Chikungunya In Brazil: An Emerging Challenge.

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The risk of chikungunya fever in a dengue-endemic area

Background. Chikungunya, an alphavirus of the Togaviridae family, causes a febrile disease transmitted to humans by the bite of infected Aedes mosquitoes. This infection is reaching endemic levels in many Southeast Asian countries. Symptoms include sudden onset of fever, chills, headache, nausea, vomiting, joint pain with or without swelling, low back pain, and rash. According to the World Health Organization, there are 2 billion people living in Aedes-infested areas. In addition, traveling to these areas is popular, making the potential risk of infections transmitted by the bite of infected Aedes mosquitoes very high. Methods. We proposed a mathematical model to estimate the risk of acquiring chikungunya fever in an Aedes-infested area by taking the prevalence of dengue fever into account. The basic reproduction number for chikungunya fever R-0chik can be written as a function of the basic reproduction number of dengue R-0dengue by calculating the ratio R-0chik/R-0dengue. From R-0chik, we estimated the force of infection and the risk of acquiring the disease both for local residents of a dengue-endemic area and for travelers to this area. Results. We calculated that R-0chik is 64.4% that of R-0dengue. The model was applied to a hypothetical situation, namely, estimating the individual risk of acquiring chikungunya fever in a dengue-endemic area, both for local inhabitants (22% in steady state) and for visiting travelers (from 0.31% to 1.23% depending on the time spent in the area). Conclusions. The method proposed based on the output of a dynamical model is innovative and provided an estimation of the risk of infection, both for local inhabitants and for visiting travelers.

Chikungunya risk for Brazil

This study aimed to show, based on the literature on the subject, the potential for dispersal and establishment of the chikungunya virus in Brazil. The chikungunya virus, a Togaviridae member of the genusAlphavirus, reached the Americas in 2013 and, the following year, more than a million cases were reported. In Brazil, indigenous transmission was registered in Amapa and Bahia States, even during the period of low rainfall, exposing the whole country to the risk of virus spreading. Brazil is historically infested by Ae. aegypti and Ae. albopictus, also dengue vectors. Chikungunya may spread, and it is important to take measures to prevent the virus from becoming endemic in the country. Adequate care for patients with chikungunya fever requires training general practitioners, rheumatologists, nurses, and experts in laboratory diagnosis. Up to November 2014, more than 1,000 cases of the virus were reported in Brazil. There is a need for experimental studies in animal models to understand the dynamics of infection and the pathogenesis as well as to identify pathophysiological mechanisms that may contribute to identifying effective drugs against the virus. Clinical trials are needed to identify the causal relationship between the virus and serious injuries observed in different organs and joints. In the absence of vaccines or effective drugs against the virus, currently the only way to prevent the disease is vector control, which will also reduce the number of cases of dengue fever.

Expressão e purificação da proteína E3 do vírus chikungunya (CHIKV)

