Surveillance of arthropod-borne viruses in Benin,West Africa 2020–2021:detection of dengue virus 3 in Aedes aegypti(Diptera:Culicidae)

Dear Editor,Dengue virus(DENV,family Flaviviridae,genus Flavivirus)serotypes 1 to 4(DENV-1,-2,-3,and-4)are responsible for more than 100 million infections per year worldwide.Symptoms of DENV infection can be diverse,reaching from an acute febrile illness to the more severe,sometimes fatal dengue haemorrhagic fever/dengue shock syndrome.

Evaluation of antiviral activities of Houttuynia cordata Thunb.extract,quercetin,quercetrin and cinanserin on murine coronavirus and dengue virus infection

Objective:To evaluate the in vitro activities of the ethyl acetate(EA) fraction of Houttuynia cordata(H.cordata) Thunb.(Saururaceae) and three of its constituent flavonoids(quercetin.quercitrin and rutin) against murine coronavirus and dengue virus(DENV).Methods:The antiviral activities of various concentrations of the EA fraction of H.cordata and flavonoids were assessed using virus neutralization tests against mouse hepatitis virus(MHV) and DENV type 2(DENV-2).Cinanserin hydrochloride was also tested against MHV.The EA fraction of H.cordata was tested for acute oral toxicity in C57BL/6 mice.Results:The EA fraction of H.cordata inhibited viral infectivity up to 6 d.Cinanserin hydrochloride was able to inhibit MHV for only 2 d.The 50%inhibitory concentrations(IC_(50)) of the EA fraction of H.cordata added before the viral adsorption stage were 0.98 μg/mL for MHV and 7.50 μg/mL for DENV-2with absence of cytotoxicity.The mice fed with the EA fraction up to 2 000 mg/kg did not induce any signs of acute toxicity,with normal histological features of major organs.Certain flavonoids exhibited comparatively weaker antiviral activity,notably quercetin which could inhibit both MHV and DENV-2.This was followed by quercitrin which could inhibit DENV-2but not MHV,whereas rutin did not exert any inhibitory effect on either virus.When quercetin was combined with quercitrin,enhancement of anti-DENV-2 activity and reduced cytotoxicity were observed.However,the synergistic efficacy of the flavonoid combination was still less than that of the EA fraction.Conclusions:The compounds in H.cordata contribute to the superior antiviral efficacy of the EA fraction which lacked cytotoxicity in vitro and acute toxicity in vim.H.cordata has much potential for the development of antiviral agents against coronavirus and dengue infections.

RNAi:antiviral therapy against dengue virus

Dengue virus infection has become a global threat affecting around 100 countries in the world.Currently,there is no licensed antiviral agent available against dengue.Thus,there is a strong need to develop therapeutic strategies that can tackle this life threatening disease.RNA interference is an important and effective gene silencing process which degrades targeted RNA by a sequence specific process.Several studies have been conducted during the last decade to evaluate the efficiency of siRNA in inhibiting dengue virus replication.This review summarizes siRNAs as a therapeutic approach against dengue virus serotypes and concludes that siRNAs against virus and host genes can be next generation treatment of dengue virus infection.

Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.Results:The assay’s sensitivity was 97.65%,specificity was 92.59% and accuracy was 95.82%when compared to conventional RT-PCR.Additionally,there was no cross-reaction between CHIKV,DENV,Japanese encephalitis virus,hepatitis C,hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.

Antiviral activity of traditional Chinese medicinal plants Dryopteris crassirhizoma and Morus alba against dengue virus

Dengue virus(DENV) has emerged as a major virus that is spread by mosquitoes. Recently, it has spread to more than a hundred nations but continues to lack specific treatable medication. Many traditional Chinese medicinal(TCM) plants are in practice for dengue fever in dengue endemic regions. These traditional medicines persevere with treatments, which modern medicines lack. The study aims to substantiate the anti-dengue potential of some traditional herbs and make them available for further studies to facilitate TCM users. Twelve TCM plants aqueous extracts were evaluated, which are described as cool herbs used for the diseases with high fever. Lead plants were established through detailed in vitro foci forming unit reduction analysis(FFURA) against all four serotypes and validated through quantitative real-time RT-PCR(q RT-PCR). Four plants potentially inhibited the virus in primary phenotypic in vitro evaluation. Two lead plants Dryopteris crassirhizoma(DC) and Morus alba(MA) were identified with half minimal inhibitory concentration(IC50) 130 and 221 μg m L^-1, respectively, while the selectivity indices(SI) were 4.21 and 4.62, respectively. Lead plants equally inhibited all four serotypes of DENV. Time-of-addition analysis suggested that, DC was active at later stages of viral replication, whereas MA was active during the early stages and even showed some prophylactic activity. Liquid chromatography-mass spectrometry(HPLC/MS) analysis revealed presence of flavonoids. DC and MA are identified as potential anti-dengue plants, active against varied stages of dengue virus replication cycle. These results may serve as the base knowledge for further investigation on their combined treatments or integrative treatment with western medicines, which may improve the overall anti-dengue activity in future.

