Dengue Virus Research Articles

Detection of encephalitis-causing viruses reveals predominance of chikungunya virus in the state of Bahia, Brazil

ObjectivesEncephalitis is a severe neurological syndrome for which herpesvirus and enteroviruses are the most common etiological agents. Arboviruses, a wildly diverse group of pathogens, are also critical epidemiological agents associated with encephalitis. In Brazil, little is known about the causative agents of encephalitis. MethodsWe conducted a hospital surveillance for encephalitis between 2020 and 2022. Molecular (RT-PCR and qPCR) and serological (virus-specific IgM and viral antigens) techniques were performed in cerebrospinal fluid and serum samples obtained from study participants. ResultsIn the 43 participants evaluated, the etiologic agent or the presence of IgM was detected in 16 (37.2%). Nine (20.9%) cases were positive for chikungunya virus (CHIKV), three (7.0%) for dengue virus, two (4.7%) for human adenovirus, one (2.3%) for varicella-zoster virus, and one (2.3%) for enterovirus. Whole-genome sequencing revealed that the CHIKV identified belongs to the East/Central/South African lineage. ConclusionHerein, CHIKV is a common pathogen identified in encephalitis cases. Our results reinforce previous evidence that chikungunya represents a significant cause of encephalitis during CHIKV outbreaks and epidemics and add to existing information on the epidemiology of encephalitis in Brazil.

Role of angiotensin II in cellular entry and replication of dengue virus.

Previous studies have demonstrated the relevance of several soluble molecules in the pathogenesis of dengue. In this regard, a possible role for angiotensin II (Ang II) in the pathophysiology of dengue has been suggested by the observation of a blockade of Ang II in patients with dengue, increased expression of molecules related to Ang II production in the plasma of dengue patients, increased expression of circulating cytokines and soluble molecules related to the action of Ang II, and an apparent relationship between DENV, Ang II effects, and miRNAs. In addition, in ex vivo experiments, the blockade of Ang II AT1 receptor and ACE-1 (angiotensin converting enzyme 1), both of which are involved in Ang II production and its function, inhibits infection of macrophages by DENV, suggesting a role of Ang II in viral entry or in intracellular viral replication of the virus. Here, we discuss the possible mechanisms of Ang II in the entry and replication of DENV. Ang II has the functions of increasing the expression of DENV entry receptors, creation of clathrin-coated vesicles, and increasing phagocytosis, all of which are involved in DENV entry. This hormone also modulates the expression of the Rab5 and Rab7 proteins, which are important in the endosomal processing of DENV during viral replication. This review summarizes the data related to the possible involvement of Ang II in the entry of DENV into cells and its replication.

Increased frequencies of highly activated regulatory T cells skewed to a T helper 1-like phenotype with reduced suppressive capacity in dengue patients.

According to the World Health Organization, dengue is the fastest-spreading, epidemic-prone infectious disease. The extent of dengue virus infections increased over the years, mainly driven by globalization-including travel and trade-and environmental changes. Dengue is an immunopathology caused by an imbalanced immune response to a secondary heterotypic infection. As regulatory T cells (Tregs) are essential in maintaining immune homeostasis and dampening excessive immune activation, this study addressed the role of Tregs in dengue immunopathology. We show that Tregs from dengue patients are highly activated, skewed to a Th1-like Treg phenotype and less suppressive compared to healthy donor Tregs. Our data suggest that Tregs fail to resolve ongoing inflammation during dengue infection and hence contribute to the immunopathology of severe dengue disease. These data clarify the role of Tregs in dengue immunopathogenesis, emphasizing the need to develop T cell-based vaccines for dengue.

Comparison of Five Serological Methods for the Detection of West Nile Virus Antibodies

The West Nile Virus (WNV), a member of the family Flaviviridae, is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.

Metabolic Resistance and Not Voltage-Gated Sodium Channel Gene Mutation Is Associated with Pyrethroid Resistance of Aedes albopictus (Skuse, 1894) from Cambodia.

