Unraveling Dengue Virus Diversity in Asia: An Epidemiological Study through Genetic Sequences and Phylogenetic Analysis.

Dengue virus (DENV) is the causative agent of dengue. Although most infected individuals are asymptomatic or present with only mild symptoms, severe manifestations could potentially devastate human populations in tropical and subtropical regions. In hyperendemic regions such as South Asia and Southeast Asia (SEA), all four DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) have been prevalent for several decades. Each DENV serotype is further divided into multiple genotypes, reflecting the extensive diversity of DENV. Historically, specific DENV genotypes were associated with particular geographical distributions within endemic regions. However, this epidemiological pattern has changed due to urbanization, globalization, and climate change. This review comprehensively traces the historical and recent genetic epidemiology of DENV in Asia from the first time DENV was identified in the 1950s to the present. We analyzed envelope sequences from a database covering 16 endemic countries across three distinct geographic regions in Asia. These countries included Bangladesh, Bhutan, India, Maldives, Nepal, Pakistan, and Sri Lanka from South Asia; Cambodia, Laos, Myanmar, Thailand, and Vietnam from Mainland SEA; and Indonesia, the Philippines, Malaysia, and Singapore from Maritime SEA. Additionally, we describe the phylogenetic relationships among DENV genotypes within each serotype, along with their geographic distribution, to enhance the understanding of DENV dynamics.

Serotype and genomic diversity of dengue virus during the 2023 outbreak in Pakistan reveals the circulation of genotype III of DENV-1 and cosmopolitan genotype of DENV-2.

Dengue, a mosquito-borne viral disease, poses a significant public health challenge in Pakistan, with a significant outbreak in 2023, prompting our investigation into the serotype and genomic diversity of the dengue virus (DENV). NS-1 positive blood samples from 153 patients were referred to the National Institute of Health, Pakistan, between July and October 2023. Among these, 98 (64.1%) tested positive using multiplex real-time PCR, with higher prevalence among males (65.8%) and individuals aged 31-40. Serotyping revealed DENV-1 as the predominant serotype (84.7%), followed by DENV-2 (15.3%). Whole-genome sequencing of 18 samples (DENV-1 = 17, DENV-2 = 01) showed that DENV-1 (genotype III) samples were closely related (>99%) to Pakistan outbreak samples (2022), and approx. > 98% with USA (2022), Singapore and China (2016), Bangladesh (2017), and Pakistan (2019). The DENV-2 sequence (cosmopolitan genotype; clade IVA) shared genetic similarity with Pakistan outbreak sequences (2022), approx. > 99% with China and Singapore (2018-2019) and showed divergence from Pakistan sequences (2008-2013). No coinfection with dengue serotypes or other viruses were observed. Comparisons with previous DENV-1 sequences highlighted genetic variations affecting viral replication efficiency (NS2B:K55R) and infectivity (E:M272T). These findings contribute to dengue epidemiology understanding and underscore the importance of ongoing genomic surveillance for future outbreak responses in Pakistan.

DENV-2 Outbreak Associated With Cosmopolitan Genotype Emergence in Western Brazilian Amazon.

Background: Dengue virus (DENV) causes an important disease and directly affects public health, being the arbovirus that presents the highest number of infections and deaths in the Western Brazilian Amazon. This virus is divided into 4 serotypes that have already circulated in the region. Methodology: Molecular characterization of a cohort containing 841 samples collected from febrile patients between 2021 and 2023 was analyzed using a commercial kit to detect the main arboviruses circulating in Brazil: Zika, DENV-1, DENV-2, DENV-3, DENV-4 and, Chikungunya. Subsequently, Sanger sequencing was performed for positive samples. Results: The cohort detected 162 positive samples, 12 for DENV-1 and 150 identified as DENV-2, indicating co-circulation of serotypes. The samples were subjected to sequencing and the analysis of the sequences that obtained good quality revealed that 5 samples belonged to the V genotype of DENV-1 and 46 were characterized as DENV-2 Cosmopolitan genotype-lineage 5. Conclusion: The results allowed us to identify for the first time the Cosmopolitan genotype in Rondônia, Brazilian Western Amazon, and its fast spread dispersion.

Case report: dengue fever associated acute macular neuroretinopathy.

