DENV-2 Strains Research Articles

Genetic characterization of dengue virus serotype 1 circulating in Reunion Island, 2019–2021, and the Seychelles, 2015–2016

BackgroundAn unprecedent increase in the number of cases and deaths reported from dengue virus (DENV) infection has occurred in the southwestern Indian ocean in recent years. From 2017 to mid-2021 more than 70,000 confirmed dengue cases were reported in Reunion Island, and 1967 cases were recorded in the Seychelles from 2015 to 2016. Both these outbreaks displayed similar trends, with the initial circulation of DENV-2 which was replaced by DENV-1. Here, we aim to determine the origin of the DENV-1 epidemic strains and to explore their genetic characteristics along the uninterrupted circulation, particularly in Reunion.MethodsNucleic acids were extracted from blood samples collected from dengue positive patients; DENV-1 was identified by RT-qPCR. Positive samples were used to infect VERO cells. Genome sequences were obtained from either blood samples or infected-cell supernatants through a combination of both Illumina or MinION technologies.ResultsPhylogenetic analyses of partial or whole genome sequences revealed that all DENV-1 sequences from Reunion formed a monophyletic cluster that belonged to genotype I and were closely related to one isolate from Sri Lanka (OL752439.1, 2020). Sequences from the Seychelles belonged to the same major phylogenetic branch of genotype V, but fell into two paraphyletic clusters, with greatest similarity for one cluster to 2016–2017 isolate from Bangladesh, Singapore and China, and for the other cluster to ancestral isolates from Singapore, dating back to 2012. Compared to publicly available DENV-1 genotype I sequences, fifteen non-synonymous mutations were identified in the Reunion strains, including one in the capsid and the others in nonstructural proteins (NS) (three in NS1, two in NS2B, one in NS3, one in NS4B, and seven in NS5).ConclusionIn contrast to what was seen in previous outbreaks, recent DENV-1 outbreaks in Reunion and the Seychelles were caused by distinct genotypes, all likely originating from Asia where dengue is (hyper)endemic in many countries. Epidemic DENV-1 strains from Reunion harbored specific non-synonymous mutations whose biological significance needs to be further investigated.

Phylogenetic Reconstructions Reveal the Circulation of a Novel Dengue Virus-1V Clade and the Persistence of a Dengue Virus-2 III Genotype in Northeast Brazil.

Dengue fever is among the most significant public health concerns in Brazil. To date, the highest number of Dengue notifications in the Americas has been reported in Brazil, with cases accounting for a total number of 3,418,796 reported cases as of mid-December 2022. Furthermore, the northeastern region of Brazil registered the second-highest incidence of Dengue fever in 2022. Due to the alarming epidemiological scenario, in this study, we used a combination of portable whole-genome sequencing, phylodynamic, and epidemiological analyses to reveal a novel DENV-1 genotype V clade and the persistence of DENV-2 genotype III in the region. We further report the presence of non-synonymous mutations associated with non-structural domains, especially the NS2A (non-structural protein 2A), as well as describe synonymous mutations in envelope and membrane proteins, distributed differently between clades. However, the absence of clinical data at the time of collection and notification, as well as the impossibility of monitoring patients in order to observe worsening or death, restricts our possibility of correlating mutational findings with possible clinical prognoses. Together, these results reinforce the crucial role of genomic surveillance to follow the evolution of circulating DENV strains and understand their spread across the region through inter-regional importation events, likely mediated by human mobility, and also the possible impacts on public health and outbreak management.

Transient Blockade of Type I Interferon Signalling Promotes Replication of Dengue Virus Strain D2Y98P in Adult Wild-Type Mice.

