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An effective dengue vaccine should induce a long-lasting immune response against all four serotypes simultaneously with a minimum number of immunizations. Our live attenuated tetravalent dengue vaccine candidate, KD-382, was developed using a classical host range mutation strategy (no addition of artificial genetic modification). In our previous study, cynomolgus monkeys immunized with a single dose of KD-382 seroconverted to all four serotypes. However, it is important to determine if neutralizing antibodies (NAbs) induced by KD-382 can work as a long-lasting immune response to prevent dengue. In this study, a single dose of KD-382 induced a strong NAb response against all four serotypes in cynomolgus monkeys. We also confirmed that NAb titers against all four serotypes persist for at least five years, indicating its high potential as a dengue vaccine candidate. Next, we evaluated the effect of pre-existing dengue immunity on NAb responses induced by KD-382. We administered KD-382 to cynomolgus monkeys pre-administered one of the monovalent parental wild-type strains 60 days before vaccination. Regardless of the pre-immunized serotype, all the monkeys showed sufficient tetravalent NAb responses, which lasted for over two years. All the KD-382 vaccinated monkeys were then challenged with different parental wild-type viruses than that used for pre-administration; viral RNA in the serum was less than the lower limit of quantification, indicating complete protection against secondary heterologous dengue infection without any harmful disease enhancement. Consequently, KD-382 successfully induced a long-lasting and protective tetravalent NAb response in monkeys, suggesting that KD-382 is a promising vaccine candidate usable for both dengue seronegative and seropositive individuals.

An Asian Zika virus (ZIKV) isolated from a Thai patient that was serially passaged in Primary Dog Kidney (PDK) cells for attenuation displayed both big and small plaque-forming viruses by the 7th passage. Two small-plaque isolates were selected and purified for characterization as attenuated ZIKV candidates. In vitro growth kinetics showed significantly reduced titers for small-plaque isolates in Vero cells early post-infection compared to the parental ZIKV and a big-plaque isolate, but no significant difference was observed in C6/36 cells. Viral entry experiments elucidate that titer reduction likely occurred due to the diminished entry capabilities of a small-plaque isolate. Additionally, a small-plaque isolate displayed lowered neurovirulence in newborn mice compared to 100% lethality from infection with the parental ZIKV. Genomic analysis revealed the same three unique non-synonymous mutations for both small-plaque isolates: two on the envelope (E) protein at residues 310, alanine to glutamic acid (A310E), and 393, glutamic acid to lysine (E393K), and one on residue 355 of NS3, histidine to tyrosine (H355Y). Three-dimensional (3D) mapping suggests that the E protein mutations located on the receptor-binding and fusion domain III likely affect cell entry, tropism, and virulence. These ZIKV isolates and genotypic markers will be beneficial for vaccine development.

A combination of Hybridization Chain Reaction (HCR) and Lateral Flow Immunoassay (LFIA) is an attractive strategy for a simple signal amplification DNA/RNA detection. The present study aimed to report a strategy used to solve a problem encountered when the target DNA contained folded secondary structure during HCR, enabling HCR hairpin probes to easily access the target site. The 24-nt conserved sequence within 3'-UTR, present only in dengue virus genome but not in other species, is an ideal target to use as a probe binding site for pan-dengue virus detection. Thus, the 105-nt target containing the 24-nt target sequence was chosen as a target with secondary structures. The 24-nucleotide (nt) synthetic target DNA successfully induced HCR reaction within 5 min at room temperature. However, the HCR detection of the 105-nt synthetic target DNA with secondary structures was problematic. The probe hybridization was prevented by the secondary structures of the target, resulting in a failure to generate HCR product. To solve this problem, two helper oligonucleotides (helper1 and helper2) were designed to linearize the folded structure of the 105-nt target through strand-displacement mechanism, allowing the HCR hairpin probes to easily access the target site. The HCR product with the labeled helper oligonucleotides and the labeled probes were successfully detected by LFIA. With this strategy, the combination of the helper-enhanced HCR and LFIA exhibited a limit of detection (LOD) in a nanomolar range of the 105-nt DENV synthetic target DNA. Our study demonstrated that signal amplification by the combination of HCR and LFIA could successfully detect the target DNA with secondary structure, but not target RNA with secondary structure. In summary, this work provided a proof of concept of two main issues including probe hybridization enhancement by helper oligonucleotide for the target with complicated secondary structure and the advantage of a combination of labeled helper and HCR probes design for LFIA to overcome the false positive result from HCR probe leakage. Our findings on the use of helper oligonucleotides may be beneficial for the development of other isothermal amplification, since the secondary structure of the target is one of the major obstacles among hybridization-based methods.

