N-Phenylpyridine-3-Carboxamide and 6-Acetyl-1H-Indazole Inhibit the RNA Replication Step of the Dengue Virus Life Cycle.

Dengue virus (DENV) is a Flavivirus that causes the most prevalent arthropod-borne viral disease. Clinical manifestation of DENV infection ranges from asymptomatic to severe symptoms that can lead to death. Unfortunately, no antiviral treatments against DENV are currently available. In order to identify novel DENV inhibitors, we screened a library of 1,604 chemically diversified fragment-based compounds using DENV reporter viruses that allowed quantification of viral replication in infected cells. Following a validation screening, the two best inhibitor candidates were N-phenylpyridine-3-carboxamide (NPP3C) and 6-acetyl-1H-indazole (6A1HI). The half maximal effective concentration of NPP3C and 6A1H1 against DENV were 7.1 µM and 6.5 µM, respectively. 6A1H1 decreased infectious DENV particle production up to 1,000-fold without any cytotoxicity at the used concentrations. While 6A1HI was DENV-specific, NPP3C also inhibited the replication of other flaviviruses such as West Nile virus and Zika virus. Structure-activity relationship (SAR) studies with 151 analogues revealed key structural elements of NPP3C and 6A1HI required for their antiviral activity. Time-of-drug-addition experiments identified a postentry step as a target of these compounds. Consistently, using a DENV subgenomic replicon, we demonstrated that these compounds specifically impede the viral RNA replication step and exhibit a high genetic barrier-to-resistance. In contrast, viral RNA translation and the de novo biogenesis of DENV replication organelles were not affected. Overall, our data unveil NPP3C and 6A1H1 as novel DENV inhibitors. The information revealed by our SAR studies will help chemically optimize NPP3C and 6A1H1 in order to improve their anti-flaviviral potency and to challenge them in in vivo models.

Quantifying heterogeneities in arbovirus transmission: Description of the rationale and methodology for a prospective longitudinal study of dengue and Zika virus transmission in Iquitos, Peru (2014-2019).

Current knowledge of dengue virus (DENV) transmission provides only a partial understanding of a complex and dynamic system yielding a public health track record that has more failures than successes. An important part of the problem is that the foundation for contemporary interventions includes a series of longstanding, but untested, assumptions based on a relatively small portion of the human population; i.e., people who are convenient to study because they manifest clinically apparent disease. Approaching dengue from the perspective of people with overt illness has produced an extensive body of useful literature. It has not, however, fully embraced heterogeneities in virus transmission dynamics that are increasingly recognized as key information still missing in the struggle to control the most important insect-transmitted viral infection of humans. Only in the last 20 years have there been significant efforts to carry out comprehensive longitudinal dengue studies. This manuscript provides the rationale and comprehensive, integrated description of the methodology for a five-year longitudinal cohort study based in the tropical city of Iquitos, in the heart of the Peruvian Amazon. Primary data collection for this study was completed in 2019. Although some manuscripts have been published to date, our principal objective here is to support subsequent publications by describing in detail the structure, methodology, and significance of a specific research program. Our project was designed to study people across the entire continuum of disease, with the ultimate goal of quantifying heterogeneities in human variables that affect DENV transmission dynamics and prevention. Because our study design is applicable to other Aedes transmitted viruses, we used it to gain insights into Zika virus (ZIKV) transmission when during the project period ZIKV was introduced and circulated in Iquitos. Our prospective contact cluster investigation design was initiated by detecttion of a person with a symptomatic DENV infection and then followed that person's immediate contacts. This allowed us to monitor individuals at high risk of DENV infection, including people with clinically inapparent and mild infections that are otherwise difficult to detect. We aimed to fill knowledge gaps by defining the contribution to DENV transmission dynamics of (1) the understudied majority of DENV-infected people with inapparent and mild infections and (2) epidemiological, entomological, and socio-behavioral sources of heterogeneity. By accounting for factors underlying variation in each person's contribution to transmission we sought to better determine the type and extent of effort needed to better prevent virus transmission and disease.

