Mechanistic Insights Into the Inhibition of Dengue Virus NS5 Methyltransferase by Herbacetin.

Chikungunya (CHIKV) and dengue (DENV) viruses pose a public health risk and lack antiviral treatments. Structure-based molecular docking of a natural MTase substrates library identified herbacetin (HC) and caffeic acid phenethyl ester (CAPE) as potential CHIKV nsP1 and DENV NS5 MTase inhibitors. Binding affinities and MTase inhibition were confirmed using purified proteins. The crystal structure of DENV 3 NS5 MTase and CAPE complex revealed CAPE binding at viral RNA capping sites. Interestingly, HC and CAPE depleted polyamines crucial for RNA virus replication and decreased viral titer with IC50 values of ~ 13.44 and ~ 0.57 μm against CHIKV, and ~ 7.24 and ~ 1.01 μm against DENV 3, respectively. Polyamine addition did not reverse the antiviral effects, suggesting a dual inhibition mechanism. Impact statement This study reveals the antiviral potential of natural small molecules, Herbacetin (HC) and Caffeic acid phenethyl ester (CAPE) against Dengue and Chikungunya viruses. The molecules deplete polyamine levels and directly inhibit viral methyltransferases. This study opens new avenues for developing antiviral strategies that target both host factors and viral components.

Risk for Emergence of Dengue and Chikungunya Virus in Israel

In India, vector-borne diseases, dengue and chikungunya are major public health concerns. In recent decades, dengue outbreaks have been reported in almost every part of India. In 2016, India recorded 101388 dengue cases and 210 deaths, including 4337 cases and six deaths in Delhi, whereas Chikungunya outbreaks were reported from several states in 2006, with 1.3 million cases. The Dengue virus (DENV) and Chikungunya virus (CHIKV) are both transmitted by the same Aedes mosquito species. DENV and CHIKV co-infections have been reported in 13 of 98 countries, with both viruses being transmitted locally. The reasons for the sudden upsurge in cases of these diseases are undetermined. From March to December 2016, a study was carried out in 66 localities of Delhi in collaboration with the Municipal Corporation of Delhi. Localities were selected on the basis of confirmed dengue cases reported during the last five years and the study area was visited once a month. A door-to-door entomological survey was conducted to identify Aedes breeding in all water-filled containers in and around houses. Both immature and mature stages of Aedes mosquitoes were collected. Mosquitoes were pooled (n≤10 each for male and female) breeding site-wise and stored in Trizol at -80°C. The Chikungunya and dengue viruses were detected using a multiplex RT-PCR. A total of 981 Aedes mosquitos were distributed among 146 Pools, and DENV and CHIKV were detected using Multiplex Reverse Transcriptase-PCR. Chikungunya virus was identified in 19 pools of females captured adults, whereas dengue virus was found in 8 pools of females captured adults. There was no evidence of coinfection in any of the pools. In endemic areas, continuous surveillance for both dengue and Chikungunya viruses is required to identify and characterize these viral pathogens. This information will also help implement effective strategies to combat outbreaks produced by these emerging viral pathogens.

The purpose of study was to isolate arboviruses from mosquitoes of different species in the cell culture and to identify them by using molecular and immunochemical techniques.Materials and methods. Viruses were isolated in C6/36 cell cultures. The pathogens were identified by using enzyme-linked immunosorbent assay (ELISA) kits for detection of antigens of dengue, Chikungunya, West Nile and Sindbis viruses as well as the reverse transcription polymerase chain reaction (RT-PCR) with specific primers and Sanger sequencing.Results. A total of 102 mosquitoes belonging to three genera, Culex spp, Culiseta spp., Aedes spp., were studied. Mosquitoes of each species or genus were divided into pools, each containing 4–5 mosquitoes. The study of suspensions of only 2 mosquito pools obtained from Aedes aegypti and Aedes albopictus, starting from the 3rd passage, showed changes in the C6/36 cell monolayer. Starting from the 4th passage, an antigen of Chikungunya virus was detected using ELISA test in the suspension obtained from the Aedes albopictus pool. Dengue virus was detected in the 5th passage from the materials obtained from the Aedes aegypti pool. Thus, antigens of the Chikungunya and dengue viruses were detected only in 2 of 23 examined pools of mosquitoes of different genera. Materials of the 5th passage were analyzed by RT-PCR with specific primers for dengue and Chikungunya viruses. It was confirmed that the isolate obtained from Aedes albopictus mosquitoes contained RNA of the Chikungunya virus and corresponded to the East/Central/South African genotype, while the isolate obtained from Aedes aegypti mosquitoes contained RNA of the dengue type 2 virus.Conclusion. The obtained nucleotide sequences of the Chikungunya virus were deposited in the GenBank international database under accession numbers MN271691 and MN271692.

