CHIKV RNA Research Articles

Evaluation of assays for nucleic acid testing for the prevention of chikungunya and dengue virus transmission by blood transfusion.

The large dengue (DENV) and chikungunya (CHIKV) outbreaks observed during the last decade across the world, as well as local transmissions in non-endemic areas are a growing concern for blood safety. The aim of this study was to evaluate and compare the sensitivity of nucleic acid tests (NAT) detecting DENV and CHIKV RNA. Using DENV 1 to 4 International Standards, the limits of detection (LODs) calculated by probit analysis of two NAT assays; the cobas CHIKV/DENV assay (Roche Diagnostics) and the Procleix Dengue Virus Assay (Grifols) were compared. In addition, CHIKV-RNA LOD of the cobas CHIKV/DENV assay was evaluated. For dengue, the 95% LOD of the cobas assay ranged between 4.10 [CI95%: 2.70-8.19] IU/mL (DENV-2) and 7.07 [CI95%: 4.34-14.89] IU/mL (DENV-4), and between 2.19 [CI95%: 1.53-3.83] IU/mL (DENV-3) and 5.84 [CI95%: 3.84-10.77] IU/mL (DENV-1) for Procleix assay. The Procleix assay had a significant lower LOD for DENV-3 (2.19 vs. 5.89 IU/mL) when compared to the cobas assay (p = 0.005). The 95% LOD for CHIKV-RNA detection of the cobas assay was 4.76 [CI95%: 3.08-8.94] IU/mL. The two NAT assays developed for blood donor screening evaluated in this study demonstrated high and similar analytical performance. Subject to an appropriate risk-benefit assessment, they can be used to support blood safety during outbreaks in endemic areas or in non-endemic areas as an alternative to deferring blood donors during local transmission likely to affect the blood supply. The development of multiplex assays is expected to optimize laboratory organization.

Molecular surveillance of arboviruses in Nigeria

Arboviral infections are fast becoming a global public health concern as a result of its high fatality rate and sporadic spread. From the outbreak of Zika virus in the Americas, the endemicity of Yellow fever in West Africa and South America, outbreaks of West Nile virus in South Africa to the year-round and national risk of Dengue fever in Mainland China and India. The war against emerging and re-emerging viral infection could probably lead to the next pandemic. To be above the pending possible arboviral pandemic, consistent surveillance of these pathogens is necessary in every society. This study was aimed at conducting a surveillance for Yellow fever virus, Zika virus, Chikungunya virus, Dengue virus and Rift Valley fever virus in four states in Nigeria using molecular techniques. A cross-sectional study involving 1600 blood samples collected from febrile patients in Lagos, Kwara, Ondo and Delta States between 2018 and 2021 was conducted using Real time polymerase chain reaction for detection of the pathogens. Extraction and purification of viral RNA were done using Qiagen Viral RNA Mini Kit. Samples were analyzed using One Step PrimeScript III RT-PCR mix (Takara Bio) alongside optimized primers and probes designed in-house. Positive samples were sequenced on MinION platform (Nanopore technologies). Bioinformatic and phylogenetic analysis were performed with DNASTAR Lasergene 17.3. All the RNA extracted from samples collected from the four states were negative for ZIKV RNA, RVFV RNA, CHIKV RNA and DENV RNA. However, twelve of the samples (2%) tested positive for YFV RNA. Three full genomes of sizes 10,751 bp, 10,500 bp and 10,715 bp were generated and deposited in GenBank with accession numbers: ON323052, ON323053 and ON323054 respectively. Phylogenetic analysis shows clustering within lineage 3 of West African genotype. This result shows an active spread of Yellow fever in Delta State, Nigeria. However, there is no emergence of a new genotype There is a need for an intense surveillance of Yellow fever virus in Nigeria to avert a major outbreak.

Phosphorylation Sites in the Hypervariable Domain in Chikungunya Virus nsP3 Are Crucial for Viral Replication.