Cross-reactivity of SARS-CoV-2 with other pathogens, especially dengue virus: A historical perspective.

Dengue is a vector-borne viral disease caused by a Flavivirus whereas the COVID-19 pandemic was caused by a highly contagious virus, SARS-CoV-2 belonging to the family Coronaviridae. However, COVID-19 severity was observably less in dengue-endemic countries and vice versa especially during the active years of the pandemic (2019-2021). We observed that dengue virus (DENV) antibodies (Abs) could cross-react with SARS-CoV-2 spike antigen. This resulted in SARS-CoV-2 false positivity by rapid Ab test kits. DENV Abs binding to SARS-CoV-2 receptor-binding domain (and the reverse scenario), as revealed by docking studies further validated DENV and SARS-CoV-2 cross-reactivity. Finally, SARS-CoV-2 Abs were found to cross-neutralize DENV1 and DENV2 in virus neutralization test (VNT). Abs to other pathogens like Plasmodium were also cross-reactive but non-neutralizing for SARS-CoV-2. Here, we analyze the existing data on SARS-CoV-2 cross-reactivity with other pathogens, especially dengue to assess its impact on health (cross-protection?) and differential sero-diagnosis/surveillance.

Rapid Diagnostic Tests for the Detection of the Four Dengue Virus Serotypes in Clinically Relevant Matrices.

The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. The identification of specific dengue virus serotype 1 (DENV-1) to DENV-4 can help in understanding the transmission dynamics and spread of dengue disease. The four rapid low-resource serotype-specific dengue tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results are obtained directly from clinical sample matrices in 35 min, requiring only a heating block and pipettes for liquid handling. In addition, we demonstrate that the rapid sample preparation step inactivates DENV, improving laboratory safety. Human plasma and serum were spiked with DENV, and DENV was detected with analytical sensitivities of 333 to 22,500 median tissue culture infectious doses (TCID50)/mL. The analytical sensitivities in blood were 94,000 to 333,000 TCID50/mL. Analytical specificity testing confirmed that each test could detect multiple serotype-specific strains but did not respond to strains of other serotypes, closely related flaviviruses, or chikungunya virus. Clinical testing on 80 human serum samples demonstrated test specificities of between 94 and 100%, with a DENV-2 test sensitivity of 100%, detecting down to 0.004 PFU/µL, similar to the sensitivity of the PCR test; the other DENV tests detected down to 0.03 to 10.9 PFU/µL. Collectively, our data suggest that some of our rapid dengue serotyping tests provide a potential alternative to conventional labor-intensive RT-quantitative PCR (RT-qPCR) detection, which requires expensive thermal cycling instrumentation, technical expertise, and prolonged testing times. Our tests provide performance and speed without compromising specificity in human plasma and serum and could become promising tools for the detection of high DENV loads in resource-limited settings. IMPORTANCE The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. This study describes the evaluation of four rapid low-resource serotype-specific dengue tests for the detection of specific DENV serotypes in clinical sample matrices. The tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. These tests have several advantages compared to RT-qPCR detection, such as a simple workflow, rapid sample processing and turnaround times (35 min from sample preparation to detection), minimal equipment needs, and improved laboratory safety through the inactivation of the virus during the sample preparation step. The low-resource formats of these rapid dengue serotyping tests have the potential to support effective dengue disease surveillance and enhance the diagnostic testing capacity in resource-limited countries with both endemic dengue and intense coronavirus disease 2019 (COVID-19) transmission.

Discovery of potent benzoxaborole inhibitors against SARS-CoV-2 main and dengue virus proteases.

