Designing one-step reverse transcriptase loop-mediated isothermal amplification for serotype O foot-and-mouth disease virus detection during the 2022 outbreak in East Java, Indonesia.

Background and Aim: Various methods can detect foot-and-mouth disease (FMD) in cows, but they necessitate resources, time, costs, laboratory facilities, and specific clinical specimen submission, often leading to FMD virus (FMDV) diagnosis delays. The 2022 FMD outbreak in East Java, Indonesia, highlighted the need for an easy, inexpensive, rapid, and accurate detection approach. This study aims to devise a one-step reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) technique and phylogenetic analysis to detect the serotype O FMDV outbreak in East Java. Materials and Methods: Swab samples were collected from the foot vesicles, nasal secretions, and saliva of five suspected FMDV-infected cows in East Java between June and July 2022. The RT-LAMP design used hydroxy naphthol blue dye or SYBR Green I dye, with confirmatory analysis through reverse transcriptase polymerase chain reaction (RT-PCR) targeting 249 base pairs. PCR products underwent purification, sequencing, and nucleotide alignment, followed by phylogenetic analysis. Results: The RT-LAMP method using hydroxy naphthol blue dye displayed a positive reaction through a color shift from purple to blue in the tube. Naked-eye observation in standard light or ultraviolet (UV) light at 365 nm, with SYBR Green I stain, also revealed color change. Specifically, using SYBR Green I dye, UV light at 365 nm revealed a color shift from yellow to green, signifying a positive reaction. Nucleotide alignment revealed mutations and deletion at the 15th sequence in the JT-INDO-K3 isolate from the East Java FMDV outbreak. Despite differing branches, the phylogenetic tree placed it in the same cluster as serotype O FMDV from Malaysia and Mongolia. Conclusion: JT-INDO-K3 exhibited distinctions from Indonesian serotype O FMDV isolates and those documented in GenBank. Then, the RT-LAMP method used in this study has a detection limit 10 times higher latter than the conventional RT-PCR limit, without any cross-reactivity among strains.

Phylogenetic analysis of feline infectious peritonitis virus, feline enteric coronavirus, and severe acute respiratory syndrome coronavirus 2 of cats in Surabaya, Indonesia.

Background and Aim: Questions about the origin of coronavirus and its introduction to human beings have persisted. The detection of a variety of coronavirus related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in bats and pangolins led to the widespread belief that SARS-CoV-2 originated from wild ani-mals and was introduced to humans through an inter-mediate animal. Thus, coronaviruses from animals, especially those in close contact with humans, have attracted particular attention. This study aimed to phylogenetically analyze feline enteric coronavirus (FECV), feline infectious peritonitis virus (FIPV), and SARS-CoV-2 found in cats in Surabaya amid the COVID-19 pandemic. The results will provide a basis for developing basic preventive and pet healthcare strategies. Materials and Methods: Samples were collected on physical examinations of domestic and Persian cats (males and females) from March 2020 to March 2022. Samples were collected if there were clinical signs of FECV and FIP based on a veterinarian's diagnosis in several clinics in Surabaya. Laboratory examinations in this study were performed by reverse-transcription-polymerase chain reaction (RT-PCR) with primers for conserved regions of FIP and FECV, DNA sequencing was performed with Applied Biosystem Genetic Analyzer protocol, homology analysis was performed using Basic Local Alignment Search Tool NCBI, phylogenetic analysis was carried out with BioEdit 7.2 software, and sequences were compared with references from GenBank. Results: Samples were collected from ten cats showing clinical signs of FECV and FIP, based on a veterinarian's diagnosis. On RT-PCR examinations performed with specifically designed primers for detecting FIPV in blood, peritoneal fluid, and feces, only one sample showed positivity for FIPV (1/10), namely, a peritoneal sample from a domestic cat in Surabaya. Homology analysis of the FIPV Surabaya isolate showed 98% similarity with FECV and FIPV reported in GenBank (MT444152 and DQ010921, respectively). In phylogenetic analysis, the FIPV Surabaya isolate was clustered together with SARS-CoV-2 of Clade A (MT198653) from Spain, SARS-CoV-2 Clade A (MT192765) from the USA, SARS-CoV-2 Clade D (039888) from the USA, and SARS-CoV-2 Clade F (MT020781) from Finland. Conclusion: This study revealed a relationship between the SARS-CoV-2 viruses that infect humans and cats (FECV), which is an important finding for those keeping cats at home. However, this finding requires further comprehensive support from laboratory studies.

Comparison Epidemiology between Tuberculosis and COVID-19 in East Java Province, Indonesia: An Analysis of Regional Surveillance Data in 2020.

