Construction of a high-yield dengue virus by replacing nonstructural proteins 3–4B without increasing virulence

Phanthanawiboon, Supranee; Pambudi, Sabar; Omokoko, Magot Diata; Hanabara, Keiko; A-nuegoonpipat, Atchareeya; Kamitani, Wataru; Ikuta, Kazuyoshi; Kurosu, Takeshi

doi:10.1016/j.bbrc.2017.11.137

Cited by 2 publications

(2 citation statements)

References 15 publications

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“…In this study, the mutation in the NS5 region in the serum-derived DENV-3 isolate (Supplementary Figure 2 ) has been hypothesized to be associated with the abrogation of viral replication in vitro . A previous study demonstrated that a chimeric virus strain with a mutation in the NS5 gene exhibited comparatively lower growth rates [ 37 ]. While differences in the clinical presentation were absent when a mouse model (A129) was inoculated with CSF-derived DENV-3 and serum-derived DENV-3 isolates, further studies are needed to determine the pathogenesis of DENV-related neurological involvement in human patients.…”

Section: Discussionmentioning

confidence: 99%

Differential Infectivity of Human Neural Cell Lines by a Dengue Virus Serotype-3 Genotype-III with a Distinct Nonstructural Protein 2A (NS2A) Amino Acid Substitution Isolated from the Cerebrospinal Fluid of a Dengue Encephalitis Patient

Nguyen

et al. 2023

Canadian Journal of Infectious Diseases and Medical Microbiolog

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Dengue encephalitis is considered as a severe but unusual clinical presentation of dengue infection. Limited molecular information is available on the neurotropism of dengue virus (DENV), highlighting the need for further research. During a dengue outbreak in Vietnam in 2013, two DENV-3 strains were isolated, in which one was isolated from cerebrospinal fluid (CSF) samples from a dengue encephalitis patient and another strain was isolated from a patient with classical dengue fever in Hai Phong, Vietnam. DENV serotype-3 (DENV-3) isolated from these samples belonged to genotype III, marking the first report of this genotype in the country at that time. Genetic variation between both strains was elucidated by using a full genome sequencing by next-generation sequencing (NGS). The infectivity of the isolated DENV-3 strains was further characterized using human and mouse neuronal cell lines. Phylogenetic analysis of the isolates demonstrated high homogeneity between the CSF-derived and serum-derived DENV-3, in which the full genome sequences of the CSF-derived DENV-3 presented a Thr-1339-Ile mutation in the nonstructural 2A (NS2A) protein. The CSF-derived DENV-3 isolate grew preferentially in human neuronal cells, with a significant proportion of cells that were positive for nonstructural 1 (NS1), nonstructural 4B (NS4B), and nonstructural 5 (NS5) antigens. These results suggest that NS2A may be a crucial region in the neuropathogenesis of DENV-3 and its growth in human neuronal cells. Taken together, our results demonstrate that a CSF-derived DENV-3 has unique infectivity characteristics for human neuronal cells, which might play a crucial role in the neuropathogenesis of DENV infection.

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Section: Discussionmentioning

confidence: 99%

Differential Infectivity of Human Neural Cell Lines by a Dengue Virus Serotype-3 Genotype-III with a Distinct Nonstructural Protein 2A (NS2A) Amino Acid Substitution Isolated from the Cerebrospinal Fluid of a Dengue Encephalitis Patient

Nguyen

et al. 2023

Canadian Journal of Infectious Diseases and Medical Microbiolog

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“…Vero cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT, USA) at 37 • C with 5% CO 2 . A DENV-4 infectious clone (DENV-4 IC, pmMW/rR05-167) was kindly provided by Professor Takeshi Kurosu (Research Institute for Microbial Diseases (RIMD), Osaka University, Japan), produced using a two-plasmid ligation strategy as described previously [22]. The primary antibody used in this study was a goat anti-mouse IgG conjugated with Alexa Fluor 488 (Thermo Fisher Scientific, USA).…”

Section: Cells Infectious Clone and Antibodymentioning

confidence: 99%

Intrahost Genetic Diversity of Dengue Virus in Human Hosts and Mosquito Vectors under Natural Conditions Which Impact Replicative Fitness In Vitro

Nonyong

Ekalaksananan

Phanthanawiboon

et al. 2023

Viruses

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Dengue virus (DENV) is an arbovirus whose transmission cycle involves disparate hosts: humans and mosquitoes. The error-prone nature of viral RNA replication drives the high mutation rates, and the consequently high genetic diversity affects viral fitness over this transmission cycle. A few studies have been performed to investigate the intrahost genetic diversity between hosts, although their mosquito infections were performed artificially in the laboratory setting. Here, we performed whole-genome deep sequencing of DENV-1 (n = 11) and DENV-4 (n = 13) derived from clinical samples and field-caught mosquitoes from the houses of naturally infected patients, in order to analyze the intrahost genetic diversity of DENV between host types. Prominent differences in DENV intrahost diversity were observed in the viral population structure between DENV-1 and DENV-4, which appear to be associated with differing selection pressures. Interestingly, three single amino acid substitutions in the NS2A (K81R), NS3 (K107R), and NS5 (I563V) proteins in DENV-4 appear to be specifically acquired during infection in Ae. aegypti mosquitoes. Our in vitro study shows that the NS2A (K81R) mutant replicates similarly to the wild-type infectious clone-derived virus, while the NS3 (K107R), and NS5 (I563V) mutants have prolonged replication kinetics in the early phase in both Vero and C6/36 cells. These findings suggest that DENV is subjected to selection pressure in both mosquito and human hosts. The NS3 and NS5 genes may be specific targets of diversifying selection that play essential roles in early processing, RNA replication, and infectious particle production, and they are potentially adaptive at the population level during host switching.

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Construction of a high-yield dengue virus by replacing nonstructural proteins 3–4B without increasing virulence

Cited by 2 publications

References 15 publications

Differential Infectivity of Human Neural Cell Lines by a Dengue Virus Serotype-3 Genotype-III with a Distinct Nonstructural Protein 2A (NS2A) Amino Acid Substitution Isolated from the Cerebrospinal Fluid of a Dengue Encephalitis Patient

Differential Infectivity of Human Neural Cell Lines by a Dengue Virus Serotype-3 Genotype-III with a Distinct Nonstructural Protein 2A (NS2A) Amino Acid Substitution Isolated from the Cerebrospinal Fluid of a Dengue Encephalitis Patient

Intrahost Genetic Diversity of Dengue Virus in Human Hosts and Mosquito Vectors under Natural Conditions Which Impact Replicative Fitness In Vitro

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