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Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro . DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.IMPORTANCEDengue is a viral disease that infects nearly 400 million people worldwide and causes dengue hemorrhagic fever, which is responsible for 10,000 deaths each year. Currently, there is no therapeutic drug licensed to treat dengue infection, which makes the development of an effective vaccine essential. Virus-like particles (VLPs) are a safe and highly immunogenic platform that can be used in young children, immunocompromised individuals, as well as healthy adults. In this study, we describe the development of a dengue VLP vaccine and demonstrate that it induces a robust immune response against the dengue virus for over 1 year in monkeys. The immunity induced by this vaccine reduced live dengue infection in both murine and non-human primate models. These results indicate that our dengue VLP vaccine is a promising vaccine candidate., Competing Interests: D.T., K.Ma., A.U., Y.W., and M.I are employees of VLP Therapeutics, Inc.; J.F.S. is an employee of and holds stocks in VLP Therapeutics, Inc. W.A. is a board member and employee of and holds stocks in VLP Therapeutics, Inc. W.A. is an inventor on a related vaccine patent. The remaining authors declare no competing interest.

Objective: The invasion of dengue virus (DENV)-2 Cosmopolitan genotype into the Philippines, where the Asian II genotype previously circulated challenges the principle of dengue serotype-specific immunity. Assessment of antibodies in this population may provide a mechanistic basis for how new genotypes emerge in dengue-endemic areas., Methods: We evaluated the neutralizing antibody (nAb) and antibody-dependent enhancement (ADE) responses against the two genotypes using archived serum samples collected from 333 patients with confirmed dengue in Metro Manila, Philippines, before, during, and after the introduction of the Cosmopolitan genotype. We quantified nAb titers in baby hamster kidney (BHK-21) cells with or without the Fcγ receptor IIA (FcγRIIA) to detect the capacity of virus-antibody complexes to neutralize or enhance DENV., Results: The nAb potency of the archived serum samples against the two genotypes was greatly affected by the presence of FcγRIIA. We found significant differences in nAb titers between the two genotypes in BHK-21 cells with FcγRIIA (P <0.0001). The archived serum samples were incapable of fully neutralizing the Cosmopolitan genotype, but instead strongly promoted its ADE compared to the Asian II genotype (P <0.0001)., Conclusion: These results reinforce the role of pre-existing immunity in driving genotype shifts. Our finding that specific genotypes exhibit differing susceptibilities to ADE by cross-reactive antibodies may have implications for dengue vaccine development., Competing Interests: Declaration of competing interests The authors have no competing interests to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

From August 27 to October 15, 2014, a dengue fever outbreak with 158 autochthonous cases occurred after nearly 70 years of no reports of autochthonous cases in Japan. The most competent mosquito vector for dengue virus (DENV) transmission in Japan is Aedes albopictus. Since A. albopictus is widely distributed throughout Japan, we examined the susceptibility of this species to infection by DENV and the relationship of the endosymbiont Wolbachia (wAlbA and wAlbB) with susceptibility to DENV. The A. albopictus YYG strain, collected from the Yoyogi Park in 2014, the epicenter of the dengue fever outbreak, was found to have lower susceptibility to DENV 1 and 3 than that of the indigenous Japanese strains A. albopictus EBN 201808 (F1 from the field) and A. albopictus ISG 201603. Furthermore, the A. albopictus EBN 201808 strain showed the same susceptibility to DENV3 as the A. albopictus ISG 201603tet strain (Wolbachia-free). Susceptibility to DENV3 was not related to Wolbachia strains wAlbA or wAlbB in the A. albopictus ISG 201603 strain.

Dengue fever, caused by the mosquito-borne dengue virus (DENV), has been endemic in Myanmar since 1970 and it has become a significant public health burden. It is crucial that circulating DENV strains are identified and monitored, and that their transmission efficiency and association with disease severity is understood. In this study, we analyzed DENV-1, DENV-2, DENV-3, and DENV-4 serotypes in 1235 serum samples collected in Myanmar between 2017 and 2019. Whole-genome sequencing of DENV-1-4 demonstrated that most DENV-1-4 strains had been circulating in Myanmar for several years. We also identified the emergence of DENV-3 genotype-I in 2017 samples, which persisted through 2018 and 2019. The emergence of the strain coincided with a period of increased DENV-3 cases and marked changes in the serotype dynamics. Nevertheless, we detected no significant differences between serum viral loads, disease severity, and infection status of individuals infected with different DENV serotypes during the 3-year study. Our results not only identify the spread of a new DENV-3 genotype into Yangon, Myanmar, but also support the importance of DENV evolution in changing the epidemic dynamics in endemic regions.

