Persistent Replication of a Chikungunya Virus Replicon in Human Cells is Associated with Presence of Stable Cytoplasmic Granules Containing Non-structural Protein 3

Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months or even years in patients. The non-structural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used sub-diffraction multi-color microscopy for spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate replicon RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and stress granule assembly factors 1 and 2 (G3BP1/2); (ii) were next to double-stranded RNA foci and nsP1-positive structures; and (iii) were close to the nuclear membrane and the nuclear pore complex protein Nup98. Analysis of protein turnover and mobility by live-cell microscopy revealed that granules could persist for hours to days, accumulated newly synthesized protein, and moved through the cytoplasm at varying speeds. Granules also had a static internal architecture and were stable in cell lysates. Refractory cells that had cleared the non-cytotoxic replicon regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein-complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus).ImportanceChikungunya virus (CHIKV) is a re-emerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of non-cytotoxic replicons (Utt, Das, Varjak, Lulla, Lulla, Merits, J Virol 89:3145-62, 2015, doi:10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged non-structural protein 3. The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms

Antiviral activity of silymarin against chikungunya virus

Citation: Lani, R., Hassandarvish, P., Chiam, C. W., Moghaddam, E., Chu, J. J. H., Rausalu, K., . . . Zandi, K. (2015). Antiviral activity of silymarin against chikungunya virus. Scientific Reports, 5, 10. doi:10.1038/srep11421The mosquito-borne chikungunya virus (CHIKV) causes chikungunya fever, with clinical presentations such as severe back and small joint pain, and debilitating arthritis associated with crippling pains that persist for weeks and even years. Although there are several studies to evaluate the efficacy of drugs against CHIKV, the treatment for chikungunya fever is mainly symptom-based and no effective licensed vaccine or antiviral are available. Here, we investigated the antiviral activity of three types of flavonoids against CHIKV in vitro replication. Three compounds: silymarin, quercetin and kaempferol were evaluated for their in vitro antiviral activities against CHIKV using a CHIKV replicon cell line and clinical isolate of CHIKV of Central/East African genotype. A cytopathic effect inhibition assay was used to determine their activities on CHIKV viral replication and quantitative reverse transcription PCR was used to calculate virus yield. Antiviral activity of effective compound was further investigated by evaluation of CHIKV protein expression using western blotting for CHIKV nsP1, nsP3, and E2E1 proteins. Briefly, silymarin exhibited significant antiviral activity against CHIKV, reducing both CHIKV replication efficiency and down-regulating production of viral proteins involved in replication. This study may have important consequence for broaden the chance of getting the effective antiviral for CHIKV infection

Chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen

Re-emergence of chikungunya virus, a mosquito-transmitted pathogen, is of serious public health concern. In the past 15 years, after decades of infrequent, sporadic outbreaks, the virus has caused major epidemic outbreaks in Africa, Asia, the Indian Ocean, and more recently the Caribbean and the Americas. Chikungunya virus is mainly transmitted by Aedes aegypti mosquitoes in tropical and subtropical regions, but the potential exists for further spread because of genetic adaptation of the virus to Aedes albopictus, a species that thrives in temperate regions. Chikungunya virus represents a substantial health burden to affected populations, with symptoms that include severe joint and muscle pain, rashes, and fever, as well as prolonged periods of disability in some patients. The inflammatory response coincides with raised levels of immune mediators and infiltration of immune cells into infected joints and surrounding tissues. Animal models have provided insights into disease pathology and immune responses. Although host innate and adaptive responses have a role in viral clearance and protection, they can also contribute to virus-induced immune pathology. Understanding the mechanisms of host immune responses is essential for the development of treatments and vaccines. Inhibitory compounds targeting key inflammatory pathways, as well as attenuated virus vaccines, have shown some success in animal models, including an attenuated vaccine strain based on an isolate from La Reunion incorporating an internal ribosome entry sequence that prevents the virus from infecting mosquitoes and a vaccine based on virus-like particles expressing envelope proteins. However, immune correlates of protection, as well as the safety of prophylactic and therapeutic candidates, are important to consider for their application in chikungunya infections. In this Review, we provide an update on chikungunya virus with regard to its epidemiology, molecular virology, virus-host interactions, immunological responses, animal models, and potential antiviral therapies and vaccines

Promoting self-disclosure from prison inmates

This study examined the effects of interviewer self-disclosure on the self-disclosure of 40 male prison inmates and 40 male university students. A four-point scale was used for rating the intimacy of selfdisclosure, the number of statements were counted for scoring the breadth of self-disclosure, and a stop watch was used to time the duration of self-disclosure. The three measures correlated highly with each others indicating all were good measures of self-disclosure. The self-disclosure reciprocity effect was demonstrated with both prison inmates and university students. It was also found that overall, prison inmates self-disclosed significantly less < .05) than university students. The personality variables of extroversion and neuroticism, as measured by the Eysenck Personality Inventory, were also examined. Prison inmates were significantly lower than university students on extroversion < .001) and no different than university students on neuroticism. It was also found that extroversion was positively related to self-disclosure. However, neuroticism was not related to either self-disclosure or to extroversion