Chikungunya virus (CHIKV, family Togaviridae) is a mosquito-transmitted alphavirus. The positive-sense RNA genome of CHIKV encodes four nonstructural proteins (nsP1 to nsP4) that are virus-specific subunits of the RNA replicase. Among nsP functions, those of nsP3 are the least understood. The C-terminal hypervariable domain (HVD) in nsP3 is disordered and serves as a platform for interactions with multiple host proteins. For Sindbis virus (SINV) and Semliki Forest virus (SFV), the nsP3 HVD has been shown to be phosphorylated. Deletion of phosphorylated regions has a mild effect on the growth of SFV and SINV in vertebrate cells. Using radiolabeling, we demonstrated that nsP3 in CHIKV and o'nyong-nyong virus is also phosphorylated. We showed that the phosphorylated residues in CHIKV nsP3 are not clustered at the beginning of the HVD. The substitution of 20 Ser/Thr residues located in the N-terminal half of the HVD or 26 Ser/Thr residues located in its C-terminal half with Ala residues reduced the activity of the CHIKV replicase and the infectivity of CHIKV in mammalian cells. Furthermore, the substitution of all 46 potentially phosphorylated residues resulted in the complete loss of viral RNA synthesis and infectivity. The mutations did not affect the interaction of the HVD in nsP3 with the host G3BP1 protein; interactions with CD2AP, BIN1, and FHL1 proteins were significantly reduced but not abolished. Thus, CHIKV differs from SFV and SINV both in the location of the phosphorylated residues in the HVD in nsP3 and, significantly, in their effect on replicase activity and virus infectivity.IMPORTANCE CHIKV outbreaks have affected millions of people, creating a need for the development of antiviral approaches. nsP3 is a component of the CHIKV RNA replicase and is involved in interactions with host proteins and signaling cascades. Phosphorylation of the HVD in nsP3 is important for the virulent alphavirus phenotype. Here, we demonstrate that nsP3 in CHIKV is phosphorylated and that the phosphorylation sites in the HVD are distributed in a unique pattern. Furthermore, the abrogation of some of the phosphorylation sites results in the attenuation of CHIKV, while abolishing all the phosphorylation sites completely blocked its replicase activity. Thus, the phosphorylation of nsP3 and/or the phosphorylation sites in nsP3 have a major impact on CHIKV infectivity. Therefore, they represent promising targets for antiviral compounds and CHIKV attenuation. In addition, this new information offers valuable insight into the vast network of virus-host interactions.

An investig-ation into the epidemiology of chikungunya virus across neglected regions of Indonesia.

Chikungunya virus (CHIKV) is an important emerging and re-emerging public health problem worldwide. In Indonesia, where the virus is endemic, epidemiological information from outside of the main islands of Java and Bali is limited. Four hundred and seventy nine acutely febrile patients presenting between September 2017-2019 were recruited from three city hospitals situated in Ambon, Maluku; Banjarmasin, Kalimantan; and Batam, Batam Island as part of a multi-site observational study. CHIKV RNA was detected in a single serum sample while a separate sample was IgM positive. IgG seroprevalence was also low across all three sites, ranging from 1.4-3.2%. The single RT-PCR positive sample from this study and 24 archived samples collected during other recent outbreaks throughout Indonesia were subjected to complete coding region sequencing to assess the genetic diversity of Indonesian strains. Phylogenetic analysis revealed all to be of a single clade, which was distinct from CHIKV strains recently reported from neighbouring regions including the Philippines and the Pacific Islands. Chikungunya virus strains from recent outbreaks across Indonesia all belong to a single clade. However, low-level seroprevalence and molecular detection of CHIKV across the three study sites appears to contrast with the generally high seroprevalences that have been reported for non-outbreak settings in Java and Bali, and may account for the relative lack of CHIKV epidemiological data from other regions of Indonesia.

Detection of dengue, chikungunya, and Zika RNA in blood donors from Southeast Asia.

BackgroundChikungunya (CHIKV), dengue (DENV), and Zika (ZIKV) viruses are of concern due to the potential of transfusion transmission in blood, especially in regions such as Southeast Asia where the viruses are endemic. The recent availability of nucleic acid testing (NAT) to screen blood donations on an automated platform provides the opportunity to detect potentially infectious units in asymptomatic donors.Study Design and MethodsThree thousand blood donations from Vietnam and 6000 from Thailand were screened with a real‐time polymerase chain reaction (PCR) test (cobas CHIKV/DENV, Roche Diagnostics, Indianapolis, IN) and equal numbers on cobas Zika (Roche Diagnostics). Reactive samples were tested by alternative NAT with resolution of discordant results by heminested PCR. Throughput of simultaneous testing of the two assays on the cobas 8800 system (Roche Diagnostics) was evaluated.ResultsIn Vietnam, 9 of 3045 samples were reactive for DENV and all were confirmed, for a prevalence (with 95% confidence interval [CI]) of 0.296% (0.135‐0.560). In Thailand, 2 of 6000 samples were reactive for CHIKV, 4 of 6000 for DENV, and 1 of 6005 for ZIKV, and all confirmed. The prevalence of CHIKV is 0.033% (0.004‐0.120), DENV 0.067% (0.018‐0.171), and ZIKV 0.017% (0.000‐0.093). The overall specificity for the cobas CHIKV/DENV and cobas Zika tests was 100% (99.959‐100).For the simultaneous assay testing, 960 test results were available in 7 hours and 53 minutes.ConclusionDetection of CHIKV, DENV, and ZIKV RNA in donor samples in Vietnam and Thailand indicate the presence of the virus in asymptomatic blood donors. The cobas 6800/8800 systems (Roche Molecular Systems, Pleasanton, CA) enable screening blood donations in endemic areas for these viruses together or separately.

