scholarly journals Detailed Analyses of Zika Virus Tropism in Culex quinquefasciatus Reveal Systemic Refractoriness

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Hannah J. MacLeod ◽  
George Dimopoulos
Keyword(s):  
Real Time ◽  
Culex Quinquefasciatus ◽  
Real Time Pcr ◽  
Zika Virus ◽  
Vector Competence ◽  
Mosquito Species ◽  
Virus Transmission ◽  
Quantitative Real Time Pcr ◽  
Content Type ◽  
Qrt Pcr

ABSTRACT The role of Culex quinquefasciatus in Zika virus transmission has been debated since the epidemic of Zika occurred in the Americas in 2015 to 2016. The majority of studies have found no evidence that C. quinquefasciatus or other Culex species are competent vectors of Zika virus, and the few studies that have proposed Zika vector status for C. quinquefasciatus have relied predominantly on quantitative real-time PCR (qRT-PCR) for viral detection. We assessed the infectious range of pre- and post-epidemic Zika virus isolates in order to classify mosquito samples based on titer infectiousness and demonstrated that two strains of C. quinquefasciatus, including one previously found to be competent, are highly resistant to infection with these Zika isolates compared to Aedes aegypti and are not competent for virus transmission. Further dissection of the dynamics of Zika exposure in both A. aegypti and C. quinquefasciatus revealed that while virus transmission by C. quinquefasciatus is blocked at the levels of the midgut and salivary glands, viral RNA persists in these tissues for prolonged periods post-exposure. We assessed Zika entry dynamics in both Aedes and Culex cells, and our results suggest that Zika virus infection in Culex cells may be blocked downstream of cell entry. These findings strongly suggest that C. quinquefasciatus is not a vector of Zika virus and additionally inform the use of qRT-PCR in vector competence assays as well as our understanding of barriers to arbovirus infection in non-susceptible mosquito species. IMPORTANCE Understanding which mosquito species transmit an emerging arbovirus is critical to effective vector control. During the Zika virus epidemic in 2015 to 2016, Aedes mosquitoes were confirmed as vectors. However, studies addressing the vector status of Culex quinquefasciatus mosquitoes presented conflicting evidence and remain an outstanding source of confusion in the field. Here, we established a robust cell-based assay to identify infectious titers of Zika virus and assessed the virus titers in C. quinquefasciatus by quantitative real-time PCR (qRT-PCR). We found that while low levels of virus were detected in C. quinquefasciatus, these titers did not correspond to infectious virus, and these mosquitoes did not transmit virus in the saliva. We also present evidence that the virus may enter Culex cells before infection is disrupted. Our findings are important for future studies incriminating vector species using qRT-PCR for virus detection and offer new information on how virus transmission is blocked by mosquitoes.

scholarly journals Vector competence ofAnophelesandCulexmosquitoes for Zika virus

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3096 ◽  
Author(s):  
Brittany L. Dodson ◽  
Jason L. Rasgon
Keyword(s):  
Anopheles Gambiae ◽  
Culex Quinquefasciatus ◽  
Zika Virus ◽  
Anopheles Stephensi ◽  
Vector Competence ◽  
Mosquito Species ◽  
Plaque Assay ◽  
Virus Transmission ◽  
Serious Disease ◽  
Blood Meals

Zika virus is a newly emergent mosquito-borne flavivirus that has caused recent large outbreaks in the new world, leading to dramatic increases in serious disease pathology including Guillain-Barre syndrome, newborn microcephaly, and infant brain damage. AlthoughAedesmosquitoes are thought to be the primary mosquito species driving infection, the virus has been isolated from dozens of mosquito species, includingCulexandAnophelesspecies, and we lack a thorough understanding of which mosquito species to target for vector control. We exposedAnopheles gambiae,Anopheles stephensi, andCulex quinquefasciatusmosquitoes to blood meals supplemented with two Zika virus strains. Mosquito bodies, legs, and saliva were collected five, seven, and 14 days post blood meal and tested for infectious virus by plaque assay. Regardless of titer, virus strain, or timepoint,Anopheles gambiae,Anopheles stephensi, andCulex quinquefasciatusmosquitoes were refractory to Zika virus infection. We conclude thatAnopheles gambiae,Anopheles stephensi, andCulex quinquefasciatusmosquitoes likely do not contribute significantly to Zika virus transmission to humans. However, future studies should continue to explore the potential for other novel potential vectors to transmit the virus.

scholarly journals Reference gene selection for quantitative real-time PCR (qRT-PCR) expression analysis in Galium aparine L.

PLoS ONE ◽  
2020 ◽  
Vol 15 (2) ◽  
pp. e0226668 ◽  
Author(s):  
Xu Su ◽  
Liuyang Lu ◽  
Yashe Li ◽  
Congai Zhen ◽  
Guilei Hu ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Expression Analysis ◽  
Real Time Pcr ◽  
Gene Selection ◽  
Quantitative Real Time Pcr ◽  
Reference Gene Selection ◽  
Qrt Pcr ◽  
Galium Aparine ◽  
Selection For

scholarly journals Extended-Interval Dosing of Rezafungin against Azole-Resistant Aspergillus fumigatus

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Nathan P. Wiederhold ◽  
Laura K. Najvar ◽  
Rosie Jaramillo ◽  
Marcos Olivo ◽  
Brian L. Wickes ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Content Type ◽  
Fungal Burden

