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      Complete genome analysis and characterization of neurotropic dengue virus 2 cosmopolitan genotype isolated from the cerebrospinal fluid of encephalitis patients

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          Abstract

          Dengue virus (DENV) infection remains a major public health concern in many parts of the world, including Southeast Asia and the Americas. Sri Lanka experienced its largest dengue outbreak in 2017. Neurological symptoms associated with DENV infection have increasingly been reported in both children and adults. Here, we characterize DENV type 2 (DENV-2) strains, which were isolated from cerebrospinal fluid (CSF) and/or serum of patients with dengue encephalitis. Acute serum and CSF samples from each patient were subjected to dengue-specific non-structural protein 1 (NS1) antigen test, IgM and IgG enzyme-linked immunosorbent assay (ELISA), virus isolation, conventional and real-time polymerase chain reaction (PCR), and next-generation sequencing (NGS). Among the 5 dengue encephalitis patients examined, 4 recovered and 1 died. DENV-2 strains were isolated from serum and/or CSF samples of 3 patients. The highest viral genome levels were detected in the CSF and serum of the patient who succumbed to the illness. A phylogenetic tree revealed that the DENV-2 isolates belonged to a new clade of cosmopolitan genotype and were genetically close to strains identified in China, South Korea, Singapore, Malaysia, Thailand, and the Philippines. According to the NGS analysis, greater frequencies of nonsynonymous and synonymous mutations per gene were identified in the nonstructural genes. The full genomes of serum- and CSF-derived DENV-2 from the same patient shared 99.7% similarity, indicating that the virus spread across the blood-brain barrier. This is the first report to describe neurotropic DENV-2 using whole-genome analysis and to provide the clinical, immunological, and virological characteristics of dengue encephalitis patients during a severe dengue outbreak in Sri Lanka in 2017.

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          Dengue, Urbanization and Globalization: The Unholy Trinity of the 21st Century

          Dengue is the most important arboviral disease of humans with over half of the world’s population living in areas of risk. The frequency and magnitude of epidemic dengue have increased dramatically in the past 40 years as the viruses and the mosquito vectors have both expanded geographically in the tropical regions of the world. There are many factors that have contributed to this emergence of epidemic dengue, but only three have been the principal drivers: 1) urbanization, 2) globalization and 3) lack of effective mosquito control. The dengue viruses have fully adapted to a human-Aedes aegypti-human transmission cycle, in the large urban centers of the tropics, where crowded human populations live in intimate association with equally large mosquito populations. This setting provides the ideal home for maintenance of the viruses and the periodic generation of epidemic strains. These cities all have modern airports through which 10s of millions of passengers pass each year, providing the ideal mechanism for transportation of viruses to new cities, regions and continents where there is little or no effective mosquito control. The result is epidemic dengue. This paper discusses this unholy trinity of drivers, along with disease burden, prevention and control and prospects for the future.
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            Dengue virus pathogenesis: an integrated view.

            Much remains to be learned about the pathogenesis of the different manifestations of dengue virus (DENV) infections in humans. They may range from subclinical infection to dengue fever, dengue hemorrhagic fever (DHF), and eventually dengue shock syndrome (DSS). As both cell tropism and tissue tropism of DENV are considered major determinants in the pathogenesis of dengue, there is a critical need for adequate tropism assays, animal models, and human autopsy data. More than 50 years of research on dengue has resulted in a host of literature, which strongly suggests that the pathogenesis of DHF and DSS involves viral virulence factors and detrimental host responses, collectively resulting in abnormal hemostasis and increased vascular permeability. Differential targeting of specific vascular beds is likely to trigger the localized vascular hyperpermeability underlying DSS. A personalized approach to the study of pathogenesis will elucidate the basis of individual risk for development of DHF and DSS as well as identify the genetic and environmental bases for differences in risk for development of severe disease.
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              Neurological manifestations of dengue infection.

              Severe forms of dengue, the most important arboviral infection of man, are associated with haemorrhagic disease and a generalised vascular leak syndrome. The importance of dengue as a cause of neurological disease is uncertain. During 1995, all patients with suspected CNS infections admitted to a referral hospital in southern Vietnam were investigated by culture, PCR, and antibody measurement in serum and CSF for dengue and other viruses. Of 378 patients, 16 (4.2%) were infected with dengue viruses, compared with four (1.4%) of 286 hospital controls (odds ratio [95% CI] 3.1 [1.7-5.8]). Five additional dengue positive patients with CNS abnormalities were studied subsequently. No other cause of CNS infection was identified. Seven infections were primary dengue, 13 secondary, and one was not classified. Ten patients had dengue viruses isolated or detected by PCR, and three had dengue antibody in the CSF. 12 of the 21 had no characteristic features of dengue on admission. The most frequent neurological manifestations were reduced consciousness and convulsions. Nine patients had encephalitis. No patient died, but six had neurological sequelae at discharge. Phylogenetic analysis of the four DEN-2 strains isolated mapped them with a DEN-2 strain isolated from a patient with dengue haemorrhagic fever, and with other strains previously isolated in southern Vietnam. In dengue endemic areas patients with encephalitis and encephalopathy should be investigated for this infection, whether or not they have other features of the disease.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: ResourcesRole: Writing – original draft
                Role: ConceptualizationRole: InvestigationRole: ResourcesRole: Writing – review & editing
                Role: Investigation
                Role: Investigation
                Role: Formal analysis
                Role: Resources
                Role: Resources
                Role: Resources
                Role: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                18 June 2020
                2020
                : 15
                : 6
                : e0234508
                Affiliations
                [1 ] Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
                [2 ] Department of Virology, Teaching Hospital Kandy, Kandy, Sri Lanka
                [3 ] Department of Immunogenetics, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
                Defense Threat Reduction Agency, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-8986-6307
                http://orcid.org/0000-0002-4072-0745
                Article
                PONE-D-20-01443
                10.1371/journal.pone.0234508
                7302667
                32555732
                a6473139-663c-45cf-95f8-67d5cd98c5d3
                © 2020 Ngwe Tun et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 16 January 2020
                : 26 May 2020
                Page count
                Figures: 2, Tables: 3, Pages: 15
                Funding
                This work was supported by grants from the Japan Initiative for Global Research Network on Infectious Diseases (JGRID) of the Japan Science and Technology Agency (JST) (Grant number JP19fm0108001), and Research Program on Emerging and Re-emerging Infectious Diseases of the Agency for Research and Development (AMED, 19fk0108035h1203).
                Categories
                Research Article
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Cerebrospinal Fluid
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Cerebrospinal Fluid
                Biology and Life Sciences
                Physiology
                Body Fluids
                Cerebrospinal Fluid
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Cerebrospinal Fluid
                Biology and Life Sciences
                Anatomy
                Nervous System
                Cerebrospinal Fluid
                Medicine and Health Sciences
                Anatomy
                Nervous System
                Cerebrospinal Fluid
                Medicine and Health Sciences
                Infectious Diseases
                Infectious Diseases of the Nervous System
                Encephalitis
                Medicine and Health Sciences
                Neurology
                Infectious Diseases of the Nervous System
                Encephalitis
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                Diagnostic Medicine
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