Chikungunya virus-vector interactions.

Chikungunya virus (CHIKV), a re-emerging infectious arbovirus, causes Chikungunya fever that is characterized by fever, skin rash, joint pain, arthralgia and occasionally death. Despite it has been described for 66 years already, neither potential vaccine nor a specific drug is available yet. During CHIKV infection, interferon type I signaling pathway is stimulated and releases hundreds of interferon stimulated genes (ISGs). Our previous study reported that IFI16, a member of ISGs, is up-regulated during CHIKV virus infection and the suppression of the gene resulted in increased virus replication. Furthermore, our group also found that inflammasome activation can inhibit CHIKV infection in human foreskin cells (HFF1). Concomitantly, it has been reported that IFI16 activates the inflammasome to suppress virus infection. Therefore, we have hypothesized that IFI16 could be involved in CHIKV infection. In this study, we confirmed the expression level of IFI16 by Western blotting analysis and found that IFI16 was up-regulated following CHIKV infection in both HFF1 and human embryonic kidney cells. We next investigated its antiviral activity and found that forced expression of IFI16 completely restricted CHIKV infection while endogenous silencing of the gene markedly increased virus replication. Furthermore, we have discovered that IFI16 inhibited CHIKV replication, at least, in cell-to-cell transmission as well as the diffusion step. Interestingly, IFI16 also exerted its antiviral activity against Zika virus (ZIKV) infection, the global threat re-emerging virus can cause microcephaly in humans. Taken together, this study provides the first evidence of an antivirus activity of IFI16 during in vitro arbovirus infection, thus expanding its antiviral spectrum that paves the way to further development of antiviral drugs and vaccines.

To the Editor: Chikungunya virus (CHIKV), an arthropod-borne virus transmitted to humans by Aedes spp. mosquitoes, was first isolated in Tanzania (Tanganyika) in 1953 (1). Various outbreaks have since occurred in Africa, Southeast Asia, and India (2). CHIKV has recently been reported in a large area in the Indian Ocean islands and the Indian subcontinent. After an outbreak in Kenya in 2004, other outbreaks occurred in early 2005 on the Comoros Islands, Reunion, and other islands in the southwestern Indian Ocean; the epidemic then spread to India (3,4). Molecular analysis showed that the epidemic was caused by a variant of the Central/East African CHIKV genotype (5,6). Internet surveillance networks provided information on epidemics in real time, alerting clinicians in the industrialized world to the spread of CHIKV and enabling them to more easily diagnose infection among travelers with fevers (7). We report results of diagnostic tests and analysis of predictors of infection among persons in Italy with symptoms suggestive of CHIKV infection who had traveled to potentially affected areas. Dengue virus (DENV) is endemic to many of these areas. We studied travelers or migrants from areas to which CHIKV infection is endemic (i.e., sub-Saharan Africa) or areas currently affected by outbreaks (i.e., the Indian Ocean islands, India) who had symptoms suggestive of infection (i.e., fever and arthralgia with or without a rash) from January 2006 through March 2007. At least 1 blood sample was collected from each patient and stored at –80°C before testing for CHIKV and DENV. Median lag between onset of symptoms and date of blood collection was 22 days (range 3–179 days). Two samples (acute phase and convalescence phase) were available from 5 patients. Serologic diagnosis of CHIKV infection was determined by hemagglutination inhibition (HI) test and confirmed by plaque-reduction neutralization test (8). Serodiagnosis of DENV infection was conducted by using the HI test and an immunoglobulin M ELISA (Focus Diagnostics, Cypress, CA, USA). A case-report form containing information about age, sex, countries visited, travel dates, and date of onset of symptoms was completed for each patient. Seventy-six persons participated in the study; 55.3% were male, median age was 39 years (range 1–69 years), and most (80.3%) were Italian (Table). A total of 29 (38.2%) were positive for CHIKV, and 13 (17.1%) were positive for DENV; 34 (44.7%) were negative for both viruses. Of the 29 CHIKV-positive persons, 22 (75.9%) had visited the Indian Ocean islands (Mauritius, Reunion, and Madagascar), 5 had visited Asia, and 2 had visited Africa. Travelers from Indian Ocean islands had a higher risk for CHIKV infection than those who had visited Africa (odds ratio [OR] 11.0, 95% confidence interval [CI] 1.60–119.13) or Asia (OR 17.05, 95% CI 4.31–73.05). Persons who had visited Asia had a higher risk for DENV infection (OR 8.36; 95% CI 1.58–81.73) than those who had visited other areas. Table Characteristics of 76 travelers studied The 5 persons who were infected with CHIKV in Asia had visited India (i.e., the most visited country [21 travelers]). However, persons who visited the Indian Ocean islands had a higher risk of being CHIKV positive than those who had visited India (OR 8.8, 95% CI 2.09–39.86). A rash was associated with CHIKV infection and was >8× more likely to be reported by CHIKV-positive persons than CHIKV-negative persons (OR 7.03, 95% CI 2.23–22.93). Moreover, rash was observed in 65% of CHIKV-positive cases and 31% of DENV-positive cases, but the difference was not statistically significant because of the small sample size (OR 4.28, 95% CI 0.88–23.23). None of the other patient’s characteristics was associated with infection with CHIKV or DENV. A limitation of our study was that only 5 patients had documented seroconversion for CHIKV. However, high titers were found in all but 1 patient (>1,280 in 21 patients and 640 in 2 patients). This patient, who had a titer of 80, was an Italian who had probably not been previously exposed to CHIKV. Thus, the risk for misclassification was low. PCR for early detection of infection was not used because only 3 persons were tested within 10 days of symptom onset. Two of these persons, who were tested 7 days after symptom onset, already had antibodies to CHIKV. In conclusion, a high proportion of travelers with symptoms of CHIKV infection who returned from areas with outbreaks of this infection or where this virus was endemic were seropositive. A lower proportion of patients had antibodies to DENV. CHIKV-positive patients were more likely to have a rash than those negative for both CHIKV and DENV. As suggested by previous studies (9), a rash was more common among CHIKV-positive patients than in DENV-infected patients, but the difference was not significant. Our study suggests that identification of predictors of infection with CHIKV is feasible, although it is complicated by cocirculation of DENV in the same areas.