Background: A periodic serosurvey of dengue seroprevalence is vital to determine the prevalence of dengue in countries where this disease is endemic. This study aimed to determine the prevalence of dengue immunoglobulin G (IgG) seropositivity among healthy Malaysian adults living in urban and rural areas., Methods: A total of 2598 serum samples (1417 urban samples, 1181 rural samples) were randomly collected from adults ages 35-74 y. The presence of the dengue IgG antibody and neutralising antibodies to dengue virus (DENV) 1-4 was determined using enzyme-linked immunosorbent assay and the plaque reduction neutralisation test assay, respectively., Results: The prevalence of dengue IgG seropositivity was 85.39% in urban areas and 83.48% in rural areas. The seropositivity increased with every 10-y increase in age. Ethnicity was associated with dengue seropositivity in urban areas but not in rural areas. The factors associated with dengue seropositivity were sex and working outdoors. In dengue IgG-positive serum samples, 98.39% of the samples had neutralising antibodies against DENV3, but only 70.97% of them had neutralising antibodies against DENV4., Conclusion: The high seroprevalence of dengue found in urban and rural areas suggests that both urban and rural communities are vital for establishing and sustaining DENV transmission in Malaysia., (© The Author(s) 2020. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Dengue remains a major public health problem in the Philippines. In this study, we determined the circulating dengue serotypes in the Philippines during the 2015-2017 outbreaks using a total of 678 serum samples from 537 individual dengue patients. Following an increase in the number of DENV-4 patients in recent years, we conducted a comprehensive molecular and epidemiology analysis on the DENV-4 strains isolated recently in the Philippines. Two genotypes of DENV-4 have been isolated in the Philippines since 1956: GI and GIIa. The GIIa DENV strains that were isolated in the present study were closely related to a distinct group of GIIa strains that were isolated from the Philippines in 2004. A majority of the isolates of this sub-group have been identified in the Philippines, suggesting that this lineage may have been introduced in the Philippines, and evolved to form the distinct sub-group within GIIa strains. The increase in DENV-4 activity also coincided with the appearance of the GIIa subgroup and the phasing-out of the GI lineage in the Philippines. Overall, our study demonstrates a shift in DENV-4 genotype and epidemic dynamics in a hyperendemic region, suggesting the importance of DENV genetic evolution in establishing and sustaining transmission.

Dengue virus (DENV) replication between mosquito and human hosts is hypothesized to be associated with viral determinants that interact in a differential manner between hosts. However, the understanding of inter-host viral determinants that drive DENV replication and growth between hosts is limited. Through the use of clinical isolates, we identified an amino acid variation of Ala, Met and Val at position 116 of DENV-1 NS4B. While the proportion of virus with the NS4B-116V variant remained constantly high in serial passages in a mosquito cell line, populations of the NS4B-116M and NS4B-116A variants became dominant after serial passages in mammalian cell lines. Using recombinant DENV-1 viruses, the Val to Ala or Met alteration at position NS4B-116 (rDENV-1-NS4B-116A and rDENV-1-NS4B-116M) resulted in enhanced virus growth in human cells in comparison to the clone with Val at NS4B-116 (rDENV-1-NS4B-116V). However, the reverse phenomenon was observed in a mosquito cell line. Additionally, in a human cell line, differential levels of IFN-α/β and IFN-stimulated gene expressions (IFIT3, IFI44L, OAS1) suggested that the enhanced viral growth was dependent on the ability of the NS4B protein to hamper host IFN response during the early phase of infection. Overall, we identified a novel and critical viral determinant at the pTMD3 of NS4B region that displayed differential effects on DENV replication and fitness in human and mosquito cell lines. Taken together, the results suggest the importance of the NS4B protein in virus replication and adaptation between hosts.

In 2014 in Japan, 162 autochthonous dengue cases were reported for the first time in nearly 70 years. Here, we report the results of the detection and isolation of dengue virus (DENV) from mosquitoes collected in Tokyo Metropolis in 2014 and 2015. The phylogenetic relationship among DENV isolates from mosquitoes and from patients based on both the entire envelope gene and whole coding sequences was evaluated. Herein, 2,298 female and 956 male Aedes albopictus mosquitoes were collected at six suspected locations of DENV infection in Tokyo Metropolis from August to October in 2014 and grouped into 124 and 35 pools, respectively, for viral genome detection and DENV isolation. Dengue virus RNA was detected using reverse transcription polymerase chain reaction and TaqMan assays from 49 female pools; 16 isolates were obtained using C6/36 and Vero cells. High minimum infection rates (11.2-66.7) persisted until mid-September. All DENV isolates belonged to the genotype I in serotype 1 (DENV-1), and its sequences demonstrated > 99% homology to the sequence of the DENV isolated from a patient in the vicinity of Tokyo Metropolis in 2014. Therefore, Ae. albopictus was a major DENV vector, and a single DENV-1 strain circulated in Tokyo Metropolis in 2014. Dengue virus was not detected from male mosquitoes in 2014 and wild larvae in April 2015. Thus, the possibility of both vertical transmission and overwintering of DENV was extremely low, even in dengue-epidemic areas. This study reports the first entomological information on a dengue outbreak in a temperate region, where no Aedes aegypti mosquitoes are distributed.

