Laboratory Evaluation of the Rapid Analyte Measurement Platform Assay to Detect Dengue Virus in Mosquito Pools

ABSTRACT We report the results of a laboratory sensitivity and specificity evaluation of the Rapid Analyte Measurement Platform (RAMP®) Dengue Virus (DENV) antigen detection assay, which is designed to detect all serotypes of DENV in mosquito pools. The RAMP DENV assay was able to detect geographically distinct strains of all 4 DENV serotypes in virus-spiked mosquito pools that contained at least 4.3 log10 plaque forming units/ml, although discrete sensitivity limits varied slightly for each serotype. The RAMP DENV assay also detected DENV 1–4 in mosquito pools containing a single infected mosquito and 24 laboratory-reared uninfected mosquitoes. No false positives were detected in negative control mosquito pools or in samples containing high titers of nontarget arboviruses. We found that while the kit-supplied RAMP buffer reduced the infectious titer of DENV, it did not completely inactivate all serotypes. We recommend adding a detergent, Triton X-100, to the buffer to ensure complete inactivation of DENV if the assay is to be conducted at a lower biosafety level than required for DENV handling.

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Releasing Intracellular NS1 from Mosquito Cells for the Detection of Dengue Virus-Infected Mosquitoes

Viruses ◽

10.3390/v12101105 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1105

Author(s):

Lie Cheng ◽

Wei-Liang Liu ◽

Hsing-Han Li ◽

Matthew P. Su ◽

Shih-Cheng Wu ◽

...

Keyword(s):

Dengue Virus ◽

Mosquito Control ◽

Detection Threshold ◽

Disease Outbreaks ◽

Rapid Test ◽

Infected Mosquito ◽

Mosquito Cells ◽

Crucial Component ◽

Sensitivity Tests ◽

Triton X

Dengue virus (DENV), the pathogen that causes dengue fever, is mainly transmitted by Aedes aegypti. Surveillance of infected mosquitoes is a major component of integrated mosquito control methods for reducing the risk of vector-born disease outbreaks. However, a specialized rapid test for DENV detection in mosquitoes is not currently available. Utilizing immunoblotting, we found that the secretion of NS1 from both a DENV-infected mosquito cell line and mosquito bodies was below the detection threshold. However, when Triton X-100 was used to lyse infected mosquitoes, intracellular NS1 was released, and could then be effectively detected by the NS1 rapid test. The distribution of DENV NS1 in intrathoracically infected mosquitoes was different from that of orally infected mosquitoes. Next, we performed sensitivity tests by bisecting mosquitoes longitudinally; one half of each mosquito was subjected to the NS1 rapid test while the other half was used for qPCR confirmation. This modified test had a sensitivity of nearly 90% from five days post-infection onwards, while DENV had escaped from the midgut barrier. This adapted test offers a valuable, easy-to-use tool for mosquito surveillance, which is a crucial component of DENV disease control.

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Development of Bacteriophage Virus-Like Particle Vaccines Displaying Conserved Epitopes of Dengue Virus Non-Structural Protein 1

Vaccines ◽

10.3390/vaccines9070726 ◽

2021 ◽

Vol 9 (7) ◽

pp. 726

Author(s):

Nikole L. Warner ◽

Kathryn M. Frietze

Keyword(s):

Dengue Virus ◽

Population Based ◽

Ns1 Protein ◽

Structural Protein ◽

Denv Serotypes ◽

Conserved Regions ◽

Virus Like Particle ◽

Dependent Cell ◽

Infected Cells ◽

Plasma Leakage

Dengue virus (DENV) is a major global health problem, with over half of the world’s population at risk of infection. Despite over 60 years of efforts, no licensed vaccine suitable for population-based immunization against DENV is available. Here, we describe efforts to engineer epitope-based vaccines against DENV non-structural protein 1 (NS1). NS1 is present in DENV-infected cells as well as secreted into the blood of infected individuals. NS1 causes disruption of endothelial cell barriers, resulting in plasma leakage and hemorrhage. Immunizing against NS1 could elicit antibodies that block NS1 function and also target NS1-infected cells for antibody-dependent cell cytotoxicity. We identified highly conserved regions of NS1 from all four DENV serotypes. We generated synthetic peptides to these regions and chemically conjugated them to bacteriophage Qβ virus-like particles (VLPs). Mice were immunized two times with the candidate vaccines and sera were tested for the presence of antibodies that bound to the cognate peptide, recombinant NS1 from all four DENV serotypes, and DENV-2-infected cells. We found that two of the candidate vaccines elicited antibodies that bound to recombinant NS1, and one candidate vaccine elicited antibodies that bound to DENV-infected cells. These results show that an epitope-specific vaccine against conserved regions of NS1 could be a promising approach for DENV vaccines or therapeutics to bind circulating NS1 protein.