Dengue virus serotypes 1 to 4 (DENV1-4) place nearly half the global population at risk of infection and the licenced tetravalent dengue vaccine fails to protect individuals who have not previously been exposed to DENV. The development of intervention strategies had long been hampered by the lack of a suitable small animal model. DENV does not replicate in wild-type mice due to its inability to antagonise the mouse type I interferon (IFN) response. Mice deficient in type I IFN signalling (Ifnar1-/- mice) are highly susceptible to DENV infection, but their immunocompromised status makes it difficult to interpret immune responses elicited by experimental vaccines. To develop an alternative mouse model for vaccine testing, we treated adult wild-type mice with MAR1-5A3-an IFNAR1-blocking, non-cell-depleting antibody-prior to infection with the DENV2 strain D2Y98P. This approach would allow for vaccination of immunocompetent mice and subsequent inhibition of type I IFN signalling prior to challenge infection. While Ifnar1-/- mice quickly succumbed to infection, MAR1-5A3-treated mice did not show any signs of illness but eventually seroconverted. Infectious virus was recovered from the sera and visceral organs of Ifnar1-/- mice, but not from those of mice treated with MAR1-5A3. However, high levels of viral RNA were detected in the samples of MAR1-5A3-treated mice, indicating productive viral replication and dissemination. This transiently immunocompromised mouse model of DENV2 infection will aid the pre-clinical assessment of next-generation vaccines as well as novel antiviral treatments.

Diagnosis of Dengue Virus Infections Imported to Hungary and Phylogenetic Analysis of Virus Isolates

Dengue virus is one of the most important arbovirus infections of public health concern. Between 2017 and June 2022, 75 imported dengue infections were confirmed by laboratory diagnostic methods in Hungary. Our study aimed to isolate the imported Dengue strains and characterize them by whole-genome sequencing. Laboratory diagnosis of imported infections was carried out using both serological and molecular methods. Virus isolation was attempted on Vero E6 cell lines. An in-house amplicon-based whole-genome sequencing method was applied for the detailed molecular characterization of the isolated virus strains. From 75 confirmed Dengue infected patients, 68 samples were used for virus isolation. Isolation and whole-genome sequencing were successful in the case of eleven specimens. Isolated strains belonged to Dengue-1,-2,-3 serotypes. The isolated strains corresponded to the circulating genotypes of the visited geographic area, and some of the genotypes were linked with more severe DENV cases in the literature. We found that multiple factors, including viral load, specimen type, and patient antibody status, influence the isolation efficacy. Analysis of imported DENV strains can help estimate the outcomes of a possible local DENV transmission in Hungary, a threat from the near future.

Stability of Dengue 2 Nonstructural Glycoprotein 1 (NS1) Is Affected by the Nature of Basic Residue at Position NS1-324.

Dengue is the most prevalent mosquito-borne viral disease. It is caused by the infection of any of the four dengue virus (DENV) serotypes DENV-1 to DENV-4. The DENV non-structural glycoprotein 1 (NS1) plays an important role in virus replication and the immunopathogenesis of virus infection. The NS1 protein has been identified as both a cell-associated homodimer and a soluble secreted lipoprotein nanoparticle. The nature of the residues at positions NS1-272 and NS1-324 in the β-ladder domain may have an effect on the biological behaviors of DENV-2 NS1 protein in human hepatoma Huh7 cells. The stability of the NS1 protein from the Reunion 2018 DENV-2 strain was affected by the presence of lysine residues at positions 272 and 324. In the present study, we evaluated the impact of mutations into lysine at positions 272 and 324 on recombinant NS1 protein from the DES-14 DENV-2 strain bearing arginine residue on these two positions. The DES-14 NS1 protein mutant bearing a lysine at position 324 was deficient in protein stability and secretion compared to wild-type protein. The defect in the DES-14 NS1 protein mutant was associated to oxidative stress and pro-inflammatory cytokine activation in Huh7 cells. The ubiquitin-proteasome proteolytic pathway might play a key role in the stability of DENV-2 protein bearing a lysine residue at position 324.

Construction and Rescue of a DNA-Launched DENV2 Infectious Clone.

