Reliable quantification of rhinovirus species C using real-time PCR

Sikazwe, Chisha; Chidlow, Glenys; Imrie, Allison; Smith, David W.

doi:10.1016/j.jviromet.2016.05.014

Cited by 9 publications

(16 citation statements)

References 37 publications

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“…RV-C45-cpz1-2013 also shares with other rhinovirus C variants the capacity to infect the lower respiratory tract and cause severe disease. Viral load values from this animal’s oropharynx (7.41 × 10 6 copies/swab), trachea (1.05 × 10 7 copies/swab), and lung (1.74 × 10 6 copies/swab) are comparable to the average viral load (7.76 × 10 6 copies/mL) found in rhinovirus C‒infected children with acute wheezing illness treated in an emergency department ( 27 ). Although published estimates of rhinovirus epidemiologic transmission parameters are sparse, the values estimated for the 2013 chimpanzee outbreak (transmission rate 0.68/d; duration of infection 3.2 days) are close to those of common cold viruses in humans (transmission rate 0.74/d; duration of infection 3.0 days) ( 39 ).…”

Section: Discussionsupporting

confidence: 57%

“…Deep sequencing of swab samples from this chimpanzee’s oropharynx, trachea, and lung yielded 10,722,035 sequence reads, of which 8,918 had high similarity to rhinovirus C. No other pathogens were detected. Subsequent qPCR confirmed infection with RV-C, with viral loads of 7.41 × 10 6 copies/swab in the oropharynx, 1.05 × 10 7 copies/swab in the trachea, and 1.74 × 10 6 copies/swab in the lung; these values are comparable to the average viral load (7.76 × 10 6 copies/mL) found in rhinovirus C‒infected children with acute wheezing illness treated in an emergency department ( 27 ). Further sequencing yielded a near-complete rhinovirus C genome consisting of a complete polyprotein gene of 6,450 bases, a complete 3′ untranslated region (UTR) of 35 bases excluding the poly(A) tail, and a partial 5′-UTR of 480 bases.…”

Section: Resultsmentioning

confidence: 55%

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Lethal Respiratory Disease Associated with Human Rhinovirus C in Wild Chimpanzees, Uganda, 2013

Scully¹,

Basnet²,

Wrangham³

et al. 2018

Emerg. Infect. Dis.

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We describe a lethal respiratory outbreak among wild chimpanzees in Uganda in 2013 for which molecular and epidemiologic analyses implicate human rhinovirus C as the cause. Postmortem samples from an infant chimpanzee yielded near-complete genome sequences throughout the respiratory tract; other pathogens were absent. Epidemiologic modeling estimated the basic reproductive number (R0) for the epidemic as 1.83, consistent with the common cold in humans. Genotyping of 41 chimpanzees and examination of 24 published chimpanzee genomes from subspecies across Africa showed universal homozygosity for the cadherin-related family member 3 CDHR3-Y529 allele, which increases risk for rhinovirus C infection and asthma in human children. These results indicate that chimpanzees exhibit a species-wide genetic susceptibility to rhinovirus C and that this virus, heretofore considered a uniquely human pathogen, can cross primate species barriers and threatens wild apes. We advocate engineering interventions and prevention strategies for rhinovirus infections for both humans and wild apes.

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Section: Discussionsupporting

confidence: 57%

Section: Resultsmentioning

confidence: 55%

Lethal Respiratory Disease Associated with Human Rhinovirus C in Wild Chimpanzees, Uganda, 2013

Scully¹,

Basnet²,

Wrangham³

et al. 2018

Emerg. Infect. Dis.

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“…Most HRV RT-PCR assays target the conserved 5= noncoding region (NCR), which exhibits the greatest sequence homology among the HRV genotypes. However, even in the 5= NCR, consensus primer and probe sets must be designed with degenerate and modified bases or multiple oligonucleotides to amplify all HRV genotypes (5)(6)(7).…”

mentioning

confidence: 99%

Superiority of Digital Reverse Transcription-PCR (RT-PCR) over Real-Time RT-PCR for Quantitation of Highly Divergent Human Rhinoviruses