The RNA viruses SARS-CoV-2 and dengue pose a major threat to human health worldwide and their proteases (Mpro; NS2B/NS3) are considered as promising targets for drug development. We present the synthesis and biological evaluation of novel benzoxaborole inhibitors of these two proteases. The most active compound achieves single-digit micromolar activity against SARS-CoV-2 Mpro in a biochemical assay. The most active substance against dengue NS2B/NS3 protease has submicromolar activity in cells (EC50 0.54 µM) and inhibits DENV-2 replication in cell culture. Most benzoxaboroles had no relevant cytotoxicity or significant off-target inhibition. Furthermore, the class demonstrated passive membrane penetration and stability against the evaluated proteases. This compound class may contribute to the development of antiviral agents with activity against DENV or SARS-CoV-2.

Adenovirus vector-based vaccines as forefront approaches in fighting the battle against flaviviruses.

Flaviviruses are arthropod-borne viruses (arboviruses) that have been recently considered among the significant public health problems in defined geographical regions. In this line, there have been vaccines approved for some flaviviruses including dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV), although the efficiency of such vaccines thought to be questionable. Surprisingly, there are no effective vaccine for many other hazardous flaviviruses, including West Nile and Zika viruses. Furthermore, in spite of approved vaccines for some flaviviruses, for example DENV, alternative prophylactic vaccines seem to be still needed for the protection of a broader population, and it originates from the unsatisfying safety, and the efficacy of vaccines that have been introduced. Thus, adenovirus vector-based vaccine candidates are suggested to be effective, safe, and reliable. Interestingly, recent widespread use of adenovirus vector-based vaccines for the COVID-19 pandemic have highlighted the importance and feasibility of their widespread application. In this review, the applicability of adenovirus vector-based vaccines, as promising approaches to harness the diseases caused by Flaviviruses, is discussed.

Covert cases of Severe Acute Respiratory Syndrome Coronavirus 2: An obscure but present danger in regions endemic for Dengue and Chikungunya viruses.

BACKGROUND: The impact of SARS-CoV-2 in regions endemic for both Dengue and Chikungunya is still not fully understood. Considering that symptoms/clinical features displayed during Dengue, Chikungunya and SARS-CoV-2 acute infections are similar, undiagnosed cases of SARS-CoV-2 in co-endemic areas may be more prevalent than expected. This study was conducted to assess the prevalence of covert cases of SARS-CoV-2 among samples from patients with clinical symptoms compatible with either Dengue or Chikungunya viral infection in the state of Espírito Santo, Brazil. METHODS: Presence of immunoglobulin G (IgG) antibody specific to SARS-CoV-2 nucleoprotein was detected using a chemiluminescent microparticle immunoassay in samples from 7,370 patients, without previous history of COVID-19 diagnosis, suspected of having either Dengue (n = 1,700) or Chikungunya (n = 7,349) from December 1st, 2019 to June 30th, 2020. FINDINGS: Covert cases of SARS-CoV-2 were detected in 210 (2.85%) out of the 7,370 serum samples tested. The earliest undiagnosed missed case of COVID-19 dated back to a sample collected on December 18, 2019, also positive for Dengue Virus. Cross-reactivity with either Dengue virus or other common coronaviruses were not observed. INTERPRETATION: Our findings demonstrate that concomitant Dengue or Chikungunya outbreaks may difficult the diagnosis of SARS-CoV-2 infections. To our knowledge, this is the first study to demonstrate, with a robust sample size (n = 7,370) and using highly specific and sensitive chemiluminescent microparticle immunoassay method, that covert SARS-CoV-2 infections are more frequent than previously expected in Dengue and Chikungunya hyperendemic regions. Moreover, our results suggest that SAR-CoV-2 cases were occurring prior to February, 2020, and that these undiagnosed missed cases may have contributed to the fast expansion of SARS-CoV-2 outbreak in Brazil. Data presented here demonstrate that in arboviral endemic regions, SARS-CoV-2 infection must be always considered, regardless of the existence of a previous positive diagnosis for Dengue or Chikungunya.

Antiviral Activity of 7-Substituted 7-Deazapurine Ribonucleosides, Monophosphate Prodrugs, and Triphoshates against Emerging RNA Viruses.

