Detection of Zika Virus in Desiccated Mosquitoes by Real-Time Reverse Transcription PCR and Plaque Assay

Burkhalter, Kristen L.; Savage, Harry M.

doi:10.3201/eid2304.161772

Cited by 13 publications

(12 citation statements)

References 6 publications

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“…Capillary tubes with saliva expectorates were placed directly into a two ml tube containing 400 μl of 2% DMEM media and centrifuged as described above. To detect ZIKV RNA, real-time RT-PCR was performed on all samples using previously reported protocol and primers (Lanciotti et al 2008, Burkhalter and Savage 2017), on a CFX96 Touch system (Bio-Rad Laboratories). Samples with a cycle threshold (i.e., the number of cycles required for the fluorescent signal to cross the threshold) of <38 were scored as positive.…”

Section: Methodsmentioning

confidence: 99%

Susceptibility and Vectorial Capacity of AmericanAedes albopictusandAedes aegypti(Diptera: Culicidae) to American Zika Virus Strains

Lozano-Fuentes

Kenney

Varnado

et al. 2018

Journal of Medical Entomology

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The rapid expansion of Zika virus (ZIKV), following the recent outbreaks of Chikungunya virus, overwhelmed the public health infrastructure in many countries and alarmed many in the scientific community. Aedes aegypti (L.) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) have previously been incriminated as the vectors of these pathogens in addition to dengue virus. In our study, we challenged low generation Ae. aegypti (Chiapas, Mexico) and Ae. albopictus (North Carolina, Mississippi), with three strains of ZIKV, Puerto Rico (GenBank: KU501215), Honduras (GenBank: KX694534), and Miami (GenBank: MF988743). Following an oral challenge with 107.5 PFU/ml of the Puerto Rico strain, we observed high infection and dissemination rates in both species (95%). We report estimated transmission rates for both species (74 and 33%, for Ae. aegypti (L.) (Diptera: Culicidae) and Ae. albopictus (Skuse) (Diptera: Culicidae), respectively), and the presence of a probable salivary gland barrier in Ae. albopictus to Zika virus. Finally, we calculated vectorial capacity for both species and found that Ae. albopictus had a slightly lower vectorial capacity when compared with Ae. aegypti.Second Language Abstract: La rápida expansión del virus Zika, poco después de las epidemias de chikungunya, rebaso la infraestructura de salud pública en muchos países y sorprendió a muchos en la comunidad científica. Notablemente, Aedes aegypti y Aedes albopictus transmiten estos patógenos además del virus del dengue. En este estudio se expusieron con tres cepas americanas de virus Zika a grupos de Aedes aegypti y Aedes albopictus de generación reciente. Encontramos altos porcentajes de infección y diseminación en ambas especies (95%). Se reporta, la transmisión viral en ambas especies (74 y 33%, para Aedes aegypti and Aedes albopictus, respectivamente) y una probable barrera a nivel de glándulas salivarías. Finalmente, calculamos la capacidad vectorial para ambas especies.

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Section: Methodsmentioning

confidence: 99%

Susceptibility and Vectorial Capacity of AmericanAedes albopictusandAedes aegypti(Diptera: Culicidae) to American Zika Virus Strains

Lozano-Fuentes

Kenney

Varnado

et al. 2018

Journal of Medical Entomology

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“…RT-qPCR reactions were analysed by Ct values being Ct ≤ 35 positive for ZIKV RNA presence. Similar studies have considered Ct values ≤ 36 ( Smartt et al 2017 ) and ≤ 38 ( Ferreira-de-Brito et al 2016 , Burkhalter & Savage 2017 ) as positive for ZIKV presence in mosquitoes. The Ct is the qPCR cycle where the reporter signal crosses the background fluorescence, and specific amplification is detected.…”