(1) Background: In Cambodia, Aedes albopictus is an important vector of the dengue virus. Vector control using insecticides is a major strategy implemented in managing mosquito-borne diseases. Resistance, however, threatens to undermine the use of insecticides. In this study, we present the levels of insecticide resistance of Ae. albopictus in Cambodia and the mechanisms involved. (2) Methods: Two Ae. albopictus populations were collected from the capital, Phnom Penh city, and from rural Pailin province. Adults were tested with diagnostic doses of malathion (0.8%), deltamethrin (0.03%), permethrin (0.25%), and DDT (4%) using WHO tube assays. Synergist assays using piperonyl butoxide (PBO) were implemented before the pyrethroid assays to detect the potential involvement of metabolic resistance mechanisms. Adult female mosquitoes collected from Phnom Penh and Pailin were tested for voltage-gated sodium channel (VGSC) kdr (knockdown resistance) mutations commonly found in Aedes sp.-resistant populations throughout Asia (S989P, V1016G, and F1534C), as well as for other mutations (V410L, L982W, A1007G, I1011M, T1520I, and D1763Y). (3) Results: The two populations showed resistance against all the insecticides tested (<90% mortality). The use of PBO (an inhibitor of P450s) strongly restored the efficacy of deltamethrin and permethrin against the two resistant populations. Sequences of regions of the vgsc gene showed a lack of kdr mutations known to be associated with pyrethroid resistance. However, four novel non-synonymous mutations (L412P/S, C983S, Q1554STOP, and R1718L) and twenty-nine synonymous mutations were detected. It remains to be determined whether these mutations contribute to pyrethroid resistance. (4) Conclusions: Pyrethroid resistance is occurring in two Ae. albopictus populations originating from urban and rural areas of Cambodia. The resistance is likely due to metabolic resistance specifically involving P450s monooxygenases. The levels of resistance against different insecticide classes are a cause for concern in Cambodia. Alternative tools and insecticides for controlling dengue vectors should be used to minimize disease prevalence in the country.

Retrospective Analysis of Severe Dengue by Dengue Virus Serotypes in a Population with Social Security, Mexico 2023

Retrospective Analysis of Severe Dengue by Dengue Virus Serotypes in a Population with Social Security, Mexico 2023

Comparing the effectiveness of adulticide application interventions on mitigating local transmission of dengue virus

AbstractThe southern US has a large presence of mosquito vector species for dengue virus (DENV) and experiences thousands of DENV importations every year, which have led to several local outbreaks. Adulticide spraying targeting active mosquitoes is one of the most common insecticide strategies used as a response to an outbreak. The aim of this study is to evaluate the effectiveness of adulticide spraying conducted at different times of the day to curb DENV transmission. Based on unique dataset of Aedes aegypti diel activity patterns in Miami-Dade County, Florida, and Brownsville, Texas, we developed a mechanistic model of DENV transmission, which simulates adulticide spraying interventions. We estimated that spraying adulticide for 14 consecutive days at 7am or 8 pm was highly effective in reducing DENV outbreak probability from 10% in the absence of interventions to 0.1% for Miami-Dade County, and from 7.8 to 0.1% for Brownsville. Moreover, in case of a local outbreak in Miami-Dade County, we estimated the median number of symptomatic infections after the identification of a local outbreak to be reduced from 67.0 (IQR: 25.5–103.0) in the absence of interventions to 1.0 (IQR: 0.0–2.0) when spraying adulticide for 14 consecutive days at 8 pm. In Brownsville, the same intervention is estimated to lead to a decrease from 15.0 (IQR: 7.0–33.0) cases to 1.0 (IQR: 0.0–2.0). Our study highlights the importance of considering diel activity patterns of vector mosquito species in arbovirus preparedness and response planning and provide quantitative evidence to guide the decision-making of mosquito control authorities.

