Genomic analysis of dengue virus serotype 1 (DENV-1) genotypes from Surabaya,Indonesia

Dengue has caused a significant public health impact globally. With the diverse genetic of the causative viruses, analysis of dengue virus (DENV) genomes is important to supplement epidemiological data with information that can be used to reconstruct the history of epidemics in time and space. We have reported the clinical and virological characteristics of dengue in Surabaya, Indonesia and revealed the presence of all four DENV serotypes and the predominance of DENV-1. The further classification of Surabaya DENV-1 into two different genotypes warrants in-depth genomic analysis to study the dynamics of both genotypes and their contribution to virus evolution, virus transmission, and disease. We performed full-length genome sequencing to nine isolates’ representatives from DENV-1 Genotype I and Genotype IV. Phylogenetic and evolutionary analyses suggested the more recent introduction of Genotype I viruses compared to the more endemic Genotype IV. Comparative analysis of Surabaya DENV-1 genomes and other sequences available publicly revealed that the majority of the DENV-1 codons were under strong purifying selection, while seven codon sites identified to be under positive selection. We highlight a unique codon site under the positive pressure in the NS1 gene of DENV-1. Our results provide additional genomic data of DENV from Indonesia that may contribute to the better understanding of dengue disease dynamics.     

Epidemiology and characteristics of the dengue outbreak in Guangdong,Southern China,in 2014

Dengue is a rapidly spreading mosquito-borne disease caused by the dengue virus (DENV) and has emerged as a severe public health problem around the world. Guangdong, one of the southern Chinese provinces, experienced a serious outbreak of dengue in 2014, which was believed to be the worst dengue epidemic in China over the last 20 years. To better understand the epidemic, we collected the epidemiological data of the outbreak and analyzed 14,594 clinically suspected dengue patients from 25 hospitals in Guangdong. Dengue cases were then laboratory-confirmed by the detection of DENV non-structural protein 1 (NS1) antigen and/or DENV RNA. Afterwards, clinical manifestations of dengue patients were analyzed and 93 laboratory-positive serum specimens were chosen for the DENV serotyping and molecular analysis. Our data showed that the 2014 dengue outbreak in Guangdong had spread to 20 cities and more than 45 thousand people suffered from dengue fever. Of 14,594 participants, 11,387 were definitively diagnosed. Most manifested with a typical non-severe clinical course, and 1.96 % developed to severe dengue. The strains isolated successfully from the serum samples were identified as DENV-1. Genetic analyses revealed that the strains were classified into genotypes I and V of DENV-1, and the dengue epidemic of Guangdong in 2014 was caused by indigenous cases and imported cases from the neighboring Southeast Asian countries of Malaysia and Singapore. Overall, our study is informative and significant to the 2014 dengue outbreak in Guangdong and will provide crucial implications for dengue prevention and control in China and elsewhere.     

2018年杭州市登革热流行病学及病原特征研究

目的:了解2018年杭州市登革热疫情的流行病学及病原特征。方法:应用RT-PCR方法检测血清标本中的登革病毒核酸及其型别,并对阳性标本进行病毒分离、 E基因扩增、序列测定及进化分析;描述病例的时间、人群和地区分布特征。 结果:2018年杭州市共检测登革热病例80例,其中输入性病例55例,本地病例2...     

Dengue viruses circulating in Indonesia: A systematic review and phylogenetic analysis of data from five decades

Although epidemiological and molecular epidemiological (serotype, genotype, and lineage information) data are available for several major cities in Indonesia, there is yet to be a comprehensive national study of dengue in Indonesia over time. This study was conducted to provide a comprehensive epidemiology of circulating dengue viruses (DENV) in Indonesia between 1973 and 2016. This was conducted through a systematic review of the literature and phylogenetic analysis of available DENV sequences. Available data from National Disease Surveillance System have indicated an increasing trend of dengue incidence in Indonesia over the past 50 years. Incidence rates appear to be cyclic, peaking approximately every 6 to 8 years. In contrast, the case fatality rate has decreased approximately by half with each decade since 1980. Over this 50‐year time span, serotype shifts, genotype displacement within DENV‐1 and DENV‐2, and introduction of DENV‐1 and DENV‐3 genotype from other countries occurred. These events were associated with increased incidence of dengue cases. Our study also provides a valuable national snapshot of DENV genetic diversity in Indonesia that may contribute to development of more effective dengue vaccine compositions for the region.     

