Internally Controlled, Generic Real-Time PCR for Quantification and Multiplex Real-Time PCR with Serotype-Specific Probes for Serotyping of Dengue Virus Infections

Menting, Sandra; Thai, Khoa T. D.; Nga, Tran Thi Thanh; Phuöng, Hoang Lan; Klatser, P. R.; Wolthers, Katja C.; Bình, Trần Quang; Vries, Peter J. de; Beld, Marcel

doi:10.1155/2011/514681

Cited by 20 publications

(18 citation statements)

References 16 publications

(26 reference statements)

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“…Briefly, viral RNA was extracted using QIAamp MinElute Virus Spin Kit (Qiagen, Netherlands) and cDNA synthesis was performed with High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. RT-PCR was performed using TaqMan Universal Master Mix (Thermo Fisher Scientific, USA) on 7500 Real-Time PCR System (Applied Biosystems) with primers and probes specifically designed for ZIKV42, CHIKV43 and DENV44.…”

Section: Methodsmentioning

confidence: 99%

Zika virus disrupts molecular fingerprinting of human neurospheres

Garcez

Nascimento

Vasconcelos

et al. 2017

Sci Rep

108

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Zika virus (ZIKV) has been associated with microcephaly and other brain abnormalities; however, the molecular consequences of ZIKV to human brain development are still not fully understood. Here we describe alterations in human neurospheres derived from induced pluripotent stem (iPS) cells infected with the strain of Zika virus that is circulating in Brazil. Combining proteomics and mRNA transcriptional profiling, over 500 proteins and genes associated with the Brazilian ZIKV infection were found to be differentially expressed. These genes and proteins provide an interactome map, which indicates that ZIKV controls the expression of RNA processing bodies, miRNA biogenesis and splicing factors required for self-replication. It also suggests that impairments in the molecular pathways underpinning cell cycle and neuronal differentiation are caused by ZIKV. These results point to biological mechanisms implicated in brain malformations, which are important to further the understanding of ZIKV infection and can be exploited as therapeutic potential targets to mitigate it.

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Section: Methodsmentioning

confidence: 99%

Zika virus disrupts molecular fingerprinting of human neurospheres

Garcez

Nascimento

Vasconcelos

et al. 2017

Sci Rep

108

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“…Two microliters of internal control, diluted 1:1000 in nuclease-free water, were added to each sample. The extraction was validated by monitoring the amplification during the RT-qPCR using primers and probes for the MS2 target, as described previously [48].…”

Section: Viral Rna Extractionmentioning

confidence: 99%

Natural Infection of Aedes aegypti by Chikungunya and Dengue type 2 Virus in a Transition Area of North-Northeast Brazil

Aragão

Pinheiro

Neto

et al. 2019

Viruses

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Dengue fever, chikungunya, and Zika are diseases caused by viruses transmitted by Aedes aegypti and Aedes albopictus. In Brazil, the number of human infections is high, but few studies are performed in mosquito vectors. This study aimed to investigate the presence of Zika, Dengue and Chikungunya viruses in Ae. aegypti and Ae. albopictus from the municipalities of Alto Alegre, Caxias, Codó, and São Mateus do Maranhão, located in the state of Maranhão, Northeast Brazil. The mosquitoes were collected with a mechanical aspirator, identified, triturated, and then submitted to RNA extraction and RT-qPCR. The positive samples were confirmed by virus isolation and genome sequencing. Three hundred and forty-eight Ae. aegypti (176 males and 172 females) and 12 Ae. albopictus (eight males and four females) were collected and tested. Ae. aegypti was the only vector positive in two municipalities—Codó, with detection of Chikungunya virus (CHIKV) belonging to the East-Central-South African genotype, and in Caxias, with detection of Dengue virus (DENV)-2 belonging to the Asian/American genotype. The detection of CHIKV and DENV-2 is evidence that those viruses are maintained in arthropod vectors, and shows the epidemiological risk in the area for chikungunya cases and a possible increase of severe dengue cases, associated with the occurrence of dengue hemorrhagic fever.

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“…The CYD viruses are grown in Vero cells, purified, and combined to produce the CYD‐TDV vaccine. Although many techniques including RT‐qPCR , ELISA , and electron microscopy , have been used to detect and quantify dengue virus in biological matrices, an immunoassay is not available for quantifying CYD4.…”

Section: Introductionmentioning

confidence: 99%

Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry

et al. 2015

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Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.

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Internally Controlled, Generic Real-Time PCR for Quantification and Multiplex Real-Time PCR with Serotype-Specific Probes for Serotyping of Dengue Virus Infections

Cited by 20 publications

References 16 publications

Zika virus disrupts molecular fingerprinting of human neurospheres

Zika virus disrupts molecular fingerprinting of human neurospheres

Natural Infection of Aedes aegypti by Chikungunya and Dengue type 2 Virus in a Transition Area of North-Northeast Brazil

Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry

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