Dengue fever (DF), which is caused by the dengue virus (DENV) and transmitted through Aedes mosquitoes, is well recognized for its systemic manifestations, with its ocular involvement gaining recent attention. We present a case of a 41-year-old Taiwanese female who developed acute macular neuroretinopathy (AMN) following a DF diagnosis related to DENV-1, emphasizing the need for awareness of this complication. The patient, with a history of completely resolved optic neuritis (ON) and comorbidities, experienced blurred vision on day 10 after the onset of DF. The ophthalmic examination revealed macular edema, ellipsoid zone (EZ) infiltration, and choriocapillaris involvement. Despite pulse therapy with corticosteroids, visual disturbances persisted, highlighting the challenge of managing ocular complications. Ocular manifestations in DF include hemorrhages, inflammation, and vascular complications. DF-associated AMN, a rare presentation, poses challenges in diagnosis and treatment response evaluation. While most patients recover spontaneously, some face persistent visual impairment, especially with AMN. Our case emphasizes the importance of recognizing ocular complications in DF, necessitating a multidisciplinary approach for optimal management and further research to delineate treatment strategies and outcomes.

Sofosbuvir Suppresses the Genome Replication of DENV1 in Human Hepatic Huh7 Cells.

Dengue virus (DENV) causes dengue fever and dengue hemorrhagic fever, and DENV infection kills 20,000 people annually worldwide. Therefore, the development of anti-DENV drugs is urgently needed. Sofosbuvir (SOF) is an effective drug for HCV-related diseases, and its triphosphorylated metabolite inhibits viral RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of HCV. (2'R)-2'-Deoxy-2'-fluoro-2'-methyluridine (FMeU) is the dephosphorylated metabolite produced from SOF. The effects of SOF and FMeU on DENV1 replication were analyzed using two DENV1 replicon-based methods that we previously established. First, a replicon-harboring cell assay showed that DENV1 replicon replication in human hepatic Huh7 cells was decreased by SOF but not by FMeU. Second, a transient replicon assay showed that DENV1 replicon replication in Huh7 cells was decreased by SOF; however, in hamster kidney BHK-21 cells, it was not suppressed by SOF. Additionally, the replicon replication in Huh7 and BHK-21 cells was not affected by FMeU. Moreover, we assessed the effects of SOF on infectious DENV1 production. SOF suppressed infectious DENV1 production in Huh7 cells but not in monkey kidney Vero cells. To examine the substrate recognition of the HCV and DENV1 RdRps, the complex conformation of SOF-containing DENV1 RdRp or HCV RdRp was predicted using AlphaFold 2. These results indicate that SOF may be used as a treatment for DENV1 infection.

Phylogenetics, Epidemiology and Temporal Patterns of Dengue Virus in Araraquara, São Paulo State.

Dengue virus (DENV) is a prominent arbovirus with global spread, causing approximately 390 million infections each year. In Brazil, yearly epidemics follow a well-documented pattern of serotype replacement every three to four years on average. Araraquara, located in the state of São Paulo, has faced significant impacts from DENV epidemics since the emergence of DENV-1 in 2010. The municipality then transitioned from low to moderate endemicity in less than 10 years. Yet, there remains an insufficient understanding of virus circulation dynamics, particularly concerning DENV-1, in the region, as well as the genetic characteristics of the virus. To address this, we sequenced 37 complete or partial DENV-1 genomes sampled from 2015 to 2022 in Araraquara. Then, using also Brazilian and worldwide DENV-1 sequences we reconstructed the evolutionary history of DENV-1 in Araraquara and estimated the time to the most recent common ancestor (tMRCA) for serotype 1, for genotype V and its main lineages. Within the last ten years, there have been at least three introductions of genotype V in Araraquara, distributed in two main lineages (L Ia and L Ib, and L II). The tMRCA for the first sampled lineage (2015/2016 epidemics) was approximately 15 years ago (in 2008). Crucially, our analysis challenges existing assumptions regarding the emergence time of the DENV-1 genotypes, suggesting that genotype V might have diverged more recently than previously described. The presence of the two lineages of genotype V in the municipality might have contributed to the extended persistence of DENV-1 in the region.

DENV-1 Titer Impacts Viral Blocking in wMel Aedes aegypti with Brazilian Genetic Background.

