Japanese Encephalitis DNA Vaccines with Epitope Modification Reduce the Induction of Cross-Reactive Antibodies against Dengue Virus and Antibody-Dependent Enhancement of Dengue Virus Infection.

Infection with viruses belonging to the genus Flavivirus, such as Japanese encephalitis virus (JEV) and dengue virus (DENV), is a worldwide health problem. Vaccines against JEV and DENV are currently available. However, the dengue vaccine possibly increases the risk of severe dengue due to antibody-dependent enhancement (ADE). Moreover, the Japanese encephalitis (JE) vaccine reportedly induces cross-reactive ADE-prone antibodies against DENV, potentially leading to symptomatic dengue. Therefore, it is necessary to eliminate the risk of ADE through vaccination. In this study, we attempted to develop a JE vaccine that does not induce ADE of DENV infection using an epitope modification strategy. We found that an ADE-prone monoclonal antibody cross-reactive to DENV and JEV recognizes the 106th amino acid residue of the E protein of JEV (E-106). The JE DNA vaccine with a mutation at E-106 (E-106 vaccine) induced comparable neutralizing antibody titers against JEV to those induced by the wild-type JE DNA vaccine. Meanwhile, the E-106 vaccine induced 64-fold less cross-reactive ADE-prone antibodies against DENV. The mutation did not compromise the protective efficacy of the vaccine in the lethal JEV challenge experiment. Altogether, the modification of a single amino acid residue identified in this study helped in the development of an ADE-free JE vaccine.

Engineered flavivirus vaccines control induction of crossreactive infection-enhancing and -neutralizing antibodies.

Flaviviruses are important human pathogens because of their global distribution and disease severity. The high structural similarity among flaviviruses induces cross-immunity, with individual flaviviruses exhibiting crossreactive infection-enhancing and/or -neutralizing activities against other flaviviruses. Unlike neutralizing antibodies, enhancing antibodies may increase the risk of disease severity. Vaccine-induced enhancement remains a concern in the development of flavivirus vaccines. Here, we immunized mice with DNA vaccine candidates (pcJEME, pcWNME or pcZIKME) against Japanese encephalitis virus (JEV), West Nile virus (WNV) or Zika virus (ZIKV), respectively, and investigated crossreactive neutralizing and enhancing antibody activities against seven flaviviruses. pcZIKME induced higher cross-neutralization against dengue viruses than against JEV and WNV. Moreover, pcZIKME with a single amino acid substitution (D87N) showed an increase in crossreactive neutralizing activity and a decrease in enhancing activities against other flaviviruses. A similar trend was observed in pcWNME. Engineered antigen might contribute to the development of safe and effective flavivirus vaccines.

Antibody-dependent enhancement representing in vitro infective progeny virus titer correlates with the viremia level in dengue patients.

Dengue virus (DENV) causes dengue fever (DF) and dengue hemorrhagic fever in humans. Some DF patients suddenly develop severe symptoms around the defervescent period. Although the pathogenic mechanism of the severe symptoms has not been fully elucidated, the viremia level in the early phase has been shown to correlate with the disease severity. One of the hypotheses is that a phenomenon called antibody-dependent enhancement (ADE) of infection leads to high level of viremia. To examine the plausibility of this hypothesis, we examined the relationship between in vitro ADE activity and in vivo viral load quantity in six patients with dengue diseases. Blood samples were collected at multiple time points between the acute and defervescent phases, and the balance between neutralizing and enhancing activities against the autologous and prototype viruses was examined. As the antibody levels against DENV were rapidly increased, ADE activity was decreased over time or partially maintained against some viruses at low serum dilution. In addition, positive correlations were observed between ADE activity representing in vitro progeny virus production and viremia levels in patient plasma samples. The measurement of ADE activity in dengue-seropositive samples may help to predict the level of viral load in the subsequent DENV infection.

A potent neutralizing mouse monoclonal antibody specific to dengue virus type 1 Mochizuki strain recognized a novel epitope around the N-67 glycan on the envelope protein: A possible explanation of dengue virus evolution regarding the acquisition of N-67 glycan.