Letter to the editor: molecular genotyping of Dengue Virus Types 2 and 4 from the Guatemalan and Honduran Epidemics of 2007 using the envelope glycoprotein gene

Eight serum specimens collected from dengue patients in Guatemala and Honduras during the Central American epidemic of 2007 were analyzed. Virus identification and serotyping performed by a nested RT-PCR assay revealed two DENV-1 isolates from Guatemala, four DENV-2 isolates, two each from Guatemala and Honduras, and two DENV-4 isolates from Honduras. Viral genotyping determined by phylogenetic analysis of the complete envelope gene sequences demonstrated that the DENV-2 isolates from Guatemala and Honduras fell into the American/Asian Genotype III, and were most closely related to DENV-2/NI/BID-V2683-1999 isolated from a dengue case in Nicaragua in 1999; and the DENV-4 F07-076 isolate from Honduras belonged to genotype II, and was most closely related to DENV-4/US/BID-V1093/1998 isolated from Puerto Rico in 1998. Our results suggest that the 2007 dengue outbreaks in Guatemala and Honduras were most likely caused by the re-emergence of earlier, indigenous DENV strains rather than by newly introduced strains and there were at least three serotypes of DENV co-circulating during the 2007 Central American epidemics.     

Molecular Characterisation and Phylogenetic Analysis of Dengue Outbreak in Pasighat,Arunachal Pradesh,Northeast India

Background and Objectives: Dengue is one of the most prevalent arboviral diseases in the world with 390 million dengue infections per year. In this study, we report the molecular characterisation of dengue outbreak in Pasighat, Arunachal Pradesh, Northeast India during 2015. Subjects and Methods: A total of 613 dengue-suspected cases were screened for dengue virus by dengue NS1 Ag and anti-dengue IgM antibody depending on the duration of sample collection and onset of symptom. Further, molecular characterisation was done by amplifying the C-PrM region by real-time polymerase chain reaction followed by phylogenetic analysis. Results: Molecular characterisation revealed that the dengue outbreak was predominantly due to dengue virus serotype-1 (DENV-1) (90.9%) while DENV-2 was detected in 7.5% of samples. Co-infection of DENV-1 and DENV-2 was detected in one case. Phylogenetic analysis of the DENV-1 strains with the prototype revealed that the DENV-1 strains were grouped within genotype III. Similarly, DENV-2 strains were clustered within genotype IV. The study revealed a change in the predominant serotype in recent years with DENV-3 in 2012 to DENV-1, 2, 3 and 4 in 2014 to DENV-1 in 2015 in the study region. A unique L24M mutation was observed in the DENV-1 strains of Arunachal Pradesh which was absent in all the circulating strains in India except one strain from the state of Kerala in South India. Marked variation within the DENV-2 strains was observed at A102V and I163V in one strain similar to earlier circulating isolates in India. Conclusions: The present study reveals a shift in the serotype dominance in the study region. As serotype shifts and secondary infection with a heterologous DENV serotype are frequently associated with disease severity, there is an urgent need for sustained monitoring of the circulating serotypes and enhanced surveillance operations, especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in this region.     

Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.     

Comprehensive mapping of immunodominant and conserved serotype- and group-specific B-cell epitopes of nonstructural protein 1 from dengue virus type 1

The dengue virus (DENV) nonstructural protein 1 (NS1) is an immunogenic protein that holds potential for the development of vaccines and diagnostic reagents; however, the epitopes of NS1 have not been comprehensively mapped. We mapped B-cell linear epitopes on NS1 using 149 monoclonal antibodies with DENV serotype specificity and cross-reactivity as well as antisera from 27 mice immunized with the four DENV serotypes. Epitope recognition analysis was performed using a set of 15-mer sequential overlapping peptides that spanned the entire NS1 protein from DENV-1. This strategy identified three regions of NS1 that are DENV-1 serotype-specific epitopes, namely amino acid residues 1-15, 71-85, and 338-352. We also identified five group-specific B-cell epitopes that were highly conserved among isolates of the four DENV serotypes. These novel immunodominant serotype- and group-specific B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and diagnostic assays.     