Several countries have been using Wolbachia deployments to replace highly competent native Aedes aegypti populations with Wolbachia-carrying mosquitoes with lower susceptibility to arboviruses such as dengue, Zika, and chikungunya. In Rio de Janeiro, Wolbachia deployments started in 2015 and still present a moderate introgression with a modest reduction in dengue cases in humans (38%). Here, we evaluated the vector competence of wild-type and wMel-infected Ae. aegypti with a Brazilian genetic background to investigate whether virus leakage could contribute to the observed outcomes in Brazil. We collected the specimens in three areas of Rio de Janeiro with distinct frequencies of mosquitoes with wMel strain and two areas with wild Ae. aegypti. The mosquitoes were orally exposed to two titers of DENV-1 and the saliva of DENV-1-infected Ae. aegypti was microinjected into wMel-free mosquitoes to check their infectivity. When infected with the high DENV-1 titer, the presence of wMel did not avoid viral infection in mosquitoes' bodies and saliva but DENV-1-infected wMel mosquitoes produced lower viral loads than wMel-free mosquitoes. On the other hand, wMel mosquitoes infected with the low DENV-1 titer were less susceptible to virus infection than wMel-free mosquitoes, although once infected, wMel and wMel-free mosquitoes exhibited similar viral loads in the body and the saliva. Our results showed viral leakage in 60% of the saliva of wMel mosquitoes with Brazilian background; thus, sustained surveillance is imperative to monitor the presence of other circulating DENV-1 strains capable of overcoming the Wolbachia blocking phenotype, enabling timely implementation of action plans.

Dengue 1 outbreak in Rosso, northern Senegal, October 2021: entomologic investigations.

Senegal has experienced periodic epidemics of dengue in urban areas with increased incidence in recent years. However, few data are available on the local ecology of the epidemic vectors. In October 2021, a dengue outbreak was reported in northern Senegal to the Institute Pasteur de Dakar. Entomologic investigations then were undertaken to identify the areas at risk of transmission and to identify the vector(s). Adult mosquitoes were collected indoors and outdoors at selected households, while containers with water were inspected for mosquito larvae. All the Aedes aegypti (L.) collected were tested for dengue virus NS1 protein using a rapid diagnostic test (RDT), and positive samples were confirmed by real-time RT-PCR. The qRT-PCR positive samples were subjected to whole genome sequencing using Nanopore technology. The majority of the larvae-positive containers (83.1%) were used for water storage. The Breteau and Container indices exceeded the WHO-recommended thresholds for the risk of dengue virus transmission except at 2 localities. Ae. aegypti, the only reputed dengue vector, was collected resting indoors as well as outdoors and biting during the day and night. The NS1 protein was detected in 22 mosquito pools, including one pool of females emerging from field-collected larvae. All NS1-positive results were confirmed by RT-PCR. Virus serotyping showed that the outbreak was caused by DENV-1. This study demonstrates the need for continuous control of adult and aquatic stages of Ae. aegypti to prevent future dengue epidemics in Senegal. RDTs appear to be a promising tool for dengue diagnostics and surveillance.

Comparative analysis of the susceptibility of Aedes aegypti and Japanese Aedes albopictus to all dengue virus serotypes.

BACKGROUND: Dengue fever, caused by the dengue virus (DENV), is the most common viral infection transmitted by Aedes mosquitoes (mainly Ae. aegypti and Ae. albopictus) worldwide. Aedes aegypti is not currently established in Japan, and Ae. albopictus is the primary vector mosquito for DENV in the country, but knowledge of its viral susceptibility is limited. Therefore, we aimed to clarify the status of DENV susceptibility by comparing the infection and dissemination dynamics of Japanese Ae. albopictus to all known DENV serotypes with those of Ae. aegypti. METHODS: After propagation of each DENV serotype in Vero cells, the culture supernatants were mixed with defibrinated rabbit blood and adenosine triphosphate, and the mixture was artificially blood-sucked by two colonies of Ae. albopictus from Japan and one colony of Ae. aegypti from a dengue-endemic country (Vietnam). After 14 days of sucking, the mosquito body was divided into two parts (thorax/abdomen and head/wings/legs) and total RNA was extracted from each sample. DENV RNA was detected in these extracted RNA samples using a quantitative RT-PCR method specific for each DENV serotype, and infection and dissemination rates were analyzed. RESULTS: The Japanese Ae. albopictus colonies were susceptible to all DENV serotypes. Its infection and dissemination rates were significantly lower than those of Ae. aegypti. However, the number of DENV RNA copies in Ae. albopictus was almost not significantly different from that in Ae. aegypti. Furthermore, Japanese Ae. albopictus differed widely in their susceptibility to each DENV serotype. CONCLUSIONS: In Japanese Ae. albopictus, once DENV overcame the midgut infection barrier, the efficiency of subsequent propagation and dissemination of the virus in the mosquito body was comparable to that of Ae. aegypti. Based on the results of this study and previous dengue outbreak trends, Ae. albopictus is predicted to be highly compatible with DENV-1, suggesting that this serotype poses a high risk for future epidemics in Japan.