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A bioinformatics approach to investigate serum and hematopoietic cell-specific therapeutic microRNAs targeting the 3' UTRs of all four Dengue virus serotypes

Pathogens and Disease ◽

10.1093/femspd/ftab050 ◽

2021 ◽

Author(s):

Mirza Sarwar Baig ◽

Anuja Krishnan

Keyword(s):

Dengue Virus ◽

Antigen Processing ◽

Target Genes ◽

Public Health Problem ◽

Hematopoietic Cell ◽

Viral Gene ◽

P Value ◽

Pathway Enrichment Analysis ◽

Global Public Health ◽

Denv Serotypes

Abstract Hyperendemic circulation of all four Dengue virus (DENV) serotypes is a severe global public health problem, so any vaccine or therapeutics should be able to target all four of them. Cells of hemopoietic origin are believed to be primary sites of DENV replication. This study aimed to identify potential host miRNAs that target 3' UTR of all 4 DENV serotypes, thereby directly regulating viral gene expression or indirectly modulating the host system at different virus infection steps. We used four prediction algorithms viz. miRanda, RNA22, RNAhybrid, and StarMir for predicting miRNA, targeting 3'UTR of all four DENV serotypes. Statistically, the most significant miRNA targets were screened based on their Log10 P-value (> 0.0001) of GO term and KEGG pathway enrichment analysis. The intersection test of at least three prediction tools identified a total of 30 miRNAs, which could bind to 3'UTR of all four DENV serotypes. Of the 30, eight miRNAs were of hematopoietic cell origin. GO term enrichment and KEGG analysis showed four hemopoietic origin miRNAs target genes of the biological processes mainly involved in the innate immune response, mRNA 3'-end processing, antigen processing, and presentation and nuclear-transcribed mRNA catabolic process.

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Use of immunodot blot and multiplex reverse transcriptase-polymerase chain reaction in dengue virus detection in macerates of Aedes aegypti larvae

Microbiology Research ◽

10.4081/mr.2012.e13 ◽

2012 ◽

Vol 3 (1) ◽

pp. 13

Author(s):

Aline T.A. Chagas ◽

Michelle D. Oliveira ◽

Jose M.S. Mezencio ◽

Eduardo A.M. Silva ◽

Leandro L. Oliveira ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Aedes Aegypti ◽

Dengue Virus ◽

Reverse Transcriptase ◽

Epidemiological Surveillance ◽

Rt Pcr ◽

Chain Reaction ◽

Denv Serotypes ◽

Polymerase Chain ◽

Pcr Techniques

The <em>Dengue virus</em> is the main arbovirus that affects man in terms of morbidity and mortality. The detection of the virus is very important for epidemiological surveillance, so here we propose to standardize and compare the immunodot blot (IDB) and multiplex reverse transcriptase-polymerase chain reaction (M-RT-PCR) techniques to detect and characterize the dengue virus (DENV) serotypes in samples of <em>Aedes aegypti</em> larvae. Thus, the IDB and M-RT-PCR techniques were standardized using macerated samples of larvae collected in nature. The use of monoclonal antibodies in IDB has not shown great results, but DENV detection through this method was possible using polyclonal antibodies. The distinction of serotypes 1, 2 and 3 was carried out by M-RT-PCR.

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Boosting can explain patterns of fluctuations of ratios of inapparent to symptomatic dengue virus infections

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2013941118 ◽

2021 ◽

Vol 118 (14) ◽

pp. e2013941118

Author(s):

Laura W. Alexander ◽

Rotem Ben-Shachar ◽

Leah C. Katzelnick ◽

Guillermina Kuan ◽

Angel Balmaseda ◽

...