RESUMO: O vírus chikungunya (CHIKV) é um vírus de RNA, com invólucro, da família Togaviridae, transmitido por mosquitos Aedes spp. Distribuído por largas regiões de África e Ásia, causa grandes epidemias de artrite grave. A semelhança de sintomas com outras doenças como a dengue e a malária e a persistência de IgM específicas, dificultam o diagnóstico da infeção por CHIKV. A deteção no sangue de E3, uma glicoproteína viral secretada, a incluir num ensaio imunoenzimático poderá melhorar o diagnóstico nos países onde as técnicas de biologia molecular são de difícil acesso. Para testar a utilidade de E3 num ensaio de diagnóstico, esta deverá ser expressa em quantidade, purificada e usada para produção de anticorpos específicos. Para expressar E3 numa forma solúvel, suscetível de ser purificada num único passo cromatográfico sem proteases, recorreu-se à estratégia da fusão com o domínio de ligação à quitina (CBD)-inteína (IMPACT™ System, NEB). A sequência codificadora de E3 foi amplificada a partir de RNA viral, clonada em pTYB21 e expressa em E. coli como uma proteína de fusão insolúvel de 64 kDa. A expressão a 12ºC induzida por IPTG 0,1 mM aumentou a solubilidade de CBD-inteína-E3. A aplicação de lisados celulares em colunas de quitina originou a retenção de CBD-inteína-E3 na matriz. Porém, a autoclivagem da inteína na coluna, induzida com reagentes tiol, foi pouco eficiente e mesmo a proteína E3 separada não eluiu da coluna. E3 foi ainda expressa em E. coli com uma cauda de seis histidinas (E3[His]6) por clonagem no vetor pET28b(+). Lisados celulares aplicados em colunas de níquel permitiram a eluição de uma proteína de 9 kDa, compatível com a massa molecular estimada para E3[His]6, ainda que com outros contaminantes proteicos. A identidade da proteína de 9 kDa será confirmada pela indução de anticorpos com esta preparação e reatividade daqueles com células infetadas com CHIKV.----------------ABSTRACT: Chikungunya virus (CHIKV) is an enveloped, positive strand RNA virus belonging to the family Togaviridae. Transmitted by Aedes spp mosquitoes, CHIKV causes large epidemics of severe arthritogenic disease in Africa and Asia and represents a serious threat in countries where vectors are present. Symptoms similarity with other diseases, e.g. dengue and malaria, along with CHIKV IgM persistence turns accurate CHIKV diagnosis a difficult task in low-income countries. Detection of E3, a small secreted viral glycoprotein, to be included in an immunoenzymatic test was envisaged as a possible improvement in CHIKV diagnosis. To test the diagnostic value of E3, recombinant E3 should be expressed and purified to generate antibodies. In order to express CHIKV E3 in a soluble form amenable to purification by a single step affinity chromatography, the chitin binding domain (CBD)-intein fusion strategy without proteases (IMPACT™ System, NEB) was employed. The E3 coding sequence was amplified from viral RNA, cloned in pTYB21 and expressed in E. coli ER2566 as an insoluble 64 kDa CBD-intein-E3 fusion protein. Solubility was partially achieved by lowering the expression temperature to 12ºC and the inducer (IPTG) concentration to 0.1 mM. Clarified cell lysate loaded onto a chitin column allowed ligation of the fusion protein but the intein-mediated cleavage efficiency was low and E3 failed to elute from the column as demonstrated by SDS-PAGE. E3 was further expressed with a six histidine tag, E3[His]6, employing the pET System (Novagen). E3[His]6 was expressed in E. coli Rosetta (30ºC, 0.4 mM IPTG) as a 9 kDa protein. Soluble cell extracts in 20-40 mM imidazole, applied onto a nickel column and eluted with 500 mM imidazole yielded a protein preparation enriched in the 9kDa protein. The 9 kDa will be used as antigen to generate antibodies that upon reaction with CHIKV infected cells will confirm its identity.

Chikungunya virus infection: report of the first case diagnosed in Rio de Janeiro, Brazil

Initially diagnosed in Africa and Asia, the Chikungunya virus has been detected in the last three years in the Caribbean, Italy, France, and the United States of America. Herein, we report the first case for Rio de Janeiro, Brazil, in 2010.

Emerging alphaviruses in the Americas: Chikungunya and Mayaro

Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are emergent arthropod-borne viruses that produce outbreaks of acute febrile illness with arthropathy. Despite their different continental origins, CHIKV and MAYV are closely related and are components of the Semliki Forest Complex of the Alphavirus (Togaviridae). MAYV and, more recently, CHIKV, which are both transmitted by Aedes mosquitoes, have resulted in severe public health problems in the Americas, including Brazil. In this review, we present aspects of the pathogenesis, clinical presentation and treatment of febrile illnesses produced by CHIKV and MAYV. We also discuss the epidemiological aspects and effects related to the prophylaxis of infections by both viruses.

Failure to demonstrate experimental vertical transmission of the epidemic strain of Chikungunya virus in Aedes albopictus from La Réunion Island, Indian Ocean

Aedes albopictus was responsible for transmission in the first outbreak of chikungunya (CHIK) on La Réunion Island, Indian Ocean, in 2005-2006. The magnitude of the outbreak on this island, which had been free of arboviral diseases for over 30 years, as well as the efficiency of Ae. albopictus as the main vector, raises questions about the maintenance of the CHIK virus (CHIKV) through vertical transmission mechanisms. Few specimens collected from the field as larvae were found to be infected. In this study, Ae. albopictus originating from La Réunion were orally infected with a blood-meal containing 10(8) pfu/mL of the CHIKV epidemic strain (CHIKV 06.21). Eggs from the first and second gonotrophic cycles were collected and raised to the adult stage. The infectious status of the progeny was checked (i) by immunofluorescence on head squashes of individual mosquitoes to detect the presence of viral particles or (ii) by quantitative RT-PCR on mosquito pools to detect viral RNA. We analysed a total of 1,675 specimens from the first gonotrophic cycle and 1,709 from the second gonotrophic cycle without detecting any viral particles or viral RNA. These laboratory results are compared to field records.