Inhibitory activities of extracts of Rumex dentatus, Commelina benghalensis, Ajuga bracteosa, Ziziphus mauritiana as well as their compounds of gallic acid and emodin against dengue virus

Objective: To investigate the inhibitory effects against dengue virus serotype 2(DENV-2) by five different fractions(extracted by methanol, ethanol, benzene, chloroform and n-hexane) of Rumex dentatus, Commelina benghalensis, Ajuga bracteosa and Ziziphus mauritiana, as well as their constituents(gallic acid, emodin, and isovanillic acid). Methods: All the samples were tested for cytotoxicity on baby hamster kidney cells by MTT assay and for anti-DENV-2 activity by plaque reduction neutralization assay using two DENV-2 doses(45 and 90 plaqueforming units or PFU). Results: All the samples except isovanillic acid exhibited significant prophylactic effects against DENV-2 infectivity(without cytotoxicity) when administered to cells before infection, but were not effective when given 6 h post-infection. The methanol extract of Rumex dentatus demonstrated the highest antiviral efficacy by inhibiting DENV-2 replication, with IC_(50) of 0.154 μg/mL and 0.234 μg/mL, when added before infection with 45 and 90 PFU of virus, respectively. Gallic acid also exhibited significant antiviral effects by prophylactic treatment prior to virus adsorption on cells, with IC_(50) of 0.191 μg/mL and 0.522 μg/mL at 45 and 90 PFU of DENV-2 infection, respectively. Conclusions: The highly potent activities of the extracts and constituent compounds of these plants against DENV-2 infectivity highlight their potential as targets for further research to identify novel antiviral agents against dengue.

Critical role of Dengue Virus NS1 protein in viral replication

Dengue virus(DENV) nonstructural protein 1(NS1) is a highly conserved 46-kDa protein that contains 2 glycosylation sites(Asn-130 and Asn-207) and 12 conserved cysteine(Cys) residues. Here, we performed site-directed mutagenesis to generate systematic mutants of viral strain TSV01. The results of the subsequent analysis showed that an alanine substitution at the second N-linked glycan Asn-207 in NS1 delayed viral RNA synthesis, reduced virus plaque size, and weakened the cytopathic effect. Three mutants at Cys sites(Cys-4, Cys-55, Cys-291) and a C-terminal deletion(ΔC) mutant significantly impaired RNA synthesis, and consequently abolished viral growth, whereas alanine mutations at Asn-130 and Glu-173 resulted in phenotypes that were similar to the wild-type(WT) virus. Further analysis showed that the Asn-207 mutation slightly delayed viral replication. These results suggest that the three conserved disulfide bonds and the second N-linked glycan in NS1 are required for DENV-2 replication.

Bioinformatics analysis and characteristics of envelop glycoprotein E epitopes of dengue virus

The major envelope glycoprotein E of dengue (DEN) virus plays a central role in the biology of flaviviruses. It is capable of inducing a protective immune response in vivo and responsible for the viral binding to the cellular receptor. The crystal structures of glycoprotein E ectodomains have already been determined. However, it is still un-clear where the well-defined B-cell epitopes for glycoprotein E which induce the neutralizing an-tibodies locates. Thus, in order to characterize the role of glycoprotein E in the pathogenesis of dengue virus infection, we first used network servers (http://bio.dfci. harvard.edu/Tools/ &http://www. imtech. res. in) to predict and analyze the well defined B-cell and T-cell epitopes of the glycoprotein of the DEN-1 HAWAII strain. Then based on the highly conserved envelop glyco-protein amino acids, the hydrophilicity, antigenic-ity, accessibility and flexibility of envelop glyco-protein E were further predicted by using Biotic softwares (DNASTAR) and network servers (http://bio. dfci.harvard.edu/Tools/), the secondary structure was putatively obtained. In our study, the sequence at 281-295 amino acid (aa) for den-gue virus type 1 HAWAII strain and the sequence at 345-359, 383-397 for dengue virus type 2 NGC strain were predicted as the more prevalent epi-topes by using multiple parameters and different analysis softwares, respectively. Two epitopes of DEN-2 and one of DEN-1 locate on the domain Ш and domainⅡ of the protein E, respectively. Sub-sequently, further studies will be carried out to examine the antigenicity and protection of the synthetic peptides with higher scores in the av-erage antigen index (AI) and better hydrophilic properties determined by our data.