The analysis of neutralizing epitope of dengue virus (DENV) is important for the development of an effective dengue vaccine. A potent neutralizing mouse monoclonal antibody named 7F4 was previously reported and, here, we further analyzed the detailed epitope of this antibody. 7F4 recognized a novel conformational epitope close to the N-67 glycan on the envelope protein. This antibody was specific to the DENV that lacks N-67 glycan, including the Mochizuki strain. Interestingly, the Mochizuki strain acquired N-67 glycan by 7F4 selective pressure. Considering that most of the currently circulating DENVs possess N-67 glycan, DENVs may have evolved to escape from antibodies targeting 7F4 epitope, suggesting the potency of this neutralizing epitope. In addition, this study demonstrated the existence of the epitopes close to 7F4 epitope and their crucial role in neutralization. In conclusion, the epitopes close to the N-67 glycan are attractive targets for the dengue vaccine antigen. Further analysis of this epitope is warranted.

Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero.

The Flaviviridae is a family of enveloped viruses with a positive-sense single-stranded RNA genome. It contains many viruses that threaten human health, such as Japanese encephalitis virus (JEV) and yellow fever virus (YFV) of the genus Flavivirus as well as hepatitis C virus of the genus Hepacivirus. Cell culture systems highly permissive for the Flaviviridae viruses are very useful for their isolation, propagation, and diagnosis, an understanding of their biology, and the development of vaccines and antiviral agents. Previously, we isolated a human hepatoma HuH-7-derived cell clone, Huh7.5.1-8, which is highly permissive to hepatitis C virus infection. Here, we have characterized flavivirus infection in the Huh7.5.1-8 cell line by comparing with that in the African green monkey kidney-derived Vero cell line, which is permissive for a wide spectrum of viruses. Upon infection with JEV, Huh7.5.1-8 cells produced a higher amount of virus particles early in infection and were more susceptible to virus-induced cell death than Vero cells. Similar outcomes were obtained when the cells were infected with another flavivirus, YFV (17D-204 strain). Quantification of cellular and extracellular viral RNA revealed that high JEV production in Huh7.5.1-8 cells can be attributed to rapid viral replication kinetics and efficient virus release early in infection. In a plaque assay, Huh7.5.1-8 cells developed JEV plaques more rapidly than Vero cells. Although this was not the case with YFV plaques, Huh7.5.1-8 cells developed higher numbers of YFV plaques than Vero cells. Sequence analysis of cDNA encoding an antiviral RNA helicase, RIG-I, showed that Huh7.5.1-8 cells expressed not only a full-length RIG-I mRNA with a known dominant-negative missense mutation but also variants without the mutation. However, the latter mRNAs lacked exon 5/6-12, indicating functional loss of RIG-I in the cells. These characteristics of the Huh7.5.1-8 cell line are helpful for flavivirus detection, titration, and propagation.

Cytokine Expression in Dengue Fever and Dengue Hemorrhagic Fever Patients with Bleeding and Severe Hepatitis.

Dengue is the most common mosquito-borne flaviviral infection in the world today. Several factors contribute and act synergistically to cause severe infection. One of these is dysregulated host immunological mediators that cause transient pathophysiology during infection. These mediators act on the endothelium to increase vascular permeability, which leads to plasma leakage compromising hemodynamics and coagulopathy. We conducted a prospective study to explore the expression of pro- and anti-inflammatory cytokines and how they relate to clinical dengue manifestations, by assessing their dynamics through acute dengue infection in adults admitted to the Hospital for Tropical Diseases, Bangkok, Thailand. We performed cytokine analysis at three phases of infection for 96 hospitalized adults together with serotyping of confirmed dengue infection during the outbreaks of 2015 and 2016. The serum concentrations of seven cytokines (interleukin [IL]-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha, and interferon gamma) were measured in duplicate using a commercial kit (Bio-Plex Human Cytokine Assay). In this study, the cytokine profile was suggestive of a T-helper 2 response. Most patients had secondary infection, and the levels of viremia were higher in patients with plasma leakage than those without plasma leakage. In addition, we observed that bleeding and hepatitis were associated with significantly higher levels of IL-8 during the early phases of infection. Furthermore, IL-6 levels in the early phase of infection were also elevated in bleeding patients with plasma leakage. These results suggest that IL-6 and IL-8 may act in synergy to cause bleeding in patients with plasma leakage.

Intraperitoneal injection with dengue virus type 1-infected K562 cells results in complete fatality among immunocompetent mice.