Introduction: Dengue and Chikungunya have re-emerged as important diseases of global concern. Co-infections with Dengue virus (DENV) and Chikungunya virus (CHIKV) could have serious outcomes if not diagnosed and managed optimally. However, the key focal points for the maintenance of CHIKV and DENV infections and the extent of their co-infection remain poorly understood in many geo-ecologically distinct parts of Tanzania. Objective: We aimed to comparatively examine the prevalence and factors for seropositivity to DENV and CHIKV and their infection rates in humans and mosquitoes Methods: A cross-sectional study was performed in the Lower Moshi area of the Kilimanjaro region from April to July 2020. DENV and CHIKV exposure was determined by detecting IgM to the viruses using enzyme linked immunosorbent assay whereas infection was determined by real time quantitative polymerase chain reaction (RT-qPCR) assay. Results: Insecticide Treated Bed Net (ITN) use (χ2=3.504; p< 0.05), being ≥7 individuals living in the same household (χ2=4.655; p<0.05) and a recent travel to an urban destination (χ2=3.39; p< 0.05) were the only factors associated with CHIKV seropositivity. ITN use was the only factor associated with CHIKV infection (χ2=5.204; p<0.05). A recent travel to an urban destination (χ2=4.401; p< 0.05) was the only factor associated with DENV seropositivity. Five (1.5%) Ae. aegypti pools were positive for CHIKV whereas 1 (0.3%) was positive for DENV. Two Cx. pipiens, pools (1.9%) were positive for CHIKV. None of the Cx. pipiens mosquitoes was positive for DENV. No associations between DENV and CHIKV seropositivity was observed in humans but DENV infection was strongly associated with CHIKV infection (χ2 = 238.45; p<0.01). CHIKV infection was observed to be consistently higher in both, humans and mosquitoes. Conclusion: Detection of DENV and CHIKV in both humans and vector mosquitoes confirms that both viruses are actively circulating in the Lower Moshi area of Kilimanjaro region in Tanzania. Our findings point out the Lower Moshi area as a potential focal point for the maintenance of the two viruses and possibly other vector borne viruses. We call upon sustained active surveillance of arboviruses and other re-emerging infections to be better prepared for possible outbreaks by the viruses.