Contribution of phylogenetics to understanding the evolution and epidemiology of dengue virus

Dengue virus(DENV)is one of the most important arboviral pathogens in the tropics and subtropics,and nearly one-third of the world's population is at risk of infection.The transmission of DENV involves a sylvatic cycle between nonhuman primates(NHP)and Aedes genus mosquitoes,and an endemic cycle between human hosts and predominantly Aedes aegypti.DENV belongs to the genus Flavivirus of the family Flaviviridae and consists of four antigenically distinct serotypes(DENV-1-4).Phylogenetic analyses of DENV have revealed its origin,epidemiology,and the drivers that determine its molecular evolution in nature.This review discusses how phyloge-netic research has improved our understanding of DENV evolution and how it affects viral ecology and improved our ability to analyze and predict future DENV emergence.

A murine model of dengue virus infection in suckling C57BL/6 and BALB/c mice

Dengue is a significant public health concern across tropical and subtropical regions worldwide,principally causing disease in children.Very young children are at increased risk of severe manifestations of dengue infection.The mechanism of dengue disease in this population is not fully understood.In this study,we present a murine model of dengue virus primary infection in suckling C57BL/6 and BALB/c mice in order to investigate disease pathogenesis.Three-day-old C57BL/6 mice intraperitoneally infected with DENV-2 NGC were more susceptible to infection than BALB/c mice,showing increased liver enzymes,extended viremia,dissemination to organs and histological alterations in liver and small intestine.Furthermore,the immune response in DENV-infected C57BL/6 mice exhibited a marked Th1 bias compared to BALB/c mice.These findings highlight the possibility of establishing an immunocompetent mouse model of DENV-2 infection in suckling mice that reproduces certain signs of disease observed in humans and that could be used to further study agerelated mechanisms of dengue pathogenesis.

Co-positivity of anti-dengue virus and anti-Japanese encephalitis virus IgM in endemic area:co-infection or cross reactivity?

Objective:To report high co-positivity of anti-dengue virus(DV)and anti-Japanese encephalitis virus(JEV)IgM in an area endemic for both the viruses and to discuss the possibilities of coinfection.Methods:Serum samples from the patients who presented with fever,suspected central nervous system infection and thrombocytopenia,were tested for anti-DV IgM and antiJEV IgM antibodies.Conventional reverse transcriptase polymerase chain reaction was done for detection of DV RNA and JEV RNA.Results:Of 1 410 patient sera tested for anti-DV and antiJEV antibodies,129(9.14%)were co-positive for both.This co-positivity was observed only in those months when anli-JEV IgM positivily was high.Tilers of both anli-DV IgM and anti-JEV IgM were high in most of the co-positive eases.Among these 129 co-positive cases,76 were lesled by conventional reverse Iranscriplase polymerase chain reaction for both flaviviruses,of which eight cases were co-positive for DV and JEV.Conclusions:Co-infection with more than one fluvivirus species can occur in hyperendemic areas.

Discovery and design of cyclic peptides as dengue virus inhibitors through structure-based molecular docking

Objective:To find potential peptide inhibitors against the NS2B/NS3 protease of DENV which in turn,can inhibit the viral replication inside host cell.Methods:Cyclic peptides were designed having combination of positively charged amino acids using ChemSketch software and were converted to 3D structures.DENV NS3 protein structure was retrieved from Protein Data Bank(PDB)using PDB Id:2FOM.DENV NS3 and cylic peptides were docked using MOE software after structural optimization.Results:Through molecular docking it was revealed that most of the peptides bound deeply in the binding pocket of DENV NS2B/NS3 protease an had interactions with catalytic triad.Peptide 2 successfully blocked the catalytic triad of NS2B/NS3 protease.Peptide 1,,4 and 6 also had potential interactions with active residues of the NS2B/NS3 protease while all other peptides were in close contact with the active sites of NS2B/NS3 protease thus,these peptides can serve as a potential drug candidate to stop viral replication.Conclusions:Thus.it can be concluded from the study that these peptides could serve as important inhibitors to inhibit the viral replication and need further in-vitro investigations to confirm their efficacy.

