Dengue Virus Type Research Articles

Construction, characterization and stability analysis of infectious clone of live attenuated dengue virus type 4 Ban18HK20 strain

Objective To construct a stable infectious clone of live attenuated dengue virus (DENV) type 4 Ban18HK20 strain for better understanding the virulence determinants of DENV and improving the development of chimeric vaccines. Methods Specific primers were constructed according to the genome of Ban18HK20 strain and used to subclone six cDNA fragments, which were linked into a high-copy plasmid pSPTM to obtain a stable full-length cDNA clone of DENV. RNA was transcribed from the full-length cDNA in vitro and electrotransfected into Vero cells to recover the virus. Biological characteristics of the recovered virus were identified using plaque assay, indirect immunofluorescence assay, growth kinetics test and pathogenicity study in mouse brain. Genetic stability of the virus passaged 15 generations was studied using RT-PCR amplification. Results Restriction enzyme digestion and sequencing analysis indicated that the infectious clone was constructed successfully. The recovered virus was consistent with the parental virus in terms of plaque morphology, DENV E protein expression, growth characteristics and pathogenicity in mouse brain. Sequencing analysis showed that the recovered virus had the same genome sequence as the parental virus with good hereditary stability. Conclusions A stable infection clone of Ban18HK20 was constructed and a reverse genetics technology platform for DENV research was established. Key words: Dengue virus; Reverse genetics; Infectious clone; Virus recovery

From the threat to the large outbreak: dengue on Reunion Island, 2015 to 2018.

BackgroundWith more than 300 million infections estimated annually worldwide, dengue is the most prevalent arboviral infection. On Reunion Island, after a large outbreak in 1977–78, only limited episodes of viral circulation or sporadic cases were reported till 2015.AimOur objective was to document and report on the circulation of dengue virus after the occurrence of a small outbreak during austral summer 2015/16 and until the large outbreak of 2018.MethodsBeside the mandatory notification of biologically confirmed dengue cases, additional systems of surveillance were set up: estimation of dengue-like syndrome in people seeking care by their family doctor, surveillance of emergency department visits related to dengue, surveillance of hospitalised dengue patients and deaths classifications.ResultsAfter a moderate outbreak during summer 2015/16 with 231 cases, 2017 was characterised by limited viral circulation (97 cases) which, however, persisted during the austral winter. By February 2018, the number of cases had increased and led to a peak at the beginning of May 2018. More than 6,000 cases were reported this year (dengue virus type 2 only). In addition, six deaths of dengue patients were notified.ConclusionIn 2017, the persistence of transmission during winter created favourable conditions for the emergence of an epidemic during summer 2018. After this moderate epidemic wave, the viral circulation persisted during winter 2018 for the second year, opening the door for the second wave in 2019 and for potential endemisation of the disease on Reunion Island in the near future.

Efficient Delivery of Dengue Virus Subunit Vaccines to the Skin by Microprojection Arrays.

Dengue virus is the most important arbovirus impacting global human health, with an estimated 390 million infections annually, and over half the world’s population at risk of infection. While significant efforts have been made to develop effective vaccines to mitigate this threat, the task has proven extremely challenging, with new approaches continually being sought. The majority of protective, neutralizing antibodies induced during infection are targeted by the envelope (E) protein, making it an ideal candidate for a subunit vaccine approach. Using truncated, recombinant, secreted E proteins (sE) of all 4 dengue virus serotypes, we have assessed their immunogenicity and protective efficacy in mice, with or without Quil-A as an adjuvant, and delivered via micropatch array (MPA) to the skin in comparison with more traditional routes of immunization. The micropatch contains an ultra-high density array (21,000/cm2) of 110 μm microprojections. Mice received 3 doses of 1 μg (nanopatch, intradermal, subcutaneous, or intra muscular injection) or 10 μg (intradermal, subcutaneous, or intra muscular injection) of tetravalent sE spaced 4 weeks apart. When adjuvanted with Quil-A, tetravalent sE vaccination delivered via MPA resulted in earlier induction of virus-neutralizing IgG antibodies for all four serotypes when compared with all of the other vaccination routes. Using the infectious dengue virus AG129 mouse infectious dengue model, these neutralizing antibodies protected all mice from lethal dengue virus type 2 D220 challenge, with protected animals showing no signs of disease or circulating virus. If these results can be translated to humans, MPA-delivered sE represents a promising approach to dengue virus vaccination.