Investigating the pathogenesis of chronic chikungunya virus infection

Abstract Chikungunya virus (CHIKV) is an arthritogenic alphavirus that acutely causes fever as well as severe joint and muscle pain. Chronic musculoskeletal pain persists in a substantial fraction of patients for months to years after infection. While replicating virus is not detected in joint-associated tissues of patients nor in various animal models at convalescent time points, viral RNA is detected months after acute infection. To identify the cells that might contribute to chronic pathogenesis, we developed a recombinant CHIKV that expresses Cre recombinase (CHIKV-3′-Cre). CHIKV-3′-Cre infection of tdTomato reporter mice resulted in a population of tdTomato+ cells that persisted for at least 112 days. Immunofluorescence and flow cytometric profiling 28 days post infection revealed that these tdTomato+ cells were predominantly myofibers and fibroblasts as well as a small population of macrophages. Further, tdTomato+ cells are enriched for CHIKV RNA. Interestingly, the population of surviving cells was dynamic over time with macrophages making up a high percentage of the cells early and then tapering off. We applied this system to determine the differential roles of the type I interferon (IFN) subtypes during chronic CHIKV infection. Results using IFN blocking antibodies revealed that mice lacking the IFNαs, but not IFNβ, show an increase in chronic RNA levels and the number of fibroblasts that survive CHIKV infection in distal sites, indicating that type I IFNs have divergent roles during chronic CHIKV disease. Ongoing studies will explore the differential functions of IFNαs and IFNβ, will determine why the surviving cells and viral RNA are not cleared by the immune system, and will elucidate their contribution to chronic inflammation.

Imported cases of Chikungunya virus in Iran

BackgroundChikungunya virus (CHIKV) is a widespread mosquito-borne virus representing a serious challenge to public health. The largest outbreak in the Middle-East was recorded in 2016–2017 in Pakistan. Sistan and Baluchistan Province of Iran shares a wide border with Pakistan; accordingly, introduction of CHIKV from Pakistan to Iran seems to be probable. The current study is aimed at investigating CHIKV infection in Sistan and Baluchistan Province.MethodsBetween April 2017 and June 2018, a total of 159 serum samples of CHIK suspected cases from 10 cities of Sistan and Baluchistan Province were tested by molecular and serological assays. Samples obtained up to 4 days after onset of illness were tested by real time PCR (n = 8). Samples collected 5–10 days after disease onset were subjected to ELISA, as well as real time PCR tests (n = 72). Samples obtained after the 10th day of disease onset were tested by only ELISA (n = 79). Phylogenetic analysis of real time PCR positive samples was carried out by sequencing of a 1014-bp region of Envelope 1 gene (E1 gene). Chi-square and independent t tests were used to evaluate the association between variables and CHIKV infection.ResultsIn total, 40 (25.1%) out of 159 samples tested positive either by real time PCR or ELISA tests.Out of 151 samples serologically analyzed, 19 (12.6%) and 28 (18.6%) cases were positive for anti-CHIKV IgM and anti-CHIKV IgG antibodies, respectively. Of 80 samples tested by real time PCR, CHIKV RNA was detected in 11 (13.7%) sera, all of them had recent travel history to Pakistan. Additionally, phylogenetic analysis of 5 samples indicated their similarity with recent isolates of Pakistan outbreak 2016–2017 belonging to Indian Ocean sub-lineage of ECSA genotype. A significant correlation between abroad travel history and CHIKV infection was observed (P < 0.001). The most common clinical symptoms included fever, arthralgia/arthritis, myalgia, headache, and chill.ConclusionsThese results present substantial evidence of CHIKV introduction to Iran from Pakistan and emphasize the need for the enhancement of surveillance system and preventive measures.

Dengue, chikungunya and zika virus coinfection: results of the national surveillance during the zika epidemic in Colombia.