Flaviviruses represent a large group of globally significant, insect-borne pathogens. For many of these viruses, there is a lack of antivirals and vaccines. Thus, there is a need to continue the development of tools to further advance our efforts to combat these pathogens, including reverse genetics techniques. Traditionally, reverse genetics methods for flaviviruses rely on producing infectious RNA from in vitro transcription reactions followed by electroporation or transfection into permissive cell lines. However, the production of Zika virus has been successful from CMV promoter-driven expression plasmids, which provides cost and time advantages. In this report, we describe the design and construction of a DNA-launched infectious clone for dengue virus (DENV) serotype 2 strain 16681. An artificial intron was introduced in the nonstructural protein 1 segment of the viral genome to promote stability in bacteria. We found that rescued viruses maintained the ability to form plaques and replicate efficiently in commonly used cell lines. Thus, we present a rapid and cost-effective method for producing DENV2 strain 16681 from plasmid DNA. This construct will be a useful platform for the continued development of anti-DENV therapeutics and vaccines.

A Six Years (2010–2016) Longitudinal Survey of the Four Serotypes of Dengue Viruses in Lao PDR

Dengue fever is the most prevalent arthropod-borne viral infection of humans in tropical and subtropical countries. Since 1979, dengue has been reported to be endemic in the Lao People's Democratic Republic (PDR), as in many countries in Southeast Asia, with a complex circulation of the four dengue viruses' serotypes (DENV-1 to DENV-4). By sequencing the complete envelope protein, we explored a panel of samples from five Lao Provinces (Vientiane capital, Luangprabang, Bolikhamxay, Saravane, Attapeu) to enrich knowledge about the co-circulation of DENVs in Lao PDR between 2010 and 2016. Phylogenetic analyses highlighted the specific circulation of DENV-1 genotype I, DENV-2 genotype Asian I, DENV-4 genotype I and the co-circulation of DENV-3 genotype II and III. The continuous co-circulation of the four serotypes was underlined, with genotype or cluster shifts among DENV-3 and DENV-1. These data suggested the emergence or re-emergence of DENV strains associated with epidemic events, potentially linked to the exchanges within the territory and with neighboring countries. Indeed, the increasing local or regional connections favored the dissemination of new isolates or new clusters around the country. Since 2012, the surveillance and alert system created in Vientiane capital by the Institut Pasteur du Laos appears to be a strategic tool for monitoring the circulation of the four serotypes, especially in this endemic country, and allows for improving dengue epidemiological knowledge to anticipate epidemic events better.

Syncytial and Congregative Effects of Dengue and Zika Viruses on the Aedes Albopictus Cell Line Differ among the Viral Strains.

Dengue viruses (DENV) and Zika viruses (ZIKV) are transmitted from human to human or from non-human primates to humans by mosquito biting, so the viral interaction with mosquito cells is one key step within the viral life cycle. Therefore, our objective is to know how DENV or ZIKV interacts with mosquito cells. Immunofluorescence assay and a direct visualization system are combined to monitor the syncytial or congregative effects of DENVs and ZIKVs on C6/36 cells. we studied the cytopathic effects of DENVs and ZIKVs on the mosquito cells, C6/36 which are widely used in the laboratory for the infections of DENV and ZIKV. Our results show that all strains of DENV-1 and DENV-2, most DENV-4 and some DENV-3 strains caused syncytial effects on C6/36 cells, while some DENV-3 and DENV-4 strains, and all the tested ZIKV strains caused cell congregation after infection but no cell fusion. In addition, we detected a range of pH environments from 6.0 to 8.0 that support the virus-caused cell fusion and figured out that the optimal pH condition is 7.5 at which the viral production is also the best. Furthermore, viral replication may be required for DENV's syncytial effects on C6/36 cells because the UV-inactivated virus failed to cause cell fusion. Syncytial and congregative effects of DENV and ZIKV on the Aedes albopictus cells differ among the viral strains. Syncytial effects of DENV on C6/36 are important for viral replication.