Background: A vaccine against all four dengue virus (DENV) serotypes includes the formulation of one genotype of each serotype. Although genetic similarities among genotypes within a serotype are higher as compared to those among serotypes, differences in the immunogenicity of the included genotypes would be a critical issue in maximizing successful dengue vaccine development. Thus, we determined the neutralizing antibody responses against three genotypes of dengue virus serotype 2 (DENV-2), namely Cosmopolitan, Asian I, and Asian/American, after primary and secondary inoculation with DENV-2 in a dengue animal model, the common marmoset (Callithrix jacchus)., Methods: A total of fifty-four plasma samples were obtained from thirty-four marmosets that were inoculated with clinically-isolated DENV strains or DENV candidate vaccines, were used in this study. Plasma samples were obtained from marmosets after primary inoculation with DENV-2 infection, secondary inoculation with homologous or heterologous genotypes, and tertiary inoculation with heterologous DENV. Neutralizing antibody titers against DENV-2 (Cosmopolitan, Asian I, and Asian/American genotypes) and DENV-1 were determined using a conventional plaque reduction neutralization assay., Results: In marmosets that were inoculated with the Cosmopolitan genotype in primary infection, neutralizing antibody neutralized 3 genotypes, and the titers to Asian I genotype were significantly higher than those to homologous Cosmopolitan genotype. After secondary DENV-2 infection with heterologous genotype (Asian I in primary and Asian/American in secondary), neutralizing antibody titers to Asian/American genotype was significantly higher than those against Cosmopolitan and Asian I genotypes. Following tertiary infection with DENV-1 following DENV-2 Asian I and Cosmopolitan genotypes, neutralizing antibody titers to Asian/American were also significantly higher than those against Cosmopolitan and Asian I genotypes., Conclusion: The present study demonstrated that different levels of neutralizing antibodies were induced against variable DENV-2 genotypes after primary, secondary and tertiary infections, and that neutralizing antibody titers to some heterologous genotypes were higher than those to homologous genotypes within a serotype. The results indicate that heterogeneity and homogeneity of infecting genotypes influence the levels and cross-reactivity of neutralizing antibodies induced in following infections. The results also suggest that certain genotypes may possess advantage in terms of breakthrough infections against vaccination.

Background: Antibodies are critical responses to protect the host from dengue virus(DENV) infection. Antibodies target DENV by two pathologic mechanisms: virus neutralization and infection enhancement. In dengue patients, the absence of neutralizing activity in the presence of FcγR implies that infection-enhancing activity hampers the neutralizing activity of antibodies, which could potentially lead to symptomatic presentations and severe clinical outcomes., Methods: A total of 100 pair serum samples from adult healthy volunteers were obtained during the dengue season in Ha Noi in 2015 for evaluation of neutralizing and infection-enhancing activity. Additionally, 20 serum samples from acute secondary DENV infection patients were also used as the patient group in this study. PRNT was performed on BHK cells and FcγR-expressing BHK cell lines for all serum samples., Results: Out of 100 residents, positive neutralizing antibodies (N.A) were found in 44.23 and 76.92% for DENV-1; 38.46 and 75% for DENV-2; 19.23 and 15.38% for DENV-3; and 1.92 and 9.62% for DENV-4 for pre and post-dengue season respectively. The percentage of post-exposure residents having positive responses against single, two, or more than three DENV serotypes were 38.46, 44.23 and 15.38%, respectively. A total of 34 residents were DENV seropositive before the dengue season and these individuals demonstrated further elevation of IgG antibodies after the dengue season. At the end of the season, 18 residents were confirmed to be new asymptomatic DENV infection cases. In both groups, N.A titers determined on BHK cells were higher than that on FcγR-expressing BHK cells. In heterotypic N.A responses, N.A titers to the infecting serotype from the samples obtained from pre-exposure group were significantly higher than those of the patient group. However, fold enhancement to the infecting serotypes from the samples in the pre-exposure group was substantially lower as compared to that of the patient group., Conclusion: Before and after the dengue season, serum samples from healthy volunteers demonstrated high levels of neutralizing antibodies and low or absence of infection-enhancement activity. The results suggest that while infection-enhancement activity hampers neutralizing activity of antibodies, high levels of DENV neutralizing antibodies set a critical threshold in facilitating the prevention of disease progression.

It has been reported that virus-antibody immune complexes formed during secondary dengue virus infection are associated with increased disease severity. Here, we describe the details of a plaque titration method that uses FcγR -expressing BHK cells to detect and quantify infectious virus-immune complexes and a quantitative real-time PCR method for the quantification of virus genome in patients with secondary dengue infection. These methods detect both viruses in free-form and in immune complexes in patients with dengue infection, and are useful for determining viremia levels and patterns that better reflect in vivo conditions.