A series of 7-deazaadenine ribonucleosides bearing alkyl, alkenyl, alkynyl, aryl, or hetaryl groups at position 7 as well as their 5'-O-triphosphates and two types of monophosphate prodrugs (phosphoramidates and S-acylthioethanol esters) were prepared and tested for antiviral activity against selected RNA viruses (Dengue, Zika, tick-borne encephalitis, West Nile, and SARS-CoV-2). The modified triphosphates inhibited the viral RNA-dependent RNA polymerases at micromolar concentrations through the incorporation of the modified nucleotide and stopping a further extension of the RNA chain. 7-Deazaadenosine nucleosides bearing ethynyl or small hetaryl groups at position 7 showed (sub)micromolar antiviral activities but significant cytotoxicity, whereas the nucleosides bearing bulkier heterocycles were still active but less toxic. Unexpectedly, the monophosphate prodrugs were similarly or less active than the corresponding nucleosides in the in vitro antiviral assays, although the bis(S-acylthioethanol) prodrug 14h was transported to the Huh7 cells and efficiently released the nucleoside monophosphate.

Presentation of fatal stroke due to SARS-CoV-2 and dengue virus coinfection.

Herein, we report a case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and dengue coinfection, presented as a fatal stroke in our hospital, in São José do Rio Preto, São Paulo State, a Brazilian city hyperendemic for dengue viruses and other arthropod-borne viruses (arboviruses) and currently facing a surge of SARS-CoV-2 cases. This case is the first described in the literature and contributes to the better understanding of clinical presentations of two important diseases in a tropical setting.

Case Report: Dengue and COVID-19 Coinfection in Thailand.

We report a 50-year-old Thai woman with recent travel to Denmark who presented with acute high-grade fever, vomiting, and myalgia for 1 day. Initial laboratory results revealed leukopenia, elevated aspartate transaminase, and elevated alanine transaminase. Chest radiograph showed no pulmonary infiltration. Reverse transcriptase-PCR (RT-PCR) of the nasopharyngeal swab detected SARS-CoV-2, and RT-PCR of the blood detected dengue virus serotype 2. COVID-19 with dengue fever co-infection was diagnosed. Her symptoms were improved with supportive treatment. Integration of clinical manifestations, history of exposure, laboratory profiles, and dynamic of disease progression assisted the physicians in precise diagnosis. Co-circulating and nonspecific presentations of dengue infection and COVID-19 challenge the healthcare system in tropical countries. To solve this threat, multi-sector strategies are required, including public health policy, development of accurate point-of-care testing, and proper prevention for both diseases.

A Versatile Reporter System To Monitor Virus-Infected Cells and Its Application to Dengue Virus and SARS-CoV-2.

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

Serological cross-reaction and coinfection of dengue and COVID-19 in Asia: Experience from Indonesia.

Similar symptoms and laboratory findings between dengue and coronavirus disease 2019 (COVID-19) pose a diagnostic challenge in some dengue-endemic countries in Asia. In this study, we reported three cases of suspected COVID-19-dengue coinfection in hospitals of Bali, Indonesia. Serological data demonstrated that patients with positive results for dengue virus (DENV) NS1 antigen and anti-dengue IgM were also reactive to COVID-19 rapid antibody tests, suggesting dengue-COVID-19 coinfection. However, two patients were later confirmed negative for SARS-COV-2 by qRT-PCR, implying a plausible cross-reactivity of anti-dengue and anti-COVID-19 antibodies in the serological test. Coinfection of dengue and COVID-19 was evident in one patient, following confirmation of SARS-COV-2 by qRT-PCR and DENV infection using the NS1 antigen serology test. This case was the first case of dengue and COVID-19 coinfection in Indonesia and revealed possible cross-reactivity between SARS-COV-2 and DENV antibodies based on rapid serological tests. Our study indicates a public health concern regarding COVID-19 and dengue detection in Indonesia as well as in other dengue-endemic countries, and it is important for these nations to manage both pathogens concurrently.

