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

Abstract

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.

Figure 1

DENV-3 growth curve of two different isolates, cerebrospinal fluid (CSF)- and serum-derived DENV-3 genotype-III, using a mosquito cell line (C6/36) and a human neural cell line (SKNSH). After infection at a multiplicity of infection (MOI) = 0.001, cell culture fluids were collected every 24 hours from 0 to 120 h and titrated by using a focus forming assay on Vero cells. P value was determined using the t test. P value ≤0.05 was considered significant.

Figure 2

DENV-3 genome levels at 0, 24, 48, 72, 96, and 120 h postinfection (P.I). Virus genome levels were quantified by using real-time qPCR after infection at a multiplicity of infection (MOI) = 0.01 ((a)–(d)) and MOI = 0.001 ((e)–(h)). DENV-3 isolated from the cerebrospinal fluid (CSF) specimen (CSF-11098) and serum specimen (HP-5528) was separately inoculated to C6/36, SKNSH, T98G, and N2A cell lines. Viral genome copies number present in infected cells was determined at different time points and compared between a CSF sample and a serum sample. P value was determined by the t-test. ^∗P value ≤0.05 was considered significant.

Figure 3

DENV-3 RNA genome levels as determined from infected cells at 0, 24, 48, 72, 96, and 120 h postinfection determined by RT-qPCR at a multiplicity of infection (MOI) of 0.01 and 0.001. DENV-3 isolated from the cerebrospinal fluid (CSF) specimen (CSF-11098) and the serum specimen (HP-5528) was separately inoculated to C6/36, SKNSH, T98G, and N2A cell lines. P value was determined by the t-test. ^∗P value ≤0.05 was considered significant.

Figure 4

Infectivity of DENV-3 was measured by the expression of viral antigen-positive cells in mosquito and neural cell lines. DENV-3 antigen-positive cells are shown as determined by using immunofluorescence (IFA) staining at 48 hours postinfection at a multiplicity of infection (MOI) of 0.01. DENV-3 isolated from the cerebrospinal fluid (CSF) specimen (CSF-11098) and the serum specimen (HP-5528) were separately inoculated to C6/36, SKNSH, T98G, and N2A cell lines, and viral antigen (E NS1, NS2B, NS3, NS4A, NS4B, and NS5) were detected, respectively, by using IFA. Infected cells (%) were determined by average values (infected/total infected and noninfected cells) × 100% in three random fields at a magnification 200x. P value was determined by the t-test. ^∗P value ≤0.05 was considered significant.

Figure 5

Infectivity of DENV-3 in mosquito and neural cell lines. DENV-3 antigen against structural (E) and nonstructural antigens (NS1, NS2B, NS3, NS4A, NS4B, and NS5) is shown as determined by using immunofluorescence (IFA) staining at 48 hours postinfection at a multiplicity of infection (MOI) of 0.01. DENV-3 isolated from the cerebrospinal fluid (CSF) specimen (CSF-11098, indicated as CSF) and the serum specimen (HP-5528, indicated as S) was separately inoculated to the C6/36, SKNSH, T98G, and N2A cell lines. Each figure represents a random field at a 200x magnification.