Interleukin 27, like interferons, activates JAK-STAT signaling and promotes pro-inflammatory and antiviral states that interfere with dengue and chikungunya viruses replication in human macrophages.

Interferons (IFNs) are a family of cytokines that activate the JAK-STAT signaling pathway to induce an antiviral state in cells. Interleukin 27 (IL-27) is a member of the IL-6 and/or IL-12 family that elicits both pro- and anti-inflammatory responses. Recent studies have reported that IL-27 also induces a robust antiviral response against diverse viruses, both in vitro and in vivo, suggesting that IFNs and IL-27 share many similarities at the functional level. However, it is still unknown how similar or different IFN- and IL-27-dependent signaling pathways are. To address this question, we conducted a comparative analysis of the transcriptomic profiles of human monocyte-derived macrophages (MDMs) exposed to IL-27 and those exposed to recombinant human IFN-α, IFN-γ, and IFN-λ. We utilized bioinformatics approaches to identify common differentially expressed genes between the different transcriptomes. To verify the accuracy of this approach, we used RT-qPCR, ELISA, flow cytometry, and microarrays data. We found that IFNs and IL-27 induce transcriptional changes in several genes, including those involved in JAK-STAT signaling, and induce shared pro-inflammatory and antiviral pathways in MDMs, leading to the common and unique expression of inflammatory factors and IFN-stimulated genes (ISGs)Importantly, the ability of IL-27 to induce those responses is independent of IFN induction and cellular lineage. Additionally, functional analysis demonstrated that like IFNs, IL-27-mediated response reduced chikungunya and dengue viruses replication in MDMs. In summary, IL-27 exhibits properties similar to those of all three types of human IFN, including the ability to stimulate a protective antiviral response. Given this similarity, we propose that IL-27 could be classified as a distinct type of IFN, possibly categorized as IFN-pi (IFN-π), the type V IFN (IFN-V).

Dengue: A review of laboratory diagnostics in the vaccine age.

Background. Dengue is an important arboviral infection of considerable public health significance. It occurs in a wide global belt within a variety of tropical regions. The timely laboratory diagnosis of Dengue infection is critical to inform both clinical management and an appropriate public health response. Vaccination against Dengue virus is being introduced in some areas.Discussion. Appropriate diagnostic strategies will vary between laboratories depending on the available resources and skills. Diagnostic methods available include viral culture, the serological detection of Dengue-specific antibodies in using enzyme immunoassays (EIAs), microsphere immunoassays, haemagglutination inhibition or in lateral flow point of care tests. The results of antibody tests may be influenced by prior vaccination and exposure to other flaviviruses. The detection of non-structural protein 1 in serum (NS1) has improved the early diagnosis of Dengue and is available in point-of-care assays in addition to EIAs. Direct detection of viral RNA from blood by PCR is more sensitive than NS1 antigen detection but requires molecular skills and resources. An increasing variety of isothermal nucleic acid detection methods are in development. Timing of specimen collection and choice of test is critical to optimize diagnostic accuracy. Metagenomics and the direct detection by sequencing of viral RNA from blood offers the ability to rapidly type isolates for epidemiologic purposes.Conclusion. The impact of vaccination on immune response must be recognized as it will impact test interpretation and diagnostic algorithms.

Antigenic distance between primary and secondary dengue infections correlates with disease risk.

Many pathogens continuously change their protein structure in response to immune-driven selection, resulting in weakened protection even in previously exposed individuals. In addition, for some pathogens, such as dengue virus, poorly targeted immunity is associated with increased risk of severe disease through a mechanism known as antibody-dependent enhancement. However, it remains unclear whether the antigenic distances between an individual's first infection and subsequent exposures dictate disease risk, explaining the observed large-scale differences in dengue hospitalizations across years. Here, we develop a framework that combines detailed antigenic and genetic characterization of viruses with details on hospitalized cases from 21 years of dengue surveillance in Bangkok, Thailand, to identify the role of the antigenic profile of circulating viruses in determining disease risk. We found that the risk of hospitalization depended on both the specific order of infecting serotypes and the antigenic distance between an individual's primary and secondary infections, with risk maximized at intermediate antigenic distances. These findings suggest that immune imprinting helps determine dengue disease risk and provide a pathway to monitor the changing risk profile of populations and to quantifying risk profiles of candidate vaccines.

A tetravalent dengue virus-like particle vaccine induces high levels of neutralizing antibodies and reduces dengue replication in non-human primates.

Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro. DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.IMPORTANCEDengue is a viral disease that infects nearly 400 million people worldwide and causes dengue hemorrhagic fever, which is responsible for 10,000 deaths each year. Currently, there is no therapeutic drug licensed to treat dengue infection, which makes the development of an effective vaccine essential. Virus-like particles (VLPs) are a safe and highly immunogenic platform that can be used in young children, immunocompromised individuals, as well as healthy adults. In this study, we describe the development of a dengue VLP vaccine and demonstrate that it induces a robust immune response against the dengue virus for over 1 year in monkeys. The immunity induced by this vaccine reduced live dengue infection in both murine and non-human primate models. These results indicate that our dengue VLP vaccine is a promising vaccine candidate.

Low-dose dengue virus 3 human challenge model: a phase 1 open-label study.

Dengue human infection models present an opportunity to explore the potential of a vaccine, anti-viral or immuno-compound for clinical benefit in a controlled setting. Here we report the outcome of a phase 1 open-label assessment of a low-dose dengue virus 3 (DENV-3) challenge model (NCT04298138), in which nine participants received a subcutaneous inoculation with 0.5 ml of a 1.4 × 103 plaque-forming unit per ml suspension of the attenuated DENV-3 strain CH53489. The primary and secondary endpoints of the study were to assess the safety of this DENV-3 strain in healthy flavivirus-seronegative individuals. All participants developed RNAaemia within 7 days after inoculation with peak titre ranging from 3.13 × 104 to 7.02 × 108 genome equivalents per ml. Solicited symptoms such as fever and rash, clinical laboratory abnormalities such as lymphopenia and thrombocytopenia, and self-reported symptoms such as myalgia were consistent with mild-to-moderate dengue in all volunteers. DENV-3-specific seroconversion and memory T cell responses were observed within 14 days after inoculation as assessed by enzyme-linked immunosorbent assay and interferon-gamma-based enzyme-linked immunospot. RNA sequencing and serum cytokine analysis revealed anti-viral responses that overlapped with the period of viraemia. The magnitude and frequency of clinical and immunologic endpoints correlated with an individual's peak viral titre.

The inflammasome pathway is activated by dengue virus non-structural protein 1 and is protective during dengue virus infection.

Dengue virus (DENV) is a medically important flavivirus causing an estimated 50-100 million dengue cases annually, some of whom progress to severe disease. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis by inducing endothelial barrier dysfunction. However, less is known about how DENV NS1 interacts with immune cells and what role these interactions play. Here we report that DENV NS1 can trigger activation of inflammasomes, a family of cytosolic innate immune sensors that respond to infectious and noxious stimuli, in mouse and human macrophages. DENV NS1 induces the release of IL-1ß in a caspase-1 dependent manner. Additionally, we find that DENV NS1-induced inflammasome activation is independent of the NLRP3, Pyrin, and AIM2 inflammasome pathways, but requires CD14. Intriguingly, DENV NS1-induced inflammasome activation does not induce pyroptosis and rapid cell death; instead, macrophages maintain cellular viability while releasing IL-1ß. Lastly, we show that caspase-1/11-deficient, but not NLRP3-deficient, mice are more susceptible to lethal DENV infection. Together, these results indicate that the inflammasome pathway acts as a sensor of DENV NS1 and plays a protective role during infection.

Systematic review and meta-analysis: assessing the accuracy of rapid immunochromatographic tests in dengue diagnosis.

The objective of this systematic review is to analyze the diagnostic accuracy of rapid dengue diagnostic tests. The search was conducted in the following databases: LILACS, Medline (Pubmed), CRD, The Cochrane Library, Trip Medical Database and Google Scholar. ELISA and PCR assays were adopted as reference methods. Thirty-four articles were included in this systematic review. Receiver operating characteristic (ROC) and Forest Plot were performed to evaluate sensitivity and specificity for each parameter analyzed (NS1, IgM and IgG). The results revealed that the combined analysis of the IgM antibody with the NS1 antigen resulted in greater sensitivity than the isolated analysis of IgM. The three analytes together showed the best performance, with a combined sensitivity of 90 % (95 % CI: 89-92 %) using ELISA as a comparator. Thus, the present review provides relevant knowledge for decision-making between the available rapid diagnostic tests.

Seroprevalence of dengue and chikungunya viruses among urban refugees in Klang Valley, Malaysia.

