Advertisement

Chikungunya Virus

Published:March 24, 2017DOI:https://doi.org/10.1016/j.cll.2017.01.008

      Keywords

      Key points

      • Chikungunya is an arboviral infection that causes debilitating arthritis and arthralgia.
      • Chikungunya virus has caused explosive epidemics in the past decade, and has spread rapidly from Africa to Asia to the Americas.
      • Improved diagnostic testing and surveillance for chikungunya infection is needed to detect and respond to future outbreaks.
      • Further investigation into the pathogenesis of chikungunya infection is needed to understand its long-term sequelae, and to develop effective therapies.

      Microbiology

      Chikungunya virus (CHIKV) belongs to the Semliki Forest antigenic group of the genus Alphaviridae, which includes other arthritogenic alphaviruses, such as o’nyong-nyong, Ross River, Barmah Forest, and Mayaro viruses.
      • Strauss J.H.
      • Strauss E.G.
      The alphaviruses: gene expression, replication, and evolution.
      • Lwande O.W.
      • Obanda V.
      • Bucht G.
      • et al.
      Global emergence of alphaviruses that cause arthritis in humans.
      Its genome is closely related to that of o’nyong-nyong virus, and consists of a single 11.8-kbp strand of positive sense RNA, which encodes a 2472 amino acid nonstructural and a 1244 amino acid structural polyprotein.
      • Khan A.H.
      • Morita K.
      • Parquet Md Mdel C.
      • et al.
      Complete nucleotide sequence of chikungunya virus and evidence for an internal polyadenylation site.
      The polyproteins give rise to the four nonstructural proteins (nsP1-4) that make up the viral replication machine, and five structural proteins. Each spherical viral particle is approximately 70 nm in diameter and is comprised of a strand of genomic RNA, encapsidated by capsid (C) proteins, surrounded by a host cell–derived lipid bilayer spiked with heterodimers of envelope proteins E1 and E2.
      • Jose J.
      • Snyder J.E.
      • Kuhn R.J.
      A structural and functional perspective of alphavirus replication and assembly.
      The other two structural proteins, 6K and E3, are leader peptides for E1 and E2, respectively, and are not observed in abundance in the mature virion.
      • Jose J.
      • Snyder J.E.
      • Kuhn R.J.
      A structural and functional perspective of alphavirus replication and assembly.
      The envelope proteins, E2 and E1, play important roles in the binding of the virus to the host cell membrane and its subsequent cellular invasion, respectively. Anti-CHIKV antibodies directed against the envelope protein that neutralize the virus in vitro also protect neonatal mice from lethal CHIKV infection in vivo, suggesting that these proteins may be important antigenic lethal targets for development of naturally acquired, or vaccine-elicited protection.
      • Weger-Lucarelli J.
      • Aliota M.T.
      • Kamlangdee A.
      • et al.
      Identifying the role of e2 domains on alphavirus neutralization and protective immune responses.
      • Fong R.H.
      • Banik S.S.
      • Mattia K.
      • et al.
      Exposure of epitope residues on the outer face of the chikungunya virus envelope trimer determines antibody neutralizing efficacy.
      • Smith S.A.
      • Silva L.A.
      • Fox J.M.
      • et al.
      Isolation and characterization of broad and ultrapotent human monoclonal antibodies with therapeutic activity against chikungunya virus.