Recognition of a multiple antigen peptide containing sequence from mimotope of the dengue type 3 virus NS4B protein by human antibodies

Objective: To evaluate the recognition of NS4 B mimotope, as multiple antigen peptide(MAP), by dengue antibodies presents in serum samples from patients with different serotype infections. Methods: A MAP containing mimotope sequence was synthesized and used to evaluate the recognition of NS4 B mimotope as MAP by a panel of 66 human sera from dengue cases by an indirect ELISA assay. Results: The MAP differentiated between sera from dengue viruses infected patients and sera from healthy individuals and the best reactivity was shown by serum from dengue type 3 virus patients. The recognition was more intense with serum from patients with secondary infection. Conclusions: The findings suggest the potential use of NS4 B mimotope on the development of a multi-epitope diagnostic tool. These results are important for further immunogenicity studies.

Dengue virus non-structural 1 protein interacts with heterogeneous nuclear ribonucleoprotein H in human monocytic cells

Objective: To study protein-protein interaction between heterogeneous nuclear ribonucleoprotein H(hn RNP H) and Dengue virus(DENV) proteins. Methods: DENV proteins were screened against the host hn RNP H protein, in order to identify the host-viral protein-protein interactions in DENV infected THP-1 cells by co-immunoprecipitation. The co-localization of the interacting proteins was further confirmed by immunofluorescence microscopy. Results: The host protein hn RNP H was found to interact with DENV nonstructural 1 protein and help the virus to multiply in the cell. Conclusions: The non-structural 1 glycoprotein is a key modulator of host immune response and is also involved in viral replication. Therefore, disruption of this key interaction between hn RNP H and DENV nonstructural 1 could be an important therapeutic strategy for management of DENV infection.

Proteomic profile of human monocytic cells infected with dengue virus

Objective: To identify the changes in the proteome of U937 cells infected with dengue virus(DENV).Methods: In this study, differentiated U937 cultures were infected with two DENV-2strains, one of which was associated with dengue(DENV-2/NG) and the other one with severe dengue(DENV-2/16681), with the aim of determining the cellular proteomic profiles under different infection conditions. Cellular proteins were extracted and separated by two-dimensional electrophoresis, and those proteins with differential expression profiles were identified by mass spectrometry. The obtained results were correlated with cellular viability, the number of infectious viral particles, and the viral DNA/protein quantity.Results: In comparison with non-infected cultures, in the cells infected with the DENV-2/NG strain, nine proteins were expressed differentially(five were upregulated and four were downregulated); in those cultures infected with the DENV-2/16681 strain, six proteins were differentially expressed(two were downregulated and four were upregulated). The downregulated proteins included fatty acid-binding protein, heterogeneous nuclear ribonucleoprotein 1, protein disulfide isomerase, enolase 1, heat shock 70 k Da protein 9, phosphotyrosyl phosphatase, and annexin IV. The upregulated proteins included heat shock 90 k Da protein AA1, tubulin beta, enolase 1, pyruvate kinase,transaldolase and phospholipase C-alpha.Conclusions: Because the monocyte/macrophage lineage is critical for disease pathogenicity, additional studies on these proteins could provide a better understanding of the cellular response to DENV infection and could help identify new therapeutic targets against infection.

IL-18 and IL-18 binding protein levels in patients with dengue virus infection

Objective:To determine the levels of IL-18 and IL-18 binding protein(BP) in patients with dengue virus infection.Methods:Acute and convalescent sera were collected from each patient. Control group was sera from blood donors.The levels of both IL-18 and IL-18BP were measured by ELISA assays.Results:It was shown that IL-18 and IL-18BP levels were significantly higher in patients when compared with controls.In addition,the level of IL-18BP was lower in convalescent than in acute sera.Conclusions:These data suggest that both IL-18 and IL-18BP production was induced following dengue virus infection.Investigating the regulation of IL-18 by its natural regulator could lead to further understanding of the immune response or immunopathogenesis following dengue virus infection.

