Potential Misdiagnosis between COVID-19 and Dengue Infection Using Rapid Serological Test.

The coronavirus disease 2019 (COVID-19) pandemic that has a significant rapid transmission is an international public health concern. Several dengue-endemic countries reported similar clinical and laboratory features between COVID-19 and dengue in the early incubation period, and thus discerning the infection is difficult. As a dengue-endemic country, Indonesia also poses the same challenge during the COVID-19 outbreak. This current study analyzed the IgG and IgM profiles from COVID-19 patients by using a serological SARS-CoV-2 and dengue rapid test. In addition, 38 sera from healthy individuals (pre-COVID-19 date) were analyzed using a dengue rapid test. Among 120 samples, 4 samples indicated dengue IgG positive. However, IgM, NS1, and RT-PCR analyses showed negative results. Interestingly, regarding seropositivity of NS1 and DENV IgG from healthy individuals (pre COVID-19 infection), two samples were positive DENV IgG, while one of them was positive NS1. This suggested that in the dengue-endemic area, many people have already experienced dengue and have immunity against dengue virus. There is also the possibility of antibody cross-reactivity between COVID-19 and dengue infection. This also emphasizes the high demand for a rapid method with high sensitivity and specificity that can distinguish between SARS-CoV-2 and dengue.

An affinity-matured human monoclonal antibody targeting fusion loop epitope of dengue virus with in vivo therapeutic potency.

Dengue virus (DENV), from the genus flavivirus of the family flaviviridae, causes serious health problems globally. Human monoclonal antibodies (HuMAb) can be used to elucidate the mechanisms of neutralization and antibody-dependent enhancement (ADE) of DENV infections, leading to the development of a vaccine or therapeutic antibodies. Here, we generated eight HuMAb clones from an Indonesian patient infected with DENV. These HuMAbs exhibited the typical characteristics of weak neutralizing antibodies including high cross-reactivity with other flaviviruses and targeting of the fusion loop epitope (FLE). However, one of the HuMAbs, 3G9, exhibited strong neutralization (NT50 < 0.1 µg/ml) and possessed a high somatic hyper-mutation rate of the variable region, indicating affinity-maturation. Administration of this antibody significantly prolonged the survival of interferon-α/ß/γ receptor knockout C57BL/6 mice after a lethal DENV challenge. Additionally, Fc-modified 3G9 that had lost their in vitro ADE activity showed enhanced therapeutic potency in vivo and competed strongly with an ADE-prone antibody in vitro. Taken together, the affinity-matured FLE-targeting antibody 3G9 exhibits promising features for therapeutic application including a low NT50 value, potential for treatment of various kinds of mosquito-borne flavivirus infection, and suppression of ADE. This study demonstrates the therapeutic potency of affinity-matured FLE-targeting antibodies.

Phylogenetic Analysis of Dengue Virus in Bangkalan, Madura Island, East Java Province, Indonesia.

Dengue virus (DENV) infection is a major health issue in tropical and subtropical areas. Indonesia is one of the biggest dengue endemic countries in the world. In the present study, the phylogenetic analysis of DENV in Bangkalan, Madura Island, Indonesia, was performed in order to obtain a clearer understanding of its dynamics in this country. A total of 359 blood samples from dengue-suspected patients were collected between 2012 and 2014. Serotyping was conducted using a multiplex Reverse Transcriptase-Polymerase Chain Reaction and a phylogenetic analysis of E gene sequences was performed using the Bayesian Markov chain Monte Carlo (MCMC) method. 17 out of 359 blood samples (4.7%) were positive for the isolation of DENV. Serotyping and the phylogenetic analysis revealed the predominance of DENV-1 genotype I (9/17, 52.9%), followed by DENV-2 Cosmopolitan type (7/17, 41.2%) and DENV-3 genotype I (1/17, 5.9%). DENV-4 was not isolated. The Madura Island isolates showed high nucleotide similarity to other Indonesian isolates, indicating frequent virus circulation in Indonesia. The results of the present study highlight the importance of continuous viral surveillance in dengue endemic areas in order to obtain a clearer understanding of the dynamics of DENV in Indonesia.

