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Antiviral Activity of the Combination of Interferon and Ribavirin Against Chikungunya Virus: Are the Results Conclusive?
... The main strength of our paper lies in the set-up of a C. trachomatis infection model using a McCoy fibroblast cell line that is known to not constitutively synthesize IFN-ε [30], allowing us to better investigate the potential anti-chlamydial effects of this cytokine [41]. Moreover, the range of concentrations (25-100 ng/mL) of IFN-ε assayed correspond to those of well characterized IFN-I, such as IFNα and β [45]. ...
... However, beside the multiple host factors involved in the activation of immune response to viral infection, CHIKV has developed mechanisms to evade early cellular immunity by, e.g., circumventing the antiviral activity of type I IFNs [42]. Interestingly, the CHIKV IOL strain with the A226V mutation obtained from patients during the epidemic of 2007 in Italy were more sensitive to type I IFNs compared to the viral strains without A226V, suggesting that level of resistance of the CHIKV to the antiviral action of IFN could actually be virusstrain dependent [43,44]. ...
Chikungunya virus (CHIKV) is a mosquito-borne infection that is emerging in temperate areas of Europe, following the expansion of one of its vector species, Aedes albopictus. Although CHIKV fever is a self-limiting disease, with a clinical syndrome often resolving within few days, it can also cause severe sequelae, including chronic polyarthralgia lasting up to 5 years. Additionally, CHIKV outbreaks may limit blood bank donations, adding economic burden on the health system. Public health authorities in Europe need to increase their preparedness against this emerging threat. Two large CHIKV outbreaks occurred in Italy in 2007 and 2017, with hundreds of cases and significant geographical spread. The aim of this paper is to review and compare the 2 Italian outbreaks in terms of available estimates of key epidemiological features, patient clinical presentation, virus and immunological characteristics, and public health response. Recommendations for public health and future directions for research are also discussed and highlighted.
Key results
Both outbreaks started in small towns, but cases were also detected in nearby larger cities where transmission was limited to small clusters. The time spans between the first and the last symptom onsets were similar between the 2 outbreaks, and the delay from the symptom onset of the index case and the first case notified was considerable. Comparable infection and transmission rates were observed in laboratory. The basic reproductive number (R0) was estimated in the range of 1.8–6 (2007) and 1.5–2.6 (2017). Clinical characteristics were similar between outbreaks, and no acute complications were reported, though a higher frequency of ocular symptoms, myalgia, and rash was observed in 2017. Very little is known about the immune mediator profile of CHIKV-infected patients during the 2 outbreaks. Regarding public health responses, after the 2007 outbreak, the Italian Ministry of Health developed national guidelines to implement surveillance and good practices to prevent and control autochthonous transmission. However, only a few regional authorities implemented it, and the perception of outbreak risk and knowledge of clinical symptoms and transmission dynamics by general practitioners remained low.
Major conclusions
Efforts should be devoted to developing suitable procedures for early detection of virus circulation in the population, possibly through the analysis of medical records in near real time. Increasing the awareness of CHIKV of general practitioners and public health officials through tailored education may be effective, especially in small coastal towns where the outbreak risk may be higher. A key element is also the shift of citizen awareness from considering Aedes mosquitoes not only as a nuisance problem but also as a public health one. We advocate the need of strengthening the surveillance and of promoting the active participation of the communities to prevent and contain future outbreaks.
... The combinations of different DAAs could be the holy grail of HCV therapy, as some researchers suggest that they have the potential to cure HCV infection completely [221]. Ribavirin and interferon alfa may also exert a strong synergistic antiviral effect against Chikungunya virus infection [235,236]. In vitro studies on Chikungunya virus-infected human cell lines (Vero, HUH-7, and A549 cells) demonstrated cell-line sensitivity to ribavirin, interferon alfa, and favipiravir drugs. ...