Discovery of a small molecule inhibitor targeting dengue virus NS5 RNA-dependent RNA polymerase

Dengue is a mosquito-borne viral infection that has spread globally in recent years. Around half of the world’s population, especially in the tropics and subtropics, is at risk of infection. Every year, 50–100 million clinical cases are reported, and more than 500,000 patients develop the symptoms of severe dengue infection: dengue haemorrhagic fever and dengue shock syndrome, which threaten life in Asia and Latin America. No antiviral drug for dengue is available. The dengue virus (DENV) non-structural protein 5 (NS5), which possesses the RNA-dependent RNA polymerase (RdRp) activity and is responsible for viral replication and transcription, is an attractive target for anti-dengue drug development. In the present study, 16,240 small-molecule compounds in a fragment library were screened for their capabilities to inhibit the DENV type 2 (DENV2) RdRp activities in vitro. Based on in cellulo antiviral and cytotoxity assays, we selected the compound RK-0404678 with the EC50 value of 6.0 μM for DENV2. Crystallographic analyses revealed two unique binding sites for RK-0404678 within the RdRp, which are conserved in flavivirus NS5 proteins. No resistant viruses emerged after nine rounds of serial passage of DENV2 in the presence of RK-0404678, suggesting the high genetic barrier of this compound to the emergence of a resistant virus. Collectively, RK-0404678 and its binding sites provide a new framework for antiviral drug development.

Analysis of nine death cases with severe dengue fever

Objective To analyze the clinical features of death cases of dengue fever and the causes of their deaths. Methods The clinical data and death reports of nine death cases of dengue fever in Guangdong Province from June 23, 2014 to September 10, 2019 were retrospectively analyzed. All of nine cases were positive for serum dengue virus RNA as confirmed by reverse transcription polymerase chain reaction, and some of the virus strains were serotyped. Results The median age of the nine patients was 57.5 (range: 18-80) years. Among them, six patients were females; eight patients were local cases and one was imported case; all of nine cases occurred in September and October. The median time from onset to visit hospital and diagnosis was three and four days, respectively. Three of the nine patients had underlying diseases. All of nine cases had fever, including three with double-peak fever. Eight of the cases had three or more severe dengue fever warning indexes before admission. Three cases had severe bleeding upon admission, one case had shock, and six cases had organ failure. Three cases underwent invasive examination, including arterial puncture catheterization, endoscopic titanium clip hemostasis, and percutaneous transluminal coronary angiography. Two cases developed malignant arrhythmia and one had massive hemorrhage of subcutaneous soft tissue after operation. The death cases included four cases of cardiogenic shock and fatal arrhythmia, three cases of encephalitis and encephalopathy, one case of refractory shock, and one case of acute liver failure. Among the six cases that underwent serological typing, four were identified as dengue virus type 1 (DEN-1) and two were DEN-2. Conclusions Severe organ failure is the major cause of dengue fever-related death, especially fulminant myocarditis, and DEN-1 is most common. Early diagnosis and treatment, and avoidance of invasive procedures can effectively reduce the mortality rate of the severe dengue fever patients. Key words: Severe dengue; Clinical features; Cause analysis

Epidemiological and clinical features of dengue fever outbreak in Jiangxi Province in 2019