3D-QSAR and molecular docking studies of peptide-hybrids as dengue virus NS2B/NS3 protease inhibitors

Global warming and climate change have made dengue disease a global health issue. More than 50 % of the world's population is at danger of dengue virus (DENV) infection, according to the World Health Organization (WHO). Therefore, a clinically approved dengue fever vaccination and effective treatment are needed. Peptide medication development is new pharmaceutical research. Here we intend to recognize the structural features inhibiting the DENV NS2B/NS3 serine protease for a series of peptide-hybrid inhibitors (R1–R2-Lys-R3-NH2) by the 3D-QSAR technique. Comparative molecular field analysis (q2 = 0.613, r2 = 0.938, r2pred = 0.820) and comparative molecular similarity indices analysis (q2 = 0.640, r2 = 0.928, r2pred = 0.693) were established, revealing minor, electropositive, H-bond acceptor groups at the R1 position, minor, electropositive, H-bond donor groups at the R2 position, and bulky, hydrophobic groups at the R3 position for higher inhibitory activity. Docking studies revealed extensive H-bond and hydrophobic interactions in the binding of tripeptide analogues to the NS2B/NS3 protease. This study provides an insight into the key structural features for the design of peptide-based inhibitors of DENV NS2B/NS3 protease.

A Comparative Study between Time Series and Machine Learning Technique to Predict Dengue Fever in Dhaka City

The dengue virus is the most dangerous one that mosquitoes may spread to people. Despite attempts by the government, dengue outbreaks are becoming increasingly common in Bangladesh. Interventions in public health rely heavily on KAP (knowledge, attitude, and practice) studies. The primary goal of this research is to forecast the occurrence of dengue disease in the city of Dhaka using methods from machine learning and time series analysis and then to compare the models in order to find the one with the lowest MAPE. From January 2016 through July 2021, monthly data were retrieved for this study from WHO and the Directorate General of Health and Services (DGHS). According to the findings of this research, neural networks outperform time series analysis when it comes to making predictions. The best-fitted neural network (NN) model was found in model 04 with 05 hidden layers which produced the minimum error model with the value of error 0.003032557, and the values of RMSE and MAPE are 7.588889e − 06 and 1.15273, respectively, for the prediction of the dengue fever in Dhaka city. In contrast, the original dengue data in the time series analysis is not stationary. Take the difference and run the unit root test by the augmented Dickey–Fuller (ADF) test to make it stationary. The dengue data series is stationary at the first-order difference, as evidenced by the ACF and PACF, which show no noticeable spike in the first-order difference. The ARIMA (6, 1, 1) model with the lowest AIC = −251.8, RMSE = 0.0310797, and MAPE = 15.2892 is the best choice model for predicting the dengue death rate. Therefore, from these two models, the NN model gives better prediction performance with the lowest value of MAPE. So, the neural network gives better prediction performance than time series analysis. The NN model forecasted 12-month death rates of dengue fever that suggest the death rate in dengue fever falling month by month. This study is more innovative than any other research because this research approach is different from any other research approach. The model selection criteria are based on the most effective performance metrics MAPE, indicating the lowest error and better prediction performance. Therefore, from this research, the author suggests machine learning gives better prediction performance than time series analysis for any other prediction performance.

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.

Tiger prowling: Distribution modelling for northward-expanding Aedes albopictus (Diptera: Culicidae) in Japan.

The Asian tiger mosquito, Aedes albopictus, is a significant public health concern owing to its expanding habitat and vector competence. Disease outbreaks attributed to this species have been reported in areas under its invasion, and its northward expansion in Japan has caused concern because of the potential for dengue virus infection in newly populated areas. Accurate prediction of Ae. albopictus distribution is crucial to prevent the spread of the disease. However, limited studies have focused on the prediction of Ae. albopictus distribution in Japan. Herein, we used the random forest model, a machine learning approach, to predict the current and potential future habitat ranges of Ae. albopictus in Japan. The model revealed that these mosquitoes prefer urban areas over forests in Japan on the current map. Under predictions for the future, the species will expand its range to the surrounding areas and eventually reach many areas of northeastern Kanto, Tohoku District, and Hokkaido, with a few variations in different scenarios. However, the affected human population is predicted to decrease owing to the declining birth rate. Anthropogenic and climatic factors contribute to range expansion, and urban size and population have profound impacts. This prediction map can guide responses to the introduction of this species in new areas, advance the spatial knowledge of diseases vectored by it, and mitigate the possible disease burden. To our knowledge, this is the first distribution-modelling prediction for Ae. albopictus with a focus on Japan.