Substance-use disorder represents a frequently hidden non-communicable chronic disease. Patients with intravenous drug addiction are at high risk of direct exposure to a variety of viral infections and are considered to be the largest subpopulation infected with the hepatitis C virus. Ribavirin is a synthetic nucleoside analog that has been used as an integral component of hepatitis C therapy. However, ribavirin medication is quite often associated with pronounced psychiatric adverse effects. It is not well understood to what extent ribavirin per se contributes to changes in drug-related neurobehavioral disturbances, especially in the case of psychostimulant drugs such as amphetamine. It is now well-known that repeated amphetamine usage produces psychosis in humans and behavioral sensitization in animals. On the other hand, ribavirin has an affinity for adenosine A1 receptors that antagonistically modulate the activity of dopamine D1 receptors, which play a critical role in the development of behavioral sensitization. This review will focus on the current knowledge of neurochemical/neurobiological changes that exist in the psychostimulant drug-addicted brain itself and the antipsychotic-like efficiency of adenosine agonists. Particular attention will be paid to the potential side effects of ribavirin therapy, and the opportunities and challenges related to its application in already existing psychostimulant-use disorder.
Chikungunya virus (CHIKV) is the mosquito-borne alphavirus that is the etiologic agent of massive outbreaks of arthralgic febrile illness that recently affected millions of people in Africa and Asia. The only CHIKV vaccine that has been tested in humans, strain 181/clone 25, is a live-attenuated derivative of Southeast Asian human isolate strain AF15561. The vaccine was immunogenic in phase I and II clinical trials; however, it induced transient arthralgia in 8% of the vaccinees. There are five amino acid differences between the vaccine and its parent, as well as five synonymous mutations, none of which involves cis-acting genome regions known to be responsible for replication or packaging. To identify the determinants of attenuation, we therefore tested the five nonsynonymous mutations by cloning them individually or in different combinations into infectious clones derived from two wild-type (WT) CHIKV strains, La Reunion and AF15561. Levels of virulence were compared with those of the WT strains and the vaccine strain in two different murine models: infant CD1 and adult A129 mice. An attenuated phenotype indistinguishable from that of the 181/clone 25 vaccine strain was obtained by the simultaneous expression of two E2 glycoprotein substitutions, with intermediate levels of attenuation obtained with the single E2 mutations. The other three amino acid mutations, in nsP1, 6K, and E1, did not have a detectable effect on CHIKV virulence. These results indicate that the attenuation of strain 181/clone 25 is mediated by two point mutations, explaining the phenotypic instability observed in human vaccinees and also in our studies.
Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus responsible for the first autochthonous Italian outbreak in 2007. A226V mutation in E1 has been associated with enhanced replication in A. albopictus vector. Possible involvement of this mutation in enhanced infection capability in primate cells and sensitivity to exogenous interferon (IFN)-a was investigated. No significant differences were observed between the two isolates in terms of replication kinetic, virus yield and cytopathic effect (CPE). Interestingly, the A226V-carrying strain was more susceptible to the antiviral action of recombinant IFN-a. The interplay between A226V mutation and innate defence mechanisms needs further investigation.
Chikungunya virus (CHIKV) is an emerging human pathogen transmitted by mosquitoes. Like that of other alphaviruses, CHIKV
replication causes general host shutoff, leading to severe cytopathicity in mammalian cells, and inhibits the ability of infected
cells to respond to interferon (IFN). Recent research, however, suggests that alphaviruses may have additional mechanisms
to circumvent the host's antiviral IFN response. Here we show that CHIKV replication is resistant to inhibition by interferon
once RNA replication has been established and that CHIKV actively suppresses the antiviral IFN response by preventing IFN-induced
gene expression. Both CHIKV infection and CHIKV replicon RNA replication efficiently blocked STAT1 phosphorylation and/or
nuclear translocation in mammalian cells induced by either type I or type II IFN. Expression of individual CHIKV nonstructural
proteins (nsPs) showed that nsP2 was a potent inhibitor of IFN-induced JAK-STAT signaling. In addition, mutations in CHIKV-nsP2
(P718S) and Sindbis virus (SINV)-nsP2 (P726S) that render alphavirus replicons noncytopathic significantly reduced JAK-STAT
inhibition. This host shutoff-independent inhibition of IFN signaling by CHIKV is likely to have an important role in viral
pathogenesis.