      Epidemiology

      The earliest report of chikungunya fever described an outbreak of a dengue-like illness that occurred in 1952 to 1953, on the Makonde Plateau in the Southern Province of Tanganyika (present day Tanzania).
      • Robinson M.C.
      An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952-53. I. Clinical features.
      Residents of all ages experienced a febrile illness with rash and arthralgia. However, certain aspects of this outbreak distinguished it from previous reports of dengue fever outbreaks. Most striking was the severity of the arthralgia that “would prevent the sufferer from changing position without help.”
      • Robinson M.C.
      An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952-53. I. Clinical features.
      The local population began to call the disease chikungunya, which is a Makonde (Bantu) term that means “that which bends up,” referring to the contorted positions of those who were affected by the sudden and severe onset of arthralgia. Additionally, many individuals affected with the disease continued to experience intermittent joint pains that persisted for months after the acute illness. The attack rate also seemed to be unusually high, often affecting entire households. Between 1952 and 1953, an estimated 60% to 80% of the population in this region developed symptoms of fever, rash, and arthralgia.
      • Robinson M.C.
      An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952-53. I. Clinical features.
      Attempts to isolate the pathologic agent from symptomatic individuals during the outbreak also diverged from previous experience with dengue virus (DENV): Inoculation of infant mice with serum samples from symptomatic individuals resulted in death of the animals. In contrast, DENV infection is difficult to establish in mice.
      • Ross R.W.
      The Newala epidemic. III. The virus: isolation, pathogenic properties and relationship to the epidemic.
      These data suggested that the cause of the syndrome termed chikungunya indeed was distinct from the cause of dengue fever.
      In Africa, CHIKV is transmitted by arboreal Aedes mosquitoes (A furcifer-taylori, A africanus, A luteocephalus, and A neoafricanus) in an enzootic cycle with nonhuman primates as the principle reservoir (Fig. 1).
      • Diallo M.
      • Thonnon J.
      • Traore-Lamizana M.
      • et al.
      Vectors of chikungunya virus in Senegal: current data and transmission cycles.
      • McIntosh B.M.
      • Paterson H.E.
      • McGillivray G.
      • et al.
      Further studies on the chikungunya outbreak in southern Rhodesia in 1962. I. Mosquitoes, wild primates and birds in relation to the epidemic.
      • Paterson H.E.
      • McIntosh B.M.
      Further studies on the chikungunya outbreak in southern Rhodesia in 1962. II. Transmission experiments with the Aedes furcifer-taylori group of mosquitoes and with a member of the Anopheles gambiae complex.
      Between the 1960s and 1990s, incidental human infection led to numerous, small-scale CHIKV outbreaks in countries throughout Central and Southern Africa, and Senegal, Guinea, and Nigeria in Western Africa (reviewed in Ref.
      • Powers A.M.
      • Logue C.H.
      Changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus.
      ). The outbreaks occurred after periods of large rainfall and associated surges in the arboreal Aedes mosquito density. In contrast, CHIKV outbreaks in Southeast Asia occurred in larger cities where Aedes aegypti mosquitoes were implicated as the primary transmission vector. A aegypti mosquitoes require very small amounts of water to lay eggs, and thrive in human urban environments, particularly in areas where residents store water in open containers or cisterns.
      Figure thumbnail gr1
      Fig. 1Transmission cycle of CHIKV in Africa. Nonhuman primates, and possibly other wild animals, serve as reservoirs of the virus. Infected arboreal Aedes mosquitoes bite and infect humans. Infected humans, in turn, infect peridomestic Aedes aegypti, perpetuating the urban cycle of CHIKV transmission.
      (From Thiboutot MM, Kannan S, Kawalekar OU, et al. Chikungunya: a potentially emerging epidemic? PLoS Negl Trop Dis 2010;4(4):e623.)
      In 2004, a large-scale CHIKV epidemic erupted, sweeping down the coast of Kenya into islands on the Indian Ocean (Comoros, Mayotte, Seychelles, Réunion, Madagascar, Sri Lanka, and the Maldives), India, Southeast Asia (Malaysia, Singapore, Thailand), and China.
      • Kariuki Njenga M.
      • Nderitu L.
      • Ledermann J.P.
      • et al.
      Tracking epidemic chikungunya virus into the Indian Ocean from East Africa.
      Although CHIKV infection in travelers returning to Europe had been reported previously, autochthonous transmission of CHIKV was observed for the first time in Italy in 2007,
      • Rezza G.
      • Nicoletti L.
      • Angelini R.
      • et al.
      Infection with chikungunya virus in Italy: an outbreak in a temperate region.
      and in France in 2009.
      • Grandadam M.
      • Caro V.
      • Plumet S.
      • et al.
      Chikungunya virus, southeastern France.
      An important factor that facilitated the rapid expansion of CHIKV infection was a novel single amino acid substitution of alanine for valine at position 226 (A226V) in the E1 envelope protein that enhanced the ability of the Aedes albopictus mosquito to transmit CHIKV to humans.
      • Tsetsarkin K.A.
      • Vanlandingham D.L.
      • McGee C.E.
      • et al.
      A single mutation in chikungunya virus affects vector specificity and epidemic potential.
      A albopictus is an anthropophilic, peridomestic species of mosquito that has an even greater geographic range than that of its relative, A aegypti, and has been implicated as having a major role in the spread of CHIKV epidemics across Asia and to the Americas.
      In December 2013, the first cases of locally transmitted CHIKV in the Americas were confirmed in St. Martin,
      • Fischer M.
      • Staples J.E.
      • Arboviral Diseases Branch, National Center for Emerging and Zoonotic Infectious Diseases, CDC
      Notes from the field: chikungunya virus spreads in the Americas - Caribbean and South America, 2013-2014.
      followed rapidly by cases identified throughout the Caribbean and Latin America. By January 2015, CHIKV infection had been identified in 42 countries or territories in the Caribbean, Central America, South America, and North America (local transmission in Florida) with more than a million suspected cases reported, and more than 25,000 laboratory-confirmed.

      The Pan American Health Organization. Number of reported cases of chikungunya fever in the Americas, by country or territory 2013-2014 cumulative cases (updated 23 Oct 2015). Number of Reported Cases of Chikungunya Fever in the Americas 2015; 2016. Available at: http://www.paho.org/hq/index.php?option=com_topics&view=readall&cid=5927&Itemid=40931&lang=en. Accessed October 1, 2016.

      Although epidemics of febrile arthralgia have been reported in the Americas since the 1700s, these outbreaks previously had been attributed to dengue fever. For example, an epidemic “break-bone fever,” which referred to modern-day dengue fever, erupted on the islands of St. Thomas and Santa Cruz in the West Indies from 1827 to 1828. Stedman, who reported this “anomalous disease” called “dandy fever” by local residents, noted that the illness “attacked almost every individual in the town,” had “extremely low mortality,” and was associated with “pains in the joints for weeks after recovery from the acute stage,” which were key differences between the 1827 and 1828 West Indies epidemic and previous descriptions of a “break-bone fever” (referring to modern-day dengue fever). He concluded that “the diseases, though somewhat alike in a few symptoms, are essentially different.”
      • Carey D.E.
      Chikungunya and dengue: a case of mistaken identity?.
      Thus, although the 2013 epidemic was the first CHIKV outbreak in the Americas to be confirmed using modern-day virologic methods, historical reports raise questions to whether this truly was the first introduction of CHIKV infection to the Americas.
      • Halstead S.B.
      Reappearance of chikungunya, formerly called dengue, in the Americas.