Our objective was to determine the frequency of zika (ZIKV), chikungunya (CHIKV) and dengue (DENV) virus coinfection and describe the mortality cases that occurred during the epidemiologic surveillance of the ZIKV epidemic in Colombia. We analysed all cases of suspected ZIKV infection that were reported to the National Institute of Health (October 2015-December 2016). DENV, CHIKV and ZIKV RNA were detected in serum or tissue samples using polymerase chain reaction assay. Medical records of the fatal cases were reviewed. We identified that 23 871 samples were processed. The frequency of viral agents was 439 (1.84%) for DENV, 257 (1.07%) for CHIKV and 10118 (42.38%) for ZIKV. Thirty-four (0.14%) cases of coinfection were identified. The CHIKV-ZIKV coinfection was present in 28 cases (82.3%), DENV-CHIKV in three (8.8%) and DENV-ZIKV in three (8.8%). Seven (20.6%) coinfection cases were fatal (two DENV-CHIKV cases and five CHIKV-ZIKV cases). Two cases were foetal deaths and the others were related to neurological syndrome and sepsis. In conclusion, the frequency of arbovirus coinfection during epidemic of ZIKV was low, and CHIKV-ZIKV coinfection was the most common. Mortality was high among coinfection patients. The role of each virus in the mortality cases of coinfection warrants further studies.

449. Post-Hurricane Maria Surveillance for Infectious Diseases in the Veterans Affairs San Juan Medical Center, Puerto Rico

BackgroundOn September 20, 2017 Category 4 Hurricane Maria made landfall in Puerto Rico (PR), causing widespread flooding, power outages, and lack of water service. Given the potential for infectious disease outbreaks, the Department of Veterans Affairs (VA) and Centers for Disease Control and Prevention established enhanced surveillance to actively monitor priority infections at VA facilities.MethodsWe queried VA data sources from August 27, 2017 to February 3, 2018 (pre-storm dates included to establish baselines). Trends in infectious disease ICD-10 syndrome groupings (respiratory illness/pneumonia, Influenza-like illness (ILI), gastrointestinal illness, conjunctivitis, rash-like Illness, jaundice) as a percent of total emergency department (ED) visits were tracked. The total number of laboratory tests performed, and percent positive per week, for influenza, hepatitis A, dengue (DENV), zika (ZIKV), leptospirosis, and chikungunya (CHIKV) were calculated.ResultsILI increased from 9.3% to 12.6% during the surveillance period (peak epi week 52: 15.7%) (Figure 1), while other ICD-10–based syndromes remained relatively stable. Weekly influenza testing increased shortly after landfall averaging 105 rapid influenza tests per week (epi weeks 41–4) (Figure 2). Influenza positivity increased in epi weeks 41 and 42 (7%), dropping the following weeks, and peaked at 15% in epi week 2 (Figure 3). Four acute infections were detected: 2 + leptospirosis DNA, 1 + CHIKV RNA, and 1 + Hepatitis A IgM. The remaining 34 positive tests were ZIKV, CHIKV, or DENV IgM positive or equivocal, awaiting confirmation (Figure 3).ConclusionWe quickly established a simple surveillance system to monitor trends in priority infectious diseases. Increases in ILI, weekly influenza testing volume, and percent positive of influenza tests coincided with onset of influenza season. Diseases of public health importance were identified through laboratory-based surveillance. The impact of Maria on VA healthcare operations, including clinic closures, power outages, and disrupted care seeking patterns limited this system. However, the timeliness and flexibility of this surveillance system provides a model for disease monitoring following future natural disasters.Figure 1.Figure 2.Figure 3.Disclosures All authors: No reported disclosures.

Evaluation of an immunochromatography rapid diagnosis kit for detection of chikungunya virus antigen in India, a dengue-endemic country

BackgroundChikungunya virus (CHIKV) and dengue virus (DENV) are arboviruses that share the same Aedes mosquito vector, and there is much overlap in endemic areas. In India, co-infection with both viruses is often reported. Clinical manifestations of Chikungunya fever is often confused with dengue fever because clinical symptoms of both infections are similar. It is, therefore, difficult to differentiate from those of other febrile illnesses, especially dengue fever. We previously developed a CHIKV antigen detection immunochromatography (IC) rapid diagnosis kit [1]. The current study examined the efficacy of previously mentioned IC kit in India, a dengue-endemic country.MethodsSera from 104 CHIKV-positive (by qRT-PCR) and/or IgM-positive (ELISA) subjects collected in 2016, were examined. Fifteen samples from individuals with CHIKV-negative/DENV-positive and 4 samples from healthy individuals were also examined. Of the 104 CHIKV-positive sera, 20 were co-infected with DENV.ResultsThe sensitivity, specificity and overall agreement of the IC assay were 93.7, 95.5 and 94.3%, respectively, using qRT-PCR as a gold standard. Also, there was a strong, statistically significant positive correlation between the IC kit device score and the CHIKV RNA copy number. The IC kit detected CHIKV antigen even in DENV-co-infected patient sera and did not cross-react with DENV NS1-positive/CHIKV-negative samples.ConclusionsThe results suggest that the IC kit is useful for rapid diagnosis of CHIKV in endemic areas in which both CHIKV and DENV are circulating.

Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease's active site cysteine residue.

Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays. First, we show that Cys478 residue in the active site of CHIKV nsP2 is indispensable for P1234 processing. Second, the substrate requirements of CHIKV nsP2 are quite similar to those of nsP2 of related Semliki Forest virus (SFV). Third, substitution of Ser482 residue, recently reported to contribute to the protease activity of nsP2, with Ala has almost no negative effect on the protease activity of CHIKV nsP2. Fourth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 completely abolished RNA replication in CHIKV and SFV trans-replication systems. In contrast, trans-replicases with Ser482 to Ala mutation were similar to wild type counterparts. Fifth, Cys478 to Ala as well as Trp479 to Ala mutations in nsP2 abolished the rescue of infectious virus from CHIKV RNA transcripts while Ser482 to Ala mutation had no effect. Thus, CHIKV nsP2 is a cysteine protease.

Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein.

Chikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) has recently caused several major outbreaks affecting millions of people. There are no licensed vaccines or antivirals, and the knowledge of the molecular biology of CHIKV, crucial for development of efficient antiviral strategies, remains fragmentary. CHIKV has a 12 kb positive-strand RNA genome, which is translated to yield a nonstructural (ns) or replicase polyprotein. CHIKV structural proteins are expressed from a subgenomic RNA synthesized in infected cells. Here we have developed CHIKV trans-replication systems, where replicase expression and RNA replication are uncoupled. Bacteriophage T7 RNA polymerase or cellular RNA polymerase II were used for production of mRNAs for CHIKV ns polyprotein and template RNAs, which are recognized by CHIKV replicase and encode for reporter proteins. CHIKV replicase efficiently amplified such RNA templates and synthesized large amounts of subgenomic RNA in several cell lines. This system was used to create tagged versions of ns proteins including nsP1 fused with enhanced green fluorescent protein and nsP4 with an immunological tag. Analysis of these constructs and a matching set of replicon vectors revealed that the replicases containing tagged ns proteins were functional and maintained their subcellular localizations. When cells were co-transfected with constructs expressing template RNA and wild type or tagged versions of CHIKV replicases, formation of characteristic replicase complexes (spherules) was observed. Analysis of mutations associated with noncytotoxic phenotype in CHIKV replicons showed that a low level of RNA replication is not a pre-requisite for reduced cytotoxicity. The CHIKV trans-replicase does not suffer from genetic instability and represents an efficient, sensitive and reliable tool for studies of different aspects of CHIKV RNA replication process.

Surveillance of dengue and chikungunya infection in Dong Thap, Vietnam: A 13-month study.

To establish a surveillance in Dong Thap, at the border with Cambodia by assessing the presence of DENV serotypes and CHIKV among patients hospitalized at Dong Thap general hospital. Cross-sectional descriptive analysis was conducted on a cohort of 131 patients hospitalized with acute fever and symptoms compatible with dengue or chikungunya. The study was conducted from January 2012 to February 2013. The full clinical picture was established as well as serological and molecular detection. Serological analysis was sequentially performed on blood samples collected on admission and an average of seven days after admission. The detection of IgM antibody to DENV was performed by IgM capture ELISA and the detection of DENV and CHIKV RNA was done by reverse-transcription multiplex PCR. 101 patients out of 131 (77%) were confirmed with dengue. All four dengue serotypes were detected with a predominance of DENV2 and DENV4. No chikungunya infection was detected although reported in neighboring Cambodia. A differential efficiency of serological dengue detection was observed. Efficiency was 29% upon admission and 53% after seven days on the same patients. 30 patients out of 131 (23%) were negative with both DENV and CHIKV. Dengue is at risk of being underestimated and chikungunya is not systematically detected. Changes in detection and surveillance procedures are therefore discussed to increase efficiency of dengue detection and continue the monitoring the emergence of CHIKV in Dong Thap province and in Vietnam.