Dengue is one of the most important mosquito-borne viral diseases. Over half of the world's population is living in dengue endemic countries, where 100 million cases are estimated to occur annually. Although one dengue vaccine is currently available commercially, unfortunately its safety and efficacy has not been demonstrated for seronegative populations. Therefore, other vaccine candidates as well as antivirals are urgently required to control dengue diseases. To contribute to the development of preventative measures, in the present study we established an immunocompetent-mouse infection model using dengue virus type 1 Mochizuki strain. Following intraperitoneal injection with K562 cells infected with Mochizuki strain, all mice injected with ≥1 × 106 cells were killed within 7-11 days. Mice injected with ≥1 × 107 cells showed viremia (~104-105 FFU/ml) within 24 h of injection. Since a higher infective titer was detected in the mouse brain, this suggested that viruses were transmitted from the blood circulation into the brain. In further experiments, mice immunized with two types of DNA vaccines were challenged with virus. In contrast to the non-immunized control mice, all vaccinated mice survived after challenge. This immunocompetent-mouse infection model using dengue virus type 1 Mochizuki strain may be a useful tool to evaluate vaccines and preventive medicines against dengue virus.

Key Amino Acid Substitution for Infection-Enhancing Activity-Free Designer Dengue Vaccines.

Dengue is a globally important disease caused by four serotypes of dengue virus. Dengue vaccine development has been hampered by antigenic cross-reactivity among serotypes, which potentially causes antibody-dependent enhancement of infection and disease severity. Here we found that a single amino acid substitution in the envelope protein at position 87 from aspartic acid to asparagine or at position 107 from leucine to phenylalanine is critical for suppressing the induction of infection-enhancing antibody in a mouse model. The site and type of amino acid substitution were determined via neutralization escape using an enhancing-activity-only monoclonal antibody that was engineered to reveal neutralizing activity. Mutated dengue type 1 DNA vaccines containing either or both amino acid substitutions induced neutralizing antibodies devoid of enhancing activity against all serotypes. The effect of substitution was further demonstrated using other serotypes and a tetravalent formulation. This finding may contribute to the development of infection-enhancing-activity-free dengue vaccines.

High-throughput neutralization assay for multiple flaviviruses based on single-round infectious particles using dengue virus type 1 reporter replicon.

Diseases caused by the genus Flavivirus, including dengue virus (DENV) and Zika virus (ZIKV), have a serious impact on public health worldwide. Due to serological cross-reactivity among flaviviruses, current enzyme-linked immunosorbent assay (ELISA) for IgM/G cannot reliably distinguish between infection by different flaviviruses. In this study, we developed a reporter-based neutralization assay using single-round infectious particles (SRIPs) derived from representative flaviviruses. SRIPs were generated by transfection of human embryonic kidney 293 T cells with a plasmid encoding premembrane and envelope (prME) proteins from DENV1-4, ZIKV, Japanese encephalitis virus, West Nile virus, yellow fever virus, Usutu virus, and tick-borne encephalitis virus, along with a plasmid carrying DENV1 replicon containing the luciferase gene and plasmid for expression of DENV1 capsid. Luciferase activity of SRIPs-infected cells was well correlated with number of infected cells, and each reporter SRIP was specifically neutralized by sera from mice immunized with each flavivirus antigen. Our high-throughput reporter SRIP-based neutralization assay for multiple flaviviruses is a faster, safer, and less laborious diagnostic method than the conventional plaque reduction neutralization test to screen the cause of primary flavivirus infection. The assay may also contribute to the evaluation of vaccine efficacy and assist in routine surveillance and outbreak response to flaviviruses.

Human monoclonal antibodies against West Nile virus from Japanese encephalitis-vaccinated volunteers.

West Nile virus (WNV) is a positive-sense single-stranded RNA flavivirus belonging to the Japanese encephalitis virus (JEV) serocomplex of the Flaviviridae family and causes mosquito-borne infections. Although most human infection cases are asymptomatic, approximately one in 150 infected individuals develops meningoencephalitis, with a mortality rate of 4-14%. While the development of human neutralizing antibody therapeutics against WNV is strongly anticipated, WNV is difficult to study in conventional laboratories due to its high safety level requirement. In this study, we established fully human WNV-neutralizing monoclonal antibodies from the peripheral blood mononuclear cells of inactivated-JEV-vaccinated individuals, and these antibodies exhibited WNV neutralization both in vitro and in vivo. Our results demonstrate a new antibody cross-reactivity strategy to develop immunological therapeutic reagents for WNV and other JEV serotype viruses.

Dengue-Immune Humans Have Higher Levels of Complement-Independent Enhancing Antibody than Complement-Dependent Neutralizing Antibody.