      Pathogenesis

      CHIKV is known to infect a variety of cell lines in vitro, including Vero cells (from green monkey kidney)
      • Higashi N.
      • Matsumoto A.
      • Tabata K.
      • et al.
      Electron microscope study of development of chikungunya virus in green monkey kidney stable (Vero) cells.
      and BHK21 baby hamster kidney cells,
      • Hahon N.
      • Zimmerman W.D.
      Chikungunya virus infection of cell monolayers by cell-to-cell and extracellular transmission.
      and various insect cell lines.
      • Buckley S.M.
      • Singh K.R.
      • Bhat U.K.
      Small- and large-plaque variants of chikungunya virus in two vertebrate and seven invertebrate cell lines.
      • Cunningham A.
      • Buckley S.M.
      • Casals J.
      • et al.
      Isolation of chikungunya virus contaminating an Aedes albopictus cell line.
      Human cellular tropism was more recently described.
      • Sourisseau M.
      • Schilte C.
      • Casartelli N.
      • et al.
      Characterization of reemerging chikungunya virus.
      Fibroblasts in the dermis, joint capsule, and muscle seem to be the major targets of CHIKV infection in humans.
      • Couderc T.
      • Chretien F.
      • Schilte C.
      • et al.
      A mouse model for chikungunya: young age and inefficient type-I interferon signaling are risk factors for severe disease.
      Human epithelial and endothelial cells
      • Sourisseau M.
      • Schilte C.
      • Casartelli N.
      • et al.
      Characterization of reemerging chikungunya virus.
      and muscle progenitors (satellite cells)
      • Ozden S.
      • Huerre M.
      • Riviere J.P.
      • et al.
      Human muscle satellite cells as targets of chikungunya virus infection.
      also have been observed to be infected by CHIKV. Lymphocytes and monocytes seem resistant, yet macrophages seem susceptible to CHIKV infection.
      • Sourisseau M.
      • Schilte C.
      • Casartelli N.
      • et al.
      Characterization of reemerging chikungunya virus.
      Investigations of disease pathogenesis during human CHIKV infection have been limited, in part, by the lack of relevant and/or accessible models of CHIKV disease. Development of nonhuman primate models of CHIKV infection has been helpful for use in evaluating potential CHIKV vaccines. Rhesus macaques immunized with an investigational CHIKV virus-like particle vaccine were protected against developing viremia after intravenous challenge with 1010 PFU of CHIKV, whereas control monkeys that received the mock vaccine developed high levels of viremia after challenge.
      • Akahata W.
      • Yang Z.Y.
      • Andersen H.
      • et al.
      A virus-like particle vaccine for epidemic chikungunya virus protects nonhuman primates against infection.
      In a separate study, cynomolgus macaques challenged with a much lower CHIKV inoculum, by either intravenous or intradermal injection, developed viremia, fever, and rash. Viral RNA remained detectable in synovial and muscle tissue for up to 1.5 months after infection, and in lymphoid tissue for up to 3 months.
      • Labadie K.
      • Larcher T.
      • Joubert C.
      • et al.
      Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages.
      Thus, this model may be useful for studying long-term sequelae of CHIKV infection, such as the prolonged arthralgia experienced by many CHIKV individuals.
      Because nonhuman primate models of CHIKV infection are not readily accessible to many investigators, some investigators have developed mouse models to study CHIKV disease. Viral challenge of neonatal wild-type mice results in fatal infection. However, this susceptibility to CHIKV infection wanes quickly and adult wild-type mice are resistant to CHIKV infection. Type I interferon receptor knock-out mice (IFN-α/βR−/-) have an impaired type I IFN pathway and, in contrast with adult wild-type mice, develop viremia after viral challenge.
      • Couderc T.
      • Chretien F.
      • Schilte C.
      • et al.
      A mouse model for chikungunya: young age and inefficient type-I interferon signaling are risk factors for severe disease.
      Thus, it seems that activation of the type I IFN pathway plays an important role in controlling CHIKV infection.
      Mice also have been used to develop models of CHIKV-related arthritis.
      • Gardner J.
      • Anraku I.
      • Le T.T.
      • et al.
      Chikungunya virus arthritis in adult wild-type mice.
      • Morrison T.E.
      • Oko L.
      • Montgomery S.A.
      • et al.
      A mouse model of chikungunya virus-induced musculoskeletal inflammatory disease: evidence of arthritis, tenosynovitis, myositis, and persistence.
      One group of investigators demonstrated that mice injected with CHIKV in the footpad developed leg swelling and weight loss, and had histologic evidence of necrotizing myositis, arthritis, tenosynovitis, and vasculitis.
      • Morrison T.E.
      • Oko L.
      • Montgomery S.A.
      • et al.
      A mouse model of chikungunya virus-induced musculoskeletal inflammatory disease: evidence of arthritis, tenosynovitis, myositis, and persistence.
      A separate group of investigators also elicited foot swelling in mice injected with CHIKV in the ventral footpad. These mice developed viremia, and histologic examination revealed large mononuclear cell infiltrates in and around synovial membranes, and in muscle tissue. Furthermore, treatment with IFN-α before CHIKV inoculation reduced viremia and prevented manifestations of arthritis,
      • Gardner J.
      • Anraku I.
      • Le T.T.
      • et al.
      Chikungunya virus arthritis in adult wild-type mice.
      again highlighting the importance of type I IFNs in CHIKV virus control.

      Clinical manifestations

      In contrast to DENV, which can cause asymptomatic infection, most individuals with CHIKV infection are symptomatic.
      • Robinson M.C.
      An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952-53. I. Clinical features.
      However, chikungunya fever shares many similarities with dengue fever. Sudden onset of high fever is typically the initial symptom reported and can appear within 2 days of infection. A rash sometimes is observed and is typically maculopapular, although bullous rashes have been noted in some infants with CHIKV infection.
      • Robin S.
      • Ramful D.
      • Zettor J.
      • et al.
      Severe bullous skin lesions associated with chikungunya virus infection in small infants.
      Both viral infections also are known to cause arthralgia and arthritis. However, the polyarthralgia caused by CHIKV frequently is characterized as debilitating and has been reported to continue well beyond the resolution of fever. For example, a third of travelers to the Caribbean who acquired CHIKV infection in 2014 while abroad reported persistent joint or muscle pain, or joint swelling at greater than or equal to 9 months after their acute infection.
      • Zeana C.
      • Kelly P.
      • Heredia W.
      • et al.
      Post-chikungunya rheumatic disorders in travelers after return from the Caribbean.
      The knees are the most commonly involved joints; however, other large or small joints may be affected. Of note, symmetric involvement of joints is frequently reported.
      • Manimunda S.P.
      • Vijayachari P.
      • Uppoor R.
      • et al.
      Clinical progression of chikungunya fever during acute and chronic arthritic stages and the changes in joint morphology as revealed by imaging.
      Additional symptoms reported include fatigue, nausea, vomiting, and conjunctivitis. Conspicuously absent among those with CHIKV infection are reports of retro-orbital headache, which are characteristic of dengue fever.
      • Robinson M.C.
      An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952-53. I. Clinical features.
      Most symptoms resolve within 7 to 10 days; however, many infected individuals have reported protracted arthralgia that has lasted weeks, months, or even years. This long-term burden of disease can be devastating to local economies and represents a significant health cost: CHIKV was responsible for 1386 to 1,081,962 nondiscounted years of life lost in 2005.
      • Labeaud A.D.
      • Bashir F.
      • King C.H.
      Measuring the burden of arboviral diseases: the spectrum of morbidity and mortality from four prevalent infections.
      These values are now significant underestimations of the true health cost burden, given its expanding distribution to the Americas since those estimates were made.
      Neurologic complications of acute CHIKV disease were observed with the 2006 outbreak on La Réunion Island, and include encephalitis
      • Lemant J.
      • Boisson V.
      • Winer A.
      • et al.
      Serious acute chikungunya virus infection requiring intensive care during the Reunion Island outbreak in 2005-2006.
      and Guillain-Barré syndrome.
      • Wielanek A.C.
      • Monredon J.D.
      • Amrani M.E.
      • et al.
      Guillain-Barre syndrome complicating a chikungunya virus infection.
      These also were observed during CHIKV outbreaks in India.
      • Rampal
      • Sharda M.
      • Meena H.
      Neurological complications in chikungunya fever.
      • Singh R.K.
      • Tiwari S.
      • Mishra V.K.
      • et al.
      Molecular epidemiology of chikungunya virus: mutation in e1 gene region.
      • Chandak N.H.
      • Kashyap R.S.
      • Kabra D.
      • et al.
      Neurological complications of chikungunya virus infection.
      Additional observations of severe manifestations of disease, including myocarditis and hepatitis,
      • Lemant J.
      • Boisson V.
      • Winer A.
      • et al.
      Serious acute chikungunya virus infection requiring intensive care during the Reunion Island outbreak in 2005-2006.
      have re-energized investigations of CHIKV disease pathogenesis.
      CHIKV infection had not been associated with increased risk of mortality before 2006. During the outbreak on La Réunion Island, however, at least 213 people with CHIKV infection died. Investigators estimated that the case-fatality rate was approximately 1:1000, and observed that the fatalities occurred mainly in persons greater than or equal to 75 years of age.
      • Josseran L.
      • Paquet C.
      • Zehgnoun A.
      • et al.
      Chikungunya disease outbreak, Reunion Island.
      During the 2006 outbreak of CHIKV infection in Ahmedebad, India, 18 of the 90 confirmed cases of CHIKV infection were fatal. Fifteen of the 18 deaths occurred in persons 60 years of age and older.
      • Tandale B.V.
      • Sathe P.S.
      • Arankalle V.A.
      • et al.
      Systemic involvements and fatalities during chikungunya epidemic in India, 2006.
      Autopsy of fatal cases of CHIKV infection in Colombia revealed evidence of hepatocellular coagulative necrosis, tubulointerstitial nephritis, and acute pericarditis.
      • Mercado M.
      • Acosta-Reyes J.
      • Parra E.
      • et al.
      Clinical and histopathological features of fatal cases with dengue and chikungunya virus co-infection in Colombia, 2014 to 2015.