Tuberculosis and COVID-19 are among the infectious diseases that constitute a public health concern. Therefore, this study aims to examine the recent epidemiology of tuberculosis and COVID-19 in East Java Province, Indonesia, in 2020. Case-based surveillance data were acquired with a retrospective design between January and December 2020 by the East Java Health Officer. The data were analyzed using Quantum Geographic Information System (QGIS) for mapping, and Microsoft Excel for recording. Furthermore, the statistical analysis (Spearman correlation test) was carried out via Statistical Package for Social Science (SPSS) applications. A total number of 38,089 confirmed cases of tuberculosis was recorded, with an incidence rate of 95.49/100,000 population, a case fatality rate (CFR) of 3.6%, and an average treatment success rate of 87.78%. COVID-19 is a new viral disease, with a total of 84,133 confirmed COVID-19 cases in East Java, with an incidence rate of 232.9/100,000 population. The highest incidence rate was found in Mojokerto city, while the lowest was found in Sampang. Furthermore, the CFR values of tuberculosis and COVID-19 were 1.4% and 6.8%, respectively. The regional survey in East Java Province showed that the incidence of tuberculosis remains high. This indicated that the search for active cases and preventive promotion was not completed. Therefore, inter-sectoral collaboration can be adapted to provide suitable tuberculosis health care.

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.

How should Indonesia consider its neglected tropical diseases in the COVID-19 era? Hopes and challenges (Review).

During the coronavirus disease 2019 (COVID-19) pandemic, some countries, including Indonesia, have faced a double burden with regards to disease control. As Indonesia is a tropical country, it serves as a suitable host for disease vectors and multiple microorganisms of causative agents of disease. In total, five of the neglected tropical diseases (NTDs) should be a consideration in Indonesia during the COVID-19 pandemic, including leprosy, yaws, filariasis, soil-transmitted helminths and schistosomiasis. The present review summarises the preparedness of Indonesia in facing NTDs during the COVID-19 pandemic. Strengthening government leadership will be a valuable factor for combating NTDs in Indonesia. For instance, strong leadership can lead to precise management, by increasing the number of health facilities, engaging in active case identification, conducting health campaigns and instituting new regulations to prevent the stigmatization faced by patients. Preventive medicine in the first level of health facilities can be prioritized and presented to the community via health campaigns, health advocacy and improvement in follow-up after active case surveillance. Government-supported integrated management is also a key component in eliminating NTD. Moreover, healthy lifestyle campaigns that include social distancing, wearing a mask and regularly washing hands should be promoted continuously to reduce the transmission of COVID-19, which is potentially associated with a poor outcome in individuals with NTDs. This review concluded that the Indonesian government should strengthen their efforts toward NTD control using alternative methods, such as involving key citizens in the collaboration of the detection of new cases and introducing mobile health as a means of detecting health problems or following up on patient progress. To reduce the transmission of COVID-19, testing, tracing and treatment must be improved, so that the gap between suspected cases and confirmed cases of COVID-19 can be closed. If the transmission of COVID-19 can be decreased, case detection and efforts toward NTD control can be conducted effectively.

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.

Detection and Serotyping of Dengue Viruses in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) Collected in Surabaya, Indonesia from 2008 to 2015.

Aedes aegypti and Aedes albopictus are the primary and secondary vectors, respectively, of dengue, the most important arboviral disease in the world. The aim of this study was to detect and serotype dengue viruses (DENV) in the vectors Ae. aegypti and Ae. albopictus in Surabaya, Indonesia. Between 2008 and 2015, 16,605 Aedes mosquitoes were collected in 15 sub-districts of Surabaya. Ae. aegypti was dominant (90.9%), whereas few Ae. albopictus were collected (9.1%). A total of 330 pools of adult Aedes mosquitoes were subjected to the serotyping of DENV by RT-PCR. DENV-1 (52.3%) was the most frequently detected serotype, followed by DENV-2 (40.3%), DENV-4 (4.6%), and DENV-3 (2.8%). The average minimum infection rate for Ae. aegypti in various sub-districts of Surabaya was 7.2 per 1,000 mosquitoes, while that for Ae. albopictus was 0.7 per 1,000 mosquitoes. The results showed that the predominantly circulating DENV serotype in mosquitoes continuously shifted from DENV-2 (2008) to DENV-1 (2009-2012), to DENV-2 again (2013-2014), and then back to DENV-1 (2015). The circulating DENV serotypes in mosquitoes were generally consistent with those in humans. Therefore, the surveillance of infected mosquitoes with DENV might provide an early warning sign for the risk of future dengue outbreaks.

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.

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.