Section: Methodsmentioning

confidence: 95%

First evidence of Zika virus venereal transmission in Aedes aegypti mosquitoes

Pereira-Silva

Nascimento

Belchior

et al. 2017

Mem. Inst. Oswaldo Cruz

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BACKGROUND Aedes aegypti is considered the main Zika virus (ZIKV) vector, and is thought to be responsible for the 2015-2016 outbreak in Brazil. Zika positive Ae. aegypti males collected in the field suggest that vertical and/or venereal transmission of ZIKV may occur.OBJECTIVES In this study, we aimed to demonstrate that venereal transmission of ZIKV by Ae. aegypti can occur under laboratory conditions.METHODS Ae. aegypti collected in the city of Manaus, confirmed as negative for Zika, Dengue and Chikungunya virus by reverse transcription real-time polymerase chain reaction (RT-qPCR) (AaM3V- strain), were reared under laboratory conditions and used for the experiments. The ZIKV used in this study was isolated from a patient presenting with symptoms; ZIKV was confirmed by RT-qPCR. Experiment 1: virgin male mosquitoes of AaM3V- strain were intrathoracically inoculated with a ZIKV suspension; four days after injection, they were transferred to a cage containing virgin females of AaM3V- strain and left to copulate for five days. Experiment 2: virgin female mosquitoes of AaM3V- strain were orally infected with a ZIKV suspension by blood feeding membrane assay; nine days after blood feeding, they were placed in cages with Ae. aegypti AaM3V- virgin males and left to copulate for four days. After copulation, all mosquitoes were individually evaluated for viral infection by RT-qPCR.FINDINGS The mean infection rate in Experiment 1 and Experiment 2 was 45% and 35%, respectively. In both experiments, cycle threshold values ranged from 13 to 35, indicating the presence of viral genomes.MAIN CONCLUSION Ae. aegypti males intrathoracically inoculated with a ZIKV suspension are infected and can transmit the virus to uninfected females by mating. Moreover, Ae. aegypti females orally infected with a ZIKV suspension can transmit the virus to uninfected males by copulation. This study shows that ZIKV infection of Ae. aegypti mosquitoes occurs not only during blood feeding, but also during copulation.

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“…The standard polymerase used in PCR can only synthesise from a DNA template, thus RNA amplification requires the use of an enzyme with reverse transcription activity (Bustin, 2000). PCR and reverse transcription-PCR are widely applied methods for the detection of both DNA and RNA viruses, respectively (Metcalf, 1995;Pring-Åkerblom et al, 1997;Burkhalter and Savage, 2017;Wadhwa et al, 2017;Liu et al, 2018;Lin et al, 2020). In standard PCR, the DNA products are generally visualised by gel electrophoresis, to check for the presence of expected DNA bands.…”

Section: Nucleic Acid-based Detection Methodsmentioning

confidence: 99%

Virus Detection: A Review of the Current and Emerging Molecular and Immunological Methods

Cassedy

Parle‐McDermott

O’Kennedy

2021

Front. Mol. Biosci.

117

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Viruses are ubiquitous in the environment. While many impart no deleterious effects on their hosts, several are major pathogens. This risk of pathogenicity, alongside the fact that many viruses can rapidly mutate highlights the need for suitable, rapid diagnostic measures. This review provides a critical analysis of widely used methods and examines their advantages and limitations. Currently, nucleic-acid detection and immunoassay methods are among the most popular means for quickly identifying viral infection directly from source. Nucleic acid-based detection generally offers high sensitivity, but can be time-consuming, costly, and require trained staff. The use of isothermal-based amplification systems for detection could aid in the reduction of results turnaround and equipment-associated costs, making them appealing for point-of-use applications, or when high volume/fast turnaround testing is required. Alternatively, immunoassays offer robustness and reduced costs. Furthermore, some immunoassay formats, such as those using lateral-flow technology, can generate results very rapidly. However, immunoassays typically cannot achieve comparable sensitivity to nucleic acid-based detection methods. Alongside these methods, the application of next-generation sequencing can provide highly specific results. In addition, the ability to sequence large numbers of viral genomes would provide researchers with enhanced information and assist in tracing infections.

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Detection of Zika Virus in Desiccated Mosquitoes by Real-Time Reverse Transcription PCR and Plaque Assay

Abstract: We assayed Zika virus–infected mosquitoes stored at room temperature for <30 days for live virus by using plaque assay and virus RNA by using real-time reverse transcription PCR. Viable virus was detected in samples stored <10 days, and virus RNA was detected in samples held for 30 days.

Cited by 13 publications

References 6 publications

Susceptibility and Vectorial Capacity of AmericanAedes albopictusandAedes aegypti(Diptera: Culicidae) to American Zika Virus Strains

Susceptibility and Vectorial Capacity of AmericanAedes albopictusandAedes aegypti(Diptera: Culicidae) to American Zika Virus Strains

First evidence of Zika virus venereal transmission in Aedes aegypti mosquitoes

Virus Detection: A Review of the Current and Emerging Molecular and Immunological Methods

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