Arboviruses in the COVID-19 era in the State of Maranhão, Brazil (2019–2021)

Chikungunya, Dengue, and Zika viruses are endemic to Brazil, with a high annual incidence rate. As COVID-19 emerged as a pandemic, the diagnosis of arboviruses was underestimated. Hence, this study aimed to characterize the sociodemographic and clinical aspects of arboviruses in the context of the COVID-19 pandemic in the state of Maranhão, Brazil. Sociodemographic and clinical data were obtained by applying questionnaires to 179 patients treated at health units in São José de Ribamar, Paço do Lumiar, Raposa, Santa Inês, and Vargem Grande municipalities. Serological and polymerase chain reaction analyses were performed do detect Chikungunya, Zika, Dengue, and COVID-19 infections. The Maranhão state registered 706 probable cases of Chikungunya from 2019 to 2020. In 2020, the cases were reduced by 74.8% compared to those reported in 2019. The test results were analyzed separately. Here, 46.3% of the Chikungunya virus test results were negative, and 36.8% were positive. For the Zika virus tests, most (79.9%) were positive, as well as the DENV1, DENV2, DENV3, and DENV4 tests (88.3%, 88.3%, 89.4%, and 65.9%, respectively). For the COVID-19 tests, of the 146 individuals tested, 52.7% were reactive when considering the anti-S test, and 17.8% were positive when considering the anti-N test. Fever and myalgia were the symptoms most frequently reported by patients. Our results emphasize the importance of continuous epidemiological surveillance of arboviruses in northeastern Brazil, and simultaneous testing for Chikungunya, Dengue, and Zika viruses among suspected patients.

Genomic Analysis of Aedes aegypti in the Northern Chihuahuan Desert of Texas and Mexico.

Background: Aedes aegypti, is the primary vector of dengue, Chikungunya, Zika, and yellow fever viruses. Both natural and human-impacted landscapes have selective pressures on Ae. aegypti, resulting in strong genomic structure even within close geographical distances. Materials and Methods: We assess the genetic structure of this medically important mosquito species at the northern leading edge of their distribution in Southwestern USA. Ae. aegypti were collected during 2017 in the urban communities of El Paso and Sparks, Texas (USA) and in the city of Ciudad Juárez, Mexico. Results: Thousands of nuclear loci were sequenced across 260 captured Ae. aegypti. First, we recovered the genetic structure of Ae. aegypti following geography, with all four major collection communities being genetically distinct. Importantly, we found population structure and genetic diversity that suggest rapid expansion through active-short distance dispersals, with Anapra being the likely source for the others. Next, tests of selection recovered eight functional genes across six outliers: calmodulin with olfactory receptor function; the protein superfamily C-type lectin with function in mosquito immune system and development; and TATA box binding protein with function in gene regulation. Conclusion: Despite these populations being documented in the early 2000s, we find that selective pressures on specific genes have already occurred and likely facilitate Ae. aegypti range expansion.

Acute liver failure resulting from coinfection with dengue and hepatitis A virus: A case report

Acute liver failure (ALF) is an infrequent and varied condition characterized by severe impairment of liver function that occurs in individuals without any preexisting liver disease. This particular case study presents a distinctive occurrence of ALF resulting from a simultaneous infection of both hepatitis A virus (HAV) and dengue virus, accompanied by dengue hemorrhagic fever. The patient, a 26-year-old male, exhibited symptoms such as fever, nausea, vomiting, abdominal pain, and generalized body aches. Subsequently, the patient developed jaundice, hepatic encephalopathy, acute cholecystitis, and acute pancreatitis. Laboratory tests confirmed the presence of markers for both dengue and HAV, along with decreased platelet count and hemoglobin levels. However, with a treatment plan focused on conservative management, the patient’s condition gradually improved, leading to eventual discharge. This case underscores the potential for coinfection with dengue and HAV to precipitate ALF and emphasizes the significance of early diagnosis and timely intervention to achieve the best possible outcomes for the patient.