A NEW COPPER (II)-IMIDAZOLE DERIVATIVE EFFECTIVELY INHIBITS REPLICATION OF DENV-2 IN VERO CELL.

BACKGROUND: Dengue is a kind of infectious disease that was distributed in the tropical and sub-tropical areas. To date, there is no clinically approved dengue vaccine or antiviral for humans, even though there have been great efforts towards this end. Therefore, finding the effective compound against dengue virus (DENV) replication is very important. Among the complex compounds, copper(II)-imidazole derivatives are of interest because of their biological and medicinal benefits. MATERIALS AND METHODS: In the present study, antiviral activity of [Cu(2,4,5-triphenylimidazole)2]n, was evaluated against different stages of dengue virus type 2 (DENV-2) replication in Vero cell using focus forming unit reduction assay and quantitative ELISA. RESULTS: [Cu(2,4,5-triphenylimidazole)2]n inhibited DENV-2 replication in Vero cells with IC50 = 2.3 µg/ml and SI= 19.42 when cells were treated 2 days after virus infection, whereas its CC50 for cytotoxicity to Vero cells was 44.174 µg/ml. CONCLUSION: The compound has high anti-DENV2 activity, less toxicity, and a high possibility to be considered a drug candidate.

Divergence of the dengue virus type 2 Cosmopolitan genotype associated with two predominant serotype shifts between 1 and 2 in Surabaya, Indonesia, 2008-2014.

Indonesia is one of the biggest dengue endemic countries, and, thus, is an important place to investigate the evolution of dengue virus (DENV). We have continuously isolated DENV in Surabaya, the second biggest city in Indonesia, since 2008. We previously reported sequential changes in the predominant serotype from DENV type 2 (DENV-2) to DENV type 1 (DENV-1) in November 2008 and from DENV-1 to DENV-2 in July 2013. The predominance of DENV-2 continued in 2014, but not in 2015. We herein phylogenetically investigated DENV-2 transitions in Surabaya between 2008 and 2014 to analyze the divergence and evolution of DENV-2 concomitant with serotype shifts. All DENV-2 isolated in Surabaya were classified into the Cosmopolitan genotype, and further divided into 6 clusters. Clusters 1-3, dominated by Surabaya strains, were defined as the "Surabaya lineage". Clusters 4-6, dominated by strains from Singapore, Malaysia, and many parts of Indonesia, were the "South East Asian lineage". The most recent common ancestor of these strains existed in 1988, coinciding with the time that an Indonesian dengue outbreak took place. Cluster 1 appeared to be unique because no other DENV-2 isolate was included in this cluster. The predominance of DENV-2 in 2008 and 2013-14 were caused by cluster 1, whereas clusters 2 and 3 sporadically emerged in 2011 and 2012. The characteristic amino acids of cluster 1, E-170V and E-282Y, may be responsible for its prevalence in Surabaya. No amino acid difference was observed in the envelope region between strains in 2008 and 2013-14, suggesting that the re-emergence of DENV-2 in Surabaya was due to the loss or decrease of herd immunity in the 5-year period when DENV-2 subsided. The South East Asian lineage primarily emerged in Surabaya in 2014, probably imported from other parts of Indonesia or foreign countries.

Dengue virus infection-enhancing antibody activities against Indonesian strains in inhabitants of central Thailand.