BACKGROUND: Mosquito-borne diseases pose a significant global public health threat, with Malaysia's Klang Valley experiencing numerous outbreaks in densely populated urban areas. METHODS: This study aimed to estimate the seroprevalence of anti-dengue and anti-chikungunya antibodies among urban refugees in the Klang Valley, Malaysia, and identify associated risk factors. RESULTS: High seroprevalence of anti-dengue immunoglobulin G (IgG) and IgM (60.0% [confidence interval {CI} 55.39 to 64.48] and 9.2% [CI 6.77 to 12.25], respectively) were observed among refugees >18 years of age (χ22=11.720, p=0.003), Kachin ethnicity (χ28=72.253, p<0.001), without formal education (χ21=3.856, p=0.050), homes near waste disposal sites (χ21=10.378, p=0.001) and refugees who have experienced flooding (χ21=5.460, p=0.019). Meanwhile, the overall seroprevalence of anti-chikungunya IgG and IgM was 9.7% (CI 7.15 to 12.73) and 10.8% (CI 8.09 to 13.93), respectively, with ages 12-18 years (χ22=6.075, p=0.048), Rohingya ethnicity (χ28=31.631, p<0.001) and homes close to waste disposal sites (χ21=3.912, p=0.048) being significant risk factors. Results showed a link to poor environmental living conditions, with an increase in the vector population with higher availability of breeding sites and thus exposure to dengue and chikungunya virus. CONCLUSIONS: Health education among the community is the key to disease prevention, as there are no specific antiviral drugs for treatment and limited vaccine availability.

Live, Attenuated, Tetravalent Butantan-Dengue Vaccine in Children and Adults.

BACKGROUND: Butantan-Dengue Vaccine (Butantan-DV) is an investigational, single-dose, live, attenuated, tetravalent vaccine against dengue disease, but data on its overall efficacy are needed. METHODS: In an ongoing phase 3, double-blind trial in Brazil, we randomly assigned participants to receive Butantan-DV or placebo, with stratification according to age (2 to 6 years, 7 to 17 years, and 18 to 59 years); 5 years of follow-up is planned. The objectives of the trial were to evaluate overall vaccine efficacy against symptomatic, virologically confirmed dengue of any serotype occurring more than 28 days after vaccination (the primary efficacy end point), regardless of serostatus at baseline, and to describe safety up to day 21 (the primary safety end point). Here, vaccine efficacy was assessed on the basis of 2 years of follow-up for each participant, and safety as solicited vaccine-related adverse events reported up to day 21 after injection. Key secondary objectives were to assess vaccine efficacy among participants according to dengue serostatus at baseline and according to the dengue viral serotype; efficacy according to age was also assessed. RESULTS: Over a 3-year enrollment period, 16,235 participants received either Butantan-DV (10,259 participants) or placebo (5976 participants). The overall 2-year vaccine efficacy was 79.6% (95% confidence interval [CI], 70.0 to 86.3) - 73.6% (95% CI, 57.6 to 83.7) among participants with no evidence of previous dengue exposure and 89.2% (95% CI, 77.6 to 95.6) among those with a history of exposure. Vaccine efficacy was 80.1% (95% CI, 66.0 to 88.4) among participants 2 to 6 years of age, 77.8% (95% CI, 55.6 to 89.6) among those 7 to 17 years of age, and 90.0% (95% CI, 68.2 to 97.5) among those 18 to 59 years of age. Efficacy against DENV-1 was 89.5% (95% CI, 78.7 to 95.0) and against DENV-2 was 69.6% (95% CI, 50.8 to 81.5). DENV-3 and DENV-4 were not detected during the follow-up period. Solicited systemic vaccine- or placebo-related adverse events within 21 days after injection were more common with Butantan-DV than with placebo (58.3% of participants, vs. 45.6%). CONCLUSIONS: A single dose of Butantan-DV prevented symptomatic DENV-1 and DENV-2, regardless of dengue serostatus at baseline, through 2 years of follow-up. (Funded by Instituto Butantan and others; DEN-03-IB ClinicalTrials.gov number, NCT02406729, and WHO ICTRP number, U1111-1168-8679.).

Internal carotid artery dissection associated with acute dengue virus infection: a case report / Dissecção da artéria carótida interna associada à infecção aguda pelo vírus da dengue: relato de caso

Cervical artery dissections (CAD) can occur spontaneously or as a direct result of significant trauma. Viral infections, such as SARS-CoV2, influenza, and Epstein Barr, are risk factors for spontaneous CAD. Dengue virus infections have dramatically increased in recent decades, and Brazil is one of the endemic areas. The dengue virus can cause headache and neurological complications such as encephalitis, myelitis, Guillain-Barré syndrome, and myositis. No report has yet been found in the literature of dissection of the internal carotid artery secondary to dengue infection. Our objective is to report the case of a patient with dissection of the internal carotid artery associated with acute dengue virus infection.