Re-Emergence of Dengue Serotype 3 in the Context of a Large Religious Gathering Event in Touba, Senegal.

Dengue virus (DENV) was detected in Senegal in 1979 for the first time. Since 2017, unprecedented frequent outbreaks of DENV were noticed yearly. In this context, epidemiological and molecular evolution data are paramount to decipher the virus diffusion route. In the current study, we focused on a dengue outbreak which occurred in Senegal in 2018 in the context of a large religious gathering with 263 confirmed DENV cases out of 832 collected samples, including 25 life-threatening cases and 2 deaths. It was characterized by a co-circulation of dengue serotypes 1 and 3. Phylogenetic analysis based on the E gene revealed that the main detected serotype in Touba was DENV-3 and belonged to Genotype III. Bayesian phylogeographic analysis was performed and suggested one viral introduction around 2017.07 (95% HPD = 2016.61-2017.57) followed by cryptic circulation before the identification of the first case on 1 October 2018. DENV-3 strains are phylogenetically related, with strong phylogenetic links between strains retrieved from Burkina Faso and other West African countries. These phylogenetic data substantiate epidemiological data of the origin of DENV-3 and its spread between African countries and subsequent diffusion after religious mass events. The study also highlighted the usefulness of a mobile laboratory during the outbreak response, allowing rapid diagnosis and resulting in improved patient management.

Analysis of a Dengue Virus Outbreak in Rosso, Senegal 2021.

Senegal is hyperendemic for dengue. Since 2017, outbreaks have been noticed annually in many regions around the country, marked by the co-circulation of DENV1-3. On 8 October 2021, a Dengue virus outbreak in the Rosso health post (sentinel site of the syndromic surveillance network) located in the north of the country was notified to the WHO Collaborating Center for arboviruses and hemorrhagic fever viruses at Institut Pasteur de Dakar. A multidisciplinary team was then sent for epidemiological and virologic investigations. This study describes the results from investigations during an outbreak in Senegal using a rapid diagnostic test (RDT) for the combined detection of dengue virus non-structural protein 1 (NS1) and IgM/IgG. For confirmation, samples were also tested by real-time RT-PCR and IgM ELISA at the reference lab in Dakar. qRT-PCR positive samples were subjected to whole genome sequencing using nanopore technology. Virologic analysis scored 102 positives cases (RT-PCR, NS1 antigen detection and/or IgM) out of 173 enrolled patients; interestingly, virus serotyping showed that the outbreak was caused by the DENV-1, a serotype different from DENV-2 involved during the outbreak in Rosso three years earlier, indicating a serotype replacement. Nearly all field-tested NS1 positives samples were confirmed by qRT-PCR with a concordance of 92.3%. Whole genome sequencing and phylogenetic analysis of strains suggested a re-introduction in Rosso of a DENV-1 strain different to the one responsible for the outbreak in the Louga area five years before. Findings call for improved dengue virus surveillance in Senegal, with a wide deployment of DENV antigenic tests, which allow easy on-site diagnosis of suspected cases and early detection of outbreaks. This work highlights the need for continuous monitoring of circulating serotypes which is crucial for a better understanding of viral epidemiology around the country.

Complete genome sequences and phylogenetic analysis of dengue virus in Southern Vietnam during 2014-2015

