Development and validation of a novel SYBR Green real-time RT-PCR assay for the detection of classical swine fever virus evaluated on different real-time PCR platforms

Pérez, Lester J.; Arce, Heidy Díaz de; Tarradas, Joan; Rosell, Rosa; Perera, Carmen Laura; Muñoz, Marta; Frı́as, Maria Teresa; Núñez, José I.; Ganges, Llilianne

doi:10.1016/j.jviromet.2011.03.022

Cited by 28 publications

(20 citation statements)

References 36 publications

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“…This is the first study using a multiplex PCR technique for the genotyping of hepatitis B virus in the state of Rio Grande do Sul, Southern Brazil. The use of melting curve analysis showed that the specificity and reproducibility were similar to previously published results for other viruses (24,25,31) . Several reports have described the use of qPCR for the identification of strains, however with a generalist approach.…”

Section: Discussionsupporting

confidence: 85%

Melting Curve Analysis for the Screening of Hepatitis B Virus Genotypes A, D and F in Patients From a General Hospital in Southern Brazil

Becker

Filho

Mattos

et al. 2013

Arq. Gastroenterol.

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-Context -Hepatitis B virus (HBV) can cause fulminant hepatitis, cirrhosis and hepatocellular carcinoma, and is one of the most common causes of acute and chronic liver failure. The genetic variants of HBV can be decisive for the evolution of these diseases as well as for the election of therapy. Objectives -The aim of this study was to evaluate and standardize an in house methodology based on the analysis of the melting curve polymerase chain reaction (PCR) of real-time (qPCR) to screen for genotypes A, D and F of HBV in patients from a hospital in Rio Grande do Sul, Brazil. Methods -We evaluated 104 patients presumably with HBV chronic infection. Viral DNA was extracted from plasma and viral genotypes and different mutations were determined using PCR-based protocols. Results -A PCR-based methodology was standardized for the analysis of genotypes A, D and F of HBV. The technique was based in a nested PCR with the final step consisting of a multiplex real-time PCR, using the melting curve as a tool for the differentiation of fragments. A higher frequency of genotype D (44.4%), followed by genotype A (22.2%) and genotype F (3.7%) was observed. Conclusion -The standardized assay, a nested PCR-multiplex qPCR using specific primers, provides a rapid and accurate method for the differentiation of HBV genotypes that are more frequent in Southern Brazil -A, D and F. This method can be applied in the clinical practice.

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Section: Discussionsupporting

confidence: 85%

Melting Curve Analysis for the Screening of Hepatitis B Virus Genotypes A, D and F in Patients From a General Hospital in Southern Brazil

Becker

Filho

Mattos

et al. 2013

Arq. Gastroenterol.

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“…cDNA synthesis was conducted by random priming, using M‐MLV reverse transcriptase as described by Diaz de Arce et al (). Specific CSFV RNA detection in all collected samples was performed by RT‐qPCR described by Perez, Arce, Tarradas, et al (). Results were considered positive when the assay yielded cycle threshold values lower than 35 (Ct < 35).…”

Section: Methodsmentioning

confidence: 99%

Positive selection pressure on E2 protein of classical swine fever virus drives variations in virulence, pathogenesis and antigenicity: Implication for epidemiological surveillance in endemic areas