ABSTRACT We evaluated extended-interval dosing of the investigational echinocandin rezafungin (1, 4, and 16 mg/kg on days 1, 4, and 7 postinoculation) for the treatment of disseminated invasive aspergillosis caused by azole-resistant Aspergillus fumigatus. Survival was significantly improved in mice treated with each dose of rezafungin and supratherapeutic posaconazole (20 mg/kg twice daily). Kidney fungal burden, as measured by quantitative real-time PCR, was also significantly reduced in mice treated with rezafungin although variability was observed.

scholarly journals A strategy for preparing non-fluorescent graphene oxide quantum dots as fluorescence quenchers in quantitative real-time PCR

RSC Advances ◽  
2020 ◽  
Vol 10 (25) ◽  
pp. 14944-14952
Author(s):  
Chenyan Hu ◽  
Zhongzhu Yang ◽  
Zhen Song ◽  
Linghui Xiao ◽  
Yang He
Keyword(s):  
Quantum Dots ◽  
Graphene Oxide ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Qrt Pcr ◽  
Taqman Probes ◽  
Specific Amplification ◽  
Graphene Oxide Quantum Dots

Non-fluorescent GOQDs quench the fluorescence of TaqMan probes and increase the specificity of qRT-PCR by reducing non-specific amplification.

Gene Expression Profiling in Multiple Myeloma Cells Treated with the Novel Anti-Myeloma Agents Zoledronate and Fluvastatin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5078-5078
Author(s):  
Timothy J. Molloy ◽  
Baulch-Brown Cindy ◽  
Yi-Mo Deng ◽  
Andrew Spencer ◽  
David F. Ma
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Real Time ◽  
Real Time Pcr ◽  
Mevalonate Pathway ◽  
Annexin V ◽  
Quantitative Real Time Pcr ◽  
Myeloma Cells ◽  
Qrt Pcr

Abstract We have shown in vitro that multiple myeloma (MM) cells can be destroyed by treating them with the mevalonate pathway inhibitors zoledronate and fluvastatin. While the efficacy of these compounds singly and combination have been demonstrated, their exact modes of action remain largely unknown. The present study aimed to use microarray and quantitative real-time PCR (QRT-PCR) techniques to analyse gene expression in treated myeloma cells to identify novel genes and pathways involved in the anti-myeloma action of these compounds. The human MM cell line NCI-H929 was treated with zoledronate and fluvastatin singly and in combination, and RNA was extracted and used to interrogate oligonucleotide microarrays consisting of 19,000 features representing known and unknown genes. Quantitative real-time PCR was subsequently used to confirm the expression of several genes of interest. Flow cytometry with Annexin V FITC staining was used to detect apoptosis. It was observed that genes related to apoptosis (caspases and p53-related genes), cell cycle control (cyclins), GTPase signalling (Rabs), and growth and proliferation (growth factors) were particularly affected by zoledronate and fluvastatin, and some of these genetic effects were synergistic when a combination of zoledronate and fluvastatin was used. QRT-PCR confirmed the effects on the caspase- and p53-related apoptotic pathways, and these effects were correlated with increased apoptosis in the myeloma cells. The mevalonate pathway inhibitors fluvastatin and zoledronate are highly efficient at killing MM cells, and their effects appear to be synergistic. Our microarray and QT-PCR analyses demonstrated that the expression of specific groups of genes important to the survival and proliferation of myeloma cells are affected by these compounds. p53 and caspase-dependent pathways appear to be the key apoptotic cascades stimulated. Insights into the mechanisms of these novel therapeutics are important as they might help to define their roles in the treatment of multiple myeloma.

scholarly journals Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting thegroELGene

2012 ◽  
Vol 78 (8) ◽  
pp. 2613-2622 ◽  
Author(s):  
Jana Junick ◽  
Michael Blaut
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Housekeeping Gene ◽  
Cell Number ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Bifidobacterium Adolescentis ◽  
Fecal Samples ◽  
Content Type ◽  
Pcr Assays

ABSTRACTQuantitative real-time PCR assays targeting thegroELgene for the specific enumeration of 12 human fecalBifidobacteriumspecies were developed. The housekeeping genegroEL(HSP60in eukaryotes) was used as a discriminative marker for the differentiation ofBifidobacterium adolescentis,B. angulatum,B. animalis,B. bifidum,B. breve,B. catenulatum,B. dentium,B. gallicum,B. longum,B. pseudocatenulatum,B. pseudolongum, andB. thermophilum. The bifidobacterial chromosome contains a single copy of thegroELgene, allowing the determination of the cell number by quantification of thegroELcopy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a givenBifidobacteriumspecies. Independent of theBifidobacteriumspecies tested, the proportion ofgroELcopies recovered from fecal samples spiked with 5 to 9 log10cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10groELcopies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3Bifidobacteriumspecies andB. longumfrequently detected. The predominant species in infant and adult fecal samples wereB. breveandB. adolescentis, respectively. It was possible to distinguishB. catenulatumandB. pseudocatenulatum. We conclude that thegroELgene is a suitable molecular marker for the specific and accurate quantification of human fecalBifidobacteriumspecies by real-time PCR.

scholarly journals Selection of reference genes for quantitative real-time PCR analysis in cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and cucumber–pumpkin grafted plants

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6536 ◽  
Author(s):  
Li Miao ◽  
Xing Qin ◽  
Lihong Gao ◽  
Qing Li ◽  
Shuzhen Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Stable Expression ◽  
Quantitative Real Time Pcr ◽  
Graft Union ◽  
Expression Levels ◽  
Qrt Pcr

Background Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber–pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber–pumpkin grafted plants. Methods In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber–pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder. Results The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber–pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber–pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. Discussion This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber–pumpkin grafted plants.

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