Dengue is an infectious disease that causes a worldwide health and economic burden despite the efforts to eradicate the disease. From 2013 to 2015, dengue epidemic significantly increased from 33,626 to 50,205 cases in Vietnam. This study aims to determine the genotype variations of dengue virus (DENV) circulating in Southern Vietnam during 2014-2015. C6/36 cells were infected with twenty-four strains of dengue virus isolated in 2014-2015 and kept frozen. The complete nucleotide sequence of dengue virus genomes was obtained by polymerase chain reaction (PCR). The genome was sequenced in the MiSeq system and analyzed by the basic local alignment search tool (BLAST) program. Data from GeneBank was used to create the phylogenetic trees. Among the 17 analyzed strains from 8 southern provinces, four (23.53%) were DENV-1, three (17.65%) were DENV-2, five (29.41%) were DENV-3, and five (29.41%) DENV-4 were isolated. Four DENV-1 isolates belong to Asia genotype. Three DENV-2 strains were concentrated in a subgroup of Asian 1 genotype. Five DENV-3 isolates were identified as belonged to Asian 2 genotype and five DENV-4 isolates were found as belong to Asia 1 genotype. There were no amino acid mutations and the transition capacity between the nucleotide among four types of DENV serotypes suggested that the probability of conversion from C to T was the highest conversion rate. These DENV isolates were genetically close to other previous strains isolated from Vietnam and its neighboring countries, including Thailand, China, Cambodia, and Singapore, Brazil, Sri Lanka due to dynamic transmission.

Evolution of inflammation and immunity in a dengue virus 1 human infection model.

Dengue virus (DENV) infections are major causes of morbidity and mortality throughout the tropics and subtropics. More than 400 million infections are estimated to occur every year, resulting in nearly 100 million symptomatic infections and more than 20,000 deaths. Early immune response kinetics to infection remain unclear, in large part due to the variable incubation period exhibited by the DENVs after introduction into a susceptible host. To fill this knowledge gap, we performed a comprehensive virologic and immunologic analysis of individuals experimentally infected with the underattenuated DENV-1 strain 45AZ5. This analysis captured both the kinetics and composition of the innate, humoral, and cellular immune responses elicited by experimental DENV-1 infection, as well as virologic and clinical features. We observed a robust DENV-specific immunoglobulin A (IgA) antibody response that manifested between the appearance of DENV-specific IgM and IgG in all challenged individuals, as well as the presence of a non-neutralizing/NS1-specific antibody response that was delayed relative to the appearance of DENV virion-specific humoral immunity. RNA sequencing analysis revealed discrete and temporally restricted gene modules that correlated with acute viremia and the induction of adaptive immunity. Our analysis provides a detailed description, in time and space, of the evolving matrix of DENV-elicited human inflammation and immunity and reveals several previously unappreciated immunological aspects of primary DENV-1 infection that can inform countermeasure development and evaluation.

A unified global genotyping framework of dengue virus serotype-1 for a stratified coordinated surveillance strategy of dengue epidemics

BackgroundDengue is the fastest spreading arboviral disease, posing great challenges on global public health. A reproduceable and comparable global genotyping framework for contextualizing spatiotemporal epidemiological data of dengue virus (DENV) is essential for research studies and collaborative surveillance.MethodsTargeting DENV-1 spreading prominently in recent decades, by reconciling all qualified complete E gene sequences of 5003 DENV-1 strains with epidemiological information from 78 epidemic countries/areas ranging from 1944 to 2018, we established and characterized a unified global high-resolution genotyping framework using phylogenetics, population genetics, phylogeography, and phylodynamics.ResultsThe defined framework was discriminated with three hierarchical layers of genotype, subgenotype and clade with respective mean pairwise distances 2–6%, 0.8–2%, and ≤ 0.8%. The global epidemic patterns of DENV-1 showed strong geographic constraints representing stratified spatial-genetic epidemic pairs of Continent-Genotype, Region-Subgenotype and Nation-Clade, thereby identifying 12 epidemic regions which prospectively facilitates the region-based coordination. The increasing cross-transmission trends were also demonstrated. The traditional endemic countries such as Thailand, Vietnam and Indonesia displayed as persisting dominant source centers, while the emerging epidemic countries such as China, Australia, and the USA, where dengue outbreaks were frequently triggered by importation, showed a growing trend of DENV-1 diffusion. The probably hidden epidemics were found especially in Africa and India. Then, our framework can be utilized in an accurate stratified coordinated surveillance based on the defined viral population compositions. Thereby it is prospectively valuable for further hampering the ongoing transition process of epidemic to endemic, addressing the issue of inadequate monitoring, and warning us to be concerned about the cross-national, cross-regional, and cross-continental diffusions of dengue, which can potentially trigger large epidemics.ConclusionsThe framework and its utilization in quantitatively assessing DENV-1 epidemics has laid a foundation and re-unveiled the urgency for establishing a stratified coordinated surveillance platform for blocking global spreading of dengue. This framework is also expected to bridge classical DENV-1 genotyping with genomic epidemiology and risk modeling. We will promote it to the public and update it periodically.Graphical