Design and use of Chikungunya virus replication templates utilizing mammalian and mosquito RNA polymerase I mediated transcription

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. It has a positive sense RNA genome that also serves as the mRNA for four non-structural proteins (nsPs) representing subunits of the viral replicase. Coupling of nsP and RNA synthesis complicates analysis of viral RNA replication. We developed trans-replication systems, where production of replication competent RNA and expression of viral replicase are uncoupled. Mammalian and mosquito RNA polymerase I promoters were used to produce non-capped RNA templates, which are poorly translated relative to CHIKV replicase generated capped RNAs. It was found that, in human cells, constructs driven by RNA polymerase I promoters of human and Chinese hamster origin performed equally well. In contrast, RNA polymerase I promoters from Aedes mosquitoes exhibited strong species specificity. In both mammalian and mosquito cells, novel trans-replicase assays had exceptional sensitivity, with up to 105-fold higher reporter expression in the presence of replicase relative to background. Using this highly sensitive assay to analyse CHIKV nsP1 functionality, several mutations that severely reduced, but did not completely block, CHIKV replicase activity were identified: (i) tagging the N-terminus of nsP1 with eGFP; (ii) mutations D63A and Y248A blocking the RNA capping; (iii) mutation R252E affecting nsP1 membrane anchoring. In contrast, a mutation in the nsP1 palmitoylation site completely inactivated CHIKV replicase in both human and mosquito cells and was lethal for the virus. Our data confirms that this novel system provides a valuable tool to study CHIKV replicase, RNA replication and virus-host interactions

Spindle-E acts antivirally against alphaviruses in mosquito cells

Mosquitoes transmit several human- and animal-pathogenic alphaviruses (Togaviridae family). In alphavirus-infected mosquito cells two different types of virus-specific small RNAs are produced as part of the RNA interference response: short-interfering (si)RNAs and PIWI-interacting (pi)RNAs. The siRNA pathway is generally thought to be the main antiviral pathway. Although an antiviral activity has been suggested for the piRNA pathway its role in host defences is not clear. Knock down of key proteins of the piRNA pathway (Ago3 and Piwi5) in Aedesaegypti-derived cells reduced the production of alphavirus chikungunya virus (CHIKV)-specific piRNAs but had no effect on virus replication. In contrast, knock down of the siRNA pathway key protein Ago2 resulted in an increase in virus replication. Similar results were obtained when expression of Piwi4 was silenced. Knock down of the helicase Spindle-E (SpnE), an essential co-factor of the piRNA pathway in Drosophila melanogaster, resulted in increased virus replication indicating that SpnE acts as an antiviral against alphaviruses such as CHIKV and the related Semliki Forest virus (SFV). Surprisingly, this effect was found to be independent of the siRNA and piRNA pathways in Ae. aegypti cells and specific for alphaviruses. This suggests a small RNA-independent antiviral function for this protein in mosquitoes

Is the ADP ribose site of the Chikungunya virus NSP3 Macro domain a target for antiviral approaches?

Chikungunya virus (CHIKV) is a mosquito-transmitted virus of special concern as it causes Chikungunya fever, characterized by an acute febrile illness, rash, and arthralgia that can progress to chronic and debilitating arthritic symptoms. The effects of climate change on the geographic distribution of the mosquito vector has the potential to expose more of the globe to this virus. No antiviral agents or vaccines are currently available against CHIKV infection and the development of novel therapies that may lead to a future treatment is therefore necessary. In this context, the ADP-ribose binding site of the CHIKV nsP3 macro domain has been reported as a potential target for the development of antivirals. Mutations in the ADP-ribose binding site demonstrated decreased viral replication in cell culture and reduced virulence. In this study, 48,750 small molecules were screened in silico for their ability to bind to the ADP-ribose binding site of the CHIKV nsP3 macro domain. From this in silico analysis, 12 molecules were selected for in vitro analysis using a CHIKV subgenomic replicon in Huh-7 cells. Cell viability and CHIKV replication were evaluated and molecules C5 and C13 demonstrated 53 and 66% inhibition of CHIKV replication, respectively. By using a CHIKV-Dual luciferase replicon contain two reporter genes, we also demonstrated that the treatment with either compounds are probably interfering in the early replication rather than after RNA replication has occurred

Semliki Forest virus strongly reduces mosquito host defence signaling

The Alphavirus genus within the Togaviridae family contains several important mosquito-borne arboviruses. Other than the antiviral activity of RNAi, relatively little is known about alphavirus interactions with insect cell defences. Here we show that Semliki Forest virus (SFV) infection of Aedes albopictus-derived U4.4 mosquito cells reduces cellular gene expression. Activation prior to SFV infection of pathways involving STAT/IMD, but not Toll signaling reduced subsequent virus gene expression and RNA levels. These pathways are therefore not only able to mediate protective responses against bacteria but also arboviruses. However, SFV infection of mosquito cells did not result in activation of any of these pathways and suppressed their subsequent activation by other stimuli