      Diagnostic evaluation

      Good laboratory testing services are important in the diagnosis of suspected CHIKV infections. Chikungunya fever can easily be confused at various stages of the disease with other arboviral infections, such as dengue and Zika virus. The clinical consequences of these three viruses are different, so specific diagnosis is important. Situations where laboratory test confirmation for presence or absence of infection are discussed next.
      Testing sporadic cases of suspected arboviral infection can provide early warning that the virus is in the community. This can help public health and medical personnel prevent a possible epidemic. This is especially important if conditions conducive to an epidemic are present, such as during the rainy season when the mosquito population is high, and with open housing conditions that enhance exposure of an immunologically naive population to infection. Diagnosis of a significant number of cases early on to establish cause of an epidemic and the characteristic symptoms enables reliable clinical diagnosis with decreased need for laboratory confirmation if an epidemic occurs. Public health departments may occasionally perform epidemiologic testing for evidence of past CHIKV infection in a community so that the immunologic history and state of susceptibility in a population are determined to better approximate risk for an outbreak.
      Focused and random spot testing is important during an epidemic to detect the entrance of a second arbovirus, such as DENV, entering the population during a CHIKV outbreak. Focused testing is important in cases with serious underlying diseases and in cases with complications or a fatal outcome.
      In the postoutbreak period, persons not previously tested for CHIKV should be tested for a recent or past infection as part of the work-up in patients presenting with new chronic arthritic problems including joint pain and swelling. During interepidemic periods, patients presenting with typical CHIKV symptoms should be tested for CHIKV and other arboviruses with similar symptoms.
      Three tests for CHIKV are useful in various situations. For diagnostic confirmation of current and recent infection, a molecular test (typically polymerase chain reaction [PCR]) for the virus and an assay for the presence of specific IgM antibody are required. The most frequently needed assay is the CHIKV IgM antibody assay. Molecular testing for the presence of the virus is required in the early stages of disease. Virus is present in the blood at the time symptoms appear and PCR testing provides reliable detection for 5 days thereafter (6 days total). During that time only a molecular test for the virus should be ordered. As IgM production rises, by Days 7 to 9, viremia falls to PCR undetectable levels. During Days 5 to 9 when the viral load is waning and IgM has not reached its peak, it is necessary to order a molecular test for the virus and the IgM antibody assay to maintain good diagnostic sensitivity. After that, only the IgM test is required. After 14 to 21 days both the IgM and IgG test are positive, and the IgM test wanes over several weeks or months, whereas IgG remains for years as a good marker of past infection and immunologic protection, and also as an epidemiologic tool to determine seroprevalence in a population.
      Using Grenada as a case study to illustrate the CHIKV diagnostic strategy, during the peak of the 2014 Grenada CHIKV epidemic, 112 samples from typical cases were tested by PCR and the IgM assay independent of the stage of disease. Although all 112 had classic symptoms, only 101 were found to be positive by at least one of these laboratory tests. In the 101 samples, IgM outperformed PCR: 92% were positive by IgM, 17% were positive by PCR, and 9% were positive by both. Reliance on PCR testing only is unlikely to accurately characterize incidence.

      Jungkind D, Myers TE, Simmons M, et al. Establishment of laboratory testing capability for chikungunya virus in Grenada, West Indies. Presented at the Caribbean Public Health Agency: 60th Annual Scientific Meeting. The University of the West Indies, June 20-24, 2015.

      • Simmons M.
      • Myers T.
      • Guevara C.
      • et al.
      Development and validation of a quantitative, one-step, multiplex, real-time reverse transcriptase PCR assay for detection of dengue and chikungunya viruses.

      Macpherson CNL, Noel T, Jungkind D, et al. Clinical, molecular and serological outcomes of the chikungunya outbreak in Grenada. Presented at the Caribbean Public Health Agency: 60th Annual Scientific Meeting. The University of the West Indies. 2015.

