Rapid Simultaneous Detection of Enterovirus and Parechovirus RNAs in Clinical Samples by One-Step Real-Time Reverse Transcription-PCR Assay

Bennett, Susan E.; Harvala, Heli; Witteveldt, Jeroen; Leitch, E. Carol McWilliam; McLeish, Nigel J.; Templeton, Kate; Gunson, Rory; Carman, William F.; Simmonds, Peter

doi:10.1128/jcm.02445-10

Cited by 43 publications

(29 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Papular eruptions lasted around two weeks. Vesicle fluid specimens taken from these individuals were negative for HSV and varicella zoster virus, but positive for enterovirus (EV) RNA [1], confirming the diagnosis of atypical hand, foot and mouth disease (HFMD).…”

Section: Identification Of Hand Foot and Mouth Disease Cases In Edimentioning

confidence: 92%

See 1 more Smart Citation

Atypical hand, foot, and mouth disease associated with coxsackievirus A6 infection, Edinburgh, United Kingdom, January to February 2014

Sinclair¹,

Gaunt

Simmonds

et al. 2014

Eurosurveillance

View full text Add to dashboard Cite

In January to February 2014, 16 hand, foot and mouth disease (HFMD) cases were identified in Edinburgh, United Kingdom. All presented with atypical features, with most (n=13) resembling eczema herpeticum or chickenpox. Coxsackievirus A6 (CV-A6) was identified in all the typed cases (n=11). As atypical forms of HFMD associated with CV-A6 are likely to emerge throughout Europe, clinicians should be alert to unusual clinical presentations of HFMD and virologists aware of effective diagnostic testing and enterovirus typing methods.

show abstract

Section: Identification Of Hand Foot and Mouth Disease Cases In Edimentioning

confidence: 92%

“…EV RNA was detected in vesicle fluid specimens by real-time RT-PCR targeting the 5'untranslated region [1]. For genotyping, the VP4 gene was amplified and sequenced as previously described [2].…”

Section: Figurementioning

confidence: 99%

Atypical hand, foot, and mouth disease associated with coxsackievirus A6 infection, Edinburgh, United Kingdom, January to February 2014

Sinclair¹,

Gaunt

Simmonds

et al. 2014

Eurosurveillance

View full text Add to dashboard Cite

show abstract

“…RNA was extracted from fecal samples as previously described (20) and tested for enterovirus (EV) by one-step reverse transcriptase (RT)-PCR assay targeting the 5= untranslated regions (5=UTRs) (see Table S3A in the supplemental material) (21). To estimate viral loads in EV-positive samples, samples were retested by real-time PCR calibrated using defined copy numbers of EV RNA transcripts (22).…”

Section: Samplesmentioning

confidence: 99%

High Rates of Infection with Novel Enterovirus Variants in Wild Populations of Mandrills and Other Old World Monkey Species