Ribavirin (RBV) is a synthetic nucleoside analog with broad spectrum antiviral activity. Although RBV is approved for the treatment of hepatitis C virus, respiratory syncytial virus, and Lassa fever virus infections, its mechanism of action and therapeutic efficacy remains highly controversial. Recent reports show that the development of cell-based resistance after continuous RBV treatment via decreased RBV uptake can greatly limit its efficacy. Here, we examined whether certain cell types are naturally resistant to RBV even without prior drug exposure. Seven different cell lines from various host species were compared for RBV antiviral activity against two nonsegmented negative-strand RNA viruses, vesicular stomatitis virus (VSV, a rhabdovirus) and Sendai virus (SeV, a paramyxovirus). Our results show striking differences between cell types in their response to RBV, ranging from virtually no antiviral effect to very effective inhibition of viral replication. Despite differences in viral replication kinetics for VSV and SeV in the seven cell lines, the observed pattern of RBV resistance was very similar for both viruses, suggesting that cellular rather than viral determinants play a major role in this resistance. While none of the tested cell lines was defective in RBV uptake, dramatic variations were observed in the long-term accumulation of RBV in different cell types, and it correlated with the antiviral efficacy of RBV. While addition of guanosine neutralized RBV only in cells already highly resistant to RBV, actinomycin D almost completely reversed the RBV effect (but not uptake) in all cell lines. Together, our data suggest that RBV may inhibit the same virus via different mechanisms in different cell types depending on the intracellular RBV metabolism. Our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro.
Chikungunya virus is a mosquito-borne arthrogenic alphavirus that has recently reemerged to produce the largest epidemic ever documented for this virus. Here we describe a new adult wild-type mouse model of chikungunya virus arthritis, which recapitulates the self-limiting arthritis, tenosynovitis, and myositis seen in humans. Rheumatic disease was associated with a prolific infiltrate of monocytes, macrophages, and NK cells and the production of monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma). Infection with a virus isolate from the recent Reunion Island epidemic induced significantly more mononuclear infiltrates, proinflammatory mediators, and foot swelling than did an Asian isolate from the 1960s. Primary mouse macrophages were shown to be productively infected with chikungunya virus; however, the depletion of macrophages ameliorated rheumatic disease and prolonged the viremia. Only 1 microg of an unadjuvanted, inactivated, whole-virus vaccine derived from the Asian isolate completely protected against viremia and arthritis induced by the Reunion Island isolate, illustrating that protection is not strain specific and that low levels of immunity are sufficient to mediate protection. IFN-alpha treatment was able to prevent arthritis only if given before infection, suggesting that IFN-alpha is not a viable therapy. Prior infection with Ross River virus, a related arthrogenic alphavirus, and anti-Ross River virus antibodies protected mice against chikungunya virus disease, suggesting that individuals previously exposed to Ross River virus should be protected from chikungunya virus disease. This new mouse model of chikungunya virus disease thus provides insights into pathogenesis and a simple and convenient system to test potential new interventions.
Chikungunya virus (CHIKV) is an emerging arbovirus associated with several recent large-scale epidemics. The 2005-2006 epidemic on Reunion island that resulted in approximately 266,000 human cases was associated with a strain of CHIKV with a mutation in the envelope protein gene (E1-A226V). To test the hypothesis that this mutation in the epidemic CHIKV (strain LR2006 OPY1) might influence fitness for different vector species, viral infectivity, dissemination, and transmission of CHIKV were compared in Aedes albopictus, the species implicated in the epidemic, and the recognized vector Ae. aegypti. Using viral infectious clones of the Reunion strain and a West African strain of CHIKV, into which either the E1-226 A or V mutation was engineered, we demonstrated that the E1-A226V mutation was directly responsible for a significant increase in CHIKV infectivity for Ae. albopictus, and led to more efficient viral dissemination into mosquito secondary organs and transmission to suckling mice. This mutation caused a marginal decrease in CHIKV Ae. aegypti midgut infectivity, had no effect on viral dissemination, and was associated with a slight increase in transmission by Ae. aegypti to suckling mice in competition experiments. The effect of the E1-A226V mutation on cholesterol dependence of CHIKV was also analyzed, revealing an association between cholesterol dependence and increased fitness of CHIKV in Ae. albopictus. Our observation that a single amino acid substitution can influence vector specificity provides a plausible explanation of how this mutant virus caused an epidemic in a region lacking the typical vector. This has important implications with respect to how viruses may establish a transmission cycle when introduced into a new area. Due to the widespread distribution of Ae. albopictus, this mutation increases the potential for CHIKV to permanently extend its range into Europe and the Americas.