Dengue is the most important arboviral disease worldwide. We previously reported that most inhabitants of dengue-endemic countries who are naturally immune to the disease have infection-enhancing antibodies whose in vitro activity does not decrease in the presence of complement (complement-independent enhancing antibodies, or CiEAb). Here, we compared levels of CiEAb and complement-dependent neutralizing antibodies (CdNAb) in dengue-immune humans. A typical antibody dose-response pattern obtained in our assay system to measure the balance between neutralizing and enhancing antibodies showed both neutralizing and enhancing activities depending on serum dilution factor. The addition of complement to the assay system increased the activity of neutralizing antibodies at lower dilutions, indicating the presence of CdNAb. In contrast, similar dose-response curves were obtained with and without complement at higher dilutions, indicating higher levels of CiEAb than CdNAb. For experimental support for the higher CiEAb levels, a cocktail of mouse monoclonal antibodies against dengue virus type 1 was prepared. The antibody dose-response curves obtained in this assay, with or without complement, were similar to those obtained with human serum samples when a high proportion of D1-V-3H12 (an antibody exhibiting only enhancing activity and thus a model for CiEAb) was used in the cocktail. This study revealed higher-level induction of CiEAb than CdNAb in humans naturally infected with dengue viruses.

Utility of Japanese encephalitis virus subgenomic replicon-based single-round infectious particles as antigens in neutralization tests for Zika virus and three other flaviviruses.

The introduction of a foreign virus into an area may cause an outbreak, as with the Zika virus (ZIKV) outbreak in the Americas. Preparedness for handling a viral outbreak involves the development of tests for the serodiagnosis of foreign virus infections. We previously established a gene-based technology to generate some flaviviral antigens useful for functional antibody assays. The technology utilizes a Japanese encephalitis virus subgenomic replicon to generate single-round infectious particles (SRIPs) that possess designed surface antigens. In the present study, we successfully expanded the capacity of SRIPs to four human-pathogenic mosquito-borne flaviviruses that could potentially be introduced from endemic to non-endemic countries: ZIKV, Sepik virus, Wesselsbron virus, and Usutu virus. Flavivirus-crossreactive monoclonal antibodies dose-dependently neutralized these SRIPs. ZIKV-SRIPs also produced antibody-dose-dependent neutralization curves equivalent to those shown by authentic ZIKV particles using sera from a Zika fever patient. The faithful expression of designed surface antigens on SRIPs will allow their use in neutralization tests to diagnose foreign flaviviral infections.

Neutralizing and enhancing antibody responses to five genotypes of dengue virus type 1 (DENV-1) in DENV-1 patients.

Dengue virus (DENV) has four distinct serotypes, DENV-1-4, with four to six genotypes in each serotype. The World Health Organization recommends tetravalent formulations including one genotype of each serotype as safe and effective dengue vaccines. Here, we investigated the impact of genotype on the neutralizing antibody responses to DENV-1 in humans. Convalescent sera collected from patients with primary infection of DENV-1 were examined for neutralizing antibody against single-round infectious particles of the five DENV-1 genotypes (GI-GV). In both GI- and GIV-infected patients, their neutralizing antibody titres against the five genotypes were similar, differing ≤4-fold from the homogenotypic responses. The enhancing activities against the five genotypes were also similar in these sera. Thus, the genotype strains of DENV-1 showed no significant antigenic differences in these patients, suggesting that GI- or GIV-derived vaccine antigens should induce equivalent levels of neutralizing antibodies against all DENV-1 genotypes.

Strategies to improve the immunogenicity of prM+E dengue virus type-2 DNA vaccine.

BACKGROUND: An important goal for dengue vaccines is to induce a high and durable level of neutralizing antibody. OBJECTIVE: Three strategies were investigated for improving the immunogenicity of a prM+E dengue serotype 2 (DENV-2) DNA vaccine: 1) expression in two different plasmids; 2) adjustment of dose; and, 3) introduction of the E sequence of Japanese encephalitis virus (JEV) at the carboxy-terminal portion of DENV-2 E. METHOD: Expression cassettes were designed to encode a full-length prM+E sequence of DENV-2 virus employing human-preferred codons (D2prMEopt), or a chimeric prM+E sequence in which the 100-residue carboxy-terminal region of E was derived from JEV (D2prMEJE20opt). pHIS and pCMVkan in the presence and absence of CpG motif, respectively, were used for cassette expression. The immunogenicity was compared in mice. RESULTS: Three injections of full-length-D2prMEopt in pHIS and pCMVkan induced a comparable neutralizing antibody titer at post-week-2-injection and post-week-4-injection. The 100-µg DNA dose induced a numerically but not statistically higher neutralizing antibody titer than the 10-µg dose. The chimeric-D2prMEJE20opt produced higher extracellular prM and E protein levels in transfected Vero cells, but had a tendency to induce a lower neutralizing antibody titer in mice when compared with the full-length-D2prMEopt. To optimize the immunogenicity of D2prMEopt-DNA candidate, both expression plasmids can be used to generate reproducible high neutralizing titer. A higher dose of DNA immunogen may induce a higher neutralizing antibody response. CONCLUSION: The strategy of the C-terminal region chimeric counterpart with JE20 did not improve but may have reduced the induction of neutralizing antibodies.