Dengue virus (DENV) is one of the major infectious diseases in tropical regions and approximately half of the world population is at risk of infection. Vaccines would offer an effective control measure against this disease. We previously reported on the utility of marmosets as an animal model for studying primary and secondary dengue infections. Infected marmosets consistently develop viraemia and antibody kinetics that reflect those of patients with dengue. Thus, it is important to determine the utility of marmosets as an animal model for demonstrating vaccine efficacy. In this study, marmosets were inoculated with candidate vaccine and parent strains and challenged with a clinical DENV strain. The viraemia and antibody kinetics in these marmosets were determined. Marmosets consistently develop lower viraemia with an attenuated vaccine strain. During secondary challenge, the IgM response was delayed, whereas the IgG levels rose rapidly, indicating a secondary antibody response. The neutralizing activities against the homotypic serotype were high; all marmosets were protected against viraemia following secondary inoculation. The viraemia markers and antibody responses were consistent with those of human DENV infection and vaccinees. These results demonstrate the utility of marmosets as an animal model for the study of vaccine efficacy.

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.

Cases of autochthonous infections of dengue virus type 1 (DENV-1) were detected in Japan after a 70-year period devoid of dengue outbreaks. We previously showed that E gene sequences are identical in 11 of the 12 DENV-1 strains autochthonous to Japan. However, the E sequence represents only 14% of the DENV-1 genome. In the present study, we have sequenced the entire genome of 6 autochthonous DENV-1 strains that were isolated from patients during the 2014 outbreak. Sequencing of 5 Yoyogi group strains with identical E sequences and 1 Shizuoka strain with a different E sequence revealed that the first Yoyogi group strain differed from the Shizuoka strain by 18 amino acid residues. Furthermore, 2 Yoyogi group strains had different genomic sequences while the other 3 had identical genomes. Phylogenetic analyses indicated that the Hyogo strain, a Yoyogi group strain, was the first to diverge from the other 4 Yoyogi group strains. The E gene sequence of the Yoyogi group strains exhibits the highest homology to those of the strains isolated in Malaysia and Singapore between 2013 and 2014. The patient infected with the Hyogo strain visited Malaysia before the onset of dengue fever, suggesting that this was a case of dengue infection imported from Malaysia.

Dengue virus (DENV) isolation from mosquitoes is necessary for providing definitive evidence of virus circulation, and is critical for further virological characterization and determination of epidemiological characteristics. By using Aedes albopictus mosquitoes captured during an outbreak in Tokyo in 2014, we compared the DENV isolation rates of a conventional virus isolation method that uses C6/36 mosquito cells as assay cells with those of a virus isolation method that relies on an antibody-dependent enhancement (ADE) mechanism by using FcγR-expressing baby hamster kidney (BHK) cells and an antibody with ADE activity. The number of DENV genome copies and infectious virus titers in cell culture supernatant fluids of FcγR-expressing BHK cells were significantly higher than those of the C6/36 cells. In addition, DENV was isolated from a mosquito pool by using FcγR-expressing BHK cells only in the presence of infection-enhancing antibody. Infectious virus was detected in six mosquito pools only by using FcγR-expressing BHK cells. The results suggest that the method that relies on ADE mechanism by using the FcγR-expressing BHK cells and an antibody with ADE activity is useful for DENV isolation from mosquitoes caught in the field.

Background: Dengue virus (DENV) and Japanese encephalitis virus (JEV) belong to the genus Flavivirus, and infection with a virus within this genus induces antibodies that are cross-reactive to other flaviviruses. Particularly in DENV infection, antibodies to DENV possess two competing activities: neutralizing activity and infection-enhancing activity. These antibody activities are considered central in modulating clinical outcomes of DENV infection. Here, we determined the neutralizing and infection-enhancing activity of DENV cross-reactive antibodies in adults before and after JE vaccination., Methods: Participants were 77 Japanese adults who had received a single dose of inactivated Vero cell-derived JE vaccine. A total of 154 serum samples were obtained either before or approximately a month after a single dose of JE vaccination. The antibody-dependent enhancement (ADE) activity to each of four DENV serotypes and the neutralizing activities to DENV and to JEV were determined in each of the serum samples by using baby hamster kidney (BHK) cells and FcγR-expressing BHK cells., Results: A total of 18 post-JE immunization samples demonstrated cross-reactivity to DENV in an anti-DENV IgG ELISA. DENV neutralizing antibodies were not detected after JE vaccination in this study. However, undiluted post-JE vaccination serum samples from 26 participants demonstrated monotypic and heterotypic ADE activity to DENV. ADE activity was also observed in 1:10-diluted samples from 35 of the JE vaccine recipients (35/77, 45 %)., Conclusion: In summary, JE vaccination induced DENV cross-reactive antibodies, and at sub-neutralizing levels, these DENV cross-reactive antibodies possess DENV infection-enhancement activity. The results also indicate that cross-reactivity to DENV is associated with high levels of JEV neutralizing antibodies and, the DENV cross-reactivity is further facilitated by JE vaccination.