Chikungunya virus: An emerging public health challenge for Pakistan

BackgroundDengue and chikungunya are arboviral diseases with overlapping clinical characteristics. Dengue virus (DENV) is endemic in Colombia, and in 2014/2015, the chikungunya virus (CHIKV) caused an epidemic that resulted in over 350,000 cases. Since then, both viruses have been actively co-circulating. The early and accurate identification of pediatric infection caused by DENV or CHIKV is essential for proper medical management. Given that subsequent infections and co-infections with DENV and CHIKV have been reported, virological and immunological factors may influence their clinical outcomes. Here, we analyzed the viremia, antigenemia, and virus-specific antibody responses in hospitalized children suspected of having dengue during the peak of CHIKV infections in Colombia.MethodsNinety-one children with a clinical diagnosis of dengue were included in the peak of the CHIKV epidemic (December 2014 to May 2015) at a reference healthcare center in Huila, south of Colombia. Multiplexed RT-qPCR for DENV, CHIKV, and ZIKV was performed, and DENV antigenemia was evaluated using an ELISA for the NS1 antigen. Commercial capture or in-house indirect NS1-based ELISAs were used to assess circulating DENV and CHIKV-IgM and IgG. Clinical and laboratory characteristics were analyzed during hospitalization, and convalescent follow-up was conducted for a fraction of children.ResultsDENV and CHIKV monoinfections were confirmed in 54% and 12% of children, respectively, with the expected virus-specific seroconversion in recovery. Overlapping infections occurred in 22% of the children, while 12% showed no detectable DENV or CHIKV infections. Abdominal pain, vomiting, hepatomegaly, and thrombocytopenia were common findings associated with DENV, while arthralgia and rash characterized CHIKV monoinfections. One fatal secondary DENV-3 monoinfection was registered, and DENV infection dominated the symptoms of overlapping infections without producing different clinical outcomes compared to monoinfections. Thirty-eight percent of children were seropositive for CHIKV-IgG, indicating a significant burden of CHIKV infection in the pediatric population shortly after its introduction in Colombia. The previous virus-specific IgG serostatus did not impact the clinical outcome of the current heterotypic arboviral infection.ConclusionThe pediatric population in southern Colombia was rapidly exposed to CHIKV infections during the first months following its arrival, with up to 12% of hospitalized children suspected of having dengue experiencing CHIKV monoinfection, supporting that complex and dynamic epidemiological patterns may lead to delayed or missed diagnoses. The overlapping infections of DENV and CHIKV were frequent and did not lead to worse clinical or fatal outcomes.

The Dengue virus (DENV) and Chikungunya virus (CHIKV) are the arboviruses that pose a threat to global public health. Coinfection and antibody-dependent enhancement are major areas of concern during DENV and CHIKV infections, which can alter the clinical severity. Acute hepatic illness is a common manifestation and major sign of disease severity upon infection with either dengue or chikungunya. Hence, in this study, we characterized the coexistence and interaction between both the viruses in human hepatic (Huh7) cells during the coinfection/superinfection scenario. We observed that prior presence of or subsequent superinfection with DENV enhanced CHIKV replication. However, prior CHIKV infection negatively affected DENV. In comparison to monoinfection, coinfection with both DENV and CHIKV resulted in lower infectivity as compared to monoinfections with modest suppression of CHIKV but dramatic suppression of DENV replication. Subsequent investigations revealed that subneutralizing levels of DENV or CHIKV anti-sera can respectively promote the ADE of CHIKV or DENV infection in FcγRII bearing human myelogenous leukemia cell line K562. Our observations suggest that CHIKV has a fitness advantage over DENV in hepatic cells and prior DENV infection may enhance CHIKV disease severity if the patient subsequently contracts CHIKV. This study highlights the natural possibility of dengue–chikungunya coinfection and their subsequent modulation in human hepatic cells. These observations have important implications in regions where both viruses are prevalent and calls for proper management of DENV-CHIKV coinfected patients.