As dissecções da artéria cervical (DAC) podem ocorrer espontaneamente ou como resultado direto de trauma significativo. Infecções virais, como SARS-CoV2, influenza e Epstein Barr, são fatores de risco para DAC espontânea. As infecções pelo vírus da dengue aumentaram dramaticamente nas últimas décadas, e o Brasil é uma das áreas endêmicas. O vírus da dengue pode causar dor de cabeça e complicações neurológicas como encefalite, mielite, síndrome de Guillain-Barré e miosite. Ainda não foi encontrado na literatura nenhum relato de dissecção da artéria carótida interna secundária à infecção por dengue. Nosso objetivo é relatar o caso de um paciente com dissecção da artéria carótida interna associada à infecção aguda pelo vírus da dengue.

Evaluation of an Immunoglobulin E Capture Enzyme-Linked Immunosorbent Assay for the Early Diagnosis of Dengue.

Dengue virus (DENV) is the etiological agent of dengue, the most important mosquito-transmitted viral disease of humans worldwide. Enzyme-linked immunosorbent assays (ELISAs) designed to detect DENV IgM are commonly used for dengue diagnosis. However, DENV IgM is not reliably detected until ≥4 days after illness onset. Reverse transcription-polymerase chain reaction (RT-PCR) can diagnose early dengue but requires specialized equipment, reagents, and trained personnel. Additional diagnostic tools are needed. Limited work has been performed to determine whether IgE-based assays can be used for the early detection of vector-borne viral diseases, including dengue. In this study, we determined the efficacy of a DENV IgE capture ELISA for the detection of early dengue. Sera were collected within the first 4 days of illness onset from 117 patients with laboratory-confirmed dengue, as determined by DENV-specific RT-PCR. The serotypes responsible for the infections were DENV-1 and DENV-2 (57 and 60 patients, respectively). Sera were also collected from 113 dengue-negative individuals with febrile illness of undetermined etiology and 30 healthy controls. The capture ELISA detected DENV IgE in 97 (82.9%) confirmed dengue patients and none of the healthy controls. There was a high false positivity rate (22.1%) among the febrile non-dengue patients. In conclusion, we provide evidence that IgE capture assays have the potential to be explored for early diagnosis of dengue, but further research is necessary to address the possible false positivity rate among patients with other febrile illnesses.

Development of monoclonal antibodies against prM of Dengue virus 4 / Desenvolvimento de anticorpos monoclonais para prM de Dengue vírus 4

Introduction: dengue is a most common mosquito-borne viral disease in the Americas and tropical countries. Objective: in this work, mice were hyperimmunized with DENV 4 antigen to produce monoclonal antibodies (mAbs). Methodology: DENV 4 (GenBank KC806069) was inoculated in C6/36 cell monolayers cultivated in Leibovitz's 15 medium supplemented with 5% fetal bovine serum and incubated at 28 oC. The virus stock was submitted to concentration and ultracentrifugation and stored at -80 oC until use (VC DENV 4). Balb/c mice were injected intraperitoneally with 50µg of DENV-4 and successive intraperitoneal injections of 25 µg of VCDENV 4 with Freund's incomplete adjuvant were performed. The spleen cells were fused to SP2/0 myeloma cells with PEG 1540 and distributed in 96-well microplates with Iscove's modified medium with Hipoxantina­Aminopterina­Timidina. Hybridoma screening by indirect ELISA showed positive results for six mAbs, and their characterization was performed by Western blotting and Indirect Immunofluorescence (IFI) techniques. Results: the six mAbs showed strong recognition of prM (24/29 kDa), and minor reaction to E protein (66 kDa), E/E protein dimer (105 kDa), and NS1 (49 kDa) protein in two mAbs. The use of mAbs anti-prM as a diagnostic tool using IFI has been demonstrated to detect DENV-4 antigen in infected cells or tissues. Conclusion: DENV 4 generate mAbs with strong reactivity to prM with potential use to confirm the presence of DENV 4 antigen in tissues or infected cells.