Computational design, docking, and molecular dynamics simulation study of RNA helicase inhibitors of dengue virus.

RNA viruses are complex pathogens in terms of their genetic makeup, mutation frequency, and transmission modes. They contain the RNA helicase enzyme, which plays a crucial role in the viral genome replication process. This work aims to develop and screen a potential molecule that could function as a dengue virus (DENV) RNA helicase inhibitor. The present study was performed by taking 26 potential derivatives of gedunin phytochemicals from the PubChem database as ligands. The binding study of the compounds were analyzed by in silico docking method considering DENV RNA helicase enzyme as the receptor. After a thorough analysis of the docking scores, toxicity, and physicochemical properties, compound tetrahydrogedunin was obtained as the best. Based on tetrahydrogedunin molecular structure, 100 drug-like molecules were designed using the Data Warrior tool. After the screening process for drug-likeness and ADMET properties, the derivative number 42 was considered as the promising. Further comparative docking of derivative 42 and a standard inhibitor molecule ST-610 with DENV RNA helicase enzyme showed binding affinity as -10.0 kcal/mol and -9.6 kcal/mol, respectively. The favorable interaction between DENV RNA helicase and derivative 42 was further validated by 50 nanoseconds molecular dynamics simulation and MM-GBSA analysis. Since the antiviral activity of derivative 42 has not been reported till date, the compound was predicted as a novel therapeutic molecule that can act against the dengue virus (DENV) RNA helicase enzyme.

Molecular docking and dynamics simulation reveal withanolides as potent antivirals against dengue virus

The prevalence of dengue remains a significant global threat to public health with millions of infections reported annually due to the lack of effective treatment options. Natural resources such as plants contain phytochemicals with desirable therapeutic effects. For instance, the Indian ginseng, Withania somnifera L. (Solanaceae), contains steroidal lactones, saponins, glycosides, and alkaloids which possessed antiviral activities against various viruses. In this study, docking analysis of seven compounds and phytochemicals were performed against the structural and non-structural (NS) proteins of all four dengue virus (DENV) serotypes as well as host cell receptors such as DC-SIGN and CLEC5A. Withanolide A (L5) was selected as the best inhibitor with high binding affinities towards ten targets consisting of DENV structural and NS proteins as well as the CLEC5A receptor. L5 also showed a fair stability with DENV1, DENV2, and DENV3 over a time span of 100 ns via molecular dynamics simulation. L5 demonstrated good drug-likeness properties, a moderate bioavailability score of 0.55, and is predicted to have high gastrointestinal absorptivity. The data from this in silico study revealed the potential of withanolide A to be further developed as an antiviral against dengue. This is the first study to report the in silico analyses of phytochemicals derived from W. somnifera L. (Solanaceae) against the viral proteins of DENV1 to 4 and their respective host cell receptors.

Regional Variation of the CD4 and CD8 T Cell Epitopes Conserved in Circulating Dengue Viruses and Shared with Potential Vaccine Candidates.

As dengue expands globally and many vaccines are under trials, there is a growing recognition of the need for assessing T cell immunity in addition to assessing the functions of neutralizing antibodies during these endeavors. While several dengue-specific experimentally validated T cell epitopes are known, less is understood about which of these epitopes are conserved among circulating dengue viruses and also shared by potential vaccine candidates. As India emerges as the epicenter of the dengue disease burden and vaccine trials commence in this region, we have here aligned known dengue specific T cell epitopes, reported from other parts of the world with published polyprotein sequences of 107 dengue virus isolates available from India. Of the 1305 CD4 and 584 CD8 epitopes, we found that 24% and 41%, respectively, were conserved universally, whereas 27% and 13% were absent in any viral isolates. With these data, we catalogued epitopes conserved in circulating dengue viruses from India and matched them with each of the six vaccine candidates under consideration (TV003, TDEN, DPIV, CYD-TDV, DENVax and TVDV). Similar analyses with viruses from Thailand, Brazil and Mexico revealed regional overlaps and variations in these patterns. Thus, our study provides detailed and nuanced insights into regional variation that should be considered for itemization of T cell responses during dengue natural infection and vaccine design, testing and evaluation.