Keyword(s):

Dengue Virus ◽

Denv Infection ◽

Protective Effects ◽

Virus Infections ◽

Denv Serotypes ◽

Symptomatic Disease ◽

Large Fluctuations ◽

Antibody Titers ◽

Dengue Control ◽

Arboviral Disease

Dengue is the most prevalent arboviral disease worldwide, and the four dengue virus (DENV) serotypes circulate endemically in many tropical and subtropical regions. Numerous studies have shown that the majority of DENV infections are inapparent, and that the ratio of inapparent to symptomatic infections (I/S) fluctuates substantially year-to-year. For example, in the ongoing Pediatric Dengue Cohort Study (PDCS) in Nicaragua, which was established in 2004, the I/S ratio has varied from 16.5:1 in 2006–2007 to 1.2:1 in 2009–2010. However, the mechanisms explaining these large fluctuations are not well understood. We hypothesized that in dengue-endemic areas, frequent boosting (i.e., exposures to DENV that do not lead to extensive viremia and result in a less than fourfold rise in antibody titers) of the immune response can be protective against symptomatic disease, and this can explain fluctuating I/S ratios. We formulate mechanistic epidemiologic models to examine the epidemiologic effects of protective homologous and heterologous boosting of the antibody response in preventing subsequent symptomatic DENV infection. We show that models that include frequent boosts that protect against symptomatic disease can recover the fluctuations in the I/S ratio that we observe, whereas a classic model without boosting cannot. Furthermore, we show that a boosting model can recover the inverse relationship between the number of symptomatic cases and the I/S ratio observed in the PDCS. These results highlight the importance of robust dengue control efforts, as intermediate dengue control may have the potential to decrease the protective effects of boosting.

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Solvent/detergent-treated plasma: a virus-inactivated substitute for fresh frozen plasma

Blood ◽

10.1182/blood.v79.3.826.826 ◽

1992 ◽

Vol 79 (3) ◽

pp. 826-831 ◽

Cited By ~ 235

Author(s):

B Horowitz ◽

R Bonomo ◽

AM Prince ◽

SN Chin ◽

B Brotman ◽

...

Keyword(s):

Sindbis Virus ◽

Animal Studies ◽

Coagulation Factor ◽

Fresh Frozen Plasma ◽

Complete Inactivation ◽

Virus Safety ◽

Fresh Frozen ◽

History Of ◽

Triton X ◽

Frozen Plasma

Abstract Fresh frozen plasma (FFP) is prepared in blood banks world-wide as a by- product of red blood cell concentrate preparation. Appropriate clinical use is for coagulation factor disorders where appropriate concentrates are unavailable and when multiple coagulation factor deficits occur such as in surgery. Viral safety depends on donor selection and screening; thus, there continues to be a small but defined risk of viral transmission comparable with that exhibited by whole blood. We have prepared a virus sterilized FFP (S/D-FFP) by treatment of FFP with 1% tri(n-butyl)phosphate (TNBP) and 1% Triton X-100 at 30 degrees C for 4 hours. Added reagents are removed by extraction with soybean oil and chromatography on insolubilized C18 resin. Treatment results in the rapid and complete inactivation of greater than or equal to 10(7.5) infectious doses (ID50) of vesicular stomatitis virus (VSV) and greater than or equal to 10(6.9) ID50 of sindbis virus (used as marker viruses), greater than or equal to 10(6.2) ID50 of human immunodeficiency virus (HIV), greater than or equal to 10(6) chimp infectious doses (CID50) of hepatitis B virus (HBV), and greater than or equal to 10(5) CID50 of hepatitis C virus (HCV). Immunization of rabbits with S/D-FFP and subsequent adsorption of elicited antibodies with untreated FFP confirmed the absence of neoimmungen formation. Coagulation factor content was comparable with that found in FFP. Based on these laboratory and animal studies, together with the extensive history of the successful use of S/D-treated coagulation factor concentrates, we conclude that replacement of FFP with S/D-FFP, prepared in a manufacturing facility, will result in improved virus safety and product uniformity with no loss of efficacy.

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Doratoxylon apetalum, an Indigenous Medicinal Plant from Mascarene Islands, Is a Potent Inhibitor of Zika and Dengue Virus Infection in Human Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20102382 ◽

2019 ◽

Vol 20 (10) ◽

pp. 2382 ◽

Cited By ~ 8

Author(s):

Juliano G. Haddad ◽

Andrea Cristine Koishi ◽

Arnaud Gaudry ◽

Claudia Nunes Duarte dos Santos ◽

Wildriss Viranaicken ◽

...