      Commercially available kits are of variable quality.
      • Soh L.T.
      • Squires R.C.
      • Tan L.K.
      • et al.
      External quality assessment of dengue and chikungunya diagnostics in the Asia Pacific region, 2015.
      PCR assays with favorable performance characteristics are documented in the literature and becoming commercially available for clinical use in other countries; however, they can only be obtained for research use in the United States. Good immunoassays for CHIKV-specific IgM in patient serum are less reliable and available.
      • Prat C.M.
      • Flusin O.
      • Panella A.
      • et al.
      Evaluation of commercially available serologic diagnostic tests for chikungunya virus.
      • Kam Y.W.
      • Pok K.Y.
      • Eng K.E.
      • et al.
      Sero-prevalence and cross-reactivity of chikungunya virus specific anti-e2ep3 antibodies in arbovirus-infected patients.
      • Parashar D.
      • Paingankar M.S.
      • Sudeep A.B.
      • et al.
      Assessment of qPCR, nested RT-PCR and ELISA techniques in diagnosis of chikungunya.
      A partial list of sources for molecular viral assays and IgM assays to document CHIKV infection is given in Table 1. These sources can serve as starting points to explore test kit choices and capabilities in what it is hoped will be an expanding menu of kits for clinical testing.
      Table 1Sources for commercially available kits for detection of chikungunya virus and IgM and IgG antibody in serum
      Partial list of companies providing PCR and ELISA kits for detection of CHIKV or human antibody to the virus. None of these are FDA approved for use as diagnostic tests at this time but are considered in the Research Use Only category. One product is CE approved. These tests may prove to be useful adjuncts to assist in the development of future FDA-approved or validated assays.
      CompanyAddressProductComments
      Altona Diagnostics, GmbHMörkenstrasse 12 22767 Hamburg, GermanyRealStar Chikungunya RT-PCR Kit
      Liferiver Bio-Tech9855 Towne Center Drive, San Diego, CA 92121Chikungunya Virus Real Time RT-PCR KitCE approved
      Primerdesign, LtdYork House School Lane, Chandler's Ford, United Kingdom, SO53 4DGChikungunya Virus Real Time RT-PCR KitDengue, Zika, and chikungunya virus multiplex kit also available
      GenWay Biotech, Inc6777 Nancy Ridge Drive, San Diego, CA 92121Real Time RT-PCR Kit

      IgM μ-capture ELISA

      IgG capture ELISA
      Separate PCR kits formatted for two commonly used PCR instrument types

      Provides PCR and ELISA assays
      Euroimmun AGSeekamp 31, D-23560 Luebeck, GermanyIgM ELISA

      IgG ELISA
      IBL Tecan US, Inc9401 Globe Center Drive, Suite 140, Morrisville, NC 27560Chikungunya IgM μ-capture ELISALaboratory ELISA automation
      NovaTec Immundiagnostica, GmbHWaldstrasse 23 A6, Dietzenbach, 63128, GermanyIgG ELISA and an IgM μ-capture ELISA
      Abbreviations: ELISA, enzyme-linked immunosorbent assay; FDA, Food and Drug Administration; RT, reverse transcriptase.
      a Partial list of companies providing PCR and ELISA kits for detection of CHIKV or human antibody to the virus. None of these are FDA approved for use as diagnostic tests at this time but are considered in the Research Use Only category. One product is CE approved. These tests may prove to be useful adjuncts to assist in the development of future FDA-approved or validated assays.
      Until reliable Food and Drug Administration–approved kits for molecular detection of virus and CHIKV antibodies are available, only the largest commercial and government reference laboratories should consider routine diagnostic testing for CHIKV. The Centers for Disease Control and Prevention has facilities for testing samples to establish transmission of CHIKV in the United States. Some city and state health departments and other government agencies also have this capability. Specific details related to collection and preserving serum for transportation to and testing at regional or national facilities can be found at: http://www.cdc.gov/chikungunya/hc/diagnostic.html.
      If an epidemic should require greater testing capacity, the Centers for Disease Control and Prevention and similar agencies in other countries may implement Emergency Use Authorization for diagnostic tools for CHIKV that could be distributed to qualified clinical laboratories that demonstrate proficiency with the assays by successfully testing verification panels for each assay.
      If CHIKV infection becomes an annual endemic threat in certain regions, there will be a need for testing capability on a more local basis. It would be ideal to have CHIKV detection as part of a routine test panel and supported by the clinical laboratory testing industry. Encouraging research studies suggest that this will be possible, first for PCR and perhaps later for the IgM assays.
      • Simmons M.
      • Myers T.
      • Guevara C.
      • et al.
      Development and validation of a quantitative, one-step, multiplex, real-time reverse transcriptase PCR assay for detection of dengue and chikungunya viruses.
      • Parashar D.
      • Paingankar M.S.
      • Sudeep A.B.
      • et al.
      Assessment of qPCR, nested RT-PCR and ELISA techniques in diagnosis of chikungunya.
      • Macpherson C.
      • Noel T.
      • Fields P.
      • et al.
      Clinical and serological insights from the Asian lineage chikungunya outbreak in Grenada, 2014: an observational study.

