Development of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA

Craggs, Joanna K.; Ball, Jonathan K.; Thomson, Brian J.; Irving, William L.; Grabowska, Anna

doi:10.1016/s0166-0934(01)00281-6

Cited by 100 publications

(89 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…One-step RT-PCR was performed according to standard protocols (One-step-RT-PCR kit; Qiagen) as described previously (Yue & Genersch, 2005), using the primer pair F1/B1 (Genersch, 2005). Viral replication was analysed via a modified two-step RT-PCR with an elevated reaction temperature (Laskus et al, 1998) and tagged primers (Craggs et al, 2001) to allow the specific detection of plus-and minus-strand DWV RNA as described previously (Yue & Genersch, 2005). In rare cases, RNA extracted from a mite collected from an asymptomatic bee gave an extremely faint signal for the negativestrand RNA as opposed to the strong signals always obtained from RNA extracted from mites collected from symptomatic bees (Fig.…”

mentioning

confidence: 99%

Deformed wing virus: replication and viral load in mites (Varroa destructor)

Gisder

Aumeier

Genersch

2009

Journal of General Virology

239

262

View full text Add to dashboard Cite

Deformed wing virus (DWV) normally causes covert infections but can have devastating effects on bees by inducing morphological deformity or even death when transmitted by the ectoparasitic mite Varroa destructor. In order to determine the role of V. destructor in the development of crippled wings, we analysed individual mites for the presence and replication of DWV. The results supported the correlation between viral replication in mites and morphologically deformed bees. Quantification of viral genome equivalents revealed that mites capable of inducing an overt DWV infection contained 10 10 -10 12 genome equivalents per mite. In contrast, mites which could not induce crippled wings contained a maximum of only 10 8 viral genome equivalents per mite.We conclude that the development of crippled wings not only depends on DWV transmission by V. destructor but also on viral replication in V. destructor and on the DWV titre in the parasitizing mites.The honeybee (Apis mellifera) is the most important pollinating insect species in agriculture and is therefore among the most important productive livestock; it is indispensable for many agricultural ecosystems. However, honeybee diseases and colony losses due to numerous pathogens, such as viruses, bacteria, fungi and metazoan parasites, negatively affect the profitability of agriculture and apiculture. Several studies implicate that the combination of Varroa destructor infestation and certain virus infections pose a serious threat to honeybee welfare (Ball, 1983;Ball & Allen, 1988;Cox-Foster et al., 2007;Hung et al., 1995Hung et al., , 1996Shen et al., 2005b;Shimanuki et al., 1994;Yang & Cox-Foster, 2007).The ectoparasitic mite V. destructor is an obligate parasite of the honeybee. Colonies infested by V. destructor develop the parasitic mite syndrome (Shimanuki et al., 1994) and ultimately collapse if left untreated. V. destructor has been confirmed as a vector in transmitting and activating bee virus infections and it is well established that viruses vectored by V. destructor play an important role in

show abstract

mentioning

confidence: 99%

Deformed wing virus: replication and viral load in mites (Varroa destructor)

Gisder

Aumeier

Genersch

2009

Journal of General Virology

239

262

View full text Add to dashboard Cite

show abstract

“…Recently, the strand-specificity of NS detection has been improved by modifying the conditions of RT-PCR for example by increasing RT reaction temperature [Laskus et al, 1997;Craggs et al, 2001;Lin et al, 2002], using tagged RT primer [Lanford et al, 1994;Craggs et al, 2001;Lin et al, 2002], selecting primers from core gene [Lérat et al, 1996;Lin et al, 2002] and removing unincorporated RT-primer [Craggs et al, 2001]. In the present study, these modifications have been applied to the TaqMan real-time PCR technique.…”

Section: Discussionmentioning

confidence: 99%

Quantitation of replication of the HCV genome in human livers with end‐stage cirrhosis by strand‐specific real‐time RT‐PCR assays: Methods and clinical relevance