Objective To investigate the epidemiological and clinical characteristics of 91 cases of dengue fever outbreak in Jiangxi Province in 2019, and to strengthen the management and prevention of dengue fever. Methods The clinical data, laboratory results and etiology tests of 91 patients with dengue fever from the Ninth Hospital of Nanchang, Zhangshu People′s Hospital, Fengcheng People′s Hospital and Nanchang County People′s Hospital from July 31, 2019 to September 27, 2019 were retrospectively collected. The t test was used for continuous variables and chi-square test was used for categorical variables. Results A total of 91 dengue fever patients were reviewed. The patients age ranged from 4 to 87 years old, and the majority were adult patients. Most patients were farmers and they all had mosquito-biting history. Local cases were the major source of this outbreak. The incubation time was (5.19±2.96) days. All patients had fever over 38 ℃. Thirty-eight (41.8%) patients developed rashes, mainly distributed on the limbs and trunk, and 15 patients (16.5%) developed myalgia and fatigue. The incidence of rashes in patients admitted in September was 15.9% (7/44), which was significantly lower than 68.1% (32/47) in July and August (χ2=25.262, P<0.01). The incidence of headache in patients admitted in September was 27.3%(12/44), which was significantly lower than 78.7% (37/47) in July and August (χ2 =24.206, P<0.01). Among the 91 patients, 64 (70.3%) patients had white blood cell counts less than 4×109/L, 14(15.4%) patients had platelet counts less than 50×109/L. The positive rate of dengue non-structural protein 1 (NS1) antigen was 100.0%(37/37), the positive rates of dengue antibody IgM and IgG were 50.0%(11/22) and 13.6%(3/22), respectively. Among the 113 serum samples tested for dengue virus RNA, 106 were identified with dengue virus type-1 and two were dengue virus type-2. In 22 patients who received tests within 7 days, the positive rate of NS1 antigen was 100.0% (22/22), the IgM and IgG positive rates were 50.0%(11/22) and 13.6% (3/22), respectively, and 19 patients were infected with dengue type-1. Among the IgM antibody positive cases, the onset days before sampling was (5.09±1.64) days, longer than that of IgM antibody negative cases ((2.82±1.83) days), with statistical significance (t=3.063, P=0.007). The onset time before sampling in dengue virus RNA-positive group was (3.53±1.87) days, which was shorter than that of RNA-negative group ((6.67±0.58) days), and the difference was statistically significant (t=2.839, P=0.013). Conclusions The dengue fever outbreak cases in Jiangxi Province in 2019 have typical clinical manifestations of dengue fever. Using the NS1 antigen test, IgM and IgG antibody tests and real-time fluorescent quantitative polymerase chain reaction for dengue virus genotyping during early onset of the disease can assistant clinicians in clinical management, controlling infectious source and stopping disease transmission. Key words: Dengue; Disease outbreaks; Epidemiology; Clinical manifestations

Sylvatic dengue virus type 4 in Aedes aegypti and Aedes albopictus mosquitoes in an urban setting in Peninsular Malaysia.

Dengue fever is endemic in Malaysia, contributing to significant economic and health burden in the country. Aedes aegypti and Ae. albopictus are the main vectors of the dengue virus (DENV), which circulates in sylvatic and human transmission cycles and has been present in Malaysia for decades. The study investigated the presence and distribution of DENV in urban localities in the Klang Valley, Peninsular Malaysia. A total of 364 Ae. aegypti and 1,025 Ae. albopictus larvae, and 10 Ae. aegypti and 42 Ae. albopictus adult mosquitoes were screened for the presence of DENV. In total, 31 (2.2%) samples were positive, of which 2 Ae. albopictus larvae were co-infected with two serotypes, one with DENV-2 and DENV-3 and the other with DENV-3 and DENV-4. Phylogenetic analysis determined that the isolates belonged to DENV-1 genotype I (1 Ae. aegypti adult), DENV-2 (1 Ae. albopictus larva), DENV-3 genotype V (3 Ae. aegypti larvae and 10 Ae. albopictus larvae) and DENV-4 genotype IV (6 Ae. aegypti larvae and 12 Ae. albopictus larvae), a sylvatic strain of DENV-4 which was most closely related with sylvatic strains isolated from arboreal mosquitoes and sentinel monkeys in Peninsular Malaysia in the 1970s. All four DENV serotypes were co-circulating throughout the study period. The detection of a sylvatic strain of DENV-4 in Ae. aegypti and Ae. albopictus mosquitoes in urban areas in Peninsular Malaysia highlights the susceptibility of these vectors to infection with sylvatic DENV. The infectivity and vector competence of these urban mosquitoes to this strain of the virus needs further investigation, as well as the possibility of the emergence of sylvatic virus into the human transmission cycle.

3-(3,4-Dimethoxyphenyl)-5-(2-fluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole

A new analogue of fluorinated pyrazoline (compound 1) has been synthesized via one-pot three-component reaction in a sealed-vessel reactor, Monowave 50. The structure of compound 1 has been established by spectroscopy analysis, including UV, FT-IR, HRMS, 1H and 13C NMR spectroscopy. Based on the in silico studies, this compound showed a good potential as an inhibitor for dengue virus type 2 (DEN2) NS2B/NS3 serine protease and can be used as a reference in the next design of an antidengue virus.