Dengue virus (DENV) infection-enhancing antibodies are a hypothetic factor to increase the dengue disease severity. In this study, we investigated the enhancing antibodies against Indonesian strains of DENV-1-4 in 50 healthy inhabitants of central Thailand (Bangkok and Uthai Thani). Indonesia and Thailand have seen the highest dengue incidence in Southeast Asia. The infection history of each subject was estimated by comparing his/her neutralizing antibody titers against prototype DENV-1-4 strains. To resolve the difficulty in obtaining foreign live viruses for use as assay antigens, we used a recombinant system to prepare single-round infectious dengue viral particles based on viral sequence information. Irrespective of the previously infecting serotype(s), most serum samples showed significantly higher enhancement titers against Indonesian DENV-2 strains than against Thai DENV-2 strains, whereas the opposite effect was observed for the DENV-3 strains. Equivalent enhancing activities were observed against both DENV-1 and DENV-4. These results suggest that the genotype has an impact on enhancing antibody activities against DENV-2 and DENV-3, because the predominant circulating genotypes of each serotype differ between Indonesia and Thailand.

Detection of chikungunya virus antigen by a novel rapid immunochromatographic test.

Chikungunya fever is a mosquito-borne disease of key public health importance in tropical and subtropical countries. Although severe joint pain is the most distinguishing feature of chikungunya fever, diagnosis remains difficult because the symptoms of chikungunya fever are shared by many pathogens, including dengue fever. The present study aimed to develop a new immunochromatographic diagnosis test for the detection of chikungunya virus antigen in serum. Mice were immunized with isolates from patients with Thai chikungunya fever, East/Central/South African genotype, to produce mouse monoclonal antibodies against chikungunya virus. Using these monoclonal antibodies, a new diagnostic test was developed and evaluated for the detection of chikungunya virus. The newly developed diagnostic test reacted with not only the East/Central/South African genotype but also with the Asian and West African genotypes of chikungunya virus. Testing of sera from patients suspected to have chikungunya fever in Thailand (n = 50), Laos (n = 54), Indonesia (n = 2), and Senegal (n = 6) revealed sensitivity, specificity, and real-time PCR (RT-PCR) agreement values of 89.4%, 94.4%, and 91.1%, respectively. In our study using serial samples, a new diagnostic test showed high agreement with the RT-PCR within the first 5 days after onset. A rapid diagnostic test was developed using mouse monoclonal antibodies that react with chikungunya virus envelope proteins. The diagnostic accuracy of our test is clinically acceptable for chikungunya fever in the acute phase.

Continuous dengue type 1 virus genotype shifts followed by co-circulation, clade shifts and subsequent disappearance in Surabaya, Indonesia, 2008-2013.

Four serotypes of dengue virus (DENV-1 to DENV-4) and their genotypes are distributed in tropical and subtropical regions. Indonesia has been recently suggested as the origin of some dengue virus genotypes. In Surabaya, the second biggest city of Indonesia, we previously reported a shift of the predominantly circulating serotype from DENV-2 to DENV-1 in November 2008, followed by a genotype shift of DENV-1 from genotype IV (GIV) to genotype I (GI) in September 2009, based on nucleotide sequences in the envelope protein coding region. Since then, GI strains had predominantly circulated until December 2010. In this report, we investigated further DENV-1 transitions in Surabaya during 2011-2013 in order to comprehend dengue dynamics during 2008-2013 in more detail. From January 2011 through December 2011, only GIV strains were isolated, indicating that a genotype shift again took place from GI to GIV. In January 2012, GI and GIV strains started co-circulating, which continued until June 2013. To further investigate this phenomenon, analysis was performed at a clade level. GI and GIV strains isolated in Surabaya formed four and three distinct clades, respectively. Concomitant with co-circulation, new clade strains appeared in both genotypes. In contrast, some previously circulating clades were not isolated during co-circulation, indicating clade shifts. Among our Surabaya isolates, nucleotide and amino acid differences in the E region were, respectively, 1.0-2.3% and 0.2-1.0% for GI isolates and 2.0-6.3% and 0.0-1.8% for GIV isolates. Several characteristic amino acid substitutions in the envelope ectodomain were observed in some clades. After July 2013, DENV-1 strains were not isolated and were replaced with DENV-2. This study showed that continuous shifts of more than one genotype resulted in their co-circulation and subsequent disappearance and suggested the relevance of clade replacement to genotype co-circulation and disappearance in Surabaya.