The whole-genome sequencing of prevalent DENV-1 strains during the largest dengue virus outbreak in Xishuangbanna Dai autonomous prefecture in 2019

In 2019, a serious dengue virus (DENV) infection broke out in the Xishuangbanna Dai Autonomous Prefecture, China. Therefore, we conducted a molecular epidemiological analysis in people that contracted DENV serotype 1 (DENV-1) during this year. We analyzed the molecular epidemiology of six DENV-1 epidemic strains in 2019 by full-length genome sequencing, amino acid mutation site analysis, evolutionary tree analysis, and recombination site comparison analysis. Through the analysis of amino acid mutation sites, it was found that DENV-1 strain (MW386867) was different from the other five epidemic DENV-1 strains in Xishuangbanna in 2019. MW386867 had unique mutation sites at six loci. The six epidemic DENV-1 strains in Xishuangbanna in 2019 were divided into two clusters. MW386867 was highly similar to the MG679800 (Myanmar 2017), MG679801 (Myanmar 2017), and KC172834 (Laos 2008), and the other five strains were highly similar to JQ045660 (Vietnam 2011), FJ176780 (GuangDong 2006). Genetic recombination analysis revealed that there was no recombination signal in the six epidemic DENV-1 strains in Xishuangbanna in 2019. We speculate that the DENV-1 epidemic in 2019 has a co-epidemic of local strains and cross-border strains.     

Intra-genotypic variation of predominant genotype II strains of dengue type-3 virus isolated during different epidemics in Thailand from 1973 to 2001

The prevalence of all four dengue virus (DENV) serotypes has increased dramatically in recent years in many tropical and sub-tropical countries accompanied by an increase in genetic diversity within each serotype. This expansion in genetic diversity is expected to give rise to viruses with altered antigenicity, virulence, and transmissibility. We previously demonstrated the co-circulation of multiple DENV genotypes in Thailand and identified a predominant genotype for each serotype. In this study, we performed a comparative analysis of the complete genomic sequences of 28 DENV-3 predominant genotype II strains previously collected during different DENV-3 epidemics in Thailand from 1973 to 2001 with the goal to define mutations that might correlate with virulence, transmission frequency, and epidemiological impact. The results revealed (1) 37 amino acid and six nucleotide substitutions adopted and fixed in the virus genome after their initial substitutions over nearly 30-year-sampling period, (2) the presence of more amino acid and nucleotide substitutions in recent virus isolates compared with earlier isolates, (3) six amino acid substitutions in capsid (C), pre-membrane (prM), envelope (E), and nonstructural (NS) proteins NS4B and NS5, which appeared to be associated with periods of high DENV-3 epidemic activity, (4) the highest degree of conservation in C, NS2B and the 5′-untranslated region (UTR), and (5) the highest percentage of amino acid substitutions in NS2A protein.     

Senescence Affects Endothelial Cells Susceptibility to Dengue Virus Infection

Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells.     

Antibodies from patients with dengue viral infection mediate cellular cytotoxicity.

Acute and late convalescent sera (collected at day 5 of disease onset and 1 year later) from dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) laboratory confirmed cases, were tested for antibody-dependent cell-mediated cytotoxicity (ADCC) activity using dengue 1 (DENV-1) or dengue 2 (DENV-2) infected cells as target. All patients experienced their first dengue virus (DENV) infection 20 years before. ADCC activity was detected in acute sera from DHF/DSS but not in sera from DF patients. However, 1 year after illness, ADCC activity was observed in all cases. This preliminary report represents one of the few studies of ADCC in dengue patients and suggests that ADCC could be implicated in dengue pathogenesis.     

Population dynamics of DENV-1 genotype V in Brazil is characterized by co-circulation and strain/lineage replacement

Following successive outbreaks of dengue fever caused predominantly by dengue virus (DENV) 2 and 3, DENV-1 is now the primary serotype circulating in Brazil. We sequenced and analyzed Brazilian DENV-1 genomes and found that all isolates belong to genotype V and are subdivided into three lineages, which were introduced during four different events. The first introduction occurred in 1984-85, the second in 1997-99, and the third and fourth occurred from 2004 to 2007. These events were associated with an increase in genetic diversity but not with positive selection. Moreover, a potential new recombinant strain derived from two distinct lineages was detected. We demonstrate that the dynamics of DENV-1 in Brazil is characterized by introduction, movement, local evolution, and lineage replacement. This study strengthens the relevance of genotype surveillance in order to identify, trace, and control virus populations circulating in Brazil and Latin America.     