Complement-independent dengue virus type 1 infection-enhancing antibody reduces complement-dependent and -independent neutralizing antibody activity.

Dengue fever and dengue hemorrhagic fever are globally important mosquito-transmitted viral diseases. However, the only licensed vaccine is not highly protective. Viremia is related to disease severity in infected humans, and it is thought to be reduced by neutralizing antibodies but increased by infection-enhancing antibodies. We established an assay system to measure the balance between neutralizing and enhancing antibodies and found that most dengue-immune individuals in endemic areas carry complement-independent enhancing antibodies (CiEAb). Studying CiEAb is important for dengue vaccine development because the enhancing activity of CiEAb does not decrease in the presence of complement, which can reduce the enhancing activity of other antibodies in vitro. Here, we investigated the effects of CiEAb on the activity of neutralizing antibodies (mainly, complement-dependent neutralizing antibodies; CdNAb) using cocktails of mouse monoclonal antibodies (MAbs) against dengue virus type 1 (DENV-1). These cocktails included MAbs with enhancing activity only (represented by D1-V-3H12 [3H12]) or neutralizing activity only (represented by D1-IV-7F4 [7F4]). Because 3H12, an IgG1 subclass antibody, is complement-independent and cross-reacted with all dengue serotypes, it is a suitable model of CiEAb. An approximately equal amount of 3H12 abolished the neutralizing activity of 7F4. The complement-dependent neutralizing activities of the IgG2a and IgG2b variants of 7F4 were also completely inhibited by ⩾3-fold concentrations of the IgG1 variant. The complement-dependent antibody activities of other anti-DENV-1 MAbs and those of MAbs directed against other serotypes were inhibited 50% by 3H12 at various mixing ratios, ranging from one-hundredth to 10-fold. The complement-dependent neutralizing activities of dengue-immune mouse ascites fluids were also effectively inhibited by 3H12. This suggests that concomitantly induced CiEAb exerts an unwanted effect on the protective capacity of a vaccine. Thus, the effective inhibition of the neutralizing activity of CdNAb by CiEAb has implications for dengue pathogenesis and vaccine development.

Unique Requirement for ESCRT Factors in Flavivirus Particle Formation on the Endoplasmic Reticulum.

Flavivirus infection induces endoplasmic reticulum (ER) membrane rearrangements to generate a compartment for replication of the viral genome and assembly of viral particles. Using quantitative mass spectrometry, we identified several ESCRT (endosomal sorting complex required for transport) proteins that are recruited to sites of virus replication on the ER. Systematic small interfering RNA (siRNA) screening revealed that release of both dengue virus and Japanese encephalitis virus was dramatically decreased by single depletion of TSG101 or co-depletion of specific combinations of ESCRT-III proteins, resulting in ≥1,000-fold titer reductions. By contrast, release was unaffected by depletion of some core ESCRTs, including VPS4. Reintroduction of ESCRT proteins to siRNA-depleted cells revealed interactions among ESCRT proteins that are crucial for flavivirus budding. Electron-microscopy studies revealed that the CHMP2 and CHMP4 proteins function directly in membrane deformation at the ER. Thus, a unique and specific subset of ESCRT contributes to ER membrane biogenesis during flavivirus infection.

Bivalent vaccine platform based on Japanese encephalitis virus (JEV) elicits neutralizing antibodies against JEV and hepatitis C virus.