      Treatment

      There is presently no licensed targeted therapy for acute CHIKV infection. Treatment is primarily supportive care and includes the use of analgesic and anti-inflammatory medication, rehydration, and rest. However, research to identify potential new antiviral therapies, or repurposing of existing compounds for treating CHIKV infection is ongoing (Table 2; reviewed in Ref.
      • Abdelnabi R.
      • Neyts J.
      • Delang L.
      Towards antivirals against chikungunya virus.
      ). For example, chloroquine has in vitro activity against several viruses, and has been found to inhibit CHIKV replication in Vero cells.
      • Khan M.
      • Santhosh S.R.
      • Tiwari M.
      • et al.
      Assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in Vero cells.
      However, it has not been shown to have anti-CHIKV effects in vivo. Compounds that may interfere with viral entry, including phenothiazines
      • Pohjala L.
      • Utt A.
      • Varjak M.
      • et al.
      Inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays.
      and flavaglines,
      • Wintachai P.
      • Thuaud F.
      • Basmadjian C.
      • et al.
      Assessment of flavaglines as potential chikungunya virus entry inhibitors.
      are being investigated as potential therapies. Ribavirin has been shown to have in vitro activity against CHIKV, and synergized with doxycycline to reduce viral load and inflammation in infected mice.
      • Rothan H.A.
      • Bahrani H.
      • Mohamed Z.
      • et al.
      A combination of doxycycline and ribavirin alleviated chikungunya infection.
      Table 2Examples of investigational strategies under development for treatment of CHIKV
      Adapted from Abdelnabi R, Neyts J, Delang L. Towards antivirals against chikungunya virus. Antiviral Res 2015;121:62; with permission.
      TherapeuticMechanismData
      In VitroIn Vivo
      ChloroquineInhibits fusion of CHIKV E1 protein with the endosomal membraneInhibited CHIKV infection in Vero A cells
      • Khan M.
      • Santhosh S.R.
      • Tiwari M.
      • et al.
      Assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in Vero cells.
      No efficacy in clinical trials in patients infected with CHIKV
      • Chopra A.
      • Saluja M.
      • Venugopalan A.
      Effectiveness of chloroquine and inflammatory cytokine response in patients with early persistent musculoskeletal pain and arthritis following chikungunya virus infection.
      siRNA targeting CHIKV genesInhibits protein synthesisInhibited CHIKV replication in Vero-E6 cells (>90%)
      • Parashar D.
      • Paingankar M.S.
      • Kumar S.
      • et al.
      Administration of e2 and ns1 siRNAs inhibit chikungunya virus replication in vitro and protects mice infected with the virus.
      Inhibited CHIKV replication in mice when administered 3 d postinfection
      • Parashar D.
      • Paingankar M.S.
      • Kumar S.
      • et al.
      Administration of e2 and ns1 siRNAs inhibit chikungunya virus replication in vitro and protects mice infected with the virus.
      RibavirinInhibits viral genome replication by depleting guanosine triphosphateInhibited CHIKV replication in Vero cells

      Synergistic inhibitory effect in combination with IFN-α2b and doxycycline
      • Briolant S.
      • Garin D.
      • Scaramozzino N.
      • et al.
      In vitro inhibition of chikungunya and Semliki forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination.
      Reduced the viral load and inflammation in infected ICR mice when combined with doxycycline
      • Rothan H.A.
      • Bahrani H.
      • Mohamed Z.
      • et al.
      A combination of doxycycline and ribavirin alleviated chikungunya infection.
      Favipiravir (T-705)Inhibits viral genome replicationInhibited CHIKV-induced cytopathic effect in Vero A cells
      • Jadav S.S.
      • Sinha B.N.
      • Hilgenfeld R.
      • et al.
      Thiazolidone derivatives as inhibitors of chikungunya virus.
      Reduced mortality of infected AG129 mice and protected from neurologic disease
      • Delang L.
      • Segura Guerrero N.
      • Tas A.
      • et al.
      Mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (T-705), a broad-spectrum antiviral.
      Monoclonal antibody C9Binds CHIKV E2 glycoproteinNeutralized CHIKV pseudovirions in HEK293T cells and CHIKV in Vero cells
      • Selvarajah S.
      • Sexton N.R.
      • Kahle K.M.
      • et al.
      A neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus e2 protects from disease.
      100% survival of CHIKV-infected mice when given at 8 or 18 h postinfection
      • Selvarajah S.
      • Sexton N.R.
      • Kahle K.M.
      • et al.
      A neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus e2 protects from disease.
      Monoclonal antibodies to the CHIKV E1 and E2 proteins have been used to protect mice and nonhuman primates from developing CHIKV infection after viral challenge.
      • Couderc T.
      • Chretien F.
      • Schilte C.
      • et al.
      A mouse model for chikungunya: young age and inefficient type-I interferon signaling are risk factors for severe disease.
      • Akahata W.
      • Yang Z.Y.
      • Andersen H.
      • et al.
      A virus-like particle vaccine for epidemic chikungunya virus protects nonhuman primates against infection.
      However, it is unclear whether passive immunization with monoclonal antibodies or hyperimmune serum can ameliorate symptomatology of CHIKV disease after infection has already been established.

      Summary

      The past decade has seen explosive viral epidemics, from severe acute respiratory syndrome to Ebola to arboviruses including Zika and CHIKV. For some diseases, the human toll is acutely evident in the form of mortality or acute morbidity. For CHIKV and others, the long-term sequelae from infection are yet ill-defined. The prolonged debilitating arthralgia associated with CHIKV infection has tremendous potential for impacting the global economy, and should be considered when evaluating the human burden of disease and the allocation of resources. There is much still unknown about CHIKV and the illnesses that it causes. Developing a better understanding of the pathogenesis of CHIKV infection is a priority and forms the basis for developing effective strategies at infection prevention and disease control.

