Chikungunya Virus RNA Research Articles

Detection of chikungunya virus in saliva and urine.

BackgroundSaliva and urine have been used for arthropod-borne viruses molecular detection but not yet for chikungunya virus (CHIKV). We investigated the use of saliva and urine for molecular detection of CHIKV during the French Polynesian outbreak.MethodsDuring the French Polynesian chikungunya outbreak (2014–2015), we collected the same day blood and saliva samples from 60 patients with probable chikungunya (47 during the 1st week post symptoms onset and 13 after), urine was available for 39 of them. All samples were tested using a CHIKV reverse-transcription PCR.ResultsForty eight patients had confirmed chikungunya. For confirmed chikungunya presenting during the 1st week post symptoms onset, CHIKV RNA was detected from 86.1 % (31/36) of blood, 58.3 % (21/36) of saliva and 8.3 % (2/24) of urine. Detection rate of CHIKV RNA was significantly higher in blood compared to saliva. For confirmed chikungunya presenting after the 1st week post symptoms onset, CHIKV RNA was detected from 8.3 % (1/12) of blood, 8.3 % (1/12) of saliva and 0 % (0/8) of urine.ConclusionsIn contrast to Zika virus (ZIKV), saliva did not increased the detection rate of CHIKV RNA during the 1st week post symptoms onset. In contrast to ZIKV, dengue virus and West Nile virus, urine did not enlarged the window of detection of CHIKV RNA after the 1st week post symptoms onset. Saliva can be used for molecular detection of CHIKV during the 1st week post symptoms onset only if blood is impossible to collect but with a lower sensitivity compared to blood.

Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

Detection of Persistent Chikungunya Virus RNA but not Infectious Virus in Experimental Vertical Transmission in Aedes aegypti from Malaysia.

Vertical transmission may contribute to the maintenance of arthropod-borne viruses, but its existence in chikungunya virus (CHIKV) is unclear. Experimental vertical transmission of infectious clones of CHIKV in Aedes aegypti mosquitoes from Malaysia was investigated. Eggs and adult progeny from the second gonotrophic cycles of infected parental mosquitoes were tested. Using polymerase chain reaction (PCR), 56.3% of pooled eggs and 10% of adult progeny had detectable CHIKV RNA, but no samples had detectable infectious virus by plaque assay. Transfected CHIKV RNA from PCR-positive eggs did not yield infectious virus in BHK-21 cells. Thus, vertical transmission of viable CHIKV was not demonstrated. Noninfectious CHIKV RNA persists in eggs and progeny of infected Ae. aegypti, but the mechanism and significance are unknown. There is insufficient evidence to conclude that vertical transmission exists in CHIKV, as positive results reported in previous studies were almost exclusively based only on viral RNA detection.

Study of viremic profile in febrile specimens of chikungunya in Bandung, Indonesia

BackgroundData regarding the viremia profile of chikungunya virus (CHIKV) infected patients especially during the pre-febrile period is limited. ObjectiveTo obtain virological kinetic data on CHIKV infections. Study designA two-week community observation for dengue transmission was conducted in Bandung, Indonesia, from 2005 to 2009. Acute specimens from non-dengue febrile patients were screened by pan-alphavirus conventional RT-PCR. The positives were confirmed for CHIKV RNA by a specific RT-PCR followed by sequencing. Simultaneously these specimens were also cultured in Vero cells and tested for anti-CHIK IgM MAC-ELISA. All the available serial specimens,including the pre-febrile specimens, from confirmed CHIK cases, were tested by virus isolation, RT-PCR, qRT-PCR, and CHIK IgM ELISA. ResultsThere were five laboratory confirmed CHIK cases identified and studied. Among these, viremia was determined to extend from as early as 6 days prior to until 13 days post fever onset. Quantitative RT-PCR showed viremia peaked at or near onset of illness. ConclusionIn this study, individuals were identified with viremia prior to fever onset and extending beyond the febrile phase. This extended viremic phase has the potential to impact transmission dynamics and thus the public health response to CHIK outbreaks.