Dengue virus capsid anchor modulates the efficiency of polyprotein processing and assembly of viral particles.

The assembly and secretion of flaviviruses are part of an elegantly regulated process. During maturation, the viral polyprotein undergoes several co- and post-translational cleavages mediated by both viral and host proteases. Among these, sequential cleavage at the N and C termini of the hydrophobic capsid anchor (Ca) is crucial in deciding the fate of viral infection. Here, using a refined dengue pseudovirus production system, along with cleavage and furin inhibition assays, immunoblotting and secondary structure prediction analysis, we show that Ca plays a key role in the processing efficiency of dengue virus type 2 (DENV2) structural proteins and viral particle assembly. Replacement of the DENV2 Ca with the homologous regions from West nile or Zika viruses or, alternatively, increasing its length, improved cleavage and hence particle assembly. Further, we showed that substitution of the Ca conserved proline residue (P110) to alanine abolishes pseudovirus production, regardless of the Ca sequence length. Besides providing the results of a biochemical analysis of DENV2 structural polyprotein processing, this study also presents a system for efficient production of dengue pseudoviruses.

Antiviral activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against dengue virus type 2 (GenBank accession no. MH488959).

This study investigated the antiviral activity of betacyanins from red pitahaya (Hylocereus polyrhizus) and red spinach (Amaranthus dubius) against dengue virus type 2 (DENV-2). The pulp of red pitahaya and the leaves of red spinach were extracted using methanol followed by sub-fractionation and Amberlite XAD16N column chromatography to obtain betacyanin fractions. The half maximum cytotoxicity concentration for betacyanin fractions from red pitahaya and red spinach on Vero cells were 4.346 and 2.287 mg ml−1, respectively. The half-maximal inhibitory concentration (IC50) of betacyanin fraction from red pitahaya was 125.8 μg ml−1 with selectivity index (SI) of 5.8. For betacyanin fraction from red spinach, the IC50 value was 14.62 µg ml−1 with SI of 28.51. Using the maximum non-toxic betacyanin concentration, direct virucidal effect against DENV-2 was obtained from betacyanin fraction from red pitahaya (IC50 of 126.70 μg ml−1; 95.0 % virus inhibition) and red spinach (IC50 value of 106.80 μg ml−1; 65.9 % of virus inhibition). Betacyanin fractions from red pitahaya and red spinach inhibited DENV-2 in vitro.

Clinical, laboratory and immune aspects of Zika virus-associated encephalitis in children

ObjectiveTo evaluate the clinical, laboratory, and immune characteristics of Zika virus (ZIKV)-associated encephalitis in pediatric patients after the epidemic in Huila, southern Colombia. MethodsA pediatric neuro-surveillance hospital study was conducted in a referral health center in southern Colombia, from October 2016 to October 2017. Cases of encephalitis were confirmed by nucleic acid amplification tests and serological methods in cerebrospinal fluid (CSF), plasma, and/or urine. Levels of six cytokines were evaluated by flow cytometry. Patients underwent daily clinical and laboratory follow-up. ResultsTwenty children with probable encephalitis were included for further studies and 16 of them were confirmed. Four cases of bacterial meningoencephalitis (Streptococcus pneumoniae, group B Streptococcus, Staphylococcus epidermidis, and Escherichia coli) and 12 cases of viral encephalitis were identified, six of them associated with ZIKV infection. Other viral encephalitis cases were caused by herpes viruses (n=3), enterovirus (n=2), and dengue virus type 2 (DENV-2; n=1) infections. ZIKV-associated encephalitis symptoms subsided faster than those of patients with encephalitis caused by other agents. CSF analysis revealed lymphocytic pleocytosis. Compared to healthy controls, children with ZIKV-associated encephalitis presented modest plasma interleukin (IL)-10 but not IL-2, IL-4, IL-6, interferon gamma (IFN-γ), or tumor necrosis factor alpha (TNF-α). Cytokine expression was differentially regulated, as dramatically elevated IL-6, IL-10, and IFN-γ levels were observed in CSF but not in paired plasma samples in one of the patients with ZIKV detectable in CSF. ConclusionsThis study provides evidence that ZIKV is responsible for pediatric encephalitis in endemic areas, and the local presence of the virus may induce cephalic but not systemic expression of cytokines.