Dengue virus serotype did not contribute to clinical severity or mortality in Taiwan's largest dengue outbreak in 2015.

BACKGROUND: Dengue virus serotype 2 (DENV-2) was the major serotype in the 2015 dengue outbreak in Taiwan, while DENV-1 and DENV-3 were dominant between 2005 and 2014. We aimed to investigate whether DENV-2 contributed to disease severity and mortality in the outbreak in Kaohsiung city, Taiwan. METHODS: We collected serum samples from dengue patients to detect the presence of DENV and determine the serotypes by using quantitative reverse transcription-polymerase chain reaction. Our cohorts comprised 105 DENV-1-infected cases and 1,550 DENV-2-infected cases. Demographic data, DENV serotype, and comorbidities were covariates for univariate and multivariate analyses to explore the association with severity and mortality. RESULTS: The results suggested that DENV-1 persisted and circulated, while DENV-2 was dominant during the dengue outbreak that occurred between September and December 2015. However, DENV-2 did not directly contribute to either severity or mortality. Aged patients and patients with diabetes mellitus (DM) or moderate to severe chronic kidney disease (CKD) had a higher risk of developing severe dengue. The mortality of dengue patients was related to a higher Charlson comorbidity index score and severe dengue. Among DENV-2-infected patients and older patients, preexisting anti-dengue IgG, DM, and moderate to severe CKD were associated with severe dengue. Moreover, female sex and severe dengue were associated with a significantly higher risk of death. CONCLUSIONS: Our findings highlight the importance of timely serological testing in elderly patients to identify potential secondary infections and focus on the meticulous management of elderly patients with DM or moderate to severe CKD to reduce dengue-related death.

Utility of CDC DENV1-4 real time PCR assay and trioplex assay for the diagnosis of dengue in patients with acute febrile illness.

Nucleic acid amplification tests (NAATs) have revolutionized reliable detection of dengue virus (DENV) during acute phase of infection. The study evaluated performance of CDC DENV-1-4 real-time assay, trioplex RT-PCR and heminested conventional RT-PCR assay in the diagnosis of DENV. The three NAATs were performed on 107 consecutive samples collected from patients suspected of DENV infection during acute phase of illness. Their performance was compared against composite reference standard, consisting of DENV NS1 antigen ELISA and DENV IgM ELISA. 88/107 study samples were positive by DENV ELISA, either NS1Ag (80), IgM (3) or both (5). The overall sensitivity of CDC DENV-1-4 RT-PCR assay, trioplex RT-PCR assay and conventional multiplex RT-PCR was 68.18%, 54.55% and 38.64%, respectively in diagnosing dengue during acute phase, with an area under the curve of 0.841, 0.773 and 0.693 respectively when compared against composite reference standard. The sensitivity was 82.93%, 73.17% and 51.22%, respectively within three days of illness and 60%, 42.86% and 28.57%, respectively between 4 and 5th day of illness. All the three molecular assays had 100% specificity. Maximum concordance values of 86.9% were recorded among CDC DENV-1-4 rRT-PCR assay and trioplex assay with kappa value of 0.74, suggestive of substantial agreement. CDC DENV-1-4 rRT-PCR assay can be used as a reliable and accurate test for diagnosis of DENV during acute phase of illness.

DENV-1 genotype V linked to the 2022 dengue epidemic in Southern Brazil.