The Dynamic Relationship between Dengue Virus and the Human Cutaneous Innate Immune Response.

Dengue virus (DENV) is a continuing global threat that puts half of the world's population at risk for infection. This mosquito-transmitted virus is endemic in over 100 countries. When a mosquito takes a bloodmeal, virus is deposited into the epidermal and dermal layers of human skin, infecting a variety of permissive cells, including keratinocytes, Langerhans cells, macrophages, dermal dendritic cells, fibroblasts, and mast cells. In response to infection, the skin deploys an array of defense mechanisms to inhibit viral replication and prevent dissemination. Antimicrobial peptides, pattern recognition receptors, and cytokines induce a signaling cascade to increase transcription and translation of pro-inflammatory and antiviral genes. Paradoxically, this inflammatory environment recruits skin-resident mononuclear cells that become infected and migrate out of the skin, spreading virus throughout the host. The details of the viral-host interactions in the cutaneous microenvironment remain unclear, partly due to the limited body of research focusing on DENV in human skin. This review will summarize the functional role of human skin, the cutaneous innate immune response to DENV, the contribution of the arthropod vector, and the models used to study DENV interactions in the cutaneous environment.

Antiviral Activities of Heparan Sulfate Mimetic RAFT Polymers Against Mosquito-borne Viruses.

Mosquito-borne viruses are a major worldwide health problem associated with high morbidity and mortality rates and significant impacts on national healthcare budgets. The development of antiviral drugs for both the treatment and prophylaxis of these diseases is thus of considerable importance. To address the need for therapeutics with antiviral activity, a library of heparan sulfate mimetic polymers was screened against dengue virus (DENV), Yellow fever virus (YFV), Zika virus (ZIKV), and Ross River virus (RRV). The polymers were prepared by RAFT polymerization of various acidic monomers with a target MW of 20 kDa (average Mn ∼ 27 kDa by GPC). Among the polymers, poly(SS), a homopolymer of sodium styrenesulfonate, was identified as a broad spectrum antiviral with activity against all the tested viruses and particularly potent inhibition of YFV (IC50 = 310 pM). Our results further uncovered that poly(SS) exhibited a robust inhibition of ZIKV infection in both mosquito and human cell lines, which points out the potential functions of poly(SS) in preventing mosquito-borne viruses associated diseases by blocking viral transmission in their mosquito vectors and mitigating viral infection in patients.

CRISPR Screen Reveals PACT as a Pro-Viral Factor for Dengue Viral Replication.

The dengue virus is a single-stranded, positive-sense RNA virus that infects ~400 million people worldwide. Currently, there are no approved antivirals available. CRISPR-based screening methods have greatly accelerated the discovery of host factors that are essential for DENV infection and that can be targeted in host-directed antiviral interventions. In the present study, we performed a focused CRISPR (Clustered Regularly Interspaced Palindromic Repeats) library screen to discover the key host factors that are essential for DENV infection in human Huh7 cells and identified the Protein Activator of Interferon-Induced Protein Kinase (PACT) as a novel pro-viral factor for DENV. PACT is a double-stranded RNA-binding protein generally known to activate antiviral responses in virus-infected cells and block viral replication. However, in our studies, we observed that PACT plays a pro-viral role in DENV infection and specifically promotes viral RNA replication. Knockout of PACT resulted in a significant decrease in DENV RNA and protein abundances in infected cells, which was rescued upon ectopic expression of full-length PACT. An analysis of global gene expression changes indicated that several ER-associated pro-viral genes such as ERN1, DDIT3, HERPUD1, and EIF2AK3 are not upregulated in DENV-infected PACT knockout cells as compared to infected wildtype cells. Thus, our study demonstrates a novel role for PACT in promoting DENV replication, possibly through modulating the expression of ER-associated pro-viral genes.