Multiplex MassTag-PCR for respiratory pathogens in pediatric nasopharyngeal washes negative by conventional diagnostic testing shows a high prevalence of viruses belonging to a newly recognized rhinovirus clade (original) (raw)
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New Respiratory Enterovirus and Recombinant Rhinoviruses among Circulating Picornaviruses
Emerging Infectious Diseases, 2009
Rhinoviruses and enteroviruses are leading causes of respiratory infections. To evaluate genotypic diversity and identify forces shaping picornavirus evolution, we screened persons with respiratory illnesses by using rhinovirus-specifi c or generic real-time PCR assays. We then sequenced the 5′ untranslated region, capsid protein VP1, and protease precursor 3CD regions of virus-positive samples. Subsequent phylogenetic analysis identifi ed the large genotypic diversity of rhinoviruses circulating in humans. We identifi ed and completed the genome sequence of a new enterovirus genotype associated with respiratory symptoms and acute otitis media, confi rming the close relationship between rhinoviruses and enteroviruses and the need to detect both viruses in respiratory specimens. Finally, we identifi ed recombinants among circulating rhinoviruses and mapped their recombination sites, thereby demonstrating that rhinoviruses can recombine in their natural host. This study clarifi es the diversity and explains the reasons for evolution of these viruses.
PLOS One, 2007
Background. Human rhinoviruses (HRVs) are the most prevalent human pathogens, and consist of 101 serotypes that are classified into groups A and B according to sequence variations. HRV infections cause a wide spectrum of clinical outcomes ranging from asymptomatic infection to severe lower respiratory symptoms. Defining the role of specific strains in various HRV illnesses has been difficult because traditional serology, which requires viral culture and neutralization tests using 101 serotype-specific antisera, is insensitive and laborious. Methods and Findings. To directly type HRVs in nasal secretions of infants with frequent respiratory illnesses, we developed a sensitive molecular typing assay based on phylogenetic comparisons of a 260-bp variable sequence in the 5' noncoding region with homologous sequences of the 101 known serotypes. Nasal samples from 26 infants were first tested with a multiplex PCR assay for respiratory viruses, and HRV was the most common virus found (108 of 181 samples). Typing was completed for 101 samples and 103 HRVs were identified. Surprisingly, 54 (52.4%) HRVs did not match any of the known serotypes and had 12-35% nucleotide divergence from the nearest reference HRVs. Of these novel viruses, 9 strains (17 HRVs) segregated from HRVA, HRVB and human enterovirus into a distinct genetic group (''C''). None of these new strains could be cultured in traditional cell lines. Conclusions. By molecular analysis, over 50% of HRV detected in sick infants were previously unrecognized strains, including 9 strains that may represent a new HRV group. These findings indicate that the number of HRV strains is considerably larger than the 101 serotypes identified with traditional diagnostic techniques, and provide evidence of a new HRV group.
Prospective genotyping of human rhinoviruses in children and adults during the winter of 2009–2010
Journal of Clinical Virology, 2012
Background: About 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. Objectives: To 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 design: Prospective virus typing was conducted by sequencing the VP4/VP2 genomic regions, and clinical data were collected. Results: Fifty-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. Conclusions: This study underlines the diversity of co-circulating strains and the potential severity of clinical presentations associated with HRV infections.
Rhinovirus Strains Circulating in Central and South America
Background: Human rhinoviruses (HRVs) are a highly prevalent cause of respiratory disease. These viruses primarily affect children, but they have also been detected in populations such as young military recruits. HRVs are one of nine genera belonging to the Picornaviridae family with high similarity to enteroviruses. There are more than 100 different serotypes that have been taxonomically grouped into 2 species: HRV-A and HRV-B. Recently, a new species named HRV-C was identified in patients with acute lower respiratory tract infections (LRTIs) in the United States, Australia, and Hong Kong. Since its detection, HRV-C has been reported to be an important etiological factor in children, causing up to 5% of LRTIs among inpatients and accounting for 73% of LRTI cases in children in Germany. Other studies have shown that HRVs possess great genetic diversity by recombination not only among different HRV species, but also with enteroviruses. Although some information exists about HRVs in A...
Journal of Clinical Microbiology, 2009
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.
