Recombination-ready Sindbis replicon expression vectors for transgene expression

<p>Abstract</p> <p>Background</p> <p>Sindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation.</p> <p>Results</p> <p>Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis.</p> <p>Conclusion</p> <p>Replication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function.</p

The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in Aedes aegypti

<p>Abstract</p> <p>Background</p> <p>The RNA interference (RNAi) pathway acts as an innate antiviral immune response in <it>Aedes aegypti</it>, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: <it>Togaviridae</it>, genus: <it>Alphavirus</it>) is an arbovirus that infects <it>Ae. aegypti </it>in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of <it>Ae. aegypti</it>. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity.</p> <p>Results</p> <p>As a novel tool for studying arbovirus-mosquito interactions, we engineered a transgenic mosquito line with an impaired RNAi pathway in the midgut of bloodfed females by silencing expression of the <it>Aa</it>-<it>dcr2 </it>gene. In midgut tissue of the transgenic Carb/dcr16 line, <it>Aa</it>-<it>dcr2 </it>expression was reduced ~50% between 1-7 days post-bloodmeal (pbm) when compared to the recipient mosquito strain. After infection with SINV-TR339EGFP, <it>Aa</it>-<it>dcr2 </it>expression levels were enhanced in both mosquito strains. In the RNAi pathway impaired mosquito strain SINV titers and midgut infection rates were significantly higher at 7 days pbm. There was also a strong tendency for increased virus dissemination rates among the transgenic mosquitoes. Between 7-14 days pbm, SINV was diminished in midgut tissue of the transgenic mosquitoes. Transgenic impairment of the RNAi pathway and/or SINV infection did not affect longevity of the mosquitoes.</p> <p>Conclusions</p> <p>We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level. Following ingestion by <it>Ae. aegypti</it>, the recombinant SINV-TR339EGFP was confronted with both MEB and a midgut infection barrier (MIB). Impairment of the RNAi pathway in the midgut strongly reduced both midgut barriers for the virus. This confirms that the endogenous RNAi pathway of <it>Ae. aegypti </it>modulates vector competence for SINV in the midgut. The RNAi pathway acts as a gatekeeper to the incoming virus by affecting infection rate of the midgut, intensity of infection, and dissemination from the midgut to secondary tissues.</p

Dual destructive and protective roles of adaptive immunity in neurodegenerative disorders.

Inappropriate T cell responses in the central nervous system (CNS) affect the pathogenesis of a broad range of neuroinflammatory and neurodegenerative disorders that include, but are not limited to, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer\u27s disease and Parkinson\u27s disease. On the one hand immune responses can exacerbate neurotoxic responses; while on the other hand, they can lead to neuroprotective outcomes. The temporal and spatial mechanisms by which these immune responses occur and are regulated in the setting of active disease have gained significant recent attention. Spatially, immune responses that affect neurodegeneration may occur within or outside the CNS. Migration of antigen-specific CD4+ T cells from the periphery to the CNS and consequent immune cell interactions with resident glial cells affect neuroinflammation and neuronal survival. The destructive or protective mechanisms of these interactions are linked to the relative numerical and functional dominance of effector or regulatory T cells. Temporally, immune responses at disease onset or during progression may exhibit a differential balance of immune responses in the periphery and within the CNS. Immune responses with predominate T cell subtypes may differentially manifest migratory, regulatory and effector functions when triggered by endogenous misfolded and aggregated proteins and cell-specific stimuli. The final result is altered glial and neuronal behaviors that influence the disease course. Thus, discovery of neurodestructive and neuroprotective immune mechanisms will permit potential new therapeutic pathways that affect neuronal survival and slow disease progression

Infectious alphavirus production from a simple plasmid transfection+

We have developed a new method for producing infectious double subgenomic alphaviruses from plasmids transfected into mammalian cells. A double subgenomic Sindbis virus (TE3'2J) was transcribed from a cytomegalovirus PolII promoter, which results in the production of infectious virus. Transfection of as little as 125 ng of plasmid is able to produce 1 × 108 plaque forming units/ml (PFU/ml) of infectious virus 48 hours post-transfection. This system represents a more efficient method for producing recombinant Sindbis viruses

Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes

<p>Abstract</p> <p>Background</p> <p>Arthropod-borne viruses (arboviruses) can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi) is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family <it>Togaviridae</it>; genus <it>Alphavirus</it>) that expresses B2 protein of Flock House virus (FHV; family <it>Nodaviridae</it>; genus <it>Alphanodavirus</it>), a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition.</p> <p>Results</p> <p>B2 protein expression from SINV (TE/3'2J) inhibited the accumulation of non-specific small RNAs in <it>Aedes aegypti </it>mosquito cell culture and virus-specific small RNAs both in infected cell culture and <it>Ae. aegypti </it>mosquitoes. More viral genomic and subgenomic RNA accumulated in cells and mosquitoes infected with TE/3'2J virus expressing B2 (TE/3'2J/B2) compared to TE/3'2J and TE/3'2J virus expressing GFP. TE/3'2J/B2 exhibited increased infection rates, dissemination rates, and infectious virus titers in mosquitoes following oral bloodmeal. Following infectious oral bloodmeal, significantly more mosquitoes died when TE/3'2J/B2 was ingested. The virus was 100% lethal following intrathoracic inoculation of multiple mosquito species and lethality was dose-dependent in <it>Ae. aegypti</it>.</p> <p>Conclusion</p> <p>We show that RNAi is active in <it>Ae. aegypti </it>cell culture and that B2 protein inhibits RNAi in mosquito cells when expressed by a recombinant SINV. Also, SINV more efficiently replicates in mosquito cells when RNAi is inhibited. Finally, TE/3'2J/B2 kills mosquitoes in a dose-dependent manner independent of infection route and mosquito species.</p

Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes

BACKGROUND: To be transmitted by its mosquito vector, dengue virus (DENV) must infect midgut epithelial cells, replicate and disseminate into the hemocoel, and finally infect the salivary glands, which is essential for transmission. The extrinsic incubation period (EIP) is very relevant epidemiologically and is the time required from the ingestion of virus until it can be transmitted to the next vertebrate host. The EIP is conditioned by the kinetics and tropisms of virus replication in its vector. Here we document the virogenesis of DENV-2 in newly-colonized Aedes aegypti mosquitoes from Chetumal, Mexico in order to understand better the effect of vector-virus interactions on dengue transmission. RESULTS: After ingestion of DENV-2, midgut infections in Chetumal mosquitoes were characterized by a peak in virus titers between 7 and 10 days post-infection (dpi). The amount of viral antigen and viral titers in the midgut then declined, but viral RNA levels remained stable. The presence of DENV-2 antigen in the trachea was positively correlated with virus dissemination from the midgut. DENV-2 antigen was found in salivary gland tissue in more than a third of mosquitoes at 4 dpi. Unlike in the midgut, the amount of viral antigen (as well as the percent of infected salivary glands) increased with time. DENV-2 antigen also accumulated and increased in neural tissue throughout the EIP. DENV-2 antigen was detected in multiple tissues of the vector, but unlike some other arboviruses, was not detected in muscle. CONCLUSION: Our results suggest that the EIP of DENV-2 in its vector may be shorter that the previously reported and that the tracheal system may facilitate DENV-2 dissemination from the midgut. Mosquito organs (e.g. midgut, neural tissue, and salivary glands) differed in their response to DENV-2 infection