Molecular Studies on Dengue Viruses Detected in Patients from Central India

Purpose: Dengue viruses (DENVs), the causative agents of dengue (DEN), are classified into four serotypes and several genotypes. Identifying circulating serotypes and genotypes has clinical and epidemiological importance; however, limited information in this regard is available from Central India. This laboratory-based study was done to fill this lacuna. Materials and Methods: The samples collected in the acute phase of illness were subjected to DEN NS1 ELISA, and NS1-positive samples (n = 80) were subjected to serotyping; representative samples from each serotype were sequenced to identify genotypes. Results: Seventy-one (88.75%) samples could be serotyped. All the four DENV serotypes with dominance of DENV-3 (n = 33; 47%) were detected. DENV-4 was detected after a gap of 3 years. Cases with multiple DENV serotype infection were identified. Genotyping showed that DENV-1 belonging to genotype III, DENV-2 cosmopolitan (IV), DENV-3 genotype III lineage C and DENV-4 genotype I were in circulation in the year 2016. Conclusion: Our study documents the molecular characteristics of DENV circulating in the area. Detection of heterologous DENV serotype with dominance of DENV-3 emphasises the need for regular molecular monitoring.     

Dengue virus serotypes and genotypic characterization from northeast India

Dengue is a rapidly spreading acute arboviral infection transmitted through a human and Aedes mosquito cycle. Though northeast region of India has been experiencing dengue outbreaks regularly for over a decade, reports on genetic characterization of the virus from this region are limited. The present study was undertaken to detect the genotype and genetic composition of circulating dengue virus (DENV) in this region. Blood samples were collected from 918 suspected dengue patients of five northeast Indian states. Serological investigations, viz, nonstructural 1 (NS1) enzyme-linked immunosorbent assay (ELISA), immunoglobulin M (IgM) ELISA, and immunoglobulin G (IgG) ELISA were performed followed by molecular detection. Sequence analysis and phylogenetic tree construction based on capsid-premembrane (C-prM) gene junction was done by BioEdit and MEGA6 software, respectively. Serological detection showed 35.34% NS1 and 18.12% IgM positivity. Secondary infection was observed in 24.53%. All four serotypes were detected. Phylogenetic analysis demonstrated circulation of genotype III of DENV-1, genotype IV of DENV-2, and genotype III of DENV-3. Sequences from this region form distinct clades in the phylogenetic tree. Characterization of the C-prM gene junction reveals divergence among the DENV strains. As genetic variation within the DENV is known to be associated with diverse clinical outcomes, information regarding the genetic composition of circulating virus could be beneficial in designing an effective intervention strategy.     

Characterization of dengue complex-reactive epitopes on dengue 3 virus envelope protein domain III

The disease dengue (DEN) is caused by four genetically and serologically related viruses termed DENV-1, -2, -3, and -4. The DENV envelope (E) protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3. The ED3 contains the DENV type-specific and DENV complex-reactive (epitopes shared by DENV 1-4) antigenic sites. In this study the epitopes recognized by four DENV complex-reactive monoclonal antibodies (MAbs) with neutralizing activity were mapped on the DENV-3 ED3 using a combination of physical and biological techniques. Amino acid residues L306, K308, G381, I387, and W389 were critical for all four MAbs, with residues V305, E309, V310, K325, D382, A384, K386, and R391 being critical for various subsets of the MAbs. A previous study by our group (Gromowski, G.D., Barrett, N.D., Barrett, A.D., 2008. Characterization of dengue complex-specific neutralizing epitopes on the envelope protein domain III of dengue 2 virus. J. Virol 82, 8828-8837) characterized the same panel of MAbs with DENV-2. The location of the DENV complex-reactive antigenic site on the DENV-2 and DENV-3 ED3s is similar; however, the critical residues for binding are not identical. Overall, this indicates that the DENV complex-reactive antigenic site on ED3 may be similar in location, but the surprising result is that DENV 2 and 3 exhibit unique sets of residues defining the energetics of interaction to the same panel of MAbs. These results imply that the amino acid sequences of DENV define a unique interaction network among these residues in spite of the fact that all flavivirus ED3s to date assume the same structural fold.