Infection, Genetics and Evolution, 2019
Background: Enteroviruses (EVs) and rhinoviruses (RVs) belong to the Enterovirus genus within the Picornaviridae family, and show genetic similarities. These viruses are related to mild diseases, but EVs infections can sometimes lead to more severe complications. Current diagnostic molecular techniques should discriminate between the four EV and the three RV species that infect humans. The aim was to revise the EV and RV PCR-confirmed specimens by sequencing for genetic characterisation. Material and methods: Respiratory tract specimens were collected from patients with suspicion of respiratory infection. Respiratory viruses' laboratory-confirmation was performed by commercial multiplex real-time RT-PCR assays. Genetic characterisation of all EV and in a selection of RV was performed based on the phylogenetic analyses of partial VP1 and VP4/2 sequences, respectively. Results: From 19,957 tested specimens, 309 (1.5%) were EV-positive, 2546 (12%) were RV-positive, and 233 (1%) were EV/RV co-detections. The phylogenetic analyses revealed that: among single EV detections, 177/309 (57%) were characterised as EV, 2/309 (1%) as RV, and 130/309 (42%) could not be typed; among single 1771 RV detections (Ct < 35), 1651/1771 (93%) were characterised as RV, 3/1771 (0.3%) as EV and 117/1771 (6.7%) could not be typed. Among EV/RV co-detections, 62/233 (27%) were characterised as EV, 130/233 (56%) as RV and 41/233 (18%) could not be typed. Conclusions: A diagnostic method well considered for routine laboratory-confirmation of respiratory viruses should discriminate EV and RV targets. RVs are usually associated with mild respiratory disease, but the potential relatedness of EVs to neurological complications makes their monitoring mandatory. Therefore, an accurate detection and differentiation should be required in commercial diagnostic solutions.
Revista Argentina de microbiología
Human rhinoviruses (HRV), the major cause of common colds, have a significant genetic diversity and are classified into 3 species (A, B, C) with more than 100 serotypes. HRV species C, described in 2006, can only be detected using molecular methods. The objectives of this paper were to adapt a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for HRV detection and to further determine the frequency of HRV in respiratory samples from children under 2 years of age, with acute respiratory infection (ARI), from Buenos Aires, Argentina. Two real-time RT-PCR assays amplifying the 207 base pair of the 5' non-coding region were compared. The original protocol includes locked nucleic acid analogues and a pyrimidine derivative in the forward primer, while the adapted protocol avoided those molecules. Of 67 respiratory samples, 17 (25.4 %) were positive with the original protocol, and 20 (29.9 %) with the adapted one. Discrepant results were confirmed by sequencing a...
Persistence of rhinovirus and enterovirus RNA after acute respiratory illness in children
Journal of Medical Virology, 2004
The persistence of rhinovirus and enterovirus RNAs was studied in the nasal secretions of children with acute expiratory wheezing (median age: 1.7 years). On admission, 84 samples from 161 (52%) children admitted to hospital were positive by reverse transcriptase-polymerase chain reaction (RT-PCR), which detects rhino- and enteroviruses simultaneously. Of the samples, 26 (16%) were positive for rhinovirus, 29 (18%) enterovirus and 29 (18%) nontypable rhino-enterovirus. After 2 weeks, 16 of these 84 (19%) samples were still positive. Rhinovirus RNA remained positive in 13 of 26 (50%) cases, whereas enterovirus RNA remained positive only in 1 of 29 (3%) cases (P = 0.0001). Respiratory symptoms at 2 weeks or systemic glucocorticoid treatment during hospital stay were not related to the persistence of viral RNA. After 5 weeks, only one sample remained PCR-positive. Thirteen of the 79 (16%) asymptomatic control children were PCR-positive for respiratory picornavirus. Five of the 13 (38%) PCR-positive children developed respiratory symptoms in the following week. The study shows that after the onset of symptomatic respiratory infection enterovirus RNA may take 2–3 weeks and rhinovirus RNA 5–6 weeks to disappear from nasal mucus. J. Med. Virol. 72:695–699, 2004. © 2004 Wiley-Liss, Inc.
Rhinovirus detection using different PCR-based strategies
Brazilian Journal of Microbiology, 2012
Human rhinoviruses (HRVs) are the major cause of the common cold. HRVs were recently reclassified into the Enterovirus genus (HEV) in the Picornaviridae family. HRVs and other members of the HEV genus share many common features, including sense RNA genomes and partial nucleotide sequence identity. The aim of this study was to evaluate different HRV detection strategies. Samples from adults with acute respiratory infection (n = 291) who were treated in Sao Paulo Hospital (2001-2003) were tested using three assays. The first assay detected picornaviruses by RT-PCR and hybridization, the second detected rhinoviruses using RT-PCR/sequencing, and the third differentiated HRV from HEV using duplex seminested-RT-PCR. Analysis of the results obtained from the first two strategies revealed 83% concordance. Discordant samples were then evaluated by the third protocol, and 82% were negative. The picornavirus detection protocol was more sensitive but less specific than the rhinovirus detection protocols. The seminested protocol utilized in the present study was less sensitive and was not useful in differentiating HRV from HEV. Sequencing assays examining different genes would address the best strategy of confirming rhinovirus and enterovirus infections.