1682. Dengue Fever Outbreak Investigation in Upper Egypt in 2015

BackgroundSurveillance is the backbone of infectious diseases control but an outbreak of a new pathogen to a developing country may have devastating consequences given less prepared healthcare systems in such countries. In October 2015, there was a sharp rise of febrile illnesses reported in Dairot Fever Hospital which prompted the general department for fever hospitals in the Egyptian ministry of health (MOH) to constitute a scientific committee for field visit study in Dairot fever hospital.MethodsThe committee held meetings at Dairot fever hospital, educated local healthcare providers, examined all isolated patients and requested samples of 118 isolated patients to be sent to central laboratories of MOH. Entomological services were also part of the committee and surveillance was started in the affected area.ResultsOut of 118 samples, 28 came back positive for Dengue virus type 1 by ELISA and PCR. Entomological surveillance revealed the presence of Aedes aegypti larvae and adult mosquito at the sites where cases were living; consequently, entomological control measures for dengue vector were immediately set leading to a dramatic decline in the density of adult mosquito (from 23% to 0%) and larvae (from 25% to 0.5%).ConclusionThe prompt response of the MOH in Egypt led to rapid control of Dengue fever outbreak but educating healthcare workers about possible imported infectious diseases would have halted the outbreak much earlier which shows the importance of Infectious Diseases training in developing countries.Disclosures All authors: No reported disclosures.

Chemical Constituents From Endiandra kingiana (Lauraceae) as Potential Inhibitors for Dengue Type 2 NS2B/NS3 Serine Protease and its Molecular Docking

A phytochemical study on the bark of Endiandra kingiana Gamble (Lauraceae) led to the isolation of a new benzofuranone, 4-hydroxy-6-(9,13,17-trimethyldodeca-8,12,16-trienyl)-2(3 H)-benzofuranone (1), together with 6 known compounds. Their structures were established on the basis of detailed spectroscopic analysis, including one- and two-dimensional nuclear magnetic resonance (NMR) and electrospray Ionisation mass spectrometry techniques. Compounds 1-3 showed moderate inhibition against dengue virus type 2 NS2B/NS3 protease with percentage inhibitions of 61.23 ± 6.96, 69.92 ± 3.34, and 62.02 ± 6.19, respectively. Molecular docking was performed to predict the binding mode of all protease inhibitor models and the results revealed that most of the essential amino acid residues such as Asp129, Ser135, Tyr161, Asn152, and His51 significantly interact with the ligands.

Glycyrrhizic acid derivatives as Dengue virus inhibitors.

Dengue virus (DENV) is one of the most geographically distributed pathogenic flaviviruses transmitted by mosquitoes Aedes sps. In this study, the structure-antiviral activity relationships of Glycyrrhizic acid (GL) derivatives was evaluated by the inhibitory assays on the cytopathic effect (CPE) and viral infectivity of DENV type 2 (DENV2) in Vero E6 cells. GL (96% purity) had a low cytotoxicity to Vero E6 cells, inhibited DENV2-induced CPE, and reduced the DENV-2 infectivity with the IC50 of 8.1 μM. Conjugation of GL with amino acids or their methyl esters and the introduction of aromatic acylhydrazide residues into the carbohydrate part strongly influenced on the antiviral activity. Among compounds tested GL conjugates with isoleucine 13 and 11-aminoundecanoic acid 17 were found as potent anti-DENV2 inhibitors (IC50 1.2-1.3 μM). Therefore, modification of GL is a perspective way in the search of new antivirals against DENV2 infection.

Cell-Fusing Agent Virus Reduces Arbovirus Dissemination in Aedes aegypti Mosquitoes In Vivo.

Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of human-pathogenic flaviviruses.IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly, A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. aegypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission.

A Dengue Virus Type 2 (DENV-2) NS4B-Interacting Host Factor, SERP1, Reduces DENV-2 Production by Suppressing Viral RNA Replication.