Directly acting antivirals recently have become available for the treatment of hepatitis C virus (HCV) infection, but there is no prophylactic vaccine for HCV. In the present study, we took advantage of the properties of Japanese encephalitis virus (JEV) to develop antigens for use in a HCV vaccine. Notably, the surface-exposed JEV envelope protein is tolerant of inserted foreign epitopes, permitting display of novel antigens. We identified 3 positions that permitted insertion of the HCV E2 neutralization epitope recognized by HCV1 antibody. JEV subviral particles (SVP) containing HCV-neutralization epitope (SVP-E2) were purified from culture supernatant by gel chromatography. Sera from mice immunized with SVP-E2 inhibited infection by JEV and by trans-complemented HCV particles (HCVtcp) derived from multi-genotypic viruses, whereas sera from mice immunized with synthetic E2 peptides did not show any neutralizing activity. Furthermore, sera from mice immunized with SVP-E2 neutralized HCVtcp with N415K escape mutation in E2. As with the SVP-E2 epitope-displaying particles, JEV SVPs with HCV E1 epitope also elicited neutralizing antibodies against HCV. Thus, this novel platform harboring foreign epitopes on the surface of the particle may facilitate the development of a bivalent vaccine against JEV and other pathogens.

Production of Japanese Encephalitis Virus-Like Particles Using Insect Cell Expression Systems.

Virus-like particles (VLPs) can be produced via the expression of virus surface proteins that self-assemble into particulate structures in recombinant protein expression systems. Expression of the DNA fragment encoding the Japanese encephalitis (JE) virus prM signal peptide, the precursor (prM) of the viral membrane protein (M), and the envelope glycoprotein (E) allows the production of a secretory form of VLPs. Expression systems that use lepidopteran insect cells, such as the baculovirus-insect cell system and stably transformed insect cells, can be used for the efficient production of JE VLPs. This chapter describes the production of JE VLPs from stably transformed lepidopteran insect cells.

Dengue virus infection-enhancing antibody activities against Indonesian strains in inhabitants of central Thailand.

Dengue virus (DENV) infection-enhancing antibodies are a hypothetic factor to increase the dengue disease severity. In this study, we investigated the enhancing antibodies against Indonesian strains of DENV-1-4 in 50 healthy inhabitants of central Thailand (Bangkok and Uthai Thani). Indonesia and Thailand have seen the highest dengue incidence in Southeast Asia. The infection history of each subject was estimated by comparing his/her neutralizing antibody titers against prototype DENV-1-4 strains. To resolve the difficulty in obtaining foreign live viruses for use as assay antigens, we used a recombinant system to prepare single-round infectious dengue viral particles based on viral sequence information. Irrespective of the previously infecting serotype(s), most serum samples showed significantly higher enhancement titers against Indonesian DENV-2 strains than against Thai DENV-2 strains, whereas the opposite effect was observed for the DENV-3 strains. Equivalent enhancing activities were observed against both DENV-1 and DENV-4. These results suggest that the genotype has an impact on enhancing antibody activities against DENV-2 and DENV-3, because the predominant circulating genotypes of each serotype differ between Indonesia and Thailand.

Divergence of the dengue virus type 2 Cosmopolitan genotype associated with two predominant serotype shifts between 1 and 2 in Surabaya, Indonesia, 2008-2014.

Indonesia is one of the biggest dengue endemic countries, and, thus, is an important place to investigate the evolution of dengue virus (DENV). We have continuously isolated DENV in Surabaya, the second biggest city in Indonesia, since 2008. We previously reported sequential changes in the predominant serotype from DENV type 2 (DENV-2) to DENV type 1 (DENV-1) in November 2008 and from DENV-1 to DENV-2 in July 2013. The predominance of DENV-2 continued in 2014, but not in 2015. We herein phylogenetically investigated DENV-2 transitions in Surabaya between 2008 and 2014 to analyze the divergence and evolution of DENV-2 concomitant with serotype shifts. All DENV-2 isolated in Surabaya were classified into the Cosmopolitan genotype, and further divided into 6 clusters. Clusters 1-3, dominated by Surabaya strains, were defined as the "Surabaya lineage". Clusters 4-6, dominated by strains from Singapore, Malaysia, and many parts of Indonesia, were the "South East Asian lineage". The most recent common ancestor of these strains existed in 1988, coinciding with the time that an Indonesian dengue outbreak took place. Cluster 1 appeared to be unique because no other DENV-2 isolate was included in this cluster. The predominance of DENV-2 in 2008 and 2013-14 were caused by cluster 1, whereas clusters 2 and 3 sporadically emerged in 2011 and 2012. The characteristic amino acids of cluster 1, E-170V and E-282Y, may be responsible for its prevalence in Surabaya. No amino acid difference was observed in the envelope region between strains in 2008 and 2013-14, suggesting that the re-emergence of DENV-2 in Surabaya was due to the loss or decrease of herd immunity in the 5-year period when DENV-2 subsided. The South East Asian lineage primarily emerged in Surabaya in 2014, probably imported from other parts of Indonesia or foreign countries.