The molecular mechanisms underlying chikungunya virus (CHIKV) infection are poorly characterized. In this study, we analyzed the host factors involved in CHIKV infection using genome-wide screening. Human haploid HAP1 cells, into which an exon-trapping vector was introduced, were challenged with a vesicular stomatitis virus pseudotype bearing the CHIKV E3 to E1 envelope proteins. Analysis of genes enriched in the cells resistant to the pseudotyped virus infection unveiled a critical role of N-sulfation of heparan sulfate (HS) for the infectivity of the clinically isolated CHIKV Thai#16856 strain to HAP1 cells. Knockout of NDST1 that catalyzes N-sulfation of HS greatly decreased the binding and infectivity of CHIKV Thai#16856 strain but not infectivity of Japanese encephalitis virus (JEV) and yellow fever virus (YFV). While glycosaminoglycans were commonly required for the efficient infectivity of CHIKV, JEV, and YFV, as shown by using B3GAT3 knockout cells, the tropism for N-sulfate was specific to CHIKV. Expression of chondroitin sulfate (CS) in NDST1-knockout HAP1 cells did not restore the binding of CHIKV Thai#16856 strain and the infectivity of its pseudotype but restored the infectivity of authentic CHIKV Thai#16856, suggesting that CS functions at later steps after CHIKV binding. Among the genes enriched in this screening, we found that TM9SF2 is critical for N-sulfation of HS and therefore for CHIKV infection because it is involved in the proper localization and stability of NDST1. Determination of the significance of and the relevant proteins to N-sulfation of HS may contribute to understanding mechanisms of CHIKV propagation, cell tropism, and pathogenesis.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Chikungunya virus (CHIKV) utilizes host glycosaminoglycans to bind efficiently to its target cells. However, the substructure in glycosaminoglycans required for CHIKV infection have not been characterized. Here, we unveil that N-sulfate in heparan sulfate is essential for the efficient infection of a clinical CHIKV strain to HAP1 cells and that chondroitin sulfate does not help the CHIKV binding but does play roles at the later steps in HAP1 cells. We show, by comparing previous reports using Chinese hamster ovary cells, along with another observation that enhanced infectivity of CHIKV bearing Arg82 in envelope E2 does not depend on glycosaminoglycans in HAP1 cells, that the infection manner of CHIKV varies among host cells. We also show that TM9SF2 is required for CHIKV infection to HAP1 cells because it is involved in the N-sulfation of heparan sulfate through ensuring NDST1 activity.

The increasing prevalence of chikungunya virus (CHIKV) in the United States and around the world, and the lack of antiviral treatments or vaccines highlight the potential threat of a global CHIKV pandemic. CHIKV is a mosquito-borne alphavirus that is currently causing outbreaks in many parts of the world, including the Americas, India, Africa, and parts of Europe. Host response against CHIKV infection is complex and remains unresolved. Here we discuss the role of the host restriction factor—bone marrow stromal antigen 2 (BST-2) also known as tetherin—as an intrinsic factor involved in CHIKV infection. Recent research reports suggest that the release of nascent CHIKV particles and replication of CHIKV in cultured cells and in animal models is impaired by the expression of BST-2. In this chapter, we discuss available published data on how BST-2 restricts CHIKV and how CHIKV in turn neutralizes BST-2.

A safe and convenient chikungunya-pseudotyped lentiviral vector for epidemiological studies on chikungunya virus in Thailand