Host cells infected with dengue virus (DENV) often trigger endoplasmic reticulum (ER) stress, a key process that allows viral reproduction, without killing the host cells until the late stage of the virus life-cycle. However, little is known regarding which DENV viral proteins interact with the ER machinery to support viral replication. In this study, we identified and characterized a novel host factor, stress-associated ER protein 1 (SERP1), which interacts with the DENV type 2 (DENV-2) NS4B protein by several assays, for example, yeast two-hybrid, subcellular localization, NanoBiT complementation, and co-immunoprecipitation. A drastic increase (34.5-fold) in the SERP1 gene expression was observed in the DENV-2-infected or replicon-transfected Huh7.5 cells. The SERP1 overexpression inhibited viral yields (37-fold) in the DENV-2-infected Huh7.5 cells. In contrast, shRNAi-knockdown and the knockout of SERP1 increased the viral yields (3.4- and 16-fold, respectively) in DENV-2-infected HEK-293 and Huh7.5 cells, respectively. DENV-2 viral RNA replication was severely reduced in stable SERP1-expressing Huh7.5 cells transfected with DENV-2 replicon plasmids. The overexpression of DENV-2 NS4B alleviated the inhibitory effect of SERP1 on DENV-2 RNA replication. Taking these results together, we hypothesized that SERP1 may serve as an antiviral player during ER stress to restrict DENV-2 infection. Our studies revealed novel anti-DENV drug targets that may facilitate anti-DENV drug discovery.

Incidence dynamics and investigation of key interventions in a dengue outbreak in Ningbo City, China.

BackgroundThe reported incidence of dengue fever increased dramatically in recent years in China. This study aimed to investigate and to assess the effectiveness of intervention implemented in a dengue outbreak in Ningbo City, Zhejiang Province, China.MethodsData of a dengue outbreak were collected in Ningbo City in China by a field epidemiological survey according to a strict protocol and case definition. Serum specimens of all cases were collected for diagnosis and the virological characteristics were detected by using polymerase chain reaction (PCR) and gene sequencing. Vector surveillance was implemented during the outbreak to collect the larva and adult mosquito densities to calculate the Breteau Index (BI) and human biting rate (HBR), respectively. Data of monthly BI and light-trap density in 2018 were built to calculate the seasonality of the vector. A transmission mathematical model was developed to dynamic the incidence of the disease. The parameters of the model were estimated by the data of the outbreak and vector surveillance data in 2018. The effectiveness of the interventions implemented during the outbreak was assessed by the data and the modelling.ResultsFrom 11 August to 8 September, 2018, a dengue outbreak was reported with 27 confirmed cases in a population of 5536-people community (community A) of Ningbo City. Whole E gene sequences were obtained from 24 cases and were confirmed as dengue virus type 1 (DENV-1). The transmission source of the outbreak was origin from community B where a dengue case having the same E gene sequence was onset on 30 July. Aedes albopictus was the only vector species in the area. The value of BI and HBR was 57.5 and 12 per person per hour respectively on 18 August, 2018 and decreased dramatically after interventions. The transmission model fitted well (χ2 = 6.324, P = 0.388) with the reported cases data. With no intervention, the total simulated number of the cases would be 1728 with a total attack rate (TAR) of 31.21% (95%CI: 29.99%– 32.43%). Case isolation and larva control (LC) have almost the same TAR and duration of outbreak (DO) as no intervention. Different levels of reducing HBR (rHBR) had different effectiveness with TARs ranging from 1.05% to 31.21% and DOs ranging from 27 days to 102 days. Adult vector control (AVC) had a very low TAR and DO. “LC+AVC” had a similar TAR and DO as that of AVC. “rHBR100%+LC”, “rHBR100%+AVC”, “rHBR100%+LC+AVC” and “rHBR100%+LC+AVC+Iso” had the same effectiveness.ConclusionsWithout intervention, DENV-1 could be transmitted rapidly within a short period of time and leads to high attack rate in community in China. AVC or rHBR should be recommended as primary interventions to control rapid transmission of the dengue virus at the early stage of an outbreak.