Introdução: a dengue é uma doença viral transmitida por mosquitos comumente das Américas e países tropicais. Objetivo: neste trabalho, camundongos foram hiperimunizados com antígeno DENV 4 para produzir anticorpos monoclonais (mAbs). Metodologia: DENV 4 (GenBank KC806069) foi inoculado em monocamadas de células C6 / 36 cultivadas em meio Leibovitz 15 suplementado com 5% de soro fetal bovino e incubadas a 28oC. O estoque viral foi submetido à concentração, ultracentrifugação e armazenado a -80 oC (VC DENV 4). Camundongos Balb / c foram injetados intraperitonealmente com 50 µg de VC DENV-4 e injeções intraperitoneais sucessivas de 25 µg de antigeno com adjuvante incompleto de Freund. As células do baço foram misturadas a células SP2/0 com PEG 1540 e distribuídas em microplacas de 96 poços com meio Iscove Modificado em presença de Hipoxantina ­ Aminopterina ­ Timidina. A triagem de hibridomas por ELISA indireto apresentou resultados positivos para seis mAbs, e sua caracterização foi realizada por técnicas de Western blotting e Imunofluorescência Indireta (IFI). Resultados: os seis mAbs mostraram forte reconhecimento de prM (24/29 kDa) e reação menor à proteína E (66 kDa), dímero de proteína E / E (105 kDa) e proteína NS1 (49 kDa) em dois mAbs. O uso de mAbs anti-prM como uma ferramenta de diagnóstico utilizando IFI demonstrou eficacia em detectar o antígeno DENV-4 em células ou tecidos infectados. Conclusão: o mAbs produzidos para DENV 4 demonstraram uma forte reatividade contra prM, e poderiam ser uma ferramenta de uso potencial no diagnóstico de DENV 4 .

Evaluation of Serologic Cross-Reactivity Between Dengue Virus and SARS-CoV-2 in Patients with Acute Febrile Illness - United States and Puerto Rico, April 2020-March 2021.

The diagnosis of dengue disease, caused by the dengue virus (DENV) (a flavivirus), often requires serologic testing during acute and early convalescent phases of the disease. Some symptoms of DENV infection, such as nonspecific fever, are similar to those caused by infection with SARS-CoV-2, the virus that causes COVID-19. In studies with few COVID-19 cases, positive DENV immunoglobulin M (IgM) results were reported with various serologic tests, indicating possible cross-reactivity in these tests for DENV and SARS-CoV-2 infections (1,2). DENV antibodies can cross-react with other flaviviruses, including Zika virus. To assess the potential cross-reactivity of SARS-CoV-2, DENV, and Zika virus IgM antibodies, serum specimens from 97 patients from Puerto Rico and 12 U.S.-based patients with confirmed COVID-19 were tested using the DENV Detect IgM Capture enzyme-linked immunosorbent assay (ELISA) (InBios International).* In addition, 122 serum specimens from patients with confirmed dengue and 121 from patients with confirmed Zika virus disease (all from Puerto Rico) were tested using the SARS-CoV-2 pan-Ig Spike Protein ELISA (CDC).† Results obtained for DENV, Zika virus IgM, and SARS-CoV-2 antibodies indicated 98% test specificity and minimal levels of cross-reactivity between the two flaviviruses and SARS-CoV-2. These findings indicate that diagnoses of dengue or Zika virus diseases with the serological assays described in this report are not affected by COVID-19, nor do dengue or Zika virus diseases interfere with the diagnosis of COVID-19.

One antibody to treat them all.

Conserved flavivirus protein holds potential as target for versatile vaccines and therapies.

Seroprevalence of dengue, Zika, chikungunya and Ross River viruses across the Solomon Islands.

Across the Pacific, and including in the Solomon Islands, outbreaks of arboviruses such as dengue, chikungunya, and Zika are increasing in frequency, scale and impact. Outbreaks of mosquito-borne disease have the potential to overwhelm the health systems of small island nations. This study mapped the seroprevalence of dengue, Zika, chikungunya and Ross River viruses in 5 study sites in the Solomon Islands. Serum samples from 1,021 participants were analysed by ELISA. Overall, 56% of participants were flavivirus-seropositive for dengue (28%), Zika (1%) or both flaviviruses (27%); and 53% of participants were alphavirus-seropositive for chikungunya (3%), Ross River virus (31%) or both alphaviruses (18%). Seroprevalence for both flaviviruses and alphaviruses varied by village and age of the participant. The most prevalent arboviruses in the Solomon Islands were dengue and Ross River virus. The high seroprevalence of dengue suggests that herd immunity may be a driver of dengue outbreak dynamics in the Solomon Islands. Despite being undetected prior to this survey, serology results suggest that Ross River virus transmission is endemic. There is a real need to increase the diagnostic capacities for each of the arboviruses to support effective case management and to provide timely information to inform vector control efforts and other outbreak mitigation interventions.