Recurrent Episodes of Some Mosquito-Borne Viral Diseases in Nigeria: A Systematic Review and Meta-Analysis.

Different ecological zones favor the breeding of Aedes species. The molecular epidemiology of dengue virus (DENV), yellow fever virus (YFV), and Chikungunya virus (CHIKV) was determined from outbreaks and surveillance activities in Nigeria. Twenty-eight DENV, twenty-five YFV, and two CHIKV sequences from Nigeria were retrieved from GenBank. Genotyping was performed with a genome detective typing tool. The evolutionary comparison was performed by the Maximum Likelihood method on MEGA. Chi-square was used to compare the association between the proportions of viral infections at different times. Six DENV-1 were detected in 1964, 1965, 1978, 2007, and 2018. Nineteen DENV-2 strains were reported, four belonging to sylvatic VI, one belonging to cosmopolitan II, and twelve to Asian I genotype V. DENV-2 genotype VI was detected in 1966, and genotypes II and V in 2019. All three DENV-3 were detected in 2018, while only one DENV-4 was identified in 2019. YFV was reported in 1946 and then in the 60s, 70s, 80s, 90s, 2018, and 2019 with reports to date. CHIKV is still circulating following its identification in 1964 and 1965. Recurrent episodes of dengue, Chikungunya, and yellow fever continue unabated. Vector control initiatives and immunization should be greatly sustained.

Evolution of inflammation and immunity in a dengue virus 1 human infection model

Abstract Dengue virus (DENV) is a significant source of morbidity and mortality throughout the tropics and subtropics. Over 400 million infections are thought to occur every year, resulting in nearly 100 million symptomatic infections and over 20,000 deaths. Early immune response kinetics to DENV infection remain unclear in large part due to the highly variable incubation period exhibited by the virus after introduction into a susceptible host. To fill this knowledge gap, we performed a comprehensive virologic and immunologic analysis of individuals experimentally infected with an under-attenuated DENV-1 strain 45AZ5. This analysis captured both the kinetics and composition of the innate, humoral, and cellular immune response elicited by experimental DENV-1 challenge, as well as the virologic and clinical feature of DENV-1 infection. Revealed in this analysis was an unexpectedly robust DENV-specific IgA antibody response that manifested between the appearance of DENV-specific IgM and IgG in all challenged individuals, as well as the presence of a non-neutralizing/NS1-specific antibody response that was delayed relative to the appearance of DENV-virion specific humoral immunity. RNAseq analysis also revealed several distinct and temporally-restricted gene modules that allowed for the identification and differentiation of the innate and adaptive immune response to DENV-infection. Our analysis provides a detailed description, in time and space, of the evolving matrix of DENV-elicited human inflammation and immunity, and reveals several previously unappreciated immunological aspects of primary DENV-1 infection.

Clinical Correlation of Dengue Strains on the Basis of Seroprevalence in a Tertiary Care Hospital.