Even though Brazil is a country where the dengue virus (DENV) is endemic, until recently, Southern states did not have significant viral circulation, such as Rio Grande do Sul (RS), and some municipalities were even considered dengue-free. During 2022, these places have shown a sharp increase in the incidence of the disease, apparently following a worldwide growth pattern. Therefore, in this study, we monitor and characterize the genetic diversity of DENV circulating in southern Brazil through next-generation sequencing during an outbreak in 2022. We generated 70 DENV-1 genome sequences, all characterized as genotype V, divided into two clade clusters in the L1 lineage. Furthermore, unique mutations have been described in each clade of L1 lineage. Our results are essential in managing outbreaks since these data provide important information during the emergence of DENV circulation in RS. Since the south of Brazil has a lower viral circulation when compared to other Brazilian states, RS still lacks data that can help in understanding the transmission, dissemination, and evolution of the dengue virus. Hence, genomic surveillance efforts are essential to increase the accuracy of preventive actions and to control viral dissemination.

Molecular Characterization of Dengue Virus Strains from the 2019-2020 Epidemic in Hanoi, Vietnam.

Dengue virus (DENV), which has circulated in Vietnam for several decades, has multiple serotypes and genotypes. A 2019 dengue outbreak resulted in a larger number of cases than any other outbreak. We conducted a molecular characterization using samples collected in 2019-2020 from dengue patients in Hanoi and nearby cities located in northern Vietnam. The circulating serotypes were DENV-1 (25%, n = 22) and DENV-2 (73%, n = 64). Phylogenetic analyses revealed that all DENV-1 (n = 13) were genotype I and clustered to local strains circulating during the previous outbreak in the 2017, whereas DENV-2 consisted of two genotypes: Asian-I (n = 5), related to local strains from 2006-2022, and cosmopolitan (n = 18), the predominant genotype in this epidemic. The current cosmopolitan virus was identified as having an Asian-Pacific lineage. The virus was closely related to strains in other recent outbreaks in Southeast Asian countries and China. Multiple introductions occurred in 2016-2017, which were possibly from maritime Southeast Asia (Indonesia, Singapore, and Malaysia), mainland Southeast Asia (Cambodia and Thailand), or China, rather than from an expansion of localized Vietnamese cosmopolitan strains that were previously detected in the 2000s. We also analyzed the genetic relationship between Vietnam's cosmopolitan strain and recent global strains reported from Asia, Oceania, Africa, and South America. This analysis revealed that viruses of Asian-Pacific lineage are not restricted to Asia but have spread to Peru and Brazil in South America.

Dengue Exposure and Wolbachia wMel Strain Affects the Fertility of Quiescent Eggs of Aedes aegypti.

(1) Background: The deployment of the bacterium Wolbachia to reduce arbovirus transmission is ongoing in several countries worldwide. When Wolbachia-carrying Aedes aegypti are released and established in the field, females may feed on dengue-infected hosts. The effects of simultaneous exposure on life-history traits of Ae. aegypti to Wolbachia wMel strain and dengue-1 virus DENV-1 remain unclear. (2) Methods: We monitored 4 groups (mosquitoes with either DENV-1 or Wolbachia, coinfected with DENV-1 and Wolbachia, as well as negative controls) to estimate Ae. aegypti survival, oviposition success, fecundity, collapsing and fertility of quiescent eggs for 12 weeks. (3) Results: Neither DENV-1 nor Wolbachia had a significant impact on mosquito survival nor on mosquito fecundity, although the last parameter showed a tendency to decrease with ageing. There was a significant decrease in oviposition success in individuals carrying Wolbachia. Wolbachia infection and storage time significantly increased egg collapse parameter on the egg viability assay, while DENV-1 had a slight protective effect on the first four weeks of storage. (4) Conclusions: Despite limitations, our results contribute to better understanding of the tripartite interaction of virus, bacteria and mosquito that may take place in field conditions and aid in guaranteeing the Wolbachia strategy success.

Recent outbreak of dengue in Bangladesh: A threat to public health.