Keyword(s):

Antiviral Activity ◽

Medicinal Plant ◽

Dengue Virus ◽

Denv Infection ◽

Antiviral Drugs ◽

Human Cells ◽

Host Cells ◽

Global Public Health ◽

Denv Serotypes ◽

Mascarene Islands

Zika virus (ZIKV) and Dengue virus (DENV) are mosquito-borne viruses of the Flavivirus genus that could cause congenital microcephaly and hemorrhage, respectively, in humans, and thus present a risk to global public health. A preventive vaccine against ZIKV remains unavailable, and no specific antiviral drugs against ZIKV and DENV are licensed. Medicinal plants may be a source of natural antiviral drugs which mostly target viral entry. In this study, we evaluate the antiviral activity of Doratoxylum apetalum, an indigenous medicinal plant from the Mascarene Islands, against ZIKV and DENV infection. Our data indicated that D. apetalum exhibited potent antiviral activity against a contemporary epidemic strain of ZIKV and clinical isolates of four DENV serotypes at non-cytotoxic concentrations in human cells. Time-of-drug-addition assays revealed that D. apetalum extract acts on ZIKV entry by preventing the internalisation of virus particles into the host cells. Our data suggest that D. apetalum-mediated ZIKV inhibition relates to virus particle inactivation. We suggest that D. apetalum could be a promising natural source for the development of potential antivirals against medically important flaviviruses.

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Low seroprevalence of Zika virus infection among adults in Southern Taiwan

BMC Infectious Diseases ◽

10.1186/s12879-019-4491-4 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

Yu-Wen Chien ◽

Tzu-Chuan Ho ◽

Pei-Wen Huang ◽

Nai-Ying Ko ◽

Wen-Chien Ko ◽

...

Keyword(s):

Dengue Virus ◽

Migrant Workers ◽

Zika Virus ◽

Southeast Asian ◽

Asian Countries ◽

Zikv Infection ◽

Serological Tests ◽

Cross Sectional ◽

Denv Serotypes ◽

Southeast Asian Countries

Abstract Background We recently conducted a serosurvey of newly arrived workers in Taiwan from four Southeast Asian countries which revealed that 1% of the migrant workers had laboratory-confirmed recent Zika virus (ZIKV) infection. Taiwan, where Aedes mosquitoes are prevalent, has a close relationship with Southeast Asian countries. Up to now, 21 imported cases of ZIKV infection have been reported in Taiwan, but there has been no confirmed indigenous case. The aim of this serosurvey was to assess whether there was unrecognized ZIKV infections in Taiwan. Methods A total of 212 serum samples collected in a cross-sectional seroepidemiologic study conducted during the end of the 2015 dengue epidemic in Tainan, Taiwan, were analyzed. Anti-ZIKV IgM and IgG were tested using commercial enzyme-linked immunosorbent assays (ELISAs). Plaque reduction neutralization tests (PRNTs) for ZIKV and four dengue virus (DENV) serotypes were performed for samples with positive anti-ZIKV antibodies. A confirmed case of ZIKV infection was defined by ZIKV PRNT90 titer ratio ≥ 4 compared to four DENV serotypes. Results The mean age of the 212 participants was 54.0 years (standard deviation 13.7 years), and female was predominant (67.0%). Anti-ZIKV IgM and IgG were detected in 0 (0%) and 9 (4.2%) of the 212 participants, respectively. For the 9 samples with anti-ZIKV IgG, only 1 sample had 4 times higher ZIKV PRNT90 titers compared to PRNT90 titers against four dengue virus serotypes; this individual denied having traveled abroad. Conclusions The results suggest that undetected indigenous ZIKV transmission might have occurred in Taiwan. The findings also suggest that the threat of epidemic transmission of ZIKV in Taiwan does exist due to extremely low-level of herd immunity. Our study also indicates that serological tests for ZIKV-specific IgG remain a big challenge due to cross-reactivity, even in dengue non-endemic countries.

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Dengue Virus and the Relationship with MicroRNAs

Dengue Fever in a One Health Perspective ◽

10.5772/intechopen.92453 ◽

2020 ◽

Author(s):

Samir Casseb ◽

Karla de Melo

Keyword(s):

Dengue Virus ◽

Viral Infection ◽

Viral Infections ◽

Immune Defense ◽

Tropical Regions ◽

Denv Serotypes ◽

History Of ◽

Infection Processes ◽

The Relationship

Dengue is an acute febrile disease caused by a virus of the genus Flavivirus, family Flaviviridae, endemic in tropical regions of the globe. The agent is a virus with single-stranded RNA, classified into four distinct dengue virus (DENV) serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. The host’s innate and adaptive immune responses play an essential role in determining the natural history of viral infections, especially in dengue. In this context, it has observed in recent years that the presence of RNA interference (RNAi) in viral infection processes is increasing, as well as immune defense. The context microRNAs (miRNAs) go for stood out, as their presence during viral infection, both in the replication of the virus and in the defense against these infections, becomes increasingly noticeable, therefore, making it increasingly necessary to better understand the role of these small RNAs within viral infection by DENV and what their consequences are in aggravating the consequences of patients affected by this disease.

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