Background Dengue is an important mosquito-borne disease. There is currently only one licensed vaccine for dengue prevention. The vaccine provides higher efficacy in pre-vaccination dengue-seropositive persons but a higher risk of subsequent more severe dengue in dengue-seronegative persons. It is recommended that the dengue vaccine may be given in dengue-seropositive individuals or as mass vaccination without individual pre-vaccination screening in areas where the dengue seroprevalence is > 80% in children aged 9 years. We evaluated a dengue specific immunoglobulin G monoclonal antibody-based capture enzyme-linked immunosorbent assay (MAb-ELISA) in the diagnosis of previous dengue infection using serum samples from the cohort study in Ratchaburi Province, Thailand. Methods The MAb-ELISA was compared to 70% plaque reduction neutralization test (PRNT70) in 453 serum samples from children aged 3–11 years in Ratchaburi Province, Thailand. Results The sensitivity and specificity of MAb-ELISA at the positive to negative (P/N) ratio cut-off level of > 3 were both 0.91 in the diagnosis of previous dengue infection, compared to PRNT70. The false positivity was mainly in Japanese encephalitis (JE) seropositive subjects. Conclusions This research provides evidence that MAb-ELISA is useful for dengue seroprevalence study and dengue pre-vaccination screening. JE seropositivity was the major cause of false positive result in the study population.

Background Children and young adults undergoing hematopoietic stem cell transplantation (HSCT) typically lose their immunity to vaccine-preventable diseases, including Japanese encephalitis (JE). Revaccination against JE in this population has not been well characterized. Methods This prospective study evaluated the immunogenicity of inactivated Vero cell culture-derived JE vaccine in children and young adults ( 1 year prior. Each patient received inactivated Vero cell culture-derived JE vaccine at enrollment and 1 month after enrollment, as well as a booster dose 13 months after enrollment. Serum JE plaque reduction neutralization test and JE-specific T lymphocyte count assay were performed at baseline, 1 month after the second dose, on the day of the booster dose, and 1 month after the booster dose. Results Thirty-seven patients were enrolled. At baseline, 15 patients (40.5%) had plaque reduction neutralization titer >10, which is considered protective. Among 22 seronegative patients, 15 (68.2%) and 19 (86.4%) exhibited seroconversion after revaccination and booster dose, respectively. Median JE-specific T lymphocyte counts also increased. Twenty of 111 (18.0%) vaccination doses resulted in self-limiting side effects. Conclusions The inactivated Vero cell culture-derived JE vaccine may be safe and effective for immunization against JE virus in children and young adults who have undergone HSCT.

Background In 2016 and 2017, Zika virus (ZIKV) infection outbreaks occurred in two communities in southern Thailand. This re-immerging infection can widely spread by mosquito bites and cause serious complications in a central nervous system among children born to infected mothers. Thus, they should be protected. This study aims to (1) To determine the prevalence of neutralizing ZIKV antibodies in the post-outbreak areas among the general population and pregnancy women residing at various distances from the houses of the nearest index patients; (2) To examine the cross-neutralizing capacity of antibodies against ZIKV on other flaviviruses commonly found in the study areas; (3) To identify factors associated with the presence of neutralizing ZIKV antibodies. Methods The two post-outbreak communities were visited at 18 months after the outbreaks. We enrolled (1) 18 confirmed ZIKV infected (index) cases, (2) sample of 554 neighbors in the outbreak areas who lived at various distances from the index patients’ houses, (3) 190 residents of non-outbreak areas, and (4) all pregnant women regardless of gestational age residing in the study areas (n = 805). All serum specimens underwent the plaque reduction neutralization test (PRNT). Ten randomly selected ZIKV seropositive and ten randomly selected seronegative specimens were tested for dengue virus serotypes 1–4 (DENV1–4) