Molecular characterization of dengue and chikungunya virus strains circulating in New Delhi,India

Dengue and chikungunya are acute viral infections with overlapping clinical symptoms. Both diseases are transmitted by common mosquito vectors resulting in their co‐circulation in a region. Molecular and serological tests specific for both dengue and chikungunya infections were performed on 87 acute phase blood samples collected from patients with suspected dengue/chikungunya infections in Delhi from September to December, 2011. RT‐PCR and IgM ELISA were performed to detect dengue virus (DENV) and chikungunya virus (CHIKV). NS1 and IgG ELISA were also performed to detect DENV specific antigen and secondary DENV infection. DENV infection was detected in 49%, CHIKV infection in 29% and co‐infection with DENV and CHIKV in 10% of the samples by RT‐PCR. DENV serotypes 1, 2 and 3 were detected in this study. Nine DENV‐1 strains, six DENV‐2 strains and 20 CHIKV strains were characterized by DNA sequencing and phylogenetic analysis of their respective envelope protein genes. DENV‐1 strains grouped in the American African genotype, DENV‐2 strains in the Cosmopolitan genotype and CHIKV strains in the East Central South African genotype by phylogenetic analysis. This is one of the few studies reporting the phylogeny of two dengue virus serotypes (DENV‐1 and DENV‐2) and CHIKV. Surveillance and monitoring of DENV and CHIKV strains are important for design of strategies to control impending epidemics.     

Dengue and Chikungunya virus co-infection in major metropolitan cities of provinces of Punjab and Khyber Pakhtunkhwa: A multi-center study

Dengue has become endemic in Pakistan with annual recurrence. A sudden increase in the dengue cases was reported from Rawalpindi in 2016, while an outbreak occurred for the first time in Peshawar in 2017. Therefore, a multi-center study was carried out to determine the circulating dengue virus (DENV) serotypes and Chikungunya virus (CHIKV) co-infection in Lahore, Rawalpindi, and Peshawar cities in 2016–18. A hospital-based cross-sectional study was carried out in Lahore and Rawalpindi in 2016–18, while a community-based study was carried out in Peshawar in 2017. The study participants were tested for dengue NS1 antigen using an immunochromatographic device while anti-dengue IgM/IgG antibodies were detected by indirect ELISA. All NS1 positive samples were used for DENV serotyping using multiplex real-time PCR assay. Additionally, dengue samples were tested for CHIKV co-infection using IgM/IgG ELISA. A total of 6291 samples were collected among which 8.11% were NS1 positive while 2.5% were PCR positive. DENV-2 was the most common serotype (75.5%) detected, followed by DENV-1 in 16.1%, DENV-3 in 3.9% and DENV-4 in 0.7% while DENV-1 and DENV-4 concurrent infections were detected in 3.9% samples. DENV-1 was the predominant serotype (62.5%) detected from Lahore and Rawalpindi, while DENV-2 was the only serotype detected from Peshawar. Comorbidities resulted in a significant increase (p-value<0.001) in the duration of hospital stay of the patients. Type 2 diabetes mellitus substantially (p-value = 0.004) contributed to the severity of the disease. Among a total of 590 dengue positive samples, 11.8% were also positive for CHIKV co-infection. Co-circulation of multiple DENV serotypes and CHIKV infection in Pakistan is a worrisome situation demanding the urgent attention of the public health experts to strengthen vector surveillance.     

Impact of the introduction of chikungunya and zika viruses on the incidence of dengue in endemic zones of Mexico

BackgroundWith the arrival of chikungunya (CHIKV) and zika (ZIKV) viruses in Mexico, there was a decrease in diagnosed dengue virus (DENV) cases. During the first years of cocirculation (2015–2017), the algorithms established by epidemiological surveillance systems and the installed capacity limited us to one diagnostic test per sample, so there was an underestimation of cases until September 2017, when a multiplex algorithm was implemented. Therefore, the objective of this study was determine the impact of the introduction of CHIKV and ZIKV on the incidence of diagnosed DENV in endemic areas of Mexico, when performing the rediagnosis, using the multiplex algorithm, in samples from the first three years of co-circulation of these arboviruses.Methodology and principal findingsFor this, 1038 samples received by the Central Laboratory of Epidemiology between 2015 and 2017 were selected for this work. Viruses were identified by multiplex RT-qPCR, and the χ² test was used to compare categorical variables. With the new multiplex algorithm, we identified 2.4 times the rate of arbovirosis as originally reported, evidencing an underestimation of the incidence of the three viruses. Even so, significantly less dengue was observed than in previous years. The high incidence rates of chikungunya and Zika coincided with periods of dengue decline. The endemic channel showed that the cases caused by DENV rose again after the circulation of CHIKV and ZIKV decreased. In addition, 23 cases of coinfection were identified, with combinations between all viruses.Conclusions and significanceThe results obtained in this study show for the first time the real impact on the detected incidence of dengue after the introduction of CHIKV and ZIKV in Mexico, the degree of underestimation of these arboviruses in the country, as well as the co-infections between these viruses, whose importance clinical and epidemiological are still unknown.     

Diagnostic performance of anti-Zika virus IgM,IgAM and IgG ELISAs during co-circulation of Zika,dengue, and chikungunya viruses in Brazil and Venezuela

BackgroundSerological diagnosis of Zika virus (ZIKV) infection is challenging because of the antibody cross-reactivity among flaviviruses. At the same time, the role of Nucleic Acid Testing (NAT) is limited by the low proportion of symptomatic infections and the low average viral load. Here, we compared the diagnostic performance of commercially available IgM, IgAM, and IgG ELISAs in sequential samples during the ZIKV and chikungunya (CHIKV) epidemics and co-circulation of dengue virus (DENV) in Brazil and Venezuela.Methodology/Principal findingsAcute (day of illness 1–5) and follow-up (day of illness ≥ 6) blood samples were collected from nine hundred and seven symptomatic patients enrolled in a prospective multicenter study between June 2012 and August 2016. Acute samples were tested by RT-PCR for ZIKV, DENV, and CHIKV. Acute and follow-up samples were tested for IgM, IgAM, and IgG antibodies to ZIKV using commercially available ELISAs. Among follow-up samples with a RT-PCR confirmed ZIKV infection, anti-ZIKV IgAM sensitivity was 93.5% (43/46), while IgM and IgG exhibited sensitivities of 30.3% (10/33) and 72% (18/25), respectively. An additional 24% (26/109) of ZIKV infections were detected via IgAM seroconversion in ZIKV/DENV/CHIKV RT-PCR negative patients. The specificity of anti-ZIKV IgM was estimated at 93% and that of IgAM at 85%.Conclusions/SignificanceOur findings exemplify the challenges of the assessment of test performance for ZIKV serological tests in the real-world setting, during co-circulation of DENV, ZIKV, and CHIKV. However, we can also demonstrate that the IgAM immunoassay exhibits superior sensitivity to detect ZIKV RT-PCR confirmed infections compared to IgG and IgM immunoassays. The IgAM assay also proves to be promising for detection of anti-ZIKV seroconversions in sequential samples, both in ZIKV PCR-positive as well as PCR-negative patients, making this a candidate assay for serological monitoring of pregnant women in future ZIKV outbreaks.     

Validation and Application of a Commercial Quantitative Real-Time Reverse Transcriptase-PCR Assay in Investigation of a Large Dengue Virus Outbreak in Southern Taiwan

BackgroundAccurate, rapid, and early diagnosis of dengue virus (DENV) infections is essential for optimal clinical care. Here, we evaluated the efficacy of the quantitative real-time PCR (qRT-PCR)-LightMix dengue virus EC kit for DENV detection using samples from a dengue outbreak in Taiwan in 2015.MethodsSera from patients with suspected DENV infection were analyzed and compared using the LightMix kit, a Dengue NS1 Ag + Ab Combo kit for detection of NS1 antigen and DENV-specific IgM and IgG antibodies, and an “in-house” qualitative DENV-specific RT-PCR assay.ResultsA total of 8,989, 8,954, and 1581 samples were subjected to NS1 antigen detection, IgM and IgG detection, and LightMix assays, respectively. The LightMix assay yielded a linear curve for viral loads (VL) between 10² and 10⁶ copies/reaction, and the minimum detection limits for DENV serotype 1 (DENV1) and DENV2, DENV3, and DENV4 were 1, 10, and 100 focus forming units (FFU)/mL, respectively. There was 88.9% concordance between the results obtained using the NS1 antigen combo kit and by LightMix analysis, and the diagnostic sensitivity and specificity of the two methods were 89.4 and 100%, and 84.7 and 100%, respectively. Notably, fatal cases were attributed to DENV2 infection, and 79.5% (27/34) of these cases occurred in patients ≥ 71 years of age. Among these older patients, 82.3% (14/17) were NS1/IgM/IgG (+/-/-), exhibiting VLs between 10⁶–10⁹ copies/mL, which was markedly higher than the rate observed in the other age groups.ConclusionsThe LightMix assay was effective for early diagnosis of DENV infection. Our data indicate that high VLs during primary infection in elderly patients may be a positive predictor for severe illness, and may contribute to high mortality rates.     

Analytical and Clinical Performance of the CDC Real Time RT-PCR Assay for Detection and Typing of Dengue Virus

Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1–4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1–4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1–4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1–4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.     

Epidemiological and Molecular Characterization of Dengue Virus Circulating in Bhutan, 2013-2014

Dengue is one of the most significant public health problems in tropical and subtropical countries, and is increasingly being detected in traditionally non-endemic areas. In Bhutan, dengue virus (DENV) has only recently been detected and limited information is available. In this study, we analyzed the epidemiological and molecular characteristics of DENV in two southern districts in Bhutan from 2013–2014. During this period, 379 patients were clinically diagnosed with suspected dengue, of whom 119 (31.4%) were positive for DENV infection by NS1 ELISA and/or nested RT-PCR. DENV serotypes 1, 2 and 3 were detected with DENV-1 being predominant. Phylogenetic analysis of DENV-1 using envelope gene demonstrated genotype V, closely related to strains from northern India.     

Vector competence of Aedes aegypti from Havana,Cuba, for dengue virus type 1, chikungunya,and Zika viruses

BackgroundLike many countries from the Americas, Cuba is threatened by Aedes aegypti-associated arboviruses such as dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) viruses. Curiously, when CHIKV was actively circulating in the region in 2013–2014, no autochthonous transmission of this virus was detected in Havana, Cuba, despite the importation of chikungunya cases into this city. To investigate if the transmission ability of local mosquito populations could explain this epidemiological scenario, we evaluated for the first time the vector competence of two Ae. aegypti populations (Pasteur and Párraga) collected from Havana for dengue virus type 1 (DENV-1), CHIKV, and ZIKV.Methodology/Principal findingsMosquito populations were fed separately using blood containing ZIKV, DENV-1, or CHIKV. Infection, dissemination, and transmission rates, were estimated at 3 (exclusively for CHIKV), 7, and 14 days post exposure (dpe) for each Ae. aegypti population-virus combination. Both mosquito populations were susceptible to DENV-1 and ZIKV, with viral infection and dissemination rates ranging from 24–97% and 6–67% respectively. In addition, CHIKV disseminated in both populations and was subsequently transmitted. Transmission rates were low (<30%) regardless of the mosquito population/virus combination and no ZIKV was detected in saliva of females from the Pasteur population at any dpe.Conclusions/SignificanceOur study demonstrated the ability of Ae. aegypti from Cuba to transmit DENV, ZIKV, and CHIKV. These results, along with the widespread distribution and high abundance of this species in the urban settings throughout the island, highlight the importance of Ae. aegypti control and arbovirus surveillance to prevent future outbreaks.     

Characteristics of a Dengue Outbreak in a Remote Pacific Island Chain – Republic of the Marshall Islands, 2011–2012

Dengue is a potentially fatal acute febrile illness caused by four mosquito-transmitted dengue viruses (DENV-1–4). Although dengue outbreaks regularly occur in many regions of the Pacific, little is known about dengue in the Republic of the Marshall Islands (RMI). To better understand dengue in RMI, we investigated an explosive outbreak that began in October 2011. Suspected cases were reported to the Ministry of Health, serum specimens were tested with a dengue rapid diagnostic test (RDT), and confirmatory testing was performed using RT-PCR and IgM ELISA. Laboratory-positive cases were defined by detection of DENV nonstructural protein 1 by RDT, DENV nucleic acid by RT-PCR, or anti-DENV IgM antibody by RDT or ELISA. Secondary infection was defined by detection of anti-DENV IgG antibody by ELISA in a laboratory-positive acute specimen. During the four months of the outbreak, 1,603 suspected dengue cases (3% of the RMI population) were reported. Of 867 (54%) laboratory-positive cases, 209 (24%) had dengue with warning signs, six (0.7%) had severe dengue, and none died. Dengue incidence was highest in residents of Majuro and individuals aged 10–29 years, and ∼95% of dengue cases were experiencing secondary infection. Only DENV-4 was detected by RT-PCR, which phylogenetic analysis demonstrated was most closely related to a virus previously identified in Southeast Asia. Cases of vertical DENV transmission, and DENV/Salmonella Typhi and DENV/Mycobacterium leprae co-infection were identified. Entomological surveys implicated water storage containers and discarded tires as the most important development sites for Aedes aegypti and Ae. albopictus, respectively. Although this is the first documented dengue outbreak in RMI, the age groups of cases and high prevalence of secondary infection demonstrate prior DENV circulation. Dengue surveillance should continue to be strengthened in RMI and throughout the Pacific to identify and rapidly respond to future outbreaks.     

Molecular Characterisation of Chikungunya Virus Infections in Trinidad and Comparison of Clinical and Laboratory Features with Dengue and Other Acute Febrile Cases

Local transmission of Chikungunya virus (CHIKV) was first documented in Trinidad and Tobago (T&T) in July 2014 preceding a large epidemic. At initial presentation, it is difficult to distinguish chikungunya fever (CHIKF) from other acute undifferentiated febrile illnesses (AUFIs), including life-threatening dengue disease. We characterised and compared dengue virus (DENV) and CHIKV infections in 158 patients presenting with suspected dengue fever (DF) and CHIKF at a major hospital in T&T, and performed phylogenetic analyses on CHIKV genomic sequences recovered from 8 individuals. The characteristics of patients with and without PCR-confirmed CHIKV were compared using Pearson’s χ² and student’s t-tests, and adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were determined using logistic regression. We then compared signs and symptoms of people with RT-qPCR-confirmed CHIKV and DENV infections using the Mann-Whitney U, Pearson’s χ² and Fisher’s exact tests. Among the 158 persons there were 8 (6%) RT-qPCR-confirmed DENV and 30 (22%) RT-qPCR-confirmed CHIKV infections. Phylogenetic analyses showed that the CHIKV strains belonged to the Asian genotype and were most closely related to a British Virgin Islands strain isolated at the beginning of the 2013/14 outbreak in the Americas. Compared to persons who were RT-qPCR-negative for CHIKV, RT-qPCR-positive individuals were significantly more likely to have joint pain (aOR: 4.52 [95% CI: 1.28–16.00]), less likely to be interviewed at a later stage of illness (days post onset of fever—aOR: 0.56 [0.40–0.78]) and had a lower white blood cell count (aOR: 0.83 [0.71–0.96]). Among the 38 patients with RT-qPCR-confirmed CHIKV or DENV, there were no significant differences in symptomatic presentation. However when individuals with serological evidence of recent DENV or CHIKV infection were included in the analyses, there were key differences in clinical presentation between CHIKF and other AUFIs including DF, which can be used to triage patients for appropriate care in the clinical setting.     

The worldwide seroprevalence of DENV,CHIKV and ZIKV infection: A systematic review and meta-analysis

BackgroundAs the three major arthropod-borne viruses, dengue virus (DENV), chikungunya virus (CHIKV), and zika virus (ZIKV) are posing a growing threat to global public health and socioeconomic development. Our study aimed to systematically review the global seroprevalences of these arboviruses from existing publications.MethodsArticles published between Jan 01, 2000 and Dec 31, 2019 in the databases of Embase, Pubmed and Web of Science were searched and collected. Countries or areas with known local presence of Aedes vector mosquitoes were included. Random effects model was utilized to estimate the pooled seroprevalences and the proportion of inapparent infection.ResultsOut of 1375, a total of 133 articles involving 176,001 subjects were included for our analysis. The pooled seroprevalences of DENV, CHIKV and ZIKV were 38%, 25% and 18%, respectively; and their corresponding proportions of inapparent infections were 80%, 40% and 50%. The South-East Asia Region had the highest seroprevalences of DENV and CHIKV, while the Region of the Americas had the highest seroprevalence of ZIKV. The seroprevalences of DENV and CHIKV were similar when comparing developed and developing countries, urban and rural areas, or among different populations. In addition, we observed a decreased global seroprevalences in the new decade (2010–2019) comparing to the decade before (2000–2009) for CHIKV. For ZIKV, the positive rates tested with the nucleic acid detection method were lower than those tested with the antibody detection method. Lastly, numerous cases of dual seropositivity for CHIKV and DENV were reported.ConclusionsOur results revealed a varied prevalence of arbovirus infections in different geographical regions and countries, and the inapparent infection accounted an unneglected portion of infections that requires more attention. This study will shed lights on our understanding of the true burden of arbovirus infections and promote appropriate vaccination in the future.     

Co-infection of Dengue,Zika and Chikungunya in a group of pregnant women from Tuxtla Gutiérrez,Chiapas: Preliminary data. 2019

IntroductionDengue, Zika and Chikungunya are RNA Arboviruses present in some areas of Mexico, mainly in the endemic state of Chiapas that is characterized by presence of the vector that transmit them and an ecology that favors high transmission. According to the national epidemiological surveillance system, Dengue has intensified since 2018 and outbreaks continue in various states while for Zika and Chikungunya a decrease in cases has been reported in recent years. The main objective of this study was to determine the incidence of Dengue, Zika and Chikungunya infections during pregnancy in the state of Chiapas.Principal findingsThe presence of previous and current infections and coinfections diagnosed by molecular (RT-PCR) and immunological (ELISA for IgG determination) techniques indicates a wide circulation of viruses in asymptomatic people, specifically in pregnant women showing that silent infections in dry season contributes to the preservation of viruses.ConclusionsFrom 136 studied samples, 27.7% tested positive for DENV, 8% for ZIKV and 24.1% for CHIKV by RTPCR and the values of IgG in sera show that 83.9% were positive for IgG antibodies against DENV, 65% against ZIKV and 59.1% against CHIKV. Results demonstrated presence of ZIKV and CHIKV, not detected by the epidemiological surveillance system, so the importance of establishing proactive epidemiological systems more strict, especially because these infections in pregnant women can cause severe health problems for newborn children.     