Introduction
We evaluated the antiviral activities of ribavirin (RBV) and interferon-α (IFN) as mono- and combination therapy against chikungunya virus (CHIKV).
Methods
Vero cells were infected with CHIKV in the presence of RBV and/or IFN and viral production was quantified by plaque assay. A mathematical model was fit to the data to identify drug interactions for effect. We ran simulations using the best-fit model parameters to predict the antiviral activity associated with clinically relevant regimens of RBV and IFN as combination therapy. The model predictions were validated using the hollow fiber infection model (HFIM) system.
Results
RBV and IFN were effective against CHIKV as monotherapy at supraphysiological concentrations. However, RBV and IFN were highly synergistic for antiviral effect when administered as combination therapy. Simulations with our mathematical model predicted that a standard clinical regimen of RBV+IFN would inhibit CHIKV burden by 2.5-log10 following 24h of treatment. In the HFIM system, RBV+IFN at clinical exposures resulted in a 2.1-log10 decrease of CHIKV burden following 24h of therapy. These findings validate the prediction made by the mathematical model.
Conclusions
These studies illustrate the promise of RBV+IFN as a potential therapeutic strategy for the treatment of CHIKV infections.
In humans, chikungunya virus (CHIKV) infection causes fever, rash, and acute and persisting polyarthralgia/arthritis associated with joint swelling. We report a new CHIKV disease model in adult mice that distinguishes the wild-type CHIKV-LR strain from the live-attenuated vaccine strain (CHIKV-181/25). Although eight-week old normal mice inoculated in the hind footpad developed no hind limb swelling with either virus, CHIKV-LR replicated in musculoskeletal tissues and caused detectable inflammation. In mice deficient in STAT1-dependent interferon (IFN) responses, CHIKV-LR caused significant swelling of the inoculated and contralateral limbs and dramatic inflammatory lesions, while CHIKV-181/25 vaccine and another arthritogenic alphavirus, Sindbis, failed to induce swelling. IFN responses suppressed CHIKV-LR and CHIKV-181/25 replication equally in dendritic cells in vitro whereas macrophages were refractory to infection independently of STAT1-mediated IFN responses. Glycosaminoglycan (GAG) binding may be a CHIKV vaccine attenuation mechanism as CHIKV-LR infectivity was not dependent upon GAG, while CHIKV-181/25 was highly dependent.
A clone of Vero cells was isolated and shown to be totally unable to synthesize interferon and insensitive to the toxic effect of poly(rI).poly(rC) treatment. Cells of this clone and mouse L cells were fused by treatment with polyethylene glycol or Sendai virus. Hybrid cell clones were isolated following selection in medium containing hypoxanthine, thymidine and ouabain. The hybrids were sensitive to the antiviral effect of poly(rI).poly(rC) and synthesized mouse, but not primate, interferon. It is proposed that in Vero cells, the gene for interferon synthesis is defective or absent.
Chikungunya virus (CHIKV), which is transmitted by Aedes spp mosquitoes, has recently caused several outbreaks on islands in the Indian Ocean and on the Indian subcontinent. We report on an outbreak in Italy.
After reports of a large number of cases of febrile illness of unknown origin in two contiguous villages in northeastern Italy, an outbreak investigation was done to identify the primary source of infection and modes of transmission. An active surveillance system was also implemented. The clinical case definition was presentation with fever and joint pain. Blood samples were gathered and analysed by PCR and serological assays to identify the causal agent. Locally captured mosquitoes were also tested by PCR. Phylogenetic analysis of the CHIKV E1 region was done.
Analysis of samples from human beings and from mosquitoes showed that the outbreak was caused by CHIKV. We identified 205 cases of infection with CHIKV between July 4 and Sept 27, 2007. The presumed index case was a man from India who developed symptoms while visiting relatives in one of the villages. Phylogenetic analysis showed a high similarity between the strains found in Italy and those identified during an earlier outbreak on islands in the Indian Ocean. The disease was fairly mild in nearly all cases, with only one reported death.
This outbreak of CHIKV disease in a non-tropical area was to some extent unexpected and emphasises the need for preparedness and response to emerging infectious threats in the era of globalisation.