In late August 2014, dengue cases were reported in Japan, and a total of 162 cases were confirmed. In the present study, the envelope (E) gene sequences of 12 specimens from the dengue patients were determined. A dengue virus serotype 1 (DENV1) strain (D1/Hu/Shizuoka/NIID181/2014), which was clearly different from the first reported strain (D1/Hu/Saitama/NIID100/2014), was identified, although the other 11 specimens showed the same nucleotide sequences as D1/Hu/Saitama/NIID100/2014. The E gene sequences of two different strains were compared with those of nine DENV1 strains of imported cases in Japan in 2014. Phylogenetic analysis based on the E gene sequences showed that the D1/Hu/Saitama/NIID100/2014 strain was closely related to a strain isolated from an imported case from Singapore. Although no strain closely related to D1/Hu/Shizuoka/NIID181/2014 was found in these imported strains, the strain was closely related to isolates in Thailand and Taiwan in 2009. These data indicate that the dengue cases in Japan were caused by two different dengue virus strains that entered Japan through different means.

In 2013 in Myanmar, dengue epidemic occurred with 20,255 cases including 84 deaths. This study aimed to determine the serological and molecular characteristics of dengue virus (DENV) infection among children with clinical diagnosis of dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) during this period. Single acute serum samples were collected from 300 children in Mandalay Children Hospital, Mandalay, Myanmar. Out of the 300 children, 175 (58.3%) and 183 (61%) were positive for anti-dengue IgM and anti-dengue IgG, respectively. Among the IgM positives, 41 (23.4%) had primary DENV infection. Thirty-nine DENV strains (23 DENV-1, 10 DENV-2 and 6 DENV-4) were successfully isolated after inoculation of the patient serum samples onto C6/36 cells. DENV 1 was the dominant serotype in the 2013 epidemic. There was no correlation between the infecting serotypes and clinical severities. The DENV-1 strains belonged to three lineages of the genotype 1; the DENV-2 strains were of the Asian I genotype and were separated into two lineages; and DENV-4 strains belonged to the same lineage of genotype I. It is of interest to note the diversity of DENV-1 and -2 circulating in the same location during June-August 2013. These DENV isolates were genetically close (98%-100%) to the other previously reported isolates from Myanmar and its neighboring countries, namely China, Thailand, Sri Lanka, Cambodia and Vietnam. Primary DENV infection was still high among the severe dengue cases. Different serotypes of DENV were co-circulating in 2013, however, genotype shift was not observed. Additionally, amino acid mutations were detected in the study strains not seen in the previously reported strains from other countries and Myanmar. This paper provided information on the circulating serotypes for the last 15years and the recent dengue situation in Mandalay, Myanmar after 2006., (Copyright © 2016 Elsevier B.V. All rights reserved.)

After 70 years with no confirmed autochthonous cases of dengue fever in Japan, 19 cases were reported during August-September 2014. Dengue virus serotype 1 was detected in 18 patients. Phylogenetic analysis of the envelope protein genome sequence from 3 patients revealed 100% identity with the strain from the first patient (2014) in Japan.

Infection with a dengue virus (DENV) serotype induces cross-reactive, weakly neutralizing antibodies to different dengue serotypes. It has been postulated that cross-reactive antibodies form a virus-antibody immune complex and enhance DENV infection of Fc gamma receptor (FcγR)-bearing cells. We determined whether infectious DENV-antibody immune complex is formed in vivo in marmosets after passive transfer of DENV-specific monoclonal antibody (mAb) and DENV inoculation and whether infectious DENV-antibody immune complex is detectable using FcγR-expressing cells. Marmosets showed that DENV-antibody immune complex was exclusively infectious to FcγR-expressing cells on days 2, 4, and 7 after passive transfer of each of the mAbs (mAb 4G2 and mAb 6B6C) and DENV inoculation. Although DENV-antibody immune complex was detected, contribution of the passively transferred antibody to overall viremia levels was limited in this study. The results indicate that DENV cross-reactive antibodies form DENV-antibody immune complex in vivo, which is infectious to FcγR-bearing cells but not FcγR-negative cells., (© The American Society of Tropical Medicine and Hygiene.)

Dengue virus (DENV) infection is a serious global health threat. For the surveillance and control of dengue, there is a need for robust diagnostic tools that are relatively easy to use and reliable in various clinical settings. We investigated the applicability of NS1 antigen detection in urine samples for the diagnosis of DENV. About 118 urine samples, obtained from 96 dengue patients at various phases of disease, were used for this study. NS1 antigen was detected by ELISA in the urine samples obtained from patients after 2-17 days of disease onset. Positive detection rates of NS1 antigen ranged between 13-43%. Based on real-time RT-PCR, positive detection rates of viral genome in the urine samples ranged between 20-33% on days 0 to ≥15. On days 11 to ≥15 after the disease onset, NS1 antigen was detected at similar rates in serum and urine samples. Additionally, NS1 antigen was detected in 2 urine samples, but not in the serum samples, on days 7 and 16 after the onset of the disease. The results confirm the applicability of NS1 antigen detection in urine samples using ELISA to diagnose acute DENV infection and suggests that the assay is potentially useful when only limited amounts of serum samples are available and in limited resource settings.