Chronic Joint Disease Caused by Persistent Chikungunya Virus Infection Is Controlled by the Adaptive Immune Response

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that causes incapacitating disease in humans characterized by intense joint pain that can persist for weeks, months, or even years. Although there is some evidence of persistent CHIKV infection in humans suffering from chronic rheumatologic disease symptoms, little is known about chronic disease pathogenesis, and no specific therapies exist for acute or chronic CHIKV disease. To investigate mechanisms of chronic CHIKV-induced disease, we utilized a mouse model and defined the duration of CHIKV infection in tissues and the associated histopathological changes. Although CHIKV RNA was readily detectable in a variety of tissues very early after infection, CHIKV RNA persisted specifically in joint-associated tissues for at least 16 weeks. Inoculation of Rag1(-/-) mice, which lack T and B cells, resulted in higher viral levels in a variety of tissues, suggesting that adaptive immunity controls the tissue specificity and persistence of CHIKV infection. The presence of CHIKV RNA in tissues of wild-type and Rag1(-/-) mice was associated with histopathological evidence of synovitis, arthritis, and tendonitis; thus, CHIKV-induced persistent arthritis is not mediated primarily by adaptive immune responses. Finally, we show that prophylactic administration of CHIKV-specific monoclonal antibodies prevented the establishment of CHIKV persistence, whereas therapeutic administration had tissue-specific efficacy. These findings suggest that chronic musculoskeletal tissue pathology is caused by persistent CHIKV infection and controlled by adaptive immune responses. Our results have significant implications for the development of strategies to mitigate the disease burden associated with CHIKV infection in humans.

Application of real-time RT-PCR in vector surveillance and assessment of replication kinetics of an emerging novel ECSA genotype of Chikungunya virus in Aedes aegypti

Chikungunya has emerged as one of the most important arboviral infection of global significance. Expansion of Chikungunya virus endemic areas can be ascribed to naive population, increasing vector population and adaptability of virus to new vector. In this study, a SYBR Green I based quantitative RT-PCR assay was developed. The assay was found to be 10-fold more sensitive than conventional RT-PCR and no cross reactivity was observed with related alphaviruses and flaviviruses. The detection efficiency of the assay was impervious to mosquitoes of different pool sizes. Vector surveillance has resulted in detection of CHIKV RNA in Aedes aegypti, confirming its vectorial potential for CHIKV in northern India. The assessment of the assay was further carried out by studying the competence of Indian Ae. aegypti for CHIKV, which revealed 100% infection rate and dissemination rate with 60% transmission rate. The replication kinetics of CHIKV in different anatomical sites of Ae. aegypti revealed highest titre at day 6 post infection in midgut and at day 10 post infection in saliva, legs and wings. The implementation of the assay in detecting lower viral load makes it a remarkable tool for surveillance of virus activity in mosquitoes.

Inhibition of Chikungunya Virus Replication by Harringtonine, a Novel Antiviral That Suppresses Viral Protein Expression

Chikungunya virus (CHIKV) is a mosquito-transmitted virus that has reemerged as a significant public health threat in the last decade. Since the 2005-2006 chikungunya fever epidemic in the Indian Ocean island of La Réunion, millions of people in more than 40 countries have been infected. Despite this, there is currently no antiviral treatment for chikungunya infection. In this study, an immunofluorescence-based screening platform was developed to identify potential inhibitors of CHIKV infection. A primary screen was performed using a highly purified natural product compound library, and 44 compounds exhibiting ≥70% inhibition of CHIKV infection were identified as positive hits. Among these, four were selected for dose-dependent inhibition assays to confirm their anti-CHIKV activity. Harringtonine, a cephalotaxine alkaloid, displayed potent inhibition of CHIKV infection (50% effective concentration [EC(50)] = 0.24 μM) with minimal cytotoxicity and was selected for elucidation of its antiviral mechanism. Time-of-addition studies, cotreatment assays, and direct transfection of viral genomic RNA indicated that harringtonine inhibited an early stage of the CHIKV replication cycle which occurred after viral entry into cells. In addition, quantitative reverse transcription-PCR (qRT-PCR) and Western blot analyses indicated that harringtonine affects CHIKV RNA production as well as viral protein expression. Treatment of harringtonine against Sindbis virus, a related alphavirus, suggested that harringtonine could inhibit other alphaviruses. This study suggests for the first time that harringtonine exerts its antiviral effects by inhibiting CHIKV viral protein synthesis.