Arbovirus epidemics are raging in tropical areas. Dengue virus (DENV), dengue shock syndrome (DSS), and dengue hemorrhagic fever (DHF) affect millions of individuals every year and cause significant mortality in Latin America, Africa, and Asia. Chikungunya virus (CHIKV) has caused recurrent epidemics in the Indian subcontinent and recent epidemics in Reunion and other islands in the Indian Ocean, with recent detection in areas of Europe. This issue of TRANFUSION includes two articles on the detection of DENV RNA in blood donors from epidemic areas, one article on the clearance of spiked DENV in the course of the manufacture of plasma derivatives, and one article on statistical modeling of transfusion risk during an epidemic of mosquito-borne CHIKV. The surprising seriousness of recurring epidemics of West Nile virus (WNV) in North America has heightened concerns about the potential for introduction and similar epidemic spread of other arbovirus infections in the United States. Dengue has received particular attention since cases have been recognized in the United States at the border between Texas and Mexico.1 The mosquito species that transmit DENV, Aedes egypti and Aedes albopictus, are present in the Southern and Southeastern parts of the United States, raising the specter of significant spread within the United States if the virus is introduced and efficiently spreads. Does dengue represent a risk to the safety of the US blood supply? Transmission by transfusion (TT) is often difficult to evaluate in the midst of an epidemic because the infection could have been acquired through a mosquito bite, through a transfusion, or even through a needle stick. Doubts about TT of WNV were eliminated when the virus was transmitted to multiple recipients of organs and, subsequently, multiple recipients of blood components obtained from the blood donors determined to be viremic.2 Despite the recognition of millions of cases of DENV infection and disease every year, there are very few published reports of transfusion transmission. One report was submitted to the public health agency of Hong Kong and, although accessible online, has not been published in a peer-reviewed journal. The other report describes a recent cluster of TT-DENV in Singapore, which is currently in press (P.A. Tambyah, National University of Singapore, personal communication, 2008). There are also reports of transmission by needle sticks and one case associated with a marrow transplant in Puerto Rico (references are included in the articles being published). Considering the available information, should precautionary measures be considered to prevent TT-DENV in the United States or even more important in countries in which DENV epidemics are occurring at expanding rates? What are the data required for decisions to implement measures that mitigate this potential risk? The articles published in the current issue of TRANSFUSION contribute data that will be needed for policy decisions that may be required in the not so distant future. Linnen and coworkers3 used a transcription-mediated amplification (TMA) assay for detection of DENV RNA to screen 13,372 specimens collected from blood donors in Honduras, Brazil, and Australia, countries with ongoing seasonal dengue epidemics. They identified 9 donors in Honduras who were repeat reactive for DENV RNA by TMA. Eight were confirmed by a polymerase chain reaction (PCR) assay that identified three different DENV serotypes, DENV-1, -2, and -4. Infectious virus could be recovered from four of these donors. Three samples from Brazil were repeatedly reactive on TMA. One was typed as DENV-1 and the other as DENV-3. None of the Australian specimens was repeatedly reactive. Samples were also tested by enzyme-linked immunosorbent assay for immunoglobulin M and immunoglobulin G antibodies to DENV and by plaque reduction neutralization assay to determine the existence and serotype of prior DENV infections in the viremic donors. Five of the donors in Honduras and one of the donors in Brazil appeared to have secondary DENV infections. The authors discuss the implications of reinfection with heterotypic subtypes because of the increased risk of DHF in these individuals. The study thus documented the presence of asymptomatic viremic donors in Honduras and Brazil who could theoretically transmit the virus to blood recipients. Mohammed and colleagues4 screened specimens from 16,521 blood donations made during an 11-week period to the American Red Cross in Puerto Rico, starting 2 weeks after the peak of dengue activity at the end of 2005. Twelve were DENV RNA repeatedly reactive using the same TMA assay used by Linnen and coworkers; 5 of the 12 were reactive in a pool of 16 (the operational pool size for WNV using TMA), and 4 of the 5 were positive on PCR. The four PCR-positive samples had viral loads ranging from 2000 to 80 million copies per mL, of which 3 were typed as DENV-2 and 1 as DENV-3. Infectious virus was recovered from all three of these donors by either mosquito inoculation or cell culture. Interestingly, the authors indicated that some of the 77,000 units of whole blood collected in Puerto Rico are exported to the continental United States each year, implying that these imported units could potentially transmit DENV to continental US recipients. A total of 722 cases of dengue have been reported in Puerto Rico in the first 6 weeks of 2008, so such transmissions could be occurring on an ongoing basis. With regard to growing concerns over potential TT-CHIKV, Brouard and colleagues5 used statistical sampling methods to estimate the risk of viremic blood donations during the CHIKV epidemic that raged through Reunion Island in the Indian Ocean from 2005 to 2007. They placed the peak risk at 1500 viremic donors per 100,000 donations between January and March 2006. Similar to DENV, there were no reports of TT despite estimates that more than 300,000 people were infected by CHIKV during this period, some of whom undoubtedly donated blood during the presymptomatic viremic phase of infection. The French government promptly suspended whole-blood collections in Reunion Island during this epidemic and provided the blood needs from the mainland. Platelet collections by apheresis continued locally, but the collected products were subjected to a process of pathogen inactivation. The statistical approach used by Brouard and coworkers5 is identical to that used by