Lin

Libbrecht

Verbeeck

et al. 2009

Journal of Medical Virology

View full text Add to dashboard Cite

HCV replicates in liver via an intermediate negative strand RNA. To study the relevance of HCV genome replication, quantitative strandspecific HCV real-time RT-PCR assays were developed and applied to livers explanted because of end-stage cirrhosis. The assays have broad ranges of determination and a high reproducibility and accuracy. Analysis of five different samples showed an even distribution of HCV genomes in four livers. Hepatic concentrations of positive (PS)-and negative (NS)-strand RNA did correlate with each other, with PS/NS ratios ranging between 3 and 340. Hepatic concentrations of HCV-PS or -NS RNA did not correlate with serum HCV-RNA levels or with genotypes. A high HCV envelope-2 protein expression correlated with a low NS concentration. HCV-PS and -NS levels, E2 protein expression and genotype did not correlate with biochemical tests or with histological changes in the explanted liver, but the ratio NS/PS, a marker of viral replication, correlated with the severity of the recurrent post-transplant hepatitis caused by HCV. This suggests the existence of an extra-hepatic location of HCV with comparable viral replication rate being responsible for the infection of the newly transplanted liver.

show abstract

“…3A). Strand specificity was needed to ensure that the transcripts detected were transcribed from the predicted DNA strand and to exclude artifacts caused from read-through transcription on the opposite strand (Craggs et al 2001).…”

Section: Experimental Evidence For Expression Of S Cerevisiae Orphanmentioning

confidence: 99%

“…Genomic DNA was extracted from yeast culture by the DNeasy blood and tissue kit (Qiagen). We modified a strand-specific RT-PCR method previously described (Craggs et al 2001), using the GeneAmp thermostable rTth reverse transcriptase RNA PCR kit (Applied Biosystems). DNase-treated poly(A) RNA sample (25 ng) was denatured for 5 min at 70°C with 2 µL of ‫ן01‬ rTth reverse transcriptase buffer and 1 µL of 10 µM reverse ORF-specific primer complementary to the ORF-coding strand (OSP R ).…”

Section: Strand-specific Rt-pcrmentioning

confidence: 99%

Revisiting the Saccharomyces cerevisiae predicted ORFeome

Carvunis²,

Yu³

et al. 2008

Genome Res.

View full text Add to dashboard Cite

Accurately defining the coding potential of an organism, i.e., all protein-encoding open reading frames (ORFs) or “ORFeome,” is a prerequisite to fully understand its biology. ORFeome annotation involves iterative computational predictions from genome sequences combined with experimental verifications. Here we reexamine a set of Saccharomyces cerevisiae “orphan” ORFs recently removed from the original ORFeome annotation due to lack of conservation across evolutionarily related yeast species. We show that many orphan ORFs produce detectable transcripts and/or translated products in various functional genomics and proteomics experiments. By combining a naïve Bayes model that predicts the likelihood of an ORF to encode a functional product with experimental verification of strand-specific transcripts, we argue that orphan ORFs should still remain candidates for functional ORFs. In support of this model, interstrain intraspecies genome sequence variation is lower across orphan ORFs than in intergenic regions, indicating that orphan ORFs endure functional constraints and resist deleterious mutations. We conclude that ORFs should be evaluated based on multiple levels of evidence and not be removed from ORFeome annotation solely based on low sequence conservation in other species. Rather, such ORFs might be important for micro-evolutionary divergence between species.

show abstract

Development of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA

Cited by 100 publications

References 19 publications

Deformed wing virus: replication and viral load in mites (Varroa destructor)

Deformed wing virus: replication and viral load in mites (Varroa destructor)

Quantitation of replication of the HCV genome in human livers with end‐stage cirrhosis by strand‐specific real‐time RT‐PCR assays: Methods and clinical relevance

Revisiting the Saccharomyces cerevisiae predicted ORFeome

Contact Info

Product

Resources

About