Multiplex detection of antibodies to Chikungunya,O’nyong-nyong,Zika, Dengue,West Nile and Usutu viruses in diverse non-human primate species from Cameroon and the Democratic Republic of Congo

BackgroundEpidemic arbovirus transmission occurs among humans by mosquito bites and the sylvatic transmission cycles involving non-human primates (NHPs) still exists. However, limited data are available on the extent in NHPs infections and their role. In this study, we have developed and validated a high-throughput serological screening tool to study the circulation of multiple arboviruses that represent a significant threat to human health, in NHPs in Central Africa.Methodology/Principal findingsRecombinant proteins NS1, envelope domain-3 (DIII) for the dengue (DENV), yellow fever (YFV), usutu (USUV), west nile (WNV) and zika (ZIKV) and envelope 2 for the chikungunya (CHIKV) and o''nyong-nyong (ONNV) were coupled to Luminex beads to detect IgG directed against these viruses. Evaluation of test performance was made using 161 human sera of known arboviral status (66 negative and 95 positive). The sensitivity and specificity of each antigen were determined by statistical methods and ROC curves (except for ONNV and USUV). All NS1 antigens (except NS1-YFV), CHIKV-E2 and WNV-DIII had sensitivities and specificities > 95%. For the other DIII antigens, the sensitivity was low, limiting the interest of their use for seroprevalence studies. Few simultaneous reactions were observed between the CHIKV+ samples and the NS1 antigens to the non-CHIKV arboviruses. On the other hand, the DENV+ samples crossed-reacted with NS1 of all the DENV serotypes (1 to 4), as well as with ZIKV, USUV and to a lesser extent with YFV. A total of 3,518 samples of 29 species of NHPs from Cameroon and the Democratic Republic of Congo (DRC) were tested against NS1 (except YFV), E2 (CHIKV/ONNV) and DIII (WNV) antigens. In monkeys (n = 2,100), the global prevalence varied between 2 and 5% for the ten antigens tested. When we stratified by monkey’s biotope, the arboreal species showed the highest reactivity. In monkeys from Cameroon, the highest IgG prevalence were observed against ONNV-E2 and DENV2-NS1 with 3.95% and 3.40% respectively and in DRC, ONNV-E2 (6.63%) and WNV-NS1 (4.42%). Overall prevalence was low in apes (n = 1,418): ranging from 0% for USUV-NS1 to 2.6% for CHIKV-E2. However, a very large disparity was observed among collection site and ape species, e.g. 18% (9/40) and 8.2% (4/49) of gorillas were reactive with CHIKV-E2 or WNV-NS1, respectively in two different sites in Cameroon.Conclusions/SignificanceWe have developed a serological assay based on Luminex technology, with high specificity and sensitivity for simultaneous detection of antibodies to 10 antigens from 6 different arboviruses. This is the first study that evaluated on a large scale the presence of antibodies to arboviruses in NHPs to evaluate their role in sylvatic cycles. The overall low prevalence (<5%) in more than 3,500 NHPs samples from Cameroon and the DRC does not allow us to affirm that NHP are reservoirs, but rather, intermediate hosts of these viruses.     

Suppression of the Arboviruses Dengue and Chikungunya Using a Dual-Acting Group-I Intron Coupled with Conditional Expression of the Bax C-Terminal Domain

In portions of South Asia, vectors and patients co-infected with dengue (DENV) and chikungunya (CHIKV) are on the rise, with the potential for this occurrence in other regions of the world, for example the United States. Therefore, we engineered an antiviral approach that suppresses the replication of both arboviruses in mosquito cells using a single antiviral group I intron. We devised unique configurations of internal, external, and guide sequences that permit homologous recognition and splicing with conserved target sequences in the genomes of both viruses using a single trans-splicing Group I intron, and examined their effectiveness to suppress infections of DENV and CHIKV in mosquito cells when coupled with a proapoptotic 3'' exon, ΔN Bax. RT-PCR demonstrated the utility of these introns in trans-splicing the ΔN Bax sequence downstream of either the DENV or CHIKV target site in transformed Aedes albopictus C6/36 cells, independent of the order in which the virus specific targeting sequences were inserted into the construct. This trans-splicing reaction forms DENV or CHIKV ΔN Bax RNA fusions that led to apoptotic cell death as evidenced by annexin V staining, caspase, and DNA fragmentation assays. TCID50-IFA analyses demonstrate effective suppression of DENV and CHIKV infections by our anti-arbovirus group I intron approach. This represents the first report of a dual-acting Group I intron, and demonstrates that we can target DENV and CHIKV RNAs in a sequence specific manner with a single, uniquely configured CHIKV/DENV dual targeting group I intron, leading to replication suppression of both arboviruses, and thus providing a promising single antiviral for the transgenic suppression of multiple arboviruses.     