Biggerstaff and Petersen6 when they estimated the risk of transmission of WNV by transfusion during the 1999 epidemic in Queens, New York City, in a prescient article published in TRANSFUSION in August 2002. There had been no reports of TT of WNV at that time. However, 2002 was the year in which WNV TT was demonstrated and the year with the highest number of documented TT cases in North America. Blood donor screening by nucleic acid testing (NAT) for WNV was implemented in July 2003 and is viewed as a great success with interdiction of thousands of viremic blood components. Finally, Xie and colleagues7 address concerns over risk of arbovirus transmission by plasma derivatives by evaluating clearance of spiked DENV serotype 2 as a result of fractionation and processing of therapeutic plasma proteins. They concluded that the multiplicity of steps, including treatment by solvent detergent, cumulatively reduced the risk of viral transmission by albumin by more than 10 log and for immunoglobulins by more than 14 log. The article speaks to the robustness of viral inactivation processes currently utilized by the plasma industry and lessens concerns about the transmission of these viruses by plasma derivatives. These results are quite similar to those that have been observed with WNV that led the FDA to the conclusion that plasma destined for further manufacture does not need to be screened for WNV. What is the reason why the number of reports of TT of DENV and CHIKV are so few? There are many differences between these viruses and WNV, but they do not clearly explain the lack of TT reports for DENV and CHIKV. For instance: WNV infects a large number of birds and mammals. Birds are highly efficient amplification vectors, presenting very high levels of viremia. Many species of mosquitoes that transmit WNV bite both animals and humans. DENV amplification occurs in the salivary glands of A. egypti and A. albopictus. The mosquito transmits DENV from human to human in densely populated areas. There may be differences between WNV, DENV, and CHIKV in the number of days with asymptomatic viremia during which an infectious donation could be given and the titers of infectious virus during this period; for WNV, the period of preseroconversion viremia is 7 days but the levels of viremia are higher for DENV or CHIKV. DENV and CHIKV epidemics currently occur primarily in developing countries. Large numbers of individuals are affected simultaneously, overwhelming hospital emergency rooms, making impossible accurate anamnesis, physical examination, and appropriate reporting. The environment is not conducive to clinical studies, even observational, that could adequately document case reports, let alone estimate rates of TT. This is likely the main reason why TT infections are not recognized. In epidemic regions, blood is diverted to the many cases with DHF and DSS. Thus, many of the patients that receive blood transfusions during the height of the epidemic are already infected with dengue. Postponement of other hospital activities like elective surgeries reduces the opportunity of transmission of infection to naïve patients by transfusions. Lookback is rarely performed in developing countries because of limited resources. If TT of DENV or CHIKV is documented to occur at “significant” rates, it will be important to establish whether disease penetrance is more or less serious than disease transmission from other vector-borne viruses. This issue is of particular interest with respect to DENV, since the more serious hemorrhagic syndromes are thought to be related to high-level anamnestic immune responses. It is possible that TT dengue in immunosuppressed patients will result in no symptoms or mild disease. There are reports suggesting that dengue infections in recipients of kidney transplants are milder than dengue infections of healthy individuals.8, 9 The authors of these reports speculate that immunosuppression interferes with the patient's immune response to the virus and reduces the opportunity for development of DHF. This clinical course is the opposite of what is observed with WNV infection. Immunosuppressed individuals like the elderly and recipients of organ transplants appear to be more susceptible to meningoencephalitis than young patients. It will be important to determine the risk of serious disease associated with TT of dengue and CHIKV because TT of these viruses almost certainly represents a very small proportion of the total infections during an epidemic (as is true of WNV). The value of implementation of donor screening or other high-cost prevention measures to protect blood safety would require careful consideration taking into account prevalence of viremia in donors, transmission rates, and disease penetrance in infected recipients. The availability of a potential donor screening assay for DENV using the same platform as the assays for WNV RNA is welcome and reassuring. It will also be wise to address issues related to potential testing algorithms in future studies, including the definition of a positive specimen, the need for minipool or individual-donation NAT, the evaluation and implications of antibody status of donors, the development of confirmatory assays, and the value of determining DENV serotype. The authors of the studies performed in Honduras and in Puerto Rico have indicated that further studies will be performed in an attempt to better document TT of dengue and estimate the actual risk. The need for a DENV assay and the benefits of introduction of blood donor screening are dependent on the outcomes of these studies. Finally, we hope that public health authorities, regulatory agencies, blood banking organizations, transfusing clinicians, and patient groups continue to support development of technologies that may be useful for pathogen reduction of cellular components. Pathogen reduction is a more generic and proactive approach to the risks associated with arboviruses, precluding the need for implementation of donor screening assays. The example of clearance by plasma fractionation and inactivation procedures is remarkable and should encourage further pursuit of methods applicable to cellular components. It would address DENV, CHIKV, and other reemerging agents like yellow fever virus that is reappearing in South America both in wild monkeys and in humans, with several reported human deaths, as well as other viral agents yet to be identified.10