Dengue viruses are icosahedral in structure and contain a single-stranded positive- sense RNA sequence of 11kb inside their capsid protein, which belongs to Flaviviridae family, genera Flavivirus. DENV a vector dependent viral virus which presents a severe health danger worldwide. Objective: To study the different strains of dengue on the basis of serotypes Methods: A cross sectional study was conducted at a tertiary care hospital Lahore, Primary and Secondary Health Department Lahore. The blood samples of 103 patients were collected from non-random sampling technique to check out the data of different parameters such as WBC’s & Platelets through Complete Blood Count (CBC), NS1 from ELISA and nature of dengue strains through RT- PCR. Results: Of 103 positive dengue patients, there were n= 58 (33.63 ± 16.54) males and n=45 (40.64± 16.00) females. Data for the total patients is subjected to statistical differences by Paired t-test (*p<0.05). The overall percentage of dengue strains within the sample population was DENV-2 (96%), DENV-3 (2%), DENV-1 & DENV-2 (2% in females, nil in males) DENV-2 & DENV-3 (2% both in males and females). According to clinical parameters the correlation of ELISA results with WBC’s was significant (<0.0001) as well as ELISA and platelets of patients had no correlation with each other. Conclusions: Leukopenia and Thrombocytopenia is found particularly in DENV-2 strain as well as we found two different strains in two patients. So, our research work is helpful for the identification in genetic similarity of dengue strains.

March 2019 dengue fever outbreak at the Kenyan south coast involving dengue virus serotype 3, genotypes III and V.

The first description of a disease resembling dengue fever (DF) was in the 15th century slave trade era by Spanish sailors visiting the Tanzania coast. The disease, then associated with evil spirits is now known to be caused by four serotypes of dengue virus (DENV1-4) that are transmitted by Aedes mosquitoes. Kenya has experienced multiple outbreaks, mostly associated with DENV-2. In this study, plasma samples obtained from 37 febrile patients during a DF outbreak at Kenya's south coast in March 2019 were screened for DENV. Total RNA was extracted and screened for the alpha- and flavi-viruses by real-time polymerase chain reaction (qPCR). DENV-3 was the only virus detected. Shotgun metagenomics and targeted sequencing were used to obtain DENV whole genomes and the complete envelope genes (E gene) respectively. Sequences were used to infer phylogenies and time-scaled genealogies. Following Maximum likelihood and Bayesian phylogenetic analysis, two DENV-3 genotypes (III, n = 15 and V, n = 2) were found. We determined that the two genotypes had been in circulation since 2015, and that both had been introduced independently. Genotype III's origin was estimated to have been from Pakistan. Although the origin of genotype V could not be ascertained due to rarity of these sequences globally, it was most related to a 2006 Brazilian isolate. Unlike genotype III that has been described in East and West Africa multiple times, this was the second description of genotype V in Kenya. Of note, there was marked amino acid variances in the E gene between study samples and the Thailand DENV-3 strain used in the approved Dengvaxia vaccine. It remains to be seen whether these variances negatively impact the efficacy of the Dengvaxia or future vaccines.

Clinical Characteristics and Risk Factors for Severe Dengue Fever in Xishuangbanna, During the Dengue Outbreak in 2019

BackgroundDengue poses a large burden on the public health systems worldwide. severe dengue (SD) could lead to more serious clinical symptoms and even death. This study aimed to identify the cause of SD in a clinical trial during the dengue outbreak in Xishuangbanna in 2019, and could provide new insights into the pathogenic mechanisms of SD.MethodsMosquito-borne viral (DENV, JEV, and CHIKV) infections were identified. The epidemiological factors and clinical symptoms of inpatients in Xishuangbanna were recorded. The IgG and IgM levels in the serum of dengue inpatients were evaluated, and secondary infections were identified. Then, the structural proteins (C/PrM/E) were sequenced and compared with those of the same type of DENV in the same area as before, and their structures were predicted by the SWISS-MODEL (expasy.org). The full-length viral genomes were sequenced and aligned with representative strains by BioEidt or MEGA 5.0.ResultsIn this outbreak, the clinical symptoms were more serious in SD. The proportion of SD inpatients of male and Han nationality was larger than that of dengue fever (DF) inpatients (p < 0.05). DENV-2 infection was the majority in DF, with 45 inpatients. However, DENV-1 infection was the most common SD, with 54 inpatients. There were 3 DENV-3-positive inpatients in the DF group and 6 ZIKV-positive inpatients in the SD group. A secondary infection accounted for 76.47% (78 cases) of SD inpatients, but secondary infections were only in 20% (17 cases) of DF inpatients. In the three-dimensional structure of protein analysis, the C/PrM/E of DENV-1 and DENV-2 showed more stability than previous epidemic strains, while DENV-3 in 2019 showed a looser spatial structure. After a complete genome sequencing and analysis, all six DENV-2 strains belonged to cosmopolitan, five of which clustered into one branch. The GC/AT of the five strains decreased from 2014 to 2018. Compared with DF strains, SD strains had no mutations of commonness.ConclusionsSD may related to secondary heteromorphic dengue in Xishuangbanna in 2019. The coinfection of ZIKV could be another related factor for SD. The currently datas were very limited and only suggestive.

