Magnetic purification of biotinylated cDNA removes false priming and ensures strand-specificity of RT-PCR for enteroviral RNAs
ABSTRACT The detection of replicative intermediate RNAs as markers of active replication of RNA viruses is an essential tool to investigate pathogenesis in acute viral infections, as well as in their long-term sequelae. In this regard, strand-specific PCR has been used widely to distinguish (−) and (+) enteroviral RNAs in pathogenesis studies of diseases such as dilated cardiomyopathy. It has been generally assumed that oligonucleotide-primed reverse transcription of a given RNA generates only the corresponding specific cDNA, thus assuring the specificity of a PCR product amplified from it. Nevertheless, such assumed strand-specificity is a fallacy, because falsely primed cDNAs can be produced by RNA reverse transcription in the absence of exogenously added primers, (cDNAprimer(−)), and such falsely primed cDNAs are amplifiable by PCR in the same way as the correctly primed cDNAs. Using as a prototype the coxsackievirus B5 (CVB5), a (+) strand RNA virus, it was shown that cDNAprimer(−) renders the differential detection of viral (−) and (+) RNAs by conventional PCR virtually impossible, due to gross non-specificity. Using in vitro transcribed CVB5 RNAs (+) and (−), it was shown that cDNAprimer(−) could be removed effectively by magnetic physical separation of correctly primed biotinylated cDNA. Such strategy enabled truly strand-specific detection of RNA (−) and (+), not only for CVB5, but also for other non-polio enteroviruses. These findings indicate that previous conclusions supporting a role for the persistence of actively replicating enterovirus in the pathogenesis of chronic myocarditis should be regarded with strong skepticism and purification of correctly primed cDNA should be used for strand-specific PCR of viral RNA in order to obtain reliable information on this important subject.
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ABSTRACT: Impaired interferon (IFN) production has been observed in various obstructive respiratory diseases. This contributes to enhanced sensitivity towards viral infections triggering acute exacerbations. To compensate for this impaired host IFN response, there is need to explore new therapeutic strategies, like exogenous administration of IFNs as prophylactic treatment. In the present study, we examined the protective potential of IFN-λ1 and compared it with the previously established protecting effect of IFN-β. A549 cells and human primary bronchial epithelial cells were first treated with either IFN-β (500 IU/ml) or IFN-λ1 (500 ng/ml) for 18 h. For infection, two approaches were adopted: i) Continuous scenario: after pre-treatment, cells were infected immediately for 24 h with human rhinovirus 1B (HRV1B) in IFN-containing medium, or were cultured for another 72 h in IFN-containing medium, and then infected for 24 h with HRV1B, ii) Pre-treatment scenario: IFN-containing medium was replaced after 18 h and cells were infected for 4 h either immediately after pre-treatment or after additional culturing for 72 h in IFN-free medium. The protective effect was evaluated in terms of reduction in the number of viral copies/infectious progeny, and enhanced expression of IFN-stimulated genes (ISGs). In both cell types and in both approaches, IFN-λ1 and IFN-β treatment resulted in pronounced and long-lasting antiviral effects exemplified by significantly reduced viral copy numbers and diminished infectious progeny. This was associated with strong up-regulation of multiple ISGs. However, in contrast to the IFN-β induced expression of ISGs, which decreased over time, expression of ISGs induced by IFN-λ1 was sustained or even increased over time. Here we demonstrate that the protective potential of IFN-λ1 is comparable to IFN-β. Yet, the long-lasting induction of ISGs by IFN-λ1 and most likely less incitement of side effects due to more localized expression of its receptors could make it an even more promising candidate for prophylactic treatment than IFN-β.PLoS ONE 04/2014; 9(4):e95134. · 3.53 Impact Factor
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ABSTRACT: Hepatitis C virus (HCV) causes liver diseases, such as hepatitis, liver cirrhosis, steatosis, and hepatocellular carcinoma. To understand the life cycle and pathogenesis of HCV, the one-step growth of HCV in a cell culture system was analyzed using a highly infectious variant of the JFH1 clone. The observed profiles of HCV RNA replication indicated that the synthesis of negative-strand RNAs occurred at 6 h (h) after infection, followed by the active synthesis of positive-strand RNAs. Our measurements of infectious virus production showed that the latent period of HCV was about 12 h. The specific infectivity of HCV particles (focus-forming unit per viral RNA molecule) secreted to the extracellular milieu early in infection was about 30-fold higher than that secreted later during infection. The buoyant densities of the infectious virion particles differed with the duration of infection, indicating changes in the compositions of the virion particles.Virology 11/2012; 433(2):462–470. · 3.35 Impact Factor
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ABSTRACT: Human respiratory syncytial virus (HRSV) is an important cause of respiratory disease. The majority of studies addressing the importance of virus co-infections to the HRSV-disease have been based on the detection of HRSV by RT-PCR, which may not distinguish current replication from prolonged shedding of remnant RNA from previous HRSV infections. To assess whether co-detections of other common respiratory viruses are associated with increased severity of HRSV illnesses from patients who were shedding viable-HRSV, nasopharyngeal aspirates from children younger than 5 years who sought medical care for respiratory infections in Ribeirão Preto (Brazil) were tested for HRSV by immunofluorescence, RT-PCR and virus isolation in cell culture. All samples with viable-HRSV were tested further by PCR for other respiratory viruses. HRSV-disease severity was assessed by a clinical score scale. A total of 266 samples from 247 children were collected and 111 (42%) were HRSV-positive. HRSV was isolated from 70 (63%), and 52 (74%) of them were positive for at least one additional virus. HRSV-positive diseases were more severe than HRSV-negative ones, but there was no difference in disease severity between patients with viable-HRSV and those HRSV-positives by RT-PCR. Co-detection of other viruses did not correlate with increased disease severity. HRSV isolation in cell culture does not seem to be superior to RT-PCR to distinguish infections associated with HRSV replication in studies of clinical impact of HRSV. A high rate of co-detection of other respiratory viruses was found in samples with viable-HRSV, but this was not associated with more severe HRSV infection. J. Med. Virol. © 2013 Wiley Periodicals, Inc.Journal of Medical Virology 07/2013; · 2.37 Impact Factor