A37 Transmission success of dengue virus type 1 lineages in a dynamic virus population: An evolutionary view

Arbovirus transmission involves an interplay between host, virus, and environmental factors. Because of the complexity of interactions, the transmission success of arboviruses could either be a function of viral fitness or be stochastic. In the present study, using 1,963 envelope (E) gene sequences and 239 whole genomes, we conducted a large-scale molecular epidemiological analysis of a dengue virus type 1 (DENV-1) population to understand the transmission success, evolution, and dispersal patterns of different lineages of DENV-1 circulating in Singapore from 2011 to 2016. The study population was highly dynamic and heterogeneous. However, only a handful of genetically distinct strains (n = 6) established sustained transmission, but at variable levels of dominance. Phylogeographic analysis revealed a weak spatial clustering and 35 well-supported diffusion pathways, implying widespread and complex dispersal of these strains in local settings. Yet, the dominant strains were neither evolving faster than less dominant ones nor under positive selection. These observations suggested that lineage dominance was likely to be stochastic and opportunistically driven by non-viral factors such as host immune pressure and vector abundance. Our findings, therefore, emphasize the implications of understanding the vector and human factors in parallel to virus dynamics on continuing efforts to control the arbovirus disease transmission in endemic regions.

Spatio-temporal distribution analysis of circulating genotypes of dengue virus type 1 in western and southern states of India by a one-step real-time RT-PCR assay

Dengue virus type 1 (DENV-1) Asian and American/African (AM/AF) genotypes were reported to be co-circulating in southern and western states of India based on envelope (E) gene sequencing of few representative samples. The objective of the present study was to develop a one-step real-time RT-PCR to discriminate between Asian and AM/AF genotypes of DENV-1 and investigate the spatio-temporal distribution of the DENV-1 genotypes in southern and western states of India. A one-step real-time RT-PCR to discriminate the Asian and AM/AF genotypes of DENV-1 was developed and validated using 40 samples (17 Asian and 23 AM/AF), for which the envelope (E) gene sequence data was available. DENV-2, DENV-3 and DENV-4 isolates, one each and DENV negative samples (n = 17) were also tested by the assay. Additional 296 samples positive for DENV-1 from selected Southern and Western states of India were genotyped using the real-time RT-PCR assay. Among the samples used for validation, the genotyping results were concordant with sequencing results for 39 samples. In the one discordant sample which was positive for AM/AF by sequencing, the genotyping assay tested positive for both Asian and AM/AF genotype. DENV-2, DENV-3 and DENV-4 isolates were not reactive in the assay. None of the DENV negative samples were positive (sensitivity 100% and specificity 98.2%). A total of 336 samples (40 samples with sequence data and 296 samples without sequence data) were used for spatio-temporal distribution analysis. The results revealed that the Asian genotype was the predominant genotype in Tamil Nadu and Kerala, the southern states. The AM/AF genotype was the predominant genotype in Maharashtra, a western state of India. In Nashik district of Maharashtra, Asian genotype was observed in 32.6% of DENV-1 samples during 2017 while the same decreased to 7.3% during 2018. In Pune district, Asian genotype was observed in 40.0% of DENV-1 samples during 2018 only. To conclude, a one step real-time RT-PCR has been developed for discriminating Asian and AM/AF genotypes of DENV-1. This assay can act as a complement to sequencing but not a substitute and can be utilized in resource limited settings for molecular surveillance of DENV-1. DENV-1 Asian genotype was the dominant genotype in South India while, AM/AF genotype was dominant in Western India.

Intraperitoneal injection with dengue virus type 1-infected K562 cells results in complete fatality among immunocompetent mice

Dengue is one of the most important mosquito-borne viral diseases. Over half of the world's population is living in dengue endemic countries, where 100 million cases are estimated to occur annually. Although one dengue vaccine is currently available commercially, unfortunately its safety and efficacy has not been demonstrated for seronegative populations. Therefore, other vaccine candidates as well as antivirals are urgently required to control dengue diseases. To contribute to the development of preventative measures, in the present study we established an immunocompetent-mouse infection model using dengue virus type 1 Mochizuki strain. Following intraperitoneal injection with K562 cells infected with Mochizuki strain, all mice injected with ≥1 × 106 cells were killed within 7–11 days. Mice injected with ≥1 × 107 cells showed viremia (~104–105 FFU/ml) within 24 h of injection. Since a higher infective titer was detected in the mouse brain, this suggested that viruses were transmitted from the blood circulation into the brain. In further experiments, mice immunized with two types of DNA vaccines were challenged with virus. In contrast to the non-immunized control mice, all vaccinated mice survived after challenge. This immunocompetent-mouse infection model using dengue virus type 1 Mochizuki strain may be a useful tool to evaluate vaccines and preventive medicines against dengue virus.