et al. 2017

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Human rhinoviruses (HRV) comprise 3 species representing more than 150 genotypes. As an important human respiratory pathogen, molecular detection is an indispensable tool for diagnosis and surveillance. However, the sequence diversity of HRV genotypes poses challenges for developing robust molecular methods that detect all genotypes with equal efficiencies. This study compares the accuracies of reverse transcription-quantitative PCR (RT-qPCR) and reverse transcription-digital PCR (RT-dPCR) for quantifying HRV RNA using genotype-specific primers and probes and a consensus primer/probe set targeting the 5= noncoding region of HRV. When using consensus primers and probes for the quantification of HRV, RT-dPCR outperformed RT-qPCR by consistently and accurately quantifying HRV RNAs across more genotype groups, despite the presence of up to 2 target-sequence mismatches within the primer or probe binding region. Because it does not rely on amplification efficiency, which can be affected by sequence mismatches in primer/probe binding regions, RT-dPCR may be the optimal molecular method for future HRV quantification studies and for quantitating other viruses with high sequence diversity.KEYWORDS human rhinovirus, digital PCR, qPCR H uman rhinoviruses (HRV) are important human respiratory pathogens that are small positive-sense RNA viruses within the family Picornaviridae. There are more than 150 genotypes of HRV that have been recognized within species A, B, and C of the genus Enterovirus (1). Molecular assays, such as reverse-transcription PCR (RT-PCR), are the most useful methods for detecting HRV in clinical samples (2-4). Most HRV RT-PCR assays target the conserved 5= noncoding region (NCR), which exhibits the greatest sequence homology among the HRV genotypes. However, even in the 5= NCR, consensus primer and probe sets must be designed with degenerate and modified bases or multiple oligonucleotides to amplify all HRV genotypes (5-7).Although qualitative detection of HRV by RT-PCR is currently sufficient for determining HRV infection, accurate quantification of HRV RNA in clinical samples is needed for studies associating HRV viral load with viral transmission and with patient symptoms and outcomes. Viral load studies of other respiratory viruses have shown that a correlation exists between viral load and disease severity (8-10). Accurate quantification of HRV will also be required for evaluating the performances of future antiviral drugs. Real-time RT-PCR assays, when accompanied by the amplification of standard curves (RT-qPCR), can be used to quantify the number of viral copies in a sample. However, RT-qPCR assays using quantification with a consensus HRV primer and probe set may not give accurate results for all genotypes due to amplification inefficiencies caused by base mismatches between the primer and probe sequences and the specific

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“…All viral quantification assays were validated in-house according to the minimum information for publication of quantitative real-time PCR experiments (MIQE) and National Pathology Accreditation Advisory Council (NPAAC) guidelines. 27 …”

Section: Methodsmentioning

confidence: 99%

Nasopharyngeal density of respiratory viruses in childhood pneumonia in a highly vaccinated setting: findings from a case–control study

et al. 2020

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BackgroundDetection of pneumonia-causing respiratory viruses in the nasopharynx of asymptomatic children has made their actual contribution to pneumonia unclear. We compared nasopharyngeal viral density between children with and without pneumonia to understand if viral density could be used to diagnose pneumonia.MethodsNasopharyngeal swabs (NPS) were collected from hospitalised pneumonia cases at Princess Margaret Hospital (PMH) and contemporaneous age-matched controls at PMH outpatient clinics and a local immunisation clinic in Perth, Australia. The density (copies/mL) of respiratory syncytial virus (RSV), influenza A virus (InfA), human metapneumovirus (HMPV) and rhinovirus in NPS was determined using quantitative PCR. Linear regression analysis was done to assess the trend between viral density and age in months. The association between viral density and disease status was examined using logistic regression. Area under receiver operating characteristic (AUROC) curves were assessed to determine optimal discriminatory viral density cut-offs.ResultsThrough May 2015 to October 2017, 230 pneumonia cases and 230 controls were enrolled. Median nasopharyngeal density for any respiratory virus was not substantially higher in cases than controls (p>0.05 for each). A decreasing density trend with increasing age was observed—the trend was statistically significant for RSV (regression coefficient −0.04, p=0.004) but not for other viruses. After adjusting for demographics and other viral densities, for every log10 copies/mL density increase, the odds of being a case increased by six times for RSV, three times for HMPV and two times for InfA. The AUROC curves were <0.70 for each virus, suggesting poor case–control discrimination based on viral density.ConclusionThe nasopharyngeal density of respiratory viruses was not significantly higher in children with pneumonia than those without; however, the odds of being a case increased with increased density for some viruses. The utility of viral density, alone, in defining pneumonia was limited.

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Reliable quantification of rhinovirus species C using real-time PCR

Cited by 9 publications

References 37 publications

Lethal Respiratory Disease Associated with Human Rhinovirus C in Wild Chimpanzees, Uganda, 2013

Lethal Respiratory Disease Associated with Human Rhinovirus C in Wild Chimpanzees, Uganda, 2013

Superiority of Digital Reverse Transcription-PCR (RT-PCR) over Real-Time RT-PCR for Quantitation of Highly Divergent Human Rhinoviruses

Nasopharyngeal density of respiratory viruses in childhood pneumonia in a highly vaccinated setting: findings from a case–control study

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