchiolitis.

BACKGROUND: Human rhinoviruses (HRVs) are some of the earliest identified and most commonly detected viruses associated with acute respiratory tract infections (ARTIs) and yet the molecular epidemiology and genomic variation of individual serotypes remains undefined. OBJECTIVES: To molecularly characterise a novel HRV and determine its prevalence and clinical impact on a predominantly paediatric population. STUDY DESIGN: Nucleotide sequencing was employed to determine the complete HRV-QPM coding sequence. Two novel real-time RT-PCR diagnostic assays were designed and employed to retrospectively screen a well-defined population of 1244 specimen extracts to identify the prevalence of HRV-QPM during 2003. RESULTS: Phylogenetic studies of complete coding sequences defined HRV-QPM as a novel member the genus Rhinovirus residing within the previously described HRV-A2 sub-lineage. Investigation of the relatively short VP1 sequence suggest that the virus is resistant to Pleconaril, setting it apart from the HRV A species. Sixteen additional HRV-QPM strains were detected (1.4% of specimens) often as the sole micro-organism present among infants with suspected bronchiolitis. HRV-QPM was also detected in Europe during 2006, and a closely related virus circulated in the United States during 2004. CONCLUSIONS: We present the molecular characterisation and preliminary clinical impact of a newly identified HRV along with sequences representing additional new HRVs.     

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.     

Subgroup prevalence and genotype circulation patterns of human respiratory syncytial virus in Belgium during ten successive epidemic seasons

Human respiratory syncytial virus (HRSV) is the leading viral cause of severe respiratory illness for infants and young children worldwide. Two major antigenic groups (A and B) of HRSV exist, and viruses from both subgroups can cocirculate during epidemics; however, their frequencies might differ between seasons. The subgroup prevalence and genotype distribution patterns of HRSV strains were investigated in a community in Belgium during 10 successive epidemic seasons (1996 to 2006). A regular 3-year cyclic pattern of subgroup dominance was observed, consisting of two predominant HRSV-A seasons, followed by a single HRSV-B-dominant year. HRSV infections with both subgroups were more prevalent among children younger than 6 months and had a peak incidence in December. The most frequently detected genotypes were GA5 and GB13, the latter including strains with the 60-nucleotide duplication in the G gene. Furthermore, GA5 remained the dominant HRSV genotype in two consecutive epidemic seasons twice during the study period. Additional variability was detected among the GB13 isolates, due to the usage of a novel termination codon in the G gene. Dual infections with both HRSV subgroups were detected for 9 patients, and subsequent infections with the heterologous HRSV subgroup were documented for 15 patients. Among five patients with homologous reinfections, only one was caused by HRSV-B. Our results support the hypothesis that the overall prevalence of HRSV-A over HRSV-B could be due to a more-transient subgroup A-specific immune protection.     

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.     

Rhinovirus frequently detected in elderly adults attending an emergency department

The general aim was to investigate the burden of respiratory virus illness in a hospital emergency department, during two different epidemic seasons. Consecutive patients attending an emergency department during two study periods (February/March 2009 and 2010) were enrolled using broad inclusion criteria (fever/preceding fever and one of a set of ICD-9 codes suggestive of respiratory illness); nasopharyngeal washes were tested for the most common respiratory viruses using PCR-based methods. Influenza A virus was detected in 24% of samples collected in February/March 2009, whereas no samples tested positive for influenza during February/March 2010 (pandemic H1N1 Influenza A having circulated earlier in October-December 2009). Rhinovirus (HRV) was detected in 16% and 8% of patients recruited over the two study periods, respectively. Other respiratory viruses were detected rarely. Patient data were then analyzed with specific PCR results, comparing the HRV-positive group with virus-positive and no virus-detected groups. Individuals over 65 years old with HRV presented with signs, symptoms and underlying conditions and were admitted to hospital as often as the other enrolled patients, mainly for dyspnoea and chronic obstructive pulmonary disease acute exacerbation. Conversely, younger individuals with HRV, although presenting with respiratory signs and symptoms, were generally diagnosed with non-respiratory conditions. HRV was detected frequently in elderly patients attending the emergency department for respiratory distress without distinguishing clinical features. Molecular diagnosis of lower respiratory tract infections and surveillance of infectious diseases should include tests for HRV, as this virus is associated frequently with hospitalization of the elderly.     