There are four dengue virus (DENV) serotypes. Primary infection with one does not confer protective immunity against the others. We have reported previously that the marmoset (Callithrix jacchus) is a useful primary DENV infection model. It has been reported that secondary DENV infection with a heterotypic serotype induces viraemia kinetics and antibody responses that differ from those in primary infection. Thus, it is important to determine the utility of the marmoset as a model for secondary DENV infection. Marmosets were infected with heterologous DENV by secondary inoculation, and viraemia kinetics and antibody responses were analysed. The marmosets consistently developed high levels of viraemia after the secondary inoculation with heterologous DENV serotypes. IgM responses were lower compared with primary inoculation responses, whilst IgG responses were rapid and high. Neutralizing activities, which possessed serotype cross-reactive activities, were detected as early as 4 days after inoculation. In addition, infectious viraemia titres were higher when assayed with Fcγ receptor-expressing baby hamster kidney (BHK) cells than when assayed with conventional BHK cells, suggesting the presence of infectious virus-antibody immune complexes. After secondary infection with heterotypic DENV, the marmosets demonstrated viraemia kinetics, IgM and IgG responses, and high levels of serotype cross-reactive neutralizing antibody responses, all of which were consistent with secondary DENV infection in humans. The results indicate the marmoset as a useful animal for studying secondary, as well as primary, DENV infection.

Dengue virus (DENV) causes a life-threatening illness, with a wide range of symptoms from mild febrile illness, dengue fever (DF), to life-threatening illness, dengue hemorrhagic fever (DHF). Antibody-dependent enhancement (ADE) is considered to be a risk factor for DHF. In the present study, we determined the parameters for ADE assays using FcγR-expressing BHK cells. Monoclonal antibodies and human serum samples were used in the assays. We examined antibody concentration and virus concentration and analyzed whether antibody concentration or DENV-antibody ratio determines ADE activity. Virus growth was quantified by a conventional plaque titration method using FcγR-expressing BHK cells. The assay allowed the detection of DENV growth with inoculation doses ranging from 10(2) PFU/ml to 10(6) PFU/ml using monoclonal antibodies and undiluted or diluted serum samples. The results indicate that antibody concentration rather than DENV-antibody ratio determines the demonstration of ADE activity. Thus, antibody concentration rather than multiplicity of infection was defined as the main determinant in ADE assays using FcγR-expressing BHK cells.

Background: Dengue virus ( DENV) nonstructural protein 1 ( NS1) has been used as a novel diagnostic marker during the early phase of DENV infection., Methods: Presence of NS1 antigen was examined using 336 serum samples obtained from 276 travelers returning to Japan from Asia, Central and South America, Pacific Islands, and Africa with dengue. Assay specificity was evaluated using 148 non-dengue samples., Results: Positive rates among four DENV serotypes were 68%-89%. NS1 antigen positive rates were at similar levels between primary infection and secondary infection. NS1 antigen positive rates were 88%-96% on days 1-5, 75%-100% on days 6-10, and 36-60% on ≥ day 11. Positive rates using real-time polymerase chain reaction (RT-PCR) were over 70% on days 1-5, but decreased thereafter., Conclusions: The results indicate that NS1 antigen positive rates were higher than those of RT-PCR during longer period of early phase in DENV infection. Thus, NS1 antigen ELISA is a useful tool for confirming DENV infection in international travelers, when it is used in combination with anti-DENV IgM ELISA., (© 2013 International Society of Travel Medicine.)

Background: Dengue virus (DENV) infection-enhancing activity of pre-existing antibody has been suggested to be one of the factors responsible for the pathogenesis of the severe form of DENV infection, dengue haemorrhagic fever. Although studies of infection-enhancement activity using diluted serum sera are informative, infection-enhancement activity determined using undiluted sera may better reflect in vivo conditions., Methods: We determined whether serum samples collected from dengue patients possessed the ability to enhance DENV infection without dilution using FcγR-expressing cells., Results: Serum samples obtained at the early phase of secondary infection enhanced DENV infection without dilution. In contrast, sera obtained at the late stage of secondary infection demonstrated low levels or absence of infection-enhancing activity., Conclusion: The results indicate that human sera obtained from patients with secondary infection possess the ability to enhance DENV infection without dilution, and suggest that antibody-dependent enhancement may occur in vivo during secondary DENV infection to infecting DENV serotypes.

Background: Marmosets are susceptible to dengue virus (DENV) infection. However, blood parameter data and clinical signs of DENV-infected marmosets are limited., Methods: Blood hematological and serum biochemical values were obtained from twelve DENV-inoculated and four mock-infected marmosets. Additionally, body temperature and activity level were determined., Results: Five DENV-inoculated marmosets demonstrated thrombocytopenia, nine demonstrated leucopenia, and five demonstrated an increase in the levels of AST, ALT, LDH, and BUN. Additionally, seven DENV-inoculated marmosets demonstrated clinical signs including fever and decreases in activity. None of the four mock-inoculated marmosets demonstrated changes in either hematological or biochemical parameters., Conclusions: Marmosets inoculated with DENV exhibited clinical signs and changes in hematological and biochemical parameters. The results suggest that blood parameter data and clinical signs could potentially be useful markers for understanding the progress of DENV infection in studies using marmosets., (© 2012 John Wiley & Sons A/S.)

