Development of a TaqMan® RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses

Pastorino, Boris; Bessaud, Maël; Grandadam, Marc; Murri, Séverine; Tolou, Hugues; Peyrefitte, Christophe N.

doi:10.1016/j.jviromet.2004.11.002

Cited by 250 publications

(192 citation statements)

References 29 publications

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“…All Ae. aegypti females that were used in human-tomosquito DENV transmission experiments were from laboratory-reared F 2 or F 3 generations derived from F 1 parental females that tested negative for DENV, JEV, and chikungunya virus (CHIKV) by virus-specific RT-PCR and for other potential flaviviruses by pan-flavivirus RT-PCR (31,(35)(36)(37).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Asymptomatic humans transmit dengue virus to mosquitoes

Duong

Lambrechts

Rácz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

408

367

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Three-quarters of the estimated 390 million dengue virus (DENV) infections each year are clinically inapparent. People with inapparent dengue virus infections are generally considered dead-end hosts for transmission because they do not reach sufficiently high viremia levels to infect mosquitoes. Here, we show that, despite their lower average level of viremia, asymptomatic people can be infectious to mosquitoes. Moreover, at a given level of viremia, DENV-infected people with no detectable symptoms or before the onset of symptoms are significantly more infectious to mosquitoes than people with symptomatic infections. Because DENV viremic people without clinical symptoms may be exposed to more mosquitoes through their undisrupted daily routines than sick people and represent the bulk of DENV infections, our data indicate that they have the potential to contribute significantly more to virus transmission to mosquitoes than previously recognized.mosquito experimental infection | Cambodia | Aedes aegypti | human-to-mosquito transmission | dengue W ith 3.97 billion people living in 128 countries currently at risk for infection, dengue viruses (DENV-1 to -4) cause more human morbidity and mortality worldwide than any other arthropod-borne virus (1, 2). Aedes aegypti mosquitoes are the primary vectors of DENV throughout the tropics (3). Dengue prevention relies on the control of Ae. aegypti populations, which is failing in most parts of the world due to lack of resources, lack of political will, and/or ineffective implementation (4).Virus transmission from infected humans to mosquitoes is a critical step in dengue epidemiology, but due to logistical constraints it has been directly examined only in a handful of studies to date (5). In initial experimental infections of human volunteers during the 1920s (6, 7), the onset of clinical symptoms occurred 4-9 d after virus inoculation by mosquito bite (8). DENV-infected humans were infectious to mosquitoes from 2 d before to 2 d after the onset of symptoms, and Ae. aegypti fed on viremic people were able to transmit virus to another person after at least 11 d of extrinsic incubation (8). Results from later studies indicated that, for naturally infected people with clinically apparent dengue, the duration of detectable viremia was on average 4-5 d after the onset of symptoms, but could range from 2 to 12 d (9, 10). Investigators in Vietnam fed Ae. aegypti directly on 208 symptomatic, hospitalized dengue patients and reported that the probability of successful human-to-mosquito DENV transmission was coincident with the kinetics of viremia (11). Dengue patients were infectious up to 5 d after the onset of symptoms, which generally corresponded with "defervescence" (11).All previous studies on human-to-mosquito DENV transmission were limited to people with overt illness and did not consider subclinical infections. An estimated 300 million of the total 390 million DENV infections per year are clinically inapparent or mildly symptomatic, i.e., no illness that disrupted a person's ...

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Section: Methodsmentioning

confidence: 99%

“…All F 1 mosquitoes were sorted by sex in a Petri dish placed on ice, pooled into vials of 10 mosquitoes (male and female) per vial, and homogenized. Extracted RNA was tested for DENV, JEV, CHIKV, and other flaviviruses (31,(35)(36)(37).…”

Section: Methodsmentioning

confidence: 99%

Asymptomatic humans transmit dengue virus to mosquitoes

Duong

Lambrechts

Rácz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

408

367

View full text Add to dashboard Cite

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“…Quantification of the extracted RNA was determined using a TaqMan assay modified from Pastorino et al (29). Viral load was estimated from a standard curve generated using serial dilutions of synthetic CHIKV E1 RNA transcripts.…”

Section: Viral Rna Extraction and Viral Load Analysismentioning

confidence: 99%

Active Infection of Human Blood Monocytes by Chikungunya Virus Triggers an Innate Immune Response

Her

Malleret

Chan

et al. 2010

The Journal of Immunology

253

254

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Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. To date, interactions between the immune system and the different stages of the virus life cycle remain poorly defined. We demonstrated for the first time that CHIKV Ags could be detected in vivo in the monocytes of acutely infected patients. Using in vitro experimental systems, whole blood and purified monocytes, we confirmed that monocytes could be infected and virus growth could be sustained. CHIKV interactions with monocytes, and with other blood leukocytes, induced a robust and rapid innate immune response with the production of specific chemokines and cytokines. In particular, high levels of IFN-α were produced rapidly after CHIKV incubation with monocytes. The identification of monocytes during the early phase of CHIKV infection in vivo is significant as infected monocyte/macrophage cells have been detected in the synovial tissues of chronically CHIKV-infected patients, and these cells may behave as the vehicles for virus dissemination. This may explain the persistence of joint symptoms despite the short duration of viremia. Our results provide a better understanding on the basic mechanisms of infection and early antiviral immune responses and will help in the development of future effective control strategies.

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“…[32] More recently, speciÞ c and sensitive one-step TaqMan RT-PCR assay has been developed as a tool for diagnosis of CHIKV and rapid indicator of active infection by quantifying viral load in clinical samples and cell culture supernatant. [33] Vector of chikungunya Aedes aegypti mosquitoes are considered as the vector of CHIKV. The vector bites humans during daytime.…”

Section: Molecular Diagnosismentioning

confidence: 99%