Background and Aims: The dengue virus is widespread throughout Bangladesh and significantly contributes to morbidity and mortality. One effective method for preventing further dengue epidemics is to reduce mosquito breeding at the most opportune period each year. This study aims to determine dengue prevalence in 2022 by comparing previous years' data and estimating the period of this disease's most significant incidence. Methods: From the beginning of 2008-December 15, 2022, we looked at the monthly reports of cases made to the Bangladesh Institute of Epidemiology, Disease Control, and Research. Results: According to our findings, there were 61089 confirmed dengue cases in 2022, with 269 fatalities - the highest annual death toll for this disease since 2000. Almost one-third (32.14%) of all dengue deaths in Bangladesh occurred in 2022 (1 January-15 December), highlighting the severity of the threat posed by this disease in the coming year. Furthermore, we observe that the months in the second half of any year in Bangladesh are the most at risk for dengue transmission. In 2022, Dhaka city and Chittagong are hit the hardest (incidence: 63.07% vs. 14.42%; morality: 63.34% vs. 24.16%), showing the relevance of population density in spreading this fatal disease. Conclusion: Statistics show an increase in dengue cases every day, and the year 2022 will be marked as the peak of the disease's death prevalence. Both the individuals and the government of Bangladesh need to take action to reduce the dissemination of this epidemic. If not, the country will soon be in great peril.

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.

Validation of a Reporter Cell Line for Flavivirus Inhibition Assays.

Here, we report the validation of a new reporter cell line, Hec1a-IFNB-Luc, for use in inhibition studies of various flaviviruses relevant to human pathology. The reporter system allows the detection of viral replication after luciferase gene activation driven by an interferon beta (IFN-ß) promoter. We found the reporter cell line to be highly responsive to all 10 flaviviruses tested, including the 4 dengue virus serotypes. The applicability of the Hec1a-IFNB-Luc reporter cell line for serodiagnostic purposes in neutralizing antibody assays was confirmed by comparison of its sensitivity and specificity to those of "gold-standard," clinically applied, cytopathic effect-based assays, showing comparable performances. The reporter cell line used for the assessment of viral inhibition by small-molecule antiviral compounds was also confirmed, and the sensitivity of the Hec1a-IFNB-Luc reporter cell line was compared to those from published data reporting on the activity of the antivirals in various other assays, indicating that the Hec1a-IFNB-Luc reporter cell line allowed the determination of the inhibitory capacity at least as sensitive as alternative assays. By measuring luciferase activity as a proxy for viral replication, the reporter cell line allows early detection, reducing the time to results from often 5 to 7 days to 3 days, without the need for optical inspection of cytopathic effects, which often differ between viruses and cell lines, streamlining the development of flavivirus assays. IMPORTANCE The Hec1a-IFNB-Luc reporter cell line allows the detection of all 10 flaviviruses tested, including the 4 dengue virus serotypes. Its use for serodiagnostic purposes, measuring neutralizing antibody activity in sera, and the assessment of the antiviral activities of small-molecule compounds was confirmed, and it was found to be comparable to clinically applied assays. The Hec1a-IFNB-Luc reporter cell line allows the rapid and quantitative determination of antiviral effects on multiple human pathological flaviviruses using a single protocol.

Genomic Characterization of Dengue Virus Outbreak in 2022 from Pakistan.

Pakistan, a dengue-endemic country, has encountered several outbreaks during the past decade. The current study aimed to explore the serotype and genomic diversity of dengue virus responsible for the 2022 outbreak in Pakistan. From August to October 2022, NS-1 positive blood samples (n = 343) were collected from dengue patients, among which, (85%; n = 293) were positive based on RT-PCR. In terms of gender and age, dengue infection was more prevalent in male patients (63%; n = 184), with more adults (21-30 years; n = 94) being infected. The serotyping results revealed DENV-2 to be the most predominant serotype (62%; n = 183), followed by DENV-1 (37%; n = 109) and DENV-3 (0.32%; n = 1). Moreover, a total of 10 samples (DENV-2; n = 8, DENV-1; n = 2) were subjected to whole-genome sequencing. Among these, four were collected in early 2022, and six were collected between August and October 2022. Phylogenetic analysis of DENV-2 sequenced samples (n = 8) revealed a monophyletic clade of cosmopolitan genotype IVA, which is closely related to sequences from China and Singapore 2018, and DENV-1 samples (n = 2) show genotype III, which is closely related to Pakistan isolates from 2019. We also reported the first whole genome sequence of a coinfection case (DENV1-DENV2) in Pakistan detected through a meta-genome approach. Thus, dengue virus dynamics reported in the current study warrant large-scale genomic surveillance to better respond to future outbreaks.

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.

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

BACKGROUND: Dengue 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. METHODS: Targeting 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. RESULTS: The 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. CONCLUSIONS: The 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.