Dengue virus (DENV) infection is a major health threat for travelers. We describe here a case of dengue virus serotype-3 (DENV-3) infection in a traveler returning to Japan from the Republic of Benin. The isolated DENV-3 genotype 3 strain exhibited high sequence similarity to those from neighboring regions., (© 2012 International Society of Travel Medicine.)

Background: Progress in dengue vaccine development has been hampered by limited understanding of protective immunity against dengue virus infection. Conventional neutralizing antibody titration assays that use FcγR-negative cells do not consider possible infection-enhancement activity. We reasoned that as FcγR-expressing cells are the major target cells of dengue virus, neutralizing antibody titration assays using FcγR-expressing cells that determine the sum of neutralizing and infection-enhancing activity, may better reflect the biological properties of antibodies in vivo., Methods and Findings: We evaluated serum samples from 80 residents of a dengue endemic country, Malaysia, for neutralizing activity, and infection-enhancing activity at 1∶10 serum dilution by using FcγR-negative BHK cells and FcγR-expressing BHK cells. The serum samples consisted of a panel of patients with acute DENV infection (31%, 25/80) and a panel of donors without acute DENV infection (69%, 55/80). A high proportion of the tested serum samples (75%, 60/80) demonstrated DENV neutralizing activity (PRNT(50)≥10) and infection-enhancing activity. Eleven of 18 serum samples from patients with acute secondary DENV infection demonstrated neutralizing activity to the infecting serotype determined by using FcγR-negative BHK cells (PRNT(50)≥10), but not when determined by using FcγR-expressing cells., Conclusion: Human serum samples with low neutralizing activity determined by using FcγR-negative cells showed DENV infection-enhancing activity using FcγR-expressing cells, whereas those with high neutralizing activity determined by using FcγR-negative cells demonstrate low or no infection-enhancing activity using FcγR-expressing cells. The results suggest an inverse relationship between neutralizing antibody titer and infection-enhancing activity, and that neutralizing activity determined by using FcγR-expressing cells, and not the activity determined by using FcγR-negative cells, may better reflect protection to DENV infection in vivo.

Background: Virus isolation is the most reliable evidence of dengue virus (DENV) infection. However, conventional virus isolation methods generally posses lower sensitivity and are time consuming as compared to other diagnostic methods such as detection of viral genome by RT-PCR, and determination of NS1 antigen and anti-DENV antibody by ELISA., Objectives: A virus isolation method relying on the antibody-dependent enhancement mechanism was established and the assay's efficacy in DENV isolation was confirmed., Study Design: FcγR-expressing BHK cells were used for DENV isolation from patient serum samples in the presence of a flavivirus-group reactive monoclonal antibody, mAb4G2, which possesses DENV infection-enhancement activity. DENV genome copy numbers in the culture supernatant fluids of FcγR-expressing BHK cells were assessed and compared to those of parent BHK cells and C6/36 mosquito cells, a cell line commonly used for DENV isolation., Results: The virus titer levels were higher in the culture supernatant fluid of FcγR-expressing BHK cells in the presence of enhancing antibody in comparison with other cell lines using laboratory-established strains and some clinical samples. DENV was isolated from 7 of 16 serum samples by using FcγR-expressing BHK cells in the presence of mAb4G2, but not by using cell lines commonly employed in conventional isolation assays, the FcγR-negative BHK cells and C6/36 cell lines., Conclusions: The results demonstrate that FcγR-expressing BHK cell line in the presence of antibodies, which possess antibody dependent enhancement (ADE) activity, is a useful tool for DENV isolation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Dengue virus (DENV) causes a wide range of illnesses in humans: dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Animal models that constantly develop high levels of viraemia are required for the development of protective and preventive measures. Common marmosets (Callithrix jacchus) demonstrated high levels of viraemia after inoculation with clinical isolates of four serotypes of DENV; in particular, over 10(6) genome copies ml(-1) after inoculation with DENV-2. Non-structural protein 1 and DENV-specific IgM and IgG antibodies were consistently detected. The DENV-2 genome was detected in lymphoid organs including the lymph nodes, spleen and thymus, and also in non-lymphoid organs. DENV antigen was detected by immunohistochemistry in the liver and spleen from inoculated marmosets. Four marmosets were reinoculated with DENV-2 at 33 weeks after primary inoculation with DENV-2. The DENV-2 genome was not detected in any of these marmosets, indicating protection from a secondary infection. The results indicate that common marmosets are highly sensitive to DENV infection, and suggest that marmosets could be a reliable primate model for the evaluation of candidate vaccines.