      References

        • Strauss J.H.
        • Strauss E.G.
        The alphaviruses: gene expression, replication, and evolution.
        Microbiol Rev. 1994; 58: 491-562
        • Lwande O.W.
        • Obanda V.
        • Bucht G.
        • et al.
        Global emergence of alphaviruses that cause arthritis in humans.
        Infect Ecol Epidemiol. 2015; 5: 29853
        • Khan A.H.
        • Morita K.
        • Parquet Md Mdel C.
        • et al.
        Complete nucleotide sequence of chikungunya virus and evidence for an internal polyadenylation site.
        J Gen Virol. 2002; 83: 3075-3084
        • Jose J.
        • Snyder J.E.
        • Kuhn R.J.
        A structural and functional perspective of alphavirus replication and assembly.
        Future Microbiol. 2009; 4: 837-856
        • Weger-Lucarelli J.
        • Aliota M.T.
        • Kamlangdee A.
        • et al.
        Identifying the role of e2 domains on alphavirus neutralization and protective immune responses.
        PLoS Negl Trop Dis. 2015; 9: e0004163
        • Fong R.H.
        • Banik S.S.
        • Mattia K.
        • et al.
        Exposure of epitope residues on the outer face of the chikungunya virus envelope trimer determines antibody neutralizing efficacy.
        J Virol. 2014; 88: 14364-14379
        • Smith S.A.
        • Silva L.A.
        • Fox J.M.
        • et al.
        Isolation and characterization of broad and ultrapotent human monoclonal antibodies with therapeutic activity against chikungunya virus.
        Cell Host Microbe. 2015; 18: 86-95
        • Robinson M.C.
        An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952-53. I. Clinical features.
        Trans R Soc Trop Med Hyg. 1955; 49: 28-32
        • Ross R.W.
        The Newala epidemic. III. The virus: isolation, pathogenic properties and relationship to the epidemic.
        J Hyg (Lond). 1956; 54: 177-191
        • Diallo M.
        • Thonnon J.
        • Traore-Lamizana M.
        • et al.
        Vectors of chikungunya virus in Senegal: current data and transmission cycles.
        Am J Trop Med Hyg. 1999; 60: 281-286
        • McIntosh B.M.
        • Paterson H.E.
        • McGillivray G.
        • et al.
        Further studies on the chikungunya outbreak in southern Rhodesia in 1962. I. Mosquitoes, wild primates and birds in relation to the epidemic.
        Ann Trop Med Parasitol. 1964; 58: 45-51
        • Paterson H.E.
        • McIntosh B.M.
        Further studies on the chikungunya outbreak in southern Rhodesia in 1962. II. Transmission experiments with the Aedes furcifer-taylori group of mosquitoes and with a member of the Anopheles gambiae complex.
        Ann Trop Med Parasitol. 1964; 58: 52-55
        • Powers A.M.
        • Logue C.H.
        Changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus.
        J Gen Virol. 2007; 88: 2363-2377
        • Kariuki Njenga M.
        • Nderitu L.
        • Ledermann J.P.
        • et al.
        Tracking epidemic chikungunya virus into the Indian Ocean from East Africa.
        J Gen Virol. 2008; 89: 2754-2760
        • Rezza G.
        • Nicoletti L.
        • Angelini R.
        • et al.
        Infection with chikungunya virus in Italy: an outbreak in a temperate region.
        Lancet. 2007; 370: 1840-1846
        • Grandadam M.
        • Caro V.
        • Plumet S.
        • et al.
        Chikungunya virus, southeastern France.
        Emerg Infect Dis. 2011; 17: 910-913
        • Tsetsarkin K.A.
        • Vanlandingham D.L.
        • McGee C.E.
        • et al.
        A single mutation in chikungunya virus affects vector specificity and epidemic potential.
        PLoS Pathog. 2007; 3: e201
        • Fischer M.
        • Staples J.E.
        • Arboviral Diseases Branch, National Center for Emerging and Zoonotic Infectious Diseases, CDC
        Notes from the field: chikungunya virus spreads in the Americas - Caribbean and South America, 2013-2014.
        MMWR Morb Mortal Wkly Rep. 2014; 63: 500-501
      1. The Pan American Health Organization. Number of reported cases of chikungunya fever in the Americas, by country or territory 2013-2014 cumulative cases (updated 23 Oct 2015). Number of Reported Cases of Chikungunya Fever in the Americas 2015; 2016. Available at: http://www.paho.org/hq/index.php?option=com_topics&view=readall&cid=5927&Itemid=40931&lang=en. Accessed October 1, 2016.

        • Carey D.E.
        Chikungunya and dengue: a case of mistaken identity?.
        J Hist Med Allied Sci. 1971; 26: 243-262
        • Halstead S.B.
        Reappearance of chikungunya, formerly called dengue, in the Americas.
        Emerg Infect Dis. 2015; 21: 557-561
        • Higashi N.
        • Matsumoto A.
        • Tabata K.
        • et al.
        Electron microscope study of development of chikungunya virus in green monkey kidney stable (Vero) cells.
        Virology. 1967; 33: 55-69
        • Hahon N.
        • Zimmerman W.D.
        Chikungunya virus infection of cell monolayers by cell-to-cell and extracellular transmission.
        Appl Microbiol. 1970; 19: 389-391
        • Buckley S.M.
        • Singh K.R.
        • Bhat U.K.
        Small- and large-plaque variants of chikungunya virus in two vertebrate and seven invertebrate cell lines.
        Acta Virol. 1975; 19: 10-18
        • Cunningham A.
        • Buckley S.M.
        • Casals J.
        • et al.
        Isolation of chikungunya virus contaminating an Aedes albopictus cell line.
        J Gen Virol. 1975; 27: 97-100
        • Sourisseau M.
        • Schilte C.
        • Casartelli N.
        • et al.
        Characterization of reemerging chikungunya virus.
        PLoS Pathog. 2007; 3: e89
        • Couderc T.
        • Chretien F.
        • Schilte C.
        • et al.
        A mouse model for chikungunya: young age and inefficient type-I interferon signaling are risk factors for severe disease.
        PLoS Pathog. 2008; 4: e29
        • Ozden S.
        • Huerre M.
        • Riviere J.P.
        • et al.
        Human muscle satellite cells as targets of chikungunya virus infection.
        PLoS One. 2007; 2: e527
        • Akahata W.
        • Yang Z.Y.
        • Andersen H.
        • et al.
        A virus-like particle vaccine for epidemic chikungunya virus protects nonhuman primates against infection.
        Nat Med. 2010; 16: 334-338
        • Labadie K.
        • Larcher T.
        • Joubert C.
        • et al.
        Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages.
        J Clin Invest. 2010; 120: 894-906
        • Gardner J.
        • Anraku I.
        • Le T.T.
        • et al.
        Chikungunya virus arthritis in adult wild-type mice.
        J Virol. 2010; 84: 8021-8032
        • Morrison T.E.
        • Oko L.
        • Montgomery S.A.
        • et al.
        A mouse model of chikungunya virus-induced musculoskeletal inflammatory disease: evidence of arthritis, tenosynovitis, myositis, and persistence.
        Am J Pathol. 2011; 178: 32-40
        • Robin S.
        • Ramful D.
        • Zettor J.
        • et al.
        Severe bullous skin lesions associated with chikungunya virus infection in small infants.
        Eur J Pediatr. 2010; 169: 67-72
        • Zeana C.
        • Kelly P.
        • Heredia W.
        • et al.
        Post-chikungunya rheumatic disorders in travelers after return from the Caribbean.
        Travel Med Infect Dis. 2016; 14: 21-25
        • Manimunda S.P.
        • Vijayachari P.
        • Uppoor R.
        • et al.
        Clinical progression of chikungunya fever during acute and chronic arthritic stages and the changes in joint morphology as revealed by imaging.
        Trans R Soc Trop Med Hyg. 2010; 104: 392-399
        • Labeaud A.D.
        • Bashir F.
        • King C.H.
        Measuring the burden of arboviral diseases: the spectrum of morbidity and mortality from four prevalent infections.
        Popul Health Metr. 2011; 9: 1
        • Lemant J.
        • Boisson V.
        • Winer A.
        • et al.
        Serious acute chikungunya virus infection requiring intensive care during the Reunion Island outbreak in 2005-2006.
        Crit Care Med. 2008; 36: 2536-2541
        • Wielanek A.C.
        • Monredon J.D.
        • Amrani M.E.
        • et al.
        Guillain-Barre syndrome complicating a chikungunya virus infection.
        Neurology. 2007; 69: 2105-2107
        • Rampal
        • Sharda M.
        • Meena H.
        Neurological complications in chikungunya fever.
        J Assoc Physicians India. 2007; 55: 765-769
        • Singh R.K.
        • Tiwari S.
        • Mishra V.K.
        • et al.
        Molecular epidemiology of chikungunya virus: mutation in e1 gene region.
        J Virol Methods. 2012; 185: 213-220
        • Chandak N.H.
        • Kashyap R.S.
        • Kabra D.
        • et al.
        Neurological complications of chikungunya virus infection.
        Neurol India. 2009; 57: 177-180
        • Josseran L.
        • Paquet C.
        • Zehgnoun A.
        • et al.
        Chikungunya disease outbreak, Reunion Island.
        Emerg Infect Dis. 2006; 12: 1994-1995
        • Tandale B.V.
        • Sathe P.S.
        • Arankalle V.A.
        • et al.
        Systemic involvements and fatalities during chikungunya epidemic in India, 2006.
        J Clin Virol. 2009; 46: 145-149
        • Mercado M.
        • Acosta-Reyes J.
        • Parra E.
        • et al.
        Clinical and histopathological features of fatal cases with dengue and chikungunya virus co-infection in Colombia, 2014 to 2015.
        Euro Surveill. 2016; ([Epub ahead of print])
      2. Jungkind D, Myers TE, Simmons M, et al. Establishment of laboratory testing capability for chikungunya virus in Grenada, West Indies. Presented at the Caribbean Public Health Agency: 60th Annual Scientific Meeting. The University of the West Indies, June 20-24, 2015.

        • Simmons M.
        • Myers T.
        • Guevara C.
        • et al.
        Development and validation of a quantitative, one-step, multiplex, real-time reverse transcriptase PCR assay for detection of dengue and chikungunya viruses.
        J Clin Microbiol. 2016; 54: 1766-1773
      3. Macpherson CNL, Noel T, Jungkind D, et al. Clinical, molecular and serological outcomes of the chikungunya outbreak in Grenada. Presented at the Caribbean Public Health Agency: 60th Annual Scientific Meeting. The University of the West Indies. 2015.

        • Soh L.T.
        • Squires R.C.
        • Tan L.K.
        • et al.
        External quality assessment of dengue and chikungunya diagnostics in the Asia Pacific region, 2015.
        Western Pac Surveill Response J. 2016; 7: 26-34
        • Prat C.M.
        • Flusin O.
        • Panella A.
        • et al.
        Evaluation of commercially available serologic diagnostic tests for chikungunya virus.
        Emerg Infect Dis. 2014; 20: 2129-2132
        • Kam Y.W.
        • Pok K.Y.
        • Eng K.E.
        • et al.
        Sero-prevalence and cross-reactivity of chikungunya virus specific anti-e2ep3 antibodies in arbovirus-infected patients.
        PLoS Negl Trop Dis. 2015; 9: e3445
        • Parashar D.
        • Paingankar M.S.
        • Sudeep A.B.
        • et al.
        Assessment of qPCR, nested RT-PCR and ELISA techniques in diagnosis of chikungunya.
        Curr Sci. 2014; 107: 2011-2013
        • Macpherson C.
        • Noel T.
        • Fields P.
        • et al.
        Clinical and serological insights from the Asian lineage chikungunya outbreak in Grenada, 2014: an observational study.
        Am J Trop Med Hyg. 2016; 95: 890-893
        • Abdelnabi R.
        • Neyts J.
        • Delang L.
        Towards antivirals against chikungunya virus.
        Antiviral Res. 2015; 121: 59-68
        • Khan M.
        • Santhosh S.R.
        • Tiwari M.
        • et al.
        Assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in Vero cells.
        J Med Virol. 2010; 82: 817-824
        • Pohjala L.
        • Utt A.
        • Varjak M.
        • et al.
        Inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays.
        PLoS One. 2011; 6: e28923
        • Wintachai P.
        • Thuaud F.
        • Basmadjian C.
        • et al.
        Assessment of flavaglines as potential chikungunya virus entry inhibitors.
        Microbiol Immunol. 2015; 59: 129-141
        • Rothan H.A.
        • Bahrani H.
        • Mohamed Z.
        • et al.
        A combination of doxycycline and ribavirin alleviated chikungunya infection.
        PLoS One. 2015; 10: e0126360
        • Chopra A.
        • Saluja M.
        • Venugopalan A.
        Effectiveness of chloroquine and inflammatory cytokine response in patients with early persistent musculoskeletal pain and arthritis following chikungunya virus infection.
        Arthritis Rheumatol. 2014; 66: 319-326
        • Parashar D.
        • Paingankar M.S.
        • Kumar S.
        • et al.
        Administration of e2 and ns1 siRNAs inhibit chikungunya virus replication in vitro and protects mice infected with the virus.
        PLoS Negl Trop Dis. 2013; 7: e2405
        • Briolant S.
        • Garin D.
        • Scaramozzino N.
        • et al.
        In vitro inhibition of chikungunya and Semliki forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination.
        Antiviral Res. 2004; 61: 111-117
        • Jadav S.S.
        • Sinha B.N.
        • Hilgenfeld R.
        • et al.
        Thiazolidone derivatives as inhibitors of chikungunya virus.
        Eur J Med Chem. 2015; 89: 172-178
        • Delang L.
        • Segura Guerrero N.
        • Tas A.
        • et al.
        Mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (T-705), a broad-spectrum antiviral.
        J Antimicrob Chemother. 2014; 69: 2770-2784
        • Selvarajah S.
        • Sexton N.R.
        • Kahle K.M.
        • et al.
        A neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus e2 protects from disease.
        PLoS Negl Trop Dis. 2013; 7: e2423