Progress 07/01/12 to 05/10/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:PD is no longer at Virginia Tech, nothing to report. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
n/a
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The PD has left the University, no further research will be conducted on this project. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The project director terminated employment in 2016 and this project is to be terminated.
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Scientific Community Changes/Problems:PD will be leaving Virginia Tech in the summer of 2016. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Results were reported at the Colloquium on Infectious Diseases & Immunity at the University of Texas Medical Branch, Galveston, TX. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Although a link between the increased susceptibility of mosquitoes for arthropod-borne viruses and exposure to lower rearing temperatures has been known for many years, the molecular basis of this has remained unknown. We investigated the role of temperature on RNA silencing using an engineered strain of mosquito where the expression of a reporter was dependent on the status of the siRNA pathway. These "sensor" strains express EGFP only when RNA silencing is inhibited. We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18oC as compared with 28oC. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA) trigger for RNA silencing, showed no change in EGFP expression when reared at 18oC. Our studies also indicated a correlation between a virus-susceptible phenotype and temperature-dependent deficiencies in antiviral RNA silencing, predisposing mosquitoes to higher levels of infection with both chikungunya virus and yellow fever virus. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNA silencing occurs downstream of Dicer processing. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. Our data suggest that cooler microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors. These findings may lead to a better understanding of how global climate change will affect the transmission of mosquito-borne viruses, and new criteria for evaluating genetic control strategies based on RNA silencing.
Publications
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Scientific Community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Findings were presented during an invited talk at the 47th Annual Meeting of the Society for Invertebrate Pathology and International Congress on Invertebrate Pathology and Microbial Control. What do you plan to do during the next reporting period to accomplish the goals? Future research will focus on validating our model of antiviral immunity and on determining the selection pressure(s) that led to the evolution of redundant antiviral defenses in vector mosquitoes.
Impacts
What was accomplished under these goals?
Mosquitoes defend themselves against viral infection with an immune response. Thus, mosquito-borne viral diseases like West Nile fever, dengue fever, and chikungunya fever are transmitted to humans only if the pathogen overcomes these defenses. Despite this, relatively little is known about the immune pathways of the mosquito. We have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is present in culicine mosquito vectors. We report here that another class of virus-derived small RNAs, exhibiting many similarities with ping-pong-dependent piwi-interacting RNAs (piRNAs), is also produced in the soma of culicine mosquitoes. However, unlike ping-pong-dependent piRNAs that have been described previously, from repetitive elements or piRNA clusters, our work suggests biogenesis in the mosquito soma. Similar to siRNAs, viral piRNAs also appear capable of modulating the pathogenesis of viral infections in mosquito cells, suggesting that mosquitoes possess redundant RNA-based antiviral responses. Our studies have provided new insights into how a mosquito's immune defenses restrict virus replication and the transmission of mosquito-borne viruses.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Research Scientists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has provided training and professional development opportunities for graduate students and post doctoral researchers. How have the results been disseminated to communities of interest? Publications and professional meetings. What do you plan to do during the next reporting period to accomplish the goals? We plan to assess whether or not mosquito-borne viruses encode proteins that suppress RNA silencing pathways.
Impacts
What was accomplished under these goals?
Mosquito-borne viruses are maintained in nature through alternating virus replication in susceptible vertebrate and mosquito hosts. While infection of the vertebrate host is typically associated with pathology and disease, a persistent non-lethal infection is established in the insect host. The mechanism by which this occurs is not well understood, but we have previously shown that RNA silencing is essential to this process. Some mammalian viruses have been shown to encode proteins capable of suppressing RNA silencing (SRS). Studies that have identified SRS proteins in virus families have often been guided by prior experimental evidence that identified a particular virus protein as a virulence factor before any suppressor activity was suspected. However, studies identifying genetic determinants of virulence in mosquito-borne viruses have primarily been conducted in vertebrate hosts, and may not be relevant in the invertebrate host where infection with the virus tends to be avirulent. However, such virulence factors might be good candidates for SRS proteins if RNA silencing also functions as an antiviral immune response in mammals. Although much of the RNA interference (RNAi) machinery appears to be conserved in mammalian cells, it remains unclear whether this pathway functions as an antiviral immune response in mammals. To address this question we cloned and sequenced small RNAs from chikungunya virus (CHIKV) infected Aedes albopictusand human embryonic kidney cells (HEK-293). The virus-derived small RNAs generated in Ae. albopictus resemble previously described products of RNA silencing. In contrast, the majority of the virus-derived small RNAs sequenced in the HEK-293 cells appear to result from RNase L cleavage of the CHIKV 26S mRNA, and not an RNA silencing response. Our results reveal some significant differences in the small RNAs present in the mosquito and mammalian cell types. These differences provide new insights into the innate antiviral responses present in both mammalian and mosquito cells.
Publications
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: We have developed and validated two new strand-specific quantitative real-time PCR (ssqPCR) assays for the medically important alphaviruses, CHIKV and ONNV. These assays will be useful in studies to determine how persistent alphavirus infections are maintained in the vector host, and in the detection and quantification of replicating virus from clinical specimens and potential reservoir hosts. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Vector Researchers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the diagnosis and study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o'nyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV minus-strand RNA with this assay when higher levels of cDNA generated from the plus-strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV minus-strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also determined the sensitivities of two different detection strategies and chemistries, SYBR Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we developed and validated ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region. This basic work on assay development has relevance to our ability to quantify RNA viruses in general.
Publications
- No publications reported this period
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