Experience of a Tertiary Hospital in Singapore with Management of a Dual Outbreak of COVID-19 and Dengue.

During the COVID-19 pandemic, distinguishing dengue from cases of COVID-19 in endemic areas can be difficult. In a tertiary hospital contending with COVID-19 during a dengue epidemic, a triage strategy of routine COVID-19 testing for febrile patients with viral prodromes was used. All febrile patients with viral prodromes and no epidemiologic risk for COVID-19 were first admitted to a designated ward for COVID-19 testing, where enhanced personal protective equipment was used by healthcare workers until COVID-19 was ruled out. From January to May 2020, 11,086 admissions were screened for COVID-19; 868 cases of COVID-19 were diagnosed in our institution, along with 380 cases of dengue. Only 8.5% (943/11,086) of suspected COVID-19 cases were concurrently tested for dengue serology due to a compatible overlapping clinical syndrome, and dengue was established as an alternative diagnosis in 2% (207/10,218) of suspected COVID-19 cases that tested negative. There were eight COVID-19 cases with likely false-positive dengue serology and one probable COVID-19/dengue coinfection. From April to May 2020, 251 admissions presenting as viral prodromes with no respiratory symptoms were screened; of those, 15 cases had COVID-19, and 2/15 had false-positive dengue IgM. Epidemiology investigations showed no healthcare-associated transmission. In a dengue epidemic season coinciding with a COVID-19 pandemic, dengue was established as an alternative diagnosis in a minority of COVID-19 suspects, likely due to early availability of basic diagnostics. Routine screening of patients with viral prodromes during a dual outbreak of COVID-19 and dengue enabled containment of COVID-19 cases masquerading as dengue with false-positive IgM.

Covid-19 and dengue: Double punches for dengue-endemic countries in Asia.

The coronavirus disease 2019 (Covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an international public health crisis with devastating effects. In particular, this pandemic has further exacerbated the burden in tropical and subtropical regions of the world, where dengue fever, caused by dengue virus (DENV), is already endemic to the population. The similar clinical manifestations shared by Covid-19 and dengue fever have raised concerns, especially in dengue-endemic countries with limited resources, leading to diagnostic challenges. In addition, cross-reactivity of the immune responses in these infections is an emerging concern, as pre-existing DENV-antibodies might potentially affect Covid-19 through antibody-dependent enhancement. In this review article, we aimed to raise the issue of Covid-19 and dengue fever misdiagnosis, not only in a clinical setting but also with regards to cross-reactivity between SARS-CoV-2 and DENV antibodies. We also have discussed the potential consequences of overlapping immunological cascades between dengue and Covid-19 on disease severity and vaccine development.

SARS-CoV-2 and Dengue virus Co-infection. A Case Report.

Coinfection of SARS-CoV-2 and dengue virus has not been previously reported. We report a confirmed case with favourable outcome, but whether the occurrence of simultaneous infections may alter the usual clinical course of each infection is still unknown.

The role of IgG Fc receptors in antibody-dependent enhancement.

Antibody-dependent enhancement (ADE) is a mechanism by which the pathogenesis of certain viral infections is enhanced in the presence of sub-neutralizing or cross-reactive non-neutralizing antiviral antibodies. In vitro modelling of ADE has attributed enhanced pathogenesis to Fcγ receptor (FcγR)-mediated viral entry, rather than canonical viral receptor-mediated entry. However, the putative FcγR-dependent mechanisms of ADE overlap with the role of these receptors in mediating antiviral protection in various viral infections, necessitating a detailed understanding of how this diverse family of receptors functions in protection and pathogenesis. Here, we discuss the diversity of immune responses mediated upon FcγR engagement and review the available experimental evidence supporting the role of FcγRs in antiviral protection and pathogenesis through ADE. We explore FcγR engagement in the context of a range of different viral infections, including dengue virus and SARS-CoV, and consider ADE in the context of the ongoing SARS-CoV-2 pandemic.