Susceptibility of Aedes flavopictus miyarai and Aedes galloisi mosquito species in Japan to dengue type 2 virus

Objective: To evaluate the potential of local mosquitoes to act as vectors for dengue transmission in Japan.Methods: Serotype 2 Th NH28/93 was used to test the dengue susceptibility profiles of Aedes flavopictus miyarai(Ae. f. miyarai), Aedes galloisi(Ae. galloisi) and Aedes albopictus(Ae.albopictus), which were collected in Japan. We used Aedes aegypti from Thailand as a positive control. The mosquitoes were infected with the virus intrathoracically or orally. At 10 or 14 days post infection, the mosquitoes were dissected and total RNA was extracted from their abdomens, thoraxes, heads and legs. Mosquito susceptibility to dengue virus was evaluated using RT-PCR with dengue virus-specific primers. Differences in the infection and mortality rates of the different mosquito species were tested using Fisher's exact probability test.Results: The infection rates for dengue virus administered intrathoracically to Ae. f. miyarai,Ae. galloisi and Aedes aegypti mosquitoes were identical by RT-PCR on Day 10 post infection.All of the body parts we tested were RT-PCR-positive for dengue virus. For the orally administered virus, the infection rates in the different body parts of the Ae. f. miyarai mosquitoes were slightly higher than those of Ae. albopictus mosquitoes, but were similar to the control mosquitoes(P > 0.05). The mortality rates for Ae. f. miyarai and Ae. albopictus mosquitoes were similar(P = 0.19). Our data indicated that dengue virus was able to replicate and disseminate to secondary infection sites in all of the four mosquito species(Japanese and Thai).Conclusions: Ae. albopictus is a well-known candidate for dengue transmission in Japan. However, our data suggest that Ae. f. miyarai from Ishigaki Island(near Okinawa Island) and Ae. galloisi from Hokkaido(Northern Japan) should also be regarded as potential vectors for dengue transmission in these regions. Further studies on these mosquitoes should be conducted.

The Study on The Immune Response Induced by Expressing Recombinant Plasmid of Dengue Virus Type 2 NS3 Protein

The PSV?NS3, an expressing recombinant plasmid of dengue virus type 2 NS3 protein, was injected directly into the quadriceps of Balb/C mice to explore whether it could inducing immune response. The splenic T cell subsets of two groups was analysed by flow cytometry. It was found that the percentage of CD4+ and CD8+ T cells of experimental group were significantly higher than those of the control group. The titer of IgG antibody was as high as 1∶5 120 in experimental group, but it couldn’t be detected in control group by ELISA. The western blot further proved that the IgG antibody was specific for NS3 protein. Those results Suggested that inoculation Balb/C mice with PSV?NS3 could inducing immune response, and the NS3 protein might be used as the candidate protein of DNA vaccine of dengue virus.

Cytokine gene expression in human hepatocytes infected with dengue virus serotype 3 (strain-16562)

Liver is a site of viral replication and liver dysfunction is a characteristic of severe dengue infection. To understand these mechanisms, we analyzed the response of a hepatic cell linage, HepG2 to infection with dengue 3 virus (strain 16562). Steady state levels of mRNA accumulation were assessed for 14 genes involved in modulation of the host immune responses, at 6, 24 and 48 hpi, by quantitative reverse transcription real-time PCR (qRT-PCR). Fourteen genes showed altered expression upon infection with D3V including;cytokines/chemokines (IL-1β, IL-6, IL-8, RANTES, MCP-2, IL-2Rα and TGF-βIIIR), type I interferon (IFN-α and IFN-β), and pattern-recognition receptors (TLR3, TLR8, RIG-1, MDA5 and MyD88). Although these genes are associated with mechanism of innate immune response and anti-viral activity, their altered expression does not inhibit D3V (strain 16562) growth kinetics and virus yield in HepG2 cells. Gene expression in liver may explain pathological changes associated with dengue virus infection.

Purification of <i>Dengue Virus</i>Particles by One-Step Ceramic Hydroxyapatite Chromatography

Dengue virus type 2 ThNH7/93 retained infectious activity after purification by ceramic hydroxyapatite chromatogra-phy. Dengue virus type 2 culture fluid was loaded onto the ceramic hydroxyapatite column and eluted with a linear gradient of sodium phosphate buffer. Culture fluid and protein contaminants derived from host cells were eluted initially, followed by elutions of dsDNA, and then dengue virus type 2. The recoveries of dengue virus type 2 were 64 ± 14% (n = 11) in the hemagglutination (HA) test and 60% (n = 2) determined by focus assay for viral infectivity. This protocol was highly reproducible, simple, rapid, and appears applicable to other virus species such as influenza virus, Japanese encephalitis virus and adenovirus.