Aedes aegypti uses RNA interference in defense against Sindbis virus infection

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is an important anti-viral defense mechanism. The <it>Aedes aegypti </it>genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use <it>Ae. aegypti </it>as a model system to determine how the mosquito's anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family <it>Togaviridae</it>, genus <it>Alphavirus</it>).</p> <p>Results</p> <p>SINV (TR339-eGFP) (+) strand RNA, infectious virus titers and infection rates transiently increased in mosquitoes following dsRNA injection to cognate <it>Ago2</it>, <it>Dcr2</it>, or <it>TSN </it>mRNAs. Detection of SINV RNA-derived small RNAs at 2 and 7 days post-infection in non-silenced mosquitoes provided important confirmation of RNAi pathway activity. Two different recombinant SINV viruses (MRE16-eGFP and TR339-eGFP) with significant differences in infection kinetics were used to delineate vector/virus interactions in the midgut. We show virus-dependent effects on RNAi component transcript and protein levels during infection. Monitoring midgut <it>Ago2</it>, <it>Dcr2</it>, and <it>TSN </it>transcript levels during infection revealed that only <it>TSN </it>transcripts were significantly increased in midguts over blood-fed controls. Ago2 protein levels were depleted immediately following a non-infectious bloodmeal and varied during SINV infection in a virus-dependent manner.</p> <p>Conclusion</p> <p>We show that silencing RNAi components in <it>Ae. aegypti </it>results in transient increases in SINV replication. Furthermore, <it>Ae. aegypti </it>RNAi is active during SINV infection as indicated by production of virus-specific siRNAs. Lastly, the RNAi response varies in a virus-dependent manner. These data define important features of RNAi anti-viral defense in <it>Ae. aegypti</it>.</p

The Impact of NLO-Corrections on the Determination of the $\bar{u},\bar{d} Content of Nucleons from Drell-Yan Production

The interpretation of Drell-Yan production in terms of the antiquark densities depends on NLO corrections. Besides the NLO corrections to the familiar annihilation $q\bar{q}\to \gamma^* \to l^+ l^-$, there is a substantial contribution from the QCD Compton subprocesses $gq \to q\gamma^* \to q l^+ l^-$ and $g\bar{q} \to q\gamma^* \to q l^+ l^-$. The beam and target dependence of the two classes of corrections is different. We discuss the impact of this difference on the determination of the $\bar{d}-\bar{u}$ asymmetry in the proton from the comparison of the $pp$ and $pn$ Drell-Yan production.Comment: 4 pages, 1 eps-figure. To be published in Proceedings of DIS'9

Small RNA profiling of Dengue virus-mosquito interactions implicates the PIWI RNA pathway in anti-viral defense

<p>Abstract</p> <p>Background</p> <p>Small RNA (sRNA) regulatory pathways (SRRPs) are important to anti-viral defence in mosquitoes. To identify critical features of the virus infection process in Dengue serotype 2 (DENV2)-infected <it>Ae. aegypti</it>, we deep-sequenced small non-coding RNAs. Triplicate biological replicates were used so that rigorous statistical metrics could be applied.</p> <p>Results</p> <p>In addition to virus-derived siRNAs (20-23 nts) previously reported for other arbovirus-infected mosquitoes, we show that PIWI pathway sRNAs (piRNAs) (24-30 nts) and unusually small RNAs (usRNAs) (13-19 nts) are produced in DENV-infected mosquitoes. We demonstrate that a major catalytic enzyme of the siRNA pathway, Argonaute 2 (Ago2), co-migrates with a ~1 megadalton complex in adults prior to bloodfeeding. sRNAs were cloned and sequenced from Ago2 immunoprecipitations. Viral sRNA patterns change over the course of infection. Host sRNAs were mapped to the published aedine transcriptome and subjected to analysis using edgeR (Bioconductor). We found that sRNA profiles are altered early in DENV2 infection, and mRNA targets from mitochondrial, transcription/translation, and transport functional categories are affected. Moreover, small non-coding RNAs (ncRNAs), such as tRNAs, spliceosomal U RNAs, and snoRNAs are highly enriched in DENV-infected samples at 2 and 4 dpi.</p> <p>Conclusions</p> <p>These data implicate the PIWI pathway in anti-viral defense. Changes to host sRNA profiles indicate that specific cellular processes are affected during DENV infection, such as mitochondrial function and ncRNA levels. Together, these data provide important progress in understanding the DENV2 infection process in <it>Ae. aegypti</it>.</p

Nonperturbative QCD Vacuum Effects in Nonlocal Quark Dynamics

A straightforward calculation reveals the essentially nonlocal character of the leading heavy $Q\bar{Q}$ interaction arising from nonperturbative gluon field correlations in the model of a fluctuating QCD vacuum. In light of this quarkonium spin splitting ratio predictions which have supported the scalar confinement ansatz are reconsidered as a specific example of possible consequences for spectroscopy.Comment: Latex, 9 page