Environmental changes are among the main factors that contribute to the emergence or re-emergence of viruses of public health importance. Here, we show the impact of environmental modifications on cases of infections by the dengue, chikungunya and Zika viruses in humans in the state of Tocantins, Brazil, between the years 2010 and 2019. We conducted a descriptive and principal component analysis (PCA) to explore the main trends in environmental modifications and in the cases of human infections caused by these arboviruses in Tocantins. Our analysis demonstrated that the occurrence of El Niño, deforestation in the Cerrado and maximum temperatures had correlations with the cases of infections by the Zika virus between 2014 and 2016. El Niño, followed by La Niña, a gradual increase in precipitation and the maximum temperature observed between 2015 and 2017 were shown to have contributed to the infections by the chikungunya virus. La Niña and precipitation were associated with infections by the dengue virus between 2010 and 2012 and El Niño contributed to the 2019 outbreak observed within the state. By PCA, deforestation, temperatures and El Niño were the most important variables related to cases of dengue in humans. We conclude from this analysis that environmental changes (deforestation and climate change) presented a strong influence on the human infections caused by the dengue, chikungunya and Zika viruses in Tocantins from 2010 to 2019.

Monitoring arbovirus in Thailand: Surveillance of dengue, chikungunya and zika virus, with a focus on coinfections

Arboviruses have been shown to circulate in Madagascar, including West Nile, dengue, and chikungunya viruses, though the extent of their circulation remains poorly documented. We estimated the seroprevalence of these three arboviruses in Madagascar and determined risk factors associated with seropositivity. Serum samples obtained from 1680 individuals surrounding the Sentinel Health Centers network in all regions of the country were analyzed using ELISA and hemagglutination inhibition assays for dengue, chikungunya, and West Nile viruses IgG antibodies, and multivariate logistic regression models were run. Overall, 6.5% [IC 95% 3.2-9.9] were seropositive for dengue virus, predominantly of Dengue serotype 1, 13.7% [IC 95% 6.5-20.9] for chikungunya virus, and 12.7% [IC 95% 9.0-16.5] for West Nile virus. There was no association with age, showing that dengue and chikungunya viruses were likely recently introduced. Eastern and Northern parts were more affected by dengue and chikungunya viruses, while West Nile virus seemed to circulate in all parts of the country. Dengue and chikungunya seropositivity were notably associated with high levels of vegetation, as well as frequent work in the forest, and West Nile seropositivity with the presence of cultivated areas, as well as standard of living. This analysis gives a new insight into arboviruses circulation and transmission patterns in Madagascar.