Molecular detection and characterization of dengue isolates circulating in north India.

Background and Objectives:In recent decades, the incidence of dengue has increased dramatically. In dengue-endemic countries, changes in dengue virus serotypes, genotypes, and lineages have been reported. This study was designed to detect and characterize the dengue virus isolates circulating in North India by serological and molecular techniques.Materials and Methods:This study was conducted at the Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. NS1 antigen and IgM antibody against dengue were detected by ELISA methods, viral RNA was extracted and amplified by conventional PCR and one-step single-tube multiplex PCR. The purified PCR products were cycle sequenced and a database search was implemented for the confirmation of the sequence product. Phylogenetic analysis was carried out with previously reported sequences.Results:Among 1509 samples, 205 (13.6%) were found positive for IgM antibodies with the highest number (n=67) among the 21 to 30 years age group with peak positivity during post-monsoon months. Among acute samples, NS1 antigen was positive in 62.9%. Seven patients out of 13 had dengue viral RNA in PCR. It comprised six DENV-2 serotypes and one DENV-3 serotype. On phylogenetic analysis, DENV-2 strains grouped with genotype IV and DENV-3 with genotype III.Conclusion:Dengue infection was found frequently during post-monsoon season. The positivity rate of the dengue NS1 antigen test was greater than that of the antibody test. The dengue isolates were characterized as genotype IV and genotype III of DENV-2 and DENV-3 respectively.

Infection of Human Retinal Pigment Epithelial Cells with Dengue Virus Strains Isolated during Outbreaks in Singapore.

Prevalence of dengue retinopathy varies across epidemics, with the disease linked to circulation of dengue virus serotype 1 (DENV-1). The retinal pigment epithelium has been implicated in the pathology. We investigated infectivity, molecular response, and barrier function of epithelial cells inoculated with DENV strains from different outbreaks in Singapore. Monolayers of human retinal pigment epithelial cells (multiple primary cell isolates and the ARPE-19 cell line) were inoculated with six DENV strains, at multiplicity of infection of 10; uninfected and recombinant strain-infected controls were included where relevant. Infectivity and cell response were assessed primarily by RT-qPCR on total cellular RNA, and barrier function was evaluated as electrical resistance across monolayers. Higher viral RNA loads were measured in human retinal pigment epithelial cells infected with DENV-1 strains from the 2005 Singapore epidemic, when retinopathy was prevalent, versus DENV-1 strains from the 2007 Singapore epidemic, when retinopathy was not observed. Type I interferon (IFN) transcripts (IFN-β and multiple IFN-stimulated genes) were up-regulated, and impact on barrier function was more pronounced, for cells infected with DENV-1 strains from the 2005 versus the 2007 Singapore epidemics. Aside from serotype, strain of DENV may determine the potential to induce retinal pathology. Identification of molecular markers of disease-associated DENV strains may provide insights into the pathogenesis of dengue retinopathy.