Chikungunya Virus Infections Among Patients with Dengue-Like Illness at a Tertiary Care Hospital in the Philippines, 2012–2013

Chikungunya virus (CHIKV) often co-circulates with dengue virus (DENV). A cross-sectional surveillance study was conducted at a tertiary hospital in Manila, Philippines, to describe the prevalence and characteristics of DENV and CHIKV infections among patients seeking care for dengue-like illness. Acute blood samples from patients ≥ 6 months of age clinically diagnosed with dengue from November 2012 to December 2013 underwent reverse transcription polymerase chain reaction (RT-PCR) to detect DENV and CHIKV RNA. A total of 118 patients with clinically diagnosed dengue (age range = 1-89 years, mean = 22 years; male-to-female ratio = 1.51) were tested by DENV RT-PCR; 40 (34%) were DENV PCR-positive (age range = 1-45 years, mean = 17 years). All DENV serotypes were detected: 11 (28%) DENV-1, 6 (15%) DENV-2, 6 (15%) DENV-3, and 17 (42%) DENV-4. Of 112 patients clinically diagnosed with dengue and tested by CHIKV RT-PCR, 11 (10%) were CHIKV PCR-positive (age range = 2-47 years, mean = 20.3 years). No coinfections were detected. Presenting signs/symptoms did not differ between DENV- and CHIKV-positive cases. Sequencing of envelope 1 gene from two CHIKV PCR-positive samples showed Asian genotype. This study highlights the potential for misdiagnosis of medically attended CHIKV infections as DENV infection and the difficulty in clinically differentiating dengue and chikungunya based on presenting signs/symptoms alone. This underscores the necessity for diagnostic laboratory tests to distinguish CHIKV infections in the background of actively co-circulating DENV.

Suramin inhibits chikungunya virus replication through multiple mechanisms

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe and often persistent arthritis. In recent years, millions of people have been infected with this virus for which registered antivirals are still lacking. Using our recently established in vitro assay, we discovered that the approved anti-parasitic drug suramin inhibits CHIKV RNA synthesis (IC50 of ∼5μM). The compound inhibited replication of various CHIKV isolates in cell culture with an EC50 of ∼80μM (CC50>5mM) and was also active against Sindbis virus and Semliki Forest virus. In vitro studies hinted that suramin interferes with (re)initiation of RNA synthesis, whereas time-of-addition studies suggested it to also interfere with a post-attachment early step in infection, possibly entry. CHIKV (nsP4) mutants resistant against favipiravir or ribavirin, which target the viral RNA polymerase, did not exhibit cross-resistance to suramin, suggesting a different mode of action. The assessment of the activity of a variety of suramin-related compounds in cell culture and the in vitro assay for RNA synthesis provided more insight into the moieties required for antiviral activity. The antiviral effect of suramin-containing liposomes was also analyzed. Its approved status makes it worthwhile to explore the use of suramin to prevent and/or treat CHIKV infections.

Seroprevalence and entomological study on Chikungunya virus at the Croatian littoral.

During 2011-2012, a total of 1008 serum samples from randomly selected inhabitants of seven Croatian counties located on the Adriatic Coast were tested for the presence of chikungunya virus (CHIKV) IgG antibodies using indirect immunofluorescence assay. Nine participants (0.9%) from four counties were found to be seropositive to CHIKV. Seroprevalence varied from 0.5% to 1.8% between counties. Additionally, a total of 3,699 mosquitoes were captured in 126 localities from August 16 to September 24, 2011. Three mosquito species were found: Ae. albopictus (3010/81.4%), Cx. pipiens (688/18.6%) and only one specimen of the Cs. longiareolata. Female mosquitoes (N = 1,748) were pooled. All pools tested negative for CHIKV RNA using a real-time RT-PCR.

Collaborative study for the characterization of a chikungunya virus RNA reference reagent for use in nucleic acid testing.

Infections with the mosquito-borne chikungunya virus (CHIKV) can cause febrile illness or be asymptomatic. Laboratory diagnosis of CHIKV is often made with laboratory-developed nucleic acid amplification technology (NAT) assays because there are no U.S. Food and Drug Administration (FDA)-approved diagnostic or blood screening assays. We aimed to produce a well-characterized CHIKV RNA reference reagent (CHIKV-RR) for use in NAT assays. A CHIKV RNA-RR consisting of cell culture-grown, heat-inactivated CHIKV diluted in human plasma was assessed by 8 laboratories in a collaborative study. The participants were asked to test the CHIKV-RR using their NAT assay(s) by qualitative testing (determination of RNA end-point by testing log and half-log dilutions followed by calculation of estimated NAT-detectable units/ml, after adjustment for the sample volume used for testing), and by quantitative testing, when available. Results from the testing showed that the CHIKV-RR had an estimated overall mean of 7.56 log10 detectable units/ml, ranging from 6.2 log10 to 8.6 log10. The Center for Biologics for Evaluation and Research/FDA CHIKV RNA-RR for NAT was established with a concentration of 7.56 log10 detectable units/ml.

Molecular Diagnosis of Chikungunya virus (CHIKV) and Dengue virus (DENV) and its concomitant circulation in South Indian population

Concomitant circulation of dengue and chikungunya viruses has been sporadically reported and needs to be individually diagnosed. Several sensitive molecular diagnostics are currently deployed for the diagnosis of both DENV and CHIKV, it is very difficult to delineate the two viral infections based on symptomology, as both share similar clinical presentation. Due to the overlapping nature of clinical signs in DENV and CHIKV infections, there is an urgent need for early and accurate diagnosis to avoid outbreaks aimed at initiating disease specific interventions. The study encompasses the diagnosis of clinically suspected 1024 DENV/CHIKV patient samples which were collected from AndhraPradesh (AP) and Kerala, India by using in-house developed RT-PCR (reverse transcription-polymerase chain reaction). The results show 43.8% DENV RNA infection and32% CHIKV RNA infection of the total samples obtained from AP. In contrast, samples from Kerala show dengue infection of 16.1%, CHIKV infection of 2.3%. In our study, we found that 23%, 0.1% of the samples were concomitant circulation for CHIKV/DENV in AP and Kerala respectively, suggesting the co-infection of these two viruses.

Stress granule components G3BP1 and G3BP2 play a proviral role early in Chikungunya virus replication.

Stress granules (SGs) are protein-mRNA aggregates that are formed in response to environmental stresses, resulting in translational inhibition. SGs are generally believed to play an antiviral role and are manipulated by many viruses, including various alphaviruses. GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is a key component and commonly used marker of SGs. Its homolog G3BP2 is a less extensively studied SG component. Here, we demonstrate that Chikungunya virus (CHIKV) infection induces cytoplasmic G3BP1- and G3BP2-containing granules that differ from bona fide SGs in terms of morphology, composition, and behavior. For several Old World alphaviruses it has been shown that nonstructural protein 3 (nsP3) interacts with G3BPs, presumably to inhibit SG formation, and we have confirmed this interaction in CHIKV-infected cells. Surprisingly, CHIKV also relied on G3BPs for efficient replication, as simultaneous depletion of G3BP1 and G3BP2 reduced viral RNA levels, CHIKV protein expression, and viral progeny titers. The G3BPs colocalized with CHIKV nsP2 and nsP3 in cytoplasmic foci, but no colocalization with nsP1, nsP4, or dsRNA was observed. Furthermore, G3BPs could not be detected in a cellular fraction enriched for CHIKV replication/transcription complexes, suggesting that they are not directly involved in CHIKV RNA synthesis. Depletion of G3BPs did not affect viral entry, translation of incoming genomes, or nonstructural polyprotein processing but resulted in severely reduced levels of negative-stranded (and consequently also positive-stranded) RNA. This suggests a role for the G3BPs in the switch from translation to genome amplification, although the exact mechanism by which they act remains to be explored. Chikungunya virus (CHIKV) causes a severe polyarthritis that has affected millions of people since its reemergence in 2004. The lack of approved vaccines or therapeutic options and the ongoing explosive outbreak in the Caribbean underline the importance of better understanding CHIKV replication. Stress granules (SGs) are cytoplasmic protein-mRNA aggregates formed in response to various stresses, including viral infection. The RNA-binding proteins G3BP1 and G3BP2 are essential SG components. SG formation and the resulting translational inhibition are generally considered an antiviral response, and many viruses manipulate or block this process. Late in infection, we and others have observed CHIKV nonstructural protein 3 in cytoplasmic G3BP1- and G3BP2-containing granules. These virally induced foci differed from true SGs and did not appear to represent replication complexes. Surprisingly, we found that G3BP1 and G3BP2 were also needed for efficient CHIKV replication, likely by facilitating the switch from translation to genome amplification early in infection.

Detection of Chikungunya virus (CHIKV) in urine of infected mice: a Potential Non-invasive Diagnostic Tool for CHIKV

Background: The reemergence of Chikungunya virus (CHIKV) infection and the spread of the virus throughout the world call for a fast, reliable, and improved laboratory diagnostic procedures. The aim of this study was to determine if CHIKV genome is present in other body fluids besides plasma as a tool for developing non-invasive diagnostic method. Methods: This study utilized a sensitive and less invasive assay that combines the specificity of immunocomplexing and the sensitivity of real-time PCR to detect CHIKV in urine and plasma of infected mice. Results: CHIKV RNA was detected by immuno-capture-quantitative reverse transcriptase PCR (IC-qPCR) in urine and plasma of infected mice. Viral RNA was detected in urine up to day 30, long after viral clearance in plasma. Furthermore, infectious CHIKV was detected in urine by cell-based immunofluorescence and 50% tissue culture infective dose (TCID50) assays, suggesting the presence of infectious virus in urine. Conclusion: We demonstrate a potential non-invasive diagnostic approach that could be adapted to screen forCHIKV infection in viremic and aviremic patients using urine.

Multiple immune factors are involved in controlling acute and chronic chikungunya virus infection.

The recent epidemic of the arthritogenic alphavirus, chikungunya virus (CHIKV) has prompted a quest to understand the correlates of protection against virus and disease in order to inform development of new interventions. Herein we highlight the propensity of CHIKV infections to persist long term, both as persistent, steady-state, viraemias in multiple B cell deficient mouse strains, and as persistent RNA (including negative-strand RNA) in wild-type mice. The knockout mouse studies provided evidence for a role for T cells (but not NK cells) in viraemia suppression, and confirmed the role of T cells in arthritis promotion, with vaccine-induced T cells also shown to be arthritogenic in the absence of antibody responses. However, MHC class II-restricted T cells were not required for production of anti-viral IgG2c responses post CHIKV infection. The anti-viral cytokines, TNF and IFNγ, were persistently elevated in persistently infected B and T cell deficient mice, with adoptive transfer of anti-CHIKV antibodies unable to clear permanently the viraemia from these, or B cell deficient, mice. The NOD background increased viraemia and promoted arthritis, with B, T and NK deficient NOD mice showing high-levels of persistent viraemia and ultimately succumbing to encephalitic disease. In wild-type mice persistent CHIKV RNA and negative strand RNA (detected for up to 100 days post infection) was associated with persistence of cellular infiltrates, CHIKV antigen and stimulation of IFNα/β and T cell responses. These studies highlight that, secondary to antibodies, several factors are involved in virus control, and suggest that chronic arthritic disease is a consequence of persistent, replicating and transcriptionally active CHIKV RNA.

Recombinant modified vaccinia virus Ankara expressing glycoprotein E2 of Chikungunya virus protects AG129 mice against lethal challenge.

Chikungunya virus (CHIKV) infection is characterized by rash, acute high fever, chills, headache, nausea, photophobia, vomiting, and severe polyarthralgia. There is evidence that arthralgia can persist for years and result in long-term discomfort. Neurologic disease with fatal outcome has been documented, although at low incidences. The CHIKV RNA genome encodes five structural proteins (C, E1, E2, E3 and 6K). The E1 spike protein drives the fusion process within the cytoplasm, while the E2 protein is believed to interact with cellular receptors and therefore most probably constitutes the target of neutralizing antibodies. We have constructed recombinant Modified Vaccinia Ankara (MVA) expressing E3E2, 6KE1, or the entire CHIKV envelope polyprotein cassette E3E26KE1. MVA is an appropriate platform because of its demonstrated clinical safety and its suitability for expression of various heterologous proteins. After completing the immunization scheme, animals were challenged with CHIV-S27. Immunization of AG129 mice with MVAs expressing E2 or E3E26KE1 elicited neutralizing antibodies in all animals and provided 100% protection against lethal disease. In contrast, 75% of the animals immunized with 6KE1 were protected against lethal infection. In conclusion, MVA expressing the glycoprotein E2 of CHIKV represents as an immunogenic and effective candidate vaccine against CHIKV infections.

An in vitro assay to study chikungunya virus RNA synthesis and the mode of action of inhibitors.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay (32)P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5' end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15,000 G: . Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3'dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.

Chikungunya outbreak in Bueng Kan Province, Thailand, 2013.

Chikungunya outbreak in Bueng Kan Province, Thailand, 2013.

Evidence of experimental vertical transmission of emerging novel ECSA genotype of Chikungunya Virus in Aedes aegypti.

BackgroundChikungunya virus (CHIKV) has emerged as one of the most important arboviruses of public health significance in the past decade. The virus is mainly maintained through human-mosquito-human cycle. Other routes of transmission and the mechanism of maintenance of the virus in nature are not clearly known. Vertical transmission may be a mechanism of sustaining the virus during inter-epidemic periods. Laboratory experiments were conducted to determine whether Aedes aegypti, a principal vector, is capable of vertically transmitting CHIKV or not.Methodology/Principal FindingsFemale Ae. aegypti were orally infected with a novel ECSA genotype of CHIKV in the 2nd gonotrophic cycle. On day 10 post infection, a non-infectious blood meal was provided to obtain another cycle of eggs. Larvae and adults developed from the eggs obtained following both infectious and non-infectious blood meal were tested for the presence of CHIKV specific RNA through real time RT-PCR. The results revealed that the larvae and adults developed from eggs derived from the infectious blood meal (2nd gonotrophic cycle) were negative for CHIKV RNA. However, the larvae and adults developed after subsequent non-infectious blood meal (3rd gonotrophic cycle) were positive with minimum filial infection rates of 28.2 (1∶35.5) and 20.2 (1∶49.5) respectively.Conclusion/SignificanceThis study is the first to confirm experimental vertical transmission of emerging novel ECSA genotype of CHIKV in Ae. aegypti from India, indicating the possibilities of occurrence of this phenomenon in nature. This evidence may have important consequence for survival of CHIKV during adverse climatic conditions and inter-epidemic periods.

Vector Competence of Aedes aegypti and Aedes vittatus (Diptera: Culicidae) from Senegal and Cape Verde Archipelago for West African Lineages of Chikungunya Virus

To assess the risk of emergence of chikungunya virus (CHIKV) in West Africa, vector competence of wild-type, urban, and non-urban Aedes aegypti and Ae. vittatus from Senegal and Cape Verde for CHIKV was investigated. Mosquitoes were fed orally with CHIKV isolates from mosquitoes (ArD30237), bats (CS13-288), and humans (HD180738). After 5, 10, and 15 days of incubation following an infectious blood meal, presence of CHIKV RNA was determined in bodies, legs/wings, and saliva using real-time reverse transcription-polymerase chain reaction. Aedes vittatus showed high susceptibility (50-100%) and early dissemination and transmission of all CHIKV strains tested. Aedes aegypti exhibited infection rates ranging from 0% to 50%. Aedes aegypti from Cape Verde and Kedougou, but not those from Dakar, showed the potential to transmit CHIKV in saliva. Analysis of biology and competence showed relatively high infective survival rates for Ae. vittatus and Ae. aegypti from Cape Verde, suggesting their efficient vector capacity in West Africa.

Chikungunya antibodies detected in non-human primates and rats in three Indian Ocean islands after the 2006 ChikV outbreak.

The role of terrestrial vertebrates in the epidemiology of chikungunya disease is poorly understood. We evaluated their exposure and amplification role during the 2006 chikungunya outbreak in the Indian Ocean. Blood samples were collected from 18 mammalian and reptile species from Reunion Island, Mauritius and Mayotte. Among the 1051 samples serologically tested for chikungunya virus (CHIKV), two crab-eating macaques (Macaca fascicularis) and two ship rats (Rattus rattus) proved to be exposed to CHIKV. CHIKV RNA was not detected in 791 analyzed sera. Our results confirm the preferential infection of simian primates and suggest that other vertebrates played a poor or no role in CHIKV transmission during the 2006 outbreak.

Evidence for Endemic Chikungunya Virus Infections in Bandung, Indonesia

Chikungunya virus (CHIKV) is known to cause sporadic or explosive outbreaks. However, little is known about the endemic transmission of CHIKV. To ascertain the endemic occurrence of CHIKV transmission, we tested blood samples from patients with a non-dengue febrile illness who participated in a prospective cohort study of factory workers in Bandung, Indonesia. From August 2000 to June 2004, and September 2006 to April 2008, 1901 febrile episodes occurred and 231 (12.2%) dengue cases were identified. The remaining febrile cases were evaluated for possible CHIKV infection by measuring anti-CHIKV IgM and IgG antibodies in acute and convalescent samples. Acute samples of serologically positive cases were subsequently tested for the presence of CHIKV RNA by RT-PCR and/or virus isolation. A total of 135 (7.1%) CHIKV infections were identified, providing an incidence rate of 10.1/1,000 person years. CHIKV infections were identified all year round and tended to increase during the rainy season (January to March). Severe illness was not found and severe arthralgia was not a prominently reported symptom. Serial post-illness samples from nine cases were tested to obtain a kinetic picture of IgM and IgG anti-CHIKV antibodies. Anti-CHIKV IgM antibodies were persistently detected in high titers for approximately one year. Three patients demonstrated evidence of possible sequential CHIKV infections. The high incidence rate and continuous chikungunya cases in this adult cohort suggests that CHIKV is endemically transmitted in Bandung. Further characterization of the circulating strains and surveillance in larger areas are needed to better understand CHIKV epidemiology in Indonesia.

Rapid Identification of Chikungunya and Dengue Virus by a Real-Time Reverse Transcription-Loop-Mediated Isothermal Amplification Method

Both Chikungunya and Dengue virus belong to the acute arthropod-borne viruses. Because of the lack of specific symptoms, it is difficult to distinguish the two infections based on clinical manifestations. To identify and quantitatively detect Chikungunya and Dengue viruses, a real-time accelerated reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) platform was developed, and 26-confirmed RNA samples, 42 suspects, and 18 healthy serum samples were evaluated by the method. The RT-polymerase chain reaction (PCR) and cDNA sequencing were used as references. The results showed that it could identify the Chikungunya and Dengue virus RNA correctly in all antibody-positive samples within 1 hour, without any cross-reactions. The virus load of the positive samples was quantitatively detected with a turbidimeter. The sensitivity was 100% and specificity was 95.25%. The findings indicate that the RT-LAMP is an effective method for rapid quantity detection of Chikungunya virus and Dengue virus in serum samples with convenient operation, high specificity, and high sensitivity.