Chikungunya virus outbreak in Kerala, India, 2007: a seroprevalence study

India was affected by a major outbreak of chikungunya fever caused by Chikungunya virus (CHIKV) during 2006-2007. Kerala was the worst affected state during 2007 with a contribution of 55.8% suspected cases in the country. However, except for clinically reported case records, no systematic information is available on infection status of CHIKV in the region. Hence, we carried out a post-epidemic survey to estimate seroprevalence status [immunoglobulin G (IgG)] in the community using commercially available indirect immunofluorescence test. This methodology had been reported to be highly specific and sensitive for CHIKV infection. The study area selected was the worst affected mid-highlands region of Kerala which harbour vast area of rubber plantations. The study evidenced 68% of the population to be seropositive for CHIKV IgG. Males were found more affected than females (χ2 = 9.86; p = 0.002). Among males, prevalence was significantly higher in the age classes 21-30 (χ2 = 5.46; p = 0.019) and 31-40 (χ2 = 5.84; p = 0.016) years. This may be due to high occupational risk of the male population engaged in plantation activities exposed to infective bites of Aedes albopictus. The current study provides an insight into the magnitude of CHIKV outbreak in Kerala.

Waiting for chikungunya fever in Argentina: spatio-temporal risk maps

Chikungunya virus (CHIKV) transmission has been detected in America in 2013 and recently reached south up to Bolivia, Brazil and Paraguay, bordering countries of Argentina. The presence of the mosquito Aedes aegyptiin half of the country together with the regional context drove us to make a rapid assessment of transmission risk. Temperature thresholds for vector breeding and for virus transmission, together with adult activity from the literature, were mapped on a monthly basis to estimate risk. Transmission of chikungunya byAe. aegyptiin the world was seen at monthly mean temperatures from 21-34ºC, with the majority occurring between 26-28ºC. In Argentina temperatures above 21ºC are observed since September in the northeast, expanding south until January and retreating back to the northeast in April. The maximum area under risk encompasses more than half the country and around 32 million inhabitants. Vector adult activity was registered where monthly means temperatures exceeded 13ºC, in the northeast all over the year and in the northern half from September-May. The models herein proposed show that conditions for transmission are already present. Considering the regional context and the historic inability to control dengue in the region, chikungunya fever illness seems unavoidable.

Phylogenetic analyses of chikungunya virus among travelers in Rio de Janeiro, Brazil, 2014-2015

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that emerged in Brazil by late 2014. In the country, two CHIKV foci characterized by the East/Central/South Africa and Asian genotypes, were established in North and Northeast regions. We characterized, by phylogenetic analyses of full and partial genomes, CHIKV from Rio de Janeiro state (2014-2015). These CHIKV strains belong to the Asian genotype, which is the determinant of the current Northern Brazilian focus, even though the genome sequence presents particular single nucleotide variations. This study provides the first genetic characterisation of CHIKV in Rio de Janeiro and highlights the potential impact of human mobility in the spread of an arthropod-borne virus.

Type I IFN controls chikungunya virus via its action on nonhematopoietic cells.

Chikungunya virus (CHIKV) is the causative agent of an outbreak that began in La Réunion in 2005 and remains a major public health concern in India, Southeast Asia, and southern Europe. CHIKV is transmitted to humans by mosquitoes and the associated disease is characterized by fever, myalgia, arthralgia, and rash. As viral load in infected patients declines before the appearance of neutralizing antibodies, we studied the role of type I interferon (IFN) in CHIKV pathogenesis. Based on human studies and mouse experimentation, we show that CHIKV does not directly stimulate type I IFN production in immune cells. Instead, infected nonhematopoietic cells sense viral RNA in a Cardif-dependent manner and participate in the control of infection through their production of type I IFNs. Although the Cardif signaling pathway contributes to the immune response, we also find evidence for a MyD88-dependent sensor that is critical for preventing viral dissemination. Moreover, we demonstrate that IFN-alpha/beta receptor (IFNAR) expression is required in the periphery but not on immune cells, as IFNAR(-/-)-->WT bone marrow chimeras are capable of clearing the infection, whereas WT-->IFNAR(-/-) chimeras succumb. This study defines an essential role for type I IFN, produced via cooperation between multiple host sensors and acting directly on nonhematopoietic cells, in the control of CHIKV.