Understanding the risk of chikungunya virus (CHIKV) infection and rheumatic sequelae across populations, including travelers and the military, is critical. We leveraged healthcare delivery data of over 9 million U.S. Military Health System (MHS) beneficiaries to identify cases, and sampled controls, to estimate the risk of post-CHIKV rheumatic sequelae. MHS beneficiary CHIKV infections diagnosed 2014-2018 were identified from the Disease Reporting System internet, TRICARE Encounter Data Non-Institutional, and Comprehensive Ambulatory/Professional Encounter Record systems. Non-CHIKV controls were matched (1:4) by age, gender, beneficiary status, and encounter date. The frequency of comorbidities and incident rheumatic diagnoses through December 2018 were derived from International Classification of Diseases codes and compared between cases and controls. Poisson regression models estimated the association of CHIKV infection with rheumatic sequelae. We further performed a nested case-control study to estimate risk factors for post-CHIKV sequelae in those with prior CHIKV. 195 CHIKV cases were diagnosed between July 2014 and December 2018. The median age was 42 years, and 43.6% were active duty. 63/195 (32.3%) of CHIKV cases had an incident rheumatic diagnosis, including arthralgia, polyarthritis, polymyalgia rheumatica, and/or rheumatoid arthritis, compared to 156/780 (20.0%) of controls (p < 0.001). CHIKV infection remained associated with rheumatic sequelae (aRR = 1.579, p = 0.008) after adjusting for prior rheumatic disease and demography. Those with rheumatic CHIKV sequelae had a median 7 healthcare encounters (IQR 3-15). Among CHIKV infections, we found no association between post-CHIKV rheumatic sequelae and demography, service characteristics, or comorbidities. CHIKV infection is uncommon but associated with rheumatic sequelae among MHS beneficiaries, with substantial healthcare requirements in a proportion of cases with such sequelae. No demographic, clinical, or occupational variables were associated with post-CHIKV rheumatic sequelae, suggesting that prediction of these complications is challenging in MHS beneficiaries. These findings are important context for future CHIKV vaccine decision making in this and other populations.

Full genomic sequence characterization of the chikungunya virus from an imported case with serum viral concentration below culturable level

To evaluate the frequency of errors in the diagnosis of medical laboratory-diagnosed Chikungunya virus (CHIKV) infections in Australia, we studied 42 laboratory-diagnosed CHIKV serum samples from one Queensland medical laboratory by ELISA IgG/IgM and measured the specific neutralization antibodies (Nab) against Barmah Forest virus (BFV), CHIKV and Ross River virus (RRV). The sero-positivity rates for the sera were as follows: anti-BFV IgG+ 19% (8/42), IgM+ 2.4% (1/42) and Nab+ 16.7% (7/42); anti-CHIKV IgG+ 90.5% (38/42), IgM+ 21.4% (9/42) and Nab+ 90.5% (38/42); anti-RRV IgG+ 88.1% (37/42), IgM+ 28.6% (12/42) and Nab+ 83.2% (35/42), respectively. Among the samples with multiple antibody positivity, 2.4% (1/42) showed triple ELISA IgM+, and 14.3% (6/42) exhibited double IgM RRV+CHIKV+; 9.5% (4/42) showed triple IgG+, 76.2% (32/42) displayed double IgG RRV+CHIKV+, 4.8% (2/42) showed IgG BFV+RRV+ and 4.8% (2/42) showed IgG BFV++CHIKV+; and 9.5% (4/42) showed triple Nab+ and 69% (29/42) exhibited double Nab RRV+CHIKV+, respectively. Our analysis of the single-virus infection control Nab results suggested no cross-neutralization between RRV and BFV, and only mild cross-neutralization between CHIKV and RRV, BFV and CHIKV, all with a ≥4-fold Nab titre ratio difference between the true virus infection and cross-reactivity counterpart virus. Subsequently, we re-diagnosed these 42 patients as 1 BFV+, 8 CHIKV+ and 23 RRV+ single-virus infections, along with five RRV+/BFV+ and four RRV+/CHIKV+ double infections, and one possible RRV+/BFV+ or RRV+CHIKV+, respectively. These findings suggests that a substantial proportion of medically attended RRV and BFV infections were misdiagnosed as CHIKV infections, highlighting the imperative need for diagnostic laboratory tests capable of distinguishing between CHIKV infections and actively co-circulating RRV and BFV. For a correct diagnosis, it is crucial to consider reliable diagnostic methods such as the neutralization assay to exclude RRV and BFV.

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced cell binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