BackgroundArboviruses are a cause of acute febrile illness and outbreaks worldwide. Recent outbreaks of Chikungunya virus (CHIKV) in dengue endemic areas have alarmed clinicians as unique clinical features differentiating CHIKV from Dengue virus (DENV) are limited. This has complicated diagnostic efforts especially in resource limited countries where lab testing is not easily available. Therefore, it is essential to analyse and compare clinical features of laboratory confirmed cases to assist clinicians in suspecting possible CHIKV infection at time of clinical presentation.MethodologyA prospective point prevalence study was conducted, with the hypothesis that not all patients presenting with clinical suspicion of dengue infections at local hospitals are suffering from dengue and that other arboviruses such as Chikungunya, West Nile viruses, Japanese Encephalitis virus and Zika virus are co-circulating in the Sindh region of Pakistan. Out-patients and hospitalized (in-patients) of selected district hospitals in different parts of Sindh province of Pakistan were recruited. Patients with presumptive dengue like illness (Syndromic diagnosis) by the treating physicians were enrolled between 2015 and 2017.Current study is a subset of larger study mentioned above. Here-in we compared laboratory confirmed cases of CHIKV and DENV to assess clinical features and laboratory findings that may help differentiate CHIKV from DENV infection at the time of clinical presentation.ResultsNinety-eight (n = 98) cases tested positive for CHIKV, by IgM and PCR and these were selected for comparative analysis with DENV confirmed cases (n = 171). On multivariable analysis, presence of musculoskeletal [OR = 2.5 (95% CI:1.6–4.0)] and neurological symptoms [OR = 4.4 (95% CI:1.9–10.2)], and thrombocytosis [OR = 2.2 (95% CI:1.1–4.0)] were associated with CHIKV infection, while atypical lymphocytes [OR = 8.3 (95% CI:4.2–16.7)] and thrombocytopenia [OR = 8.1 (95% CI:1.7–38.8)] were associated with DENV cases at time of presentation. These findings may help clinicians in differentiating CHIKV from DENV infection.ConclusionCHIKV is an important cause of illness amongst patients presenting with acute febrile illness in Sindh region of Pakistan. Arthralgia and encephalitis at time of presentation among patients with dengue-like illness should prompt suspicion of CHIKV infection, and laboratory confirmation must be sought.

Aedes albopictus as well as Aedes aegypti is an important vector of chikungunya and dengue viruses. Electron microscopic observations on the salivary glands of Ae. albopictus infected with chikungunya virus were performed in comparing with those of Ae. aegypti infected with dengue virus. No virus budding from the cell surface of the chikungunya-infected mosquito's salivary glands was found as shown in dengue-infected ones, in contrast to the findings of the mammalian cells such as Vero, KB, IMR, J-111 and BHK-21 cells infected with chikungunya and/or dengue virus(es).

RIG-I is a cytosolic sensor critically involved in the activation of the innate immune response to RNA virus infection. In the present study, we evaluated the inhibitory effect of a RIG-I agonist on the replication of two emerging arthropod-borne viral pathogens, dengue virus (DENV) and chikungunya virus (CHIKV), for which no therapeutic options currently exist. We demonstrate that when a low, noncytotoxic dose of an optimized 5'triphosphorylated RNA (5'pppRNA) molecule was administered, RIG-I stimulation generated a robust antiviral response against these two viruses. Strikingly, 5'pppRNA treatment before or after challenge with DENV or CHIKV provided protection against infection. In primary human monocytes and monocyte-derived dendritic cells, the RIG-I agonist blocked both primary infection and antibody-dependent enhancement of DENV infection. The protective response against DENV and CHIKV induced by 5'pppRNA was dependent on an intact RIG-I/MAVS/TBK1/IRF3 axis and was largely independent of the type I IFN response. Altogether, this in vitro analysis of the antiviral efficacy of 5'pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV. DENV and CHIKV are two reemerging mosquito-borne viruses for which no therapeutic options currently exist. Both viruses overlap geographically in tropical regions of the world, produce similar fever-like symptoms, and are difficult to diagnose. This study investigated the inhibitory effect of a RIG-I agonist on the replication of these two viruses. RIG-I stimulation using 5'pppRNA before or after DENV or CHIKV infection generated a protective antiviral response against both pathogens in immune and nonimmune cells; interestingly, the protective response against the viruses was largely independent of the classical type I interferon response. The antiviral efficacy of 5'pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV.

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