Molecular characterization of a variant rhinovirus from an outbreak associated with uncommonly high mortality

Kiang, David T.; Yagi, Shigeo; Kantardjieff, Katherine A.; Kim, Euna J.; Louie, Janice K.; Schnurr, David P.

doi:10.1016/j.jcv.2006.12.016

Cited by 51 publications

(47 citation statements)

References 59 publications

(72 reference statements)

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“…16 To amplify the 5′UTR region, a first round PCR targeting a 913-nucleotide fragment was performed using reverse primer SRHI1 (GCATCIGGYARYTTCCACCACCANCC) and forward primer DK001 (CAAGCACTTCTGTTTCCC). 17,18 In brief, 10.7 μL H 2 O ribonuclease (RNase) free, 6 μL of reaction buffer 5×, 1.2 μL of deoxyribonucleotide triphosphates (dNTPs) mix (10 mM each), 1.8 μL of each primer (diluted at 10 μM), 3 μL of Q solution, five units of taq DNA polymerase (Qiagen, Hilden, Germany), and 5 μL of cDNA were mixed in a PCR tube. After denaturation for 15 minutes at 95°C, the PCR was performed as follows: 40 cycles at 95°C for 30 seconds, 55°C for 60 seconds, and 72°C for 60 seconds, followed by an extension step at 72°C for 10 minutes and a 4°C hold.…”

Section: Methodsmentioning

confidence: 99%

Enteroviruses and Rhinoviruses: Molecular Epidemiology of the Most Influenza-Like Illness Associated Viruses in Senegal

Fall

Dia

Kébé

et al. 2016

The American Society of Tropical Medicine and Hygiene

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Abstract. Different viruses have been identified as etiologic agents of respiratory tract infections, including severe cases. Among these, human rhinoviruses (HRVs) and human enteroviruses (HEVs) are recognized as leading causes. The present study describes the molecular epidemiology of HRVs and HEVs in Senegal over a 3-year surveillance period. From January 2012 to December 2014, nasopharyngeal and oropharyngeal swabs specimen were collected from patients with influenza-like illness (ILI). A real-time reverse transcription polymerase chain reaction was performed for HRV and HEV detection using the RV16 kit. Two regions were targeted for the molecular characterization of RVs: 5′ untranslated region (5′UTR) and viral protein 4/viral protein 2 (VP4/VP2) transition region. For enteroviruses (EVs) phylogeny, VP1 gene was targeted. A total of 4,194 samples were collected. Children up to 5 years accounted for 52.9%. Among them, 1,415 (33.7%) were positive for HRV, 857 (20.4%) for HEV, and 437 cases of dual infections HRV/HEV. HRVs and HEVs were identified significantly in children aged 5 years or less. Only cough and vomiting signs were observed with significant association with viral infection. Both viruses co-circulated all year long with a marked increase of activity during rainy and cold period. All HRV types circulate in Senegal. HRV-A and C groups were the most common. HEV serotyping identified coxsackie B viruses (CBV) only. VP1 region revealed different CBV (CBV1, CBV2, CBV3, CBV4, and CBV5), echoviruses, coxsackieviruses A4-like strains and a poliovirus 2. The results suggest strong year-round respiratory picornavirus activity in children up to 5 years of age. Molecular studies identified a wide variety of RVs along with diverse EVs in samples from patients with ILI.

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Section: Methodsmentioning

confidence: 99%

Enteroviruses and Rhinoviruses: Molecular Epidemiology of the Most Influenza-Like Illness Associated Viruses in Senegal

Fall

Dia

Kébé

et al. 2016

The American Society of Tropical Medicine and Hygiene

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“…Primers for PCR amplification of a fragment within the 5Ј NCR were designed based on an alignment of the complete 5Ј NCR sequences from available full-length HRV sequences from the GenBank database (NCBI) and analysis of conserved regions within the 5Ј NCR. The forward primer DK001 (11) and reverse primer DK004 (5Ј-CACGGACACCCAAAGTAGT-3Ј) were used to PCR amplify a region within the 5Ј NCR as previously described (11). The PCR conditions were as follows: hot start at 95°C (5 min), followed by 40 cycles of denaturation at 95°C (15 s), annealing at 55°C (15 s), and elongation at 72°C (60 s), resulting in amplification of a fragment approximately 400 bp in length.…”

Section: Vol 46 2008 5ј Ncr Analysis Of All Hrv Prototype Strains 3737mentioning

confidence: 99%

“…These viruses have been implicated as causes of asthma exacerbations (9,25) and severe respiratory tract illnesses in children, the immunosuppressed, and the elderly (3,5,7,21,29). HRV-associated mortalities have also been recently reported (6,11,21,35). Perhaps because HRV strains are often difficult to culture, few epidemiologic data exist on the relationship between the pattern and severity of clinical manifestations associated with individual serotypes (33), and no data are available regarding the biological impact of the serotype.…”

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confidence: 99%

Assay for 5′ Noncoding Region Analysis of All Human Rhinovirus Prototype Strains

et al. 2008

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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).

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“…Thanks to numerous clinical studies, data have been gathered regarding implication of HRV in the exacerbation of underlying diseases, such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease (1,3,7,8,10,17,18,20,21,25), as well as concerning the possible impact of a given HRV species on disease severity (9,(11)(12)(13)23). However, there are inconsistent data about whether viral load is correlated with disease severity, and the threshold cycle (C T ) values from real-time RT-PCR are converted too frequently into viral copies per milliliter without any validation.…”

mentioning

confidence: 99%

Critical Analysis of Rhinovirus RNA Load Quantification by Real-Time Reverse Transcription-PCR

et al. 2012

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bRhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5=-untranslated regions (5=UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5=UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ؎10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible. Human rhinoviruses (HRVs) are small nonenveloped viruses containing a positive-strand RNA genome. They belong to the Enterovirus genus, which is part of the Picornaviridae family. HRVs are important human pathogens and the most frequent cause of respiratory infections. Their tropism is not restricted to the upper respiratory tract, and they can cause complications in the lower respiratory tract, especially in children, the elderly, and immunocompromised patients (5-7, 15, 16). Many HRVs, in particular those belonging to the newly discovered HRV-C species, do not grow under traditional cell culture conditions. Therefore, molecular diagnostic tools, such as real-time reverse transcription-PCR (RT-PCR), are the methods of choice for diagnosing HRVs.Thanks to numerous clinical studies, data have been gathered regarding implication of HRV in the exacerbation of underlying diseases, such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease (1, 3,7,8,10,17,18,20,21,25), as well as concerning the possible impact of a given HRV species on disease severity (9, 11-13, 23). However, there are inconsistent data about whether viral load is correlated with disease severity, and the threshold cycle (C T ) values from real-time RT-PCR are converted too frequently into viral copies per milliliter without any validation. This is particularly important because many published assays include primers with degenerate nucleotides or probe sequences that are biased toward a given genotype or species (4,14,22,24).We validated previously a two-step real-time RT-...

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Molecular characterization of a variant rhinovirus from an outbreak associated with uncommonly high mortality

Cited by 51 publications

References 59 publications

Enteroviruses and Rhinoviruses: Molecular Epidemiology of the Most Influenza-Like Illness Associated Viruses in Senegal

Enteroviruses and Rhinoviruses: Molecular Epidemiology of the Most Influenza-Like Illness Associated Viruses in Senegal

Assay for 5′ Noncoding Region Analysis of All Human Rhinovirus Prototype Strains

Critical Analysis of Rhinovirus RNA Load Quantification by Real-Time Reverse Transcription-PCR

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