As outbreaks of chikungunya virus (CHIKV), a mosquito-borne alphavirus, continue to present public health challenges, additional research is needed to generate protective and safe vaccines and effective therapeutics. Prior research has established a role for antibodies in mediating protection against CHIKV infection, and the early appearance of CHIKV-specific IgG or IgG neutralizing antibodies protects against progression to chronic CHIKV disease in humans. However, the importance of epitope specificity for these protective antibodies and how skewed responses contribute to development of acute and chronic CHIKV-associated joint disease remains poorly understood. Here, we describe the deep mutational scanning of one of the dominant targets of neutralizing antibodies during CHIKV infection, the E3/E2 (also known as p62) glycoprotein complex, to simultaneously test thousands of p62 mutants against selective pressures of interest in a high throughput manner. Characterization of the virus library revealed achievement of high diversity while also selecting out non-functional virus variants. Furthermore, this study provides evidence that this virus library system can comprehensively map sites critical for the neutralization function of antibodies of both known and unknown p62 domain specificities.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes rash, fever and severe polyarthritis that can last for years in humans. Murine models display inflammation and macrophage infiltration only in the adjacent tissues at the site of inoculation, showing no signs of systemic polyarthritis. Monocyte-derived macrophages are one cell type suspected to contribute to a systemic CHIKV infection. The purpose of this study was to analyze differences in CHIKV infection in two different cell lines, human U937 and murine RAW264.7 monocyte derived macrophages. PMA-differentiated U937 and RAW264.7 macrophages were infected with CHIKV, and infectious virus production was measured by plaque assay and by reverse transcriptase quantitative PCR at various time points. Secreted cytokines in the supernatants were measured using cytometric bead arrays. Cytokine mRNA levels were also measured to supplement expression data. Here we show that CHIKV replicates more efficiently in human macrophages compared to murine macrophages. In addition, infected human macrophages produced around 10-fold higher levels of infectious virus when compared to murine macrophages. Cytokine induction by CHIKV infection differed between human and murine macrophages; IL-1, IL-6, IFN-γ, and TNF were significantly upregulated in human macrophages. This evidence suggests that CHIKV replicates more efficiently and induces a much greater pro-inflammatory cytokine profile in human macrophages, when compared to murine macrophages. This may shed light on the critical role that macrophages play in the CHIKV inflammatory response.

Vitamin D modulates the expression of Toll-like receptors and pro-inflammatory cytokines without affecting Chikungunya virus replication, in monocytes and macrophages

BackgroundMany studies have demonstrated the association between intestinal microbiota and joint diseases. The “gut-joint axis” also has potential roles in chikungunya virus (CHIKV) infection. Pro-inflammatory arthritis after CHIKV infection might disrupt host homeostasis and lead to dysbacteriosis. This study investigated the characteristics of fecal and gut microbiota, intestinal metabolites, and the changes in gene regulation of intestinal tissues after CHIKV infection using multi-omics analysis to explore the involvement of gut microbiota in the pathogenesis of CHIKV infection.ResultsCHIKV infection increases the systemic burden of inflammation in the GI system of infected animals. Moreover, infection-induced alterations in GI microbiota and metabolites may be indirectly involved in the modulation of GI and bone inflammation after CHIKV infection, including the modulation of inflammasomes and interleukin-17 inflammatory cytokine levels.ConclusionOur results suggest that the GI tract and its microbes are involved in the modulation of CHIKV infection, which could serve as an indicator for the adjuvant treatment of CHIKV infection.AFFfAffYoiCj9mCxSF8PoJVideo

BackgroundSince 2006, Chikungunya virus (CHIKV) has re-emerged as an important pathogen of global concern. However, individual and household factors associated with the acquisition and the magnitude of clinically silent CHIKV infections remain poorly understood. In this present study, we aimed to investigate the seroprevalence, estimate the proportion of symptomatic illness and identify the risk factors for CHIKV infection in the primo-exposed population of Mayotte.Methods/ Principal FindingsWe conducted a household-based cross sectional serosurvey in Mayotte in November and December 2006 using complex multistage cluster sampling. To produce the results representative of the island population aged 2 years or older, sample data were adjusted with sample weights. Explanatory and multiple logistic regression analyses were performed to investigate associations between CHIKV infection seropositivity (presence of IgM and/or IgG to CHIKV by enzyme-linked immunoabsorbent assay) and risk factors. A total of 1154 individuals were analyzed. The overall seroprevalence of CHIKV infection was 37·2% (95% CI = 33·9–40·5), 318 (72·3%) of the seropositive participants reported symptoms consistent with a CHIKV infection during the epidemic period. Risk factors for CHIKV seropositivity among adults (aged 15 years and older) were male gender, low socioeconomic index, schooling ≤6 years and living in makeshift housing.ConclusionsOur findings indicate that roughly one out of four CHIKV infections is asymptomatic. Conditions associated with poverty may be considered as critical in CHIKV acquisition. Thus, these conditions should be taken into account in the development of future prevention strategies of CHIKV disease.