Nguyen

Harvala

Ngole

et al. 2014

J Virol

Self Cite

View full text Add to dashboard Cite

Enteroviruses (EVs) are a genetically and antigenically diverse group of viruses infecting humans. A mostly distinct set of EV variants have additionally been documented to infect wild apes and several, primarily captive, Old World monkey (OWM) species. To investigate the prevalence and genetic characteristics of EVs infecting OWMs in the wild, fecal samples from mandrills (Mandrillus sphinx) and other species collected in remote regions of southern Cameroon were screened for EV RNA. Remarkably high rates of EV positivity were detected in M. sphinx (100 of 102 screened), Cercocebus torquatus (7/7), and Cercopithecus cephus (2/4), with high viral loads indicative of active infection. Genetic characterization in VP4/VP2 and VP1 regions allowed EV variants to be assigned to simian species H (EV-H) and EV-J (including one or more new types), while seven matched simian EV-B variants, SA5 and EV110 (chimpanzee). Sequences from the remaining 70 formed a new genetic group distinct in VP4/2 and VP1 region from all currently recognized human or simian EV species. Complete genome sequences were obtained from three to determine their species assignment. In common with EV-J and the EV-A A13 isolate, new group sequences were chimeric, being most closely related to EV-A in capsid genes and to EV-B in the nonstructural gene region. Further recombination events created different groupings in 5= and 3= untranslated regions. While clearly a distinct EV group, the hybrid nature of new variants prevented their unambiguous classification as either members of a new species or as divergent members of EV-A using current International Committee on Taxonomy of Viruses (ICTV) assignment criteria. IMPORTANCEThis study is the first large-scale investigation of the frequency of infection and diversity of enteroviruses (EVs) infecting monkeys (primarily mandrills) in the wild. Our findings demonstrate extremely high frequencies of active infection (95%) among mandrills and other Old World monkey species inhabiting remote regions of Cameroon without human contact. EV variants detected were distinct from those infecting human populations, comprising members of enterovirus species B, J, and H and a large novel group of viruses most closely related to species A in the P1 region. The viral sequences obtained contribute substantially to our growing understanding of the genetic diversity of EVs and the existence of interspecies chimerism that characterizes the novel variants in the current study, as well as in previously characterized species A and J viruses infecting monkeys. The latter findings will contribute to future development of consensus criteria for species assignments in enteroviruses and other picornavirus genera.

show abstract

“…RNA transcripts were treated with DNase I (Invitrogen) and purified (RNeasy minikit; Qiagen), and the integrity of the resulting RNA was assessed by agarose gel electrophoresis. The RNA concentration was determined using a Nanodrop spectrophotometer and converted to genome copies per l as described previously (30), and 10-fold dilutions were prepared (31). The sensitivity of the RT-PCR was assessed by performing eight parallel amplifications of suitable transcript dilutions and subsequent probit analysis (31).…”

Section: Isolation Of Individual Huh7 Cellsmentioning

confidence: 99%

Determining the Cellular Diversity of Hepatitis C Virus Quasispecies by Single-Cell Viral Sequencing

Leitch

McLauchlan

2013

J Virol

Self Cite

View full text Add to dashboard Cite

Single-cell genomics is emerging as an important tool in cellular biology. We describe for the first time a system to investigate RNA virus quasispecies diversity at the cellular level utilizing hepatitis C virus (HCV) replicons. A high-fidelity nested reverse transcription (RT)-PCR assay was developed, and validation using control transcripts of known copy number indicated a detection limit of 3 copies of viral RNA/reaction. This system was used to determine the cellular diversity of subgenomic JFH-1 HCV replicons constitutively expressed in Huh7 cells. Each cell contained a unique quasispecies that was much less diverse than the quasispecies of the bulk cell population from which the single cells were derived, suggesting the occurrence of independent evolution at the cellular level. An assessment of the replicative fitness of the predominant single-cell quasispecies variants indicated a modest reduction in fitness compared to the wild type. Real-time RT-PCR methods capable of determining single-cell viral loads were developed and indicated an average of 113 copies of replicon RNA per cell, correlating with calculated RNA copy numbers in the bulk cell population. This study introduces a single-cell RNA viral-sequencing method with numerous potential applications to explore host-virus interactions during infection. HCV quasispecies diversity varied greatly between cells in vitro, suggesting different within-cell evolutionary pathways. Such divergent trajectories in vivo could have implications for the evolution and establishment of antiviral-resistant variants and host immune escape mutants.

show abstract

Rapid Simultaneous Detection of Enterovirus and Parechovirus RNAs in Clinical Samples by One-Step Real-Time Reverse Transcription-PCR Assay

Cited by 43 publications

References 24 publications

Atypical hand, foot, and mouth disease associated with coxsackievirus A6 infection, Edinburgh, United Kingdom, January to February 2014

Atypical hand, foot, and mouth disease associated with coxsackievirus A6 infection, Edinburgh, United Kingdom, January to February 2014

High Rates of Infection with Novel Enterovirus Variants in Wild Populations of Mandrills and Other Old World Monkey Species

Determining the Cellular Diversity of Hepatitis C Virus Quasispecies by Single-Cell Viral Sequencing

Contact Info

Product

Resources

About