It has been reported that levels of viremia reflect the severity of illness in dengue virus infection. We assessed the levels of viremia in patients with primary and secondary infections, using 2 cell lines: FcγR-expressing BHK cells and FcγR-negative cells. In primary infection, virus titers were at similar levels between FcγR-expressing and FcγR-negative cells. In secondary infection, however, virus titers were ∼10 times higher in FcγR-expressing cells on days 1-6 when compared with FcγR-negative cells, indicating discrepancy in viremia titers between FcγR-expressing and FcγR-negative cells. The results suggest that dengue virus-antibody complexes with infectious capacity exist in patients with secondary infection, and these immune complexes can be detected by using FcγR-expressing cells. As it has been reported that principal target cells of dengue virus infection are FcγR-positive, monocyte/macrophage lineage cells, virus titers determined using FcγR-expressing cells may better reflect the actual viremic conditions in vivo.

Travelers can introduce viruses from disease-endemic to non-disease-endemic areas. Serologic and virologic tests confirmed dengue virus infections in 3 travelers returning to Japan: 2 from Tanzania and 1 from Cote d'Ivoire. Phylogenetic analysis of the envelope gene showed that 2 genetically related virus isolates belonged to dengue virus type 3 genotype III.

Dengue virus (DENV) causes a wide range of symptoms, from mild febrile illness, dengue fever (DF), to severe life threatening illness, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Subneutralizing concentrations of antibody to DENV enhance DENV infection in Fc gammaR positive cells. This phenomenon is known as antibody-dependent enhancement (ADE). ADE is considered to be a risk factor for DHF and DSS. To develop an ADE assay for DENV, two stable BHK-21 cell lines were established that express Fc gammaRIIA (BHK-Fc gammaRIIA). The BHK-Fc gammaRIIA cell lines were used in an ADE assay with monoclonal antibody (4G2) to DENV, and DENV antibody-positive human sera. Virus growth was quantified directly in BHK-Fc gammaRIIA cells with a standard plaque assay procedure. ADE was detected with monoclonal antibody (4G2) to DENV. ADE was also detected with DENV antibody-positive human sera, but not with DENV antibody-negative human sera. The new ADE assay using BHK-Fc gammaRIIA cells is simple and practical, and is useful for defining the role of ADE in the pathogenesis of DENV infection., (2009 Elsevier B.V. All rights reserved.)

Sub-neutralizing concentrations of antibody to dengue virus (DENV) enhance DENV infection of Fc gamma receptor-expressing cells. This phenomenon, referred to as antibody-dependent enhancement (ADE), has been hypothesized to be responsible for the severe form of DENV infection, including dengue haemorrhagic fever and dengue shock syndrome. To analyse further the mechanisms of ADE in vitro, this study introduced a series of cytoplasmic mutants into human Fc gammaRIIA. The mutated Fc gammaRIIA was then expressed on COS-7 cells to see whether these mutants could enhance DENV infection. Wild-type Fc gammaRIIA enhanced DENV infection, consistent with previous reports using Fc gammaR-positive monocytes. Disruption of the immune tyrosine activation motif (ITAM) in the cytoplasmic domain of Fc gammaRIIA or removing the sequences between the two ITAM regions eliminated ADE. These findings suggest that the specific structure of the Fc gammaRIIA cytoplasmic domain is essential for the ability of Fc gammaRIIA to mediate ADE.

Dengue virus (DENV) isolation from mosquitoes is necessary for providing definitive evidence of virus circulation, and is critical for further virological characterization and determination of epidemiological characteristics. By using Aedes albopictus mosquitoes captured during an outbreak in Tokyo in 2014, we compared the DENV isolation rates of a conventional virus isolation method that uses C6/36 mosquito cells as assay cells with those of a virus isolation method that relies on an antibody-dependent enhancement (ADE) mechanism by using FcγR-expressing baby hamster kidney (BHK) cells and an antibody with ADE activity. The number of DENV genome copies and infectious virus titers in cell culture supernatant fluids of FcγR-expressing BHK cells were significantly higher than those of the C6/36 cells. In addition, DENV was isolated from a mosquito pool by using FcγR-expressing BHK cells only in the presence of infection-enhancing antibody. Infectious virus was detected in six mosquito pools only by using FcγR-expressing BHK cells. The results suggest that the method that relies on ADE mechanism by using the FcγR-expressing BHK cells and an antibody with ADE activity is useful for DENV isolation from mosquitoes caught in the field. [ABSTRACT FROM AUTHOR]

Due to the global burden of dengue disease, a vaccine is urgently needed. One of the key points in vaccine development is the development of a robust and reliable animal model of dengue virus infection. Characteristics including the ability to sustain viral replication, demonstration of clinical signs, and immune response that resemble those of human dengue virus infection are vital in animal models. Preclinical studies in vaccine development usually include parameters such as safety evaluation, induction of viremia and antigenemia, immunogenicity, and vaccine effectiveness. Although mice have been used as a model, non-human primates have an advantage over mice because of their relative similarity to humans in their genetic composition and immune responses. This review compares the viremia kinetics and antibody responses of cynomolgus macaques (Macaca fasicularis), common marmosets (Callithrix jacchus), and tamarins (Saguinus midas and Saguinus labitus) and summarize the perspectives and the usefulness along with challenges in dengue vaccine development. [ABSTRACT FROM AUTHOR]