Coronado

Rios

Frı́as

et al. 2019

Transbound Emerg Dis

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Classical swine fever (CSF), caused by CSF virus (CSFV), is considered one of the most important infectious diseases with devasting consequences for the pig industry. Recent reports describe the emergence of new CSFV strains resulting from the action of positive selection pressure, due mainly to the bottleneck effect generated by ineffective vaccination. Even though a decrease in the genetic diversity of the positively selected CSFV strains has been observed by several research groups, there is little information about the effect of this selective force on the virulence degree, antigenicity and pathogenicity of this type of strains. Hence, the aim of the current study was to determine the effect of the positive selection pressure on these three parameters of CSFV strains, emerged as result of the bottleneck effects induced by improper vaccination in a CSF‐endemic area. Moreover, the effect of the positively selected strains on the epidemiological surveillance system was assessed. By the combination of in vitro, in vivo and immunoinformatic approaches, we revealed that the action of the positive selection pressure induces a decrease in virulence and alteration in pathogenicity and antigenicity. However, we also noted that the evolutionary process of CSFV, especially in segregated microenvironments, could contribute to the gain‐fitness event, restoring the highly virulent pattern of the circulating strains. Besides, we denoted that the presence of low virulent strains selected by bottleneck effect after inefficient vaccination can lead to a relevant challenge for the epidemiological surveillance of CSF, contributing to under‐reports of the disease, favouring the perpetuation of the virus in the field. In this study, B‐cell and CTL epitopes on the E2 3D‐structure model were also identified. Thus, the current study provides novel and significant insights into variation in virulence, pathogenesis and antigenicity experienced by CSFV strains after the positive selection pressure effect.

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“…The synthesis of cDNA was performed by random priming, using M-MLV reverse transcriptase as described previously (Díaz de Arce et al, 2009). CSFV RNA in the serum and nasal and rectal swabs, as well as tonsil, spleen, lymph node and ileum samples, was detected using the qRT-PCR assay previously described (Pérez et al, 2011). This test has been used for inter-laboratory comparisons of CSFV diagnoses, organised by the EURL-CSF.…”

Section: Rna Extraction and Qrt-pcr For The Detection Of Csfv Rnamentioning

confidence: 99%

Novel poly-uridine insertion in the 3′UTR and E2 amino acid substitutions in a low virulent classical swine fever virus

Coronado

Liniger²,

Muñoz-González

et al. 2017

Veterinary Microbiology

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In this study, we compared the virulence in weaner pigs of the Pinar del Rio isolate and the virulent Margarita strain. The latter caused the Cuban classical swine fever (CSF) outbreak of 1993. Our results showed that the Pinar del Rio virus isolated during an endemic phase is clearly of low virulence. We analysed the complete nucleotide sequence of the Pinar del Rio virus isolated after persistence in newborn piglets, as well as the genome sequence of the inoculum. The consensus genome sequence of the Pinar del Rio virus remained completely unchanged after 28days of persistent infection in swine. More importantly, a unique poly-uridine tract was discovered in the 3'UTR of the Pinar del Rio virus, which was not found in the Margarita virus or any other known CSFV sequences. Based on RNA secondary structure prediction, the poly-uridine tract results in a long single-stranded intervening sequence (SS) between the stem-loops I and II of the 3'UTR, without major changes in the stem- loop structures when compared to the Margarita virus. The possible implications of this novel insertion on persistence and attenuation remain to be investigated. In addition, comparison of the amino acid sequence of the viral proteins E, E1, E2 and p7 of the Margarita and Pinar del Rio viruses showed that all non-conservative amino acid substitutions acquired by the Pinar del Rio isolate clustered in E2, with two of them being located within the B/C domain. Immunisation and cross-neutralisation experiments in pigs and rabbits suggest differences between these two viruses, which may be attributable to the amino acid differences observed in E2. Altogether, these data provide fresh insights into viral molecular features which might be associated with the attenuation and adaptation of CSFV for persistence in the field.

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Development and validation of a novel SYBR Green real-time RT-PCR assay for the detection of classical swine fever virus evaluated on different real-time PCR platforms

Cited by 28 publications

References 36 publications

Melting Curve Analysis for the Screening of Hepatitis B Virus Genotypes A, D and F in Patients From a General Hospital in Southern Brazil

Melting Curve Analysis for the Screening of Hepatitis B Virus Genotypes A, D and F in Patients From a General Hospital in Southern Brazil

Positive selection pressure on E2 protein of classical swine fever virus drives variations in virulence, pathogenesis and antigenicity: Implication for epidemiological surveillance in endemic areas

Novel poly-uridine insertion in the 3′UTR and E2 amino acid substitutions in a low virulent classical swine fever virus

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