Dengue Viral RNA Levels in Peripheral Blood Mononuclear Cells Are Associated with Disease Severity and Preexisting Dengue Immune Status

Background

Infection with dengue viruses (DENV) causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF), to dengue hemorrhagic fever (DHF). The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known.

Method

The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR.

Results

Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells.

Conclusions

B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC). Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis.     

Development of a Specific CHIKV-E2 Monoclonal Antibody for Chikungunya Diagnosis

Chikungunya fever is a vector-borne viral disease transmitted to humans by chikungunya virus(CHIKV)-infected mosquitoes. There have been many outbreaks of CHIKV infection worldwide, and the virus poses ongoing risks to global health. To prevent and control CHIKV infection, it is important to improve the current CHIKV diagnostic approaches to allow for the detection of low CHIKV concentrations and to correctly distinguish CHIKV infections from those due to other mosquito-transmitted viruses, including dengue virus(DENV), Japanese encephalitis virus(JEV), and Zika virus(ZIKV). Here, we produced monoclonal antibodies(mAbs) against the CHIKV envelope 2 protein(CHIKV-E2) and compared their sensitivity and specificity with commercially available m Abs using enzyme-linked immunosorbent assays(ELISA). Two anti-CHIKV-E2 mAbs, 19-1 and 21-1, showed higher binding affinities to CHIKV-E2 protein than the commercial mAbs did. In particular, the 19-1 m Ab had the strongest binding affinity to inactivated CHIKV. Moreover, the 19-1 mAb had very little cross-reactivity with other mosquito-borne viruses, such as ZIKV, JEV, and DENV. These results suggest that the newly produced anti-CHIKV-E2 mAb, 19-1, could be used for CHIKV diagnostic approaches.     

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.     

Performance of Simplexa Dengue Molecular Assay Compared to Conventional and SYBR Green RT-PCR for Detection of Dengue Infection in Indonesia

Diagnostic tests based on detection of dengue virus (DENV) genome are available with varying sensitivities and specificities. The Simplexa Dengue assay (Focus Diagnostics) is a newly developed real-time RT-PCR method designed to detect and serotype DENV simultaneously. To assess the performance of the Simplexa Dengue assay, we performed comparison with conventional RT-PCR and SYBR Green real-time RT-PCR on patients sera isolated from eight cities across Indonesia, a dengue endemic country. A total of 184 sera that were confirmed using NS1 and/or IgM and IgG ELISA were examined. Using conventional and SYBR Green real-time RT-PCR, we detected DENV in 53 (28.8%) and 81 (44.0%) out of 184 sera, respectively. When the Simplexa Dengue assay was employed, the detection rate was increased to 76.6% (141 out of 184 samples). When tested in 40 sera that were confirmed by virus isolation as the gold standard, the conventional RT-PCR yielded 95% sensitivity while the sensitivity of SYBR Green real-time RT-PCR and Simplexa Dengue assay reached 97.5% and 100%, respectively. The specificities of all methods were 100% when tested in 43 non-dengue illness and 20 healthy human samples. Altogether, our data showed the higher detection rate of Simplexa Dengue compared to conventional and SYBR Green real-time RT-PCR in field/surveillance setting. In conclusion, Simplexa Dengue offers rapid and accurate detection and typing of dengue infection and is suitable for both routine diagnostic and surveillance.