The use of insect cells to identify potent and selective inhibitors of the replication of the Dengue and Chikungunya viruses and unravel their molecular mechanism of action

Dengue and Chikungunya viruses cause the most important arthropod-borne viral infections for humans. These viruses are predominant in tropical and subtropical regions. In addition, these viruses are predominant in tropical and subtropical regions. Dengue mortality rate is around 1.2 to 3.5% and deaths due to chikungunya fever are around 1 in 1000; however, half of chikungunya-infected patients evolve into a chronic state that can persist for months up to years. There are no antiviral drugs available for DENV and CHIKV treatment and prevention. Moreover, vector control strategies have failed so far. Thus, the development of potent inhibitors for a broad spectrum of RNA viruses is urgently needed. We established and characterized a new embryonic insect cell line from Culex quinquefasciatus mosquito. Also we established the flaviviruses and alphavirus replication, both in C6/36 and Lulo insect cell lines, as well as in Vero cell line. In addition we carried out a reference compound library and reference panel of assays and data for DENV, which provides a benchmark for further studies. During this study, a panel of 9 antiviral molecules, with proven in vitro anti-dengue virus activity and that act at different stages of the DENV life cycle, was selected. Finally, Favipiravir or T-705, was identified as inhibitor in vitro and in vivo of alphaviruses and the mutation K291R in nsP4, which is responsible of the polymerase activity, was found as the mode of action in CHIKV. Interestingly, lysine in motif F1 is also highly conserved in positive-stranded RNA viruses and this might explain the broad spectrum of T-705 antiviral activity.

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing >= 50% inhibition property against CHIKV at 10 mu M were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 mu M and 7.1 mu M. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity -inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.

Chronic pain associated with the Chikungunya Fever: long lasting burden of an acute illness

Abstract Background Chikungunya virus (CHIKV) is responsible for major epidemics worldwide. Autochthonous cases were recently reported in several European countries. Acute infection is thought to be monophasic. However reports on chronic pain related to CHIKV infection have been made. In particular, the fact that many of these patients do not respond well to usual analgesics suggests that the nature of chronic pain may be not only nociceptive but also neuropathic. Neuropathic pain syndromes require specific treatment and the identification of neuropathic characteristics (NC) in a pain syndrome is a major step towards pain control. Methods We carried out a cross-sectional study at the end of the major two-wave outbreak lasting 17 months in Réunion Island. We assessed pain in 106 patients seeking general practitioners with confirmed infection with the CHIK virus, and evaluated its impact on quality of life (QoL). Results The mean intensity of pain on the visual-analogical scale (VAS) was 5.8 ± 2.1, and its mean duration was 89 ± 2 days. Fifty-six patients fulfilled the definition of chronic pain. Pain had NC in 18.9% according to the DN4 questionnaire. Conversely, about two thirds (65%) of patients with NC had chronic pain. The average pain intensity was similar between patients with or without NC (6.0 ± 1.7 vs 6.1 ± 2.0). However, the total score of the Short Form-McGill Pain Questionnaire (SF-MPQ)(15.5 ± 5.2 vs 11.6 ± 5.2; p < 0.01) and both the affective (18.8 ± 6.2 vs 13.4 ± 6.7; p < 0.01) and sensory subscores (34.3 ± 10.7 vs 25.0 ± 9.9; p < 0.01) were significantly higher in patients with NC. The mean pain interference in life activities calculated from the Brief Pain Inventory (BPI) was significantly higher in patients with chronic pain than in patients without it (6.8 ± 1.9 vs 5.9 ± 1.9, p < 0.05). This score was also significantly higher in patients with NC than in those without such a feature (7.2 ± 1.5 vs 6.1 ± 1.9, p < 0.05). Conclusions There exists a specific chronic pain condition associated to CHIKV. Pain with NC seems to be associated with more aggressive clinical picture, more intense impact in QoL and more challenging pharmacological treatment.

The 2014 chikungunya virus outbreak in the U.S. Virgin Islands: epidemiology, long-term health outcomes, and economic burden

Thesis (Ph.D.)--University of Washington, 2016-06