Epidemiology of viral respiratory infections in a tertiary care centre in the era of molecular diagnosis,Geneva, Switzerland, 2011–2012

Few studies have examined the epidemiology of respiratory viral infections in large tertiary centres over more than one season in the era of molecular diagnosis. Respiratory clinical specimens received between 1 January 2011 and 31 December 2012 were analysed. Respiratory virus testing was performed using a large panel of real-time PCR or RT-PCR. Results were analysed according to sample type (upper versus lower respiratory tract) and age group. In all, 2996 (2469 (82.4%) upper; 527 (17.6%) lower) specimens were analysed. Overall positivity rate was 47.4% and 23.7% for upper and lower respiratory samples, respectively. The highest positivity rate was observed in patients under 18 years old (p <0.001); picornaviruses were the most frequent viruses detected over the year. Influenza virus, respiratory syncytial virus, human metapneumovirus and coronaviruses showed a seasonal peak during the winter season, while picornaviruses and adenoviruses were less frequently detected in these periods. Multiple viral infections were identified in 12% of positive cases and were significantly more frequent in children (p <0.001). In conclusion, we observed significant differences in viral infection rates and virus types among age groups, clinical sample types and seasons. Follow-up of viral detection over several seasons allows a better understanding of respiratory viral epidemiology.     

Online-Only Abstracts

Accurate HPV typing is essential for evaluation and monitoring of HPV vaccines, for second-line testing in cervical cancer screening, and in epidemiological surveys. In this study, we set up and assessed in clinical samples a new HPV typing method based on 454 next-generation sequencing (NGS) of HPV L1 amplicons, generated by using a modified PGMY primer set with improved sensitivity for some HPV types that are not targeted by standard PGMY primers. By using a median 12 800-fold coverage, the NGS method allowed us to correctly identify all high-risk HPV types, in either single or multiple infections, with a sensitivity of 50 genome equivalents, as demonstrated by testing WHO LabNet EQA sample panels. Analysis of mixtures of HPV16- and HPV18-positive cell lines demonstrated that the NGS method could reproducibly quantify the proportion of each HPV type in multiple infections in a wide dynamic range. Testing of HPV-positive clinical samples showed that NGS could correctly identify a high number of HPV types in multiple infections. The NGS method was also effective in the analysis of a set of cervical specimens with discordant results at hybrid capture 2 and line probe assays. In conclusion, a new HPV typing method based on 454 pyrosequencing was set up. This method was sensitive, specific, quantitative and precise in both single and multiple infections. It could identify a wide range of HPV types and might potentially discover new HPV types.

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

J. C. Rahamat-Langendoen¹ A. Riezebos-Brilman¹ E. Hak² E. H. Schölvinck³ H. G. M. Niesters¹Division of Clinical Virology, Department of Medical Microbiology, University of Groningen, University Medical Center Groningen 2) PharmacoEpidemiology and PharmacoEconomics, University of Groningen, University Center for Pharmacy 3) Beatrix Children's Hospital, University of Groningen, University Medical Center Groningen, Groningen, the NetherlandsOriginal Submission: 28 December 2012; Revised Submission: 13 March 2013; Accepted: 4 April 2013Editor: L. KaiserArticle published online: 8 April 2013Clin Microbiol Infect 2013; 19: E435–E44210.1111/1469-0691.12242

Abstract

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.     

Epidemiological, molecular, and clinical features of enterovirus respiratory infections in French children between 1999 and 2005

Enteroviruses (EVs) can induce nonspecific respiratory tract infections in children, but their epidemiological, virological, and clinical features remain to be assessed. In the present study, we analyzed 252 EV-related infection cases (median age of subjects, 5.1 years) diagnosed among 11,509 consecutive children visiting emergency departments within a 7-year period in the north of France. EV strains were isolated from nasopharyngeal samples by viral cell culture, identified by seroneutralization assay, and genetically compared by partial amplification and sequencing of the VP1 gene. The respiratory syndromes (79 [31%] of 252 EV infections) appeared as the second most common EV-induced pediatric pathology after meningitis (111 [44%] of 252 cases) (44 versus 31%, P < 10⁻³), contributing to lower respiratory tract infection (LRTI) in 43 (54%) of 79 EV respiratory infection cases. Bronchiolitis was the most common EV-induced LRTI (34 [43%] of 79 cases, P < 10⁻³) occurring more often in infants aged 1 to 12 months (P = 0.0002), with spring-fall seasonality. Viruses ECHO 11, 6, and 13 were the more frequently identified respiratory strains (24, 13, and 11%, respectively). The VP1 gene phylogenetic analysis showed the concomitant or successive circulation of genetically distinct EV respiratory strains (species A or B) during the same month or annual epidemic period. Our findings indicated that respiratory tract infections accounted for the 30% of EV-induced pediatric pathologies, contributing to LRTIs in 54% of these cases. Moreover, the concomitant or successive circulation of genetically distinct EV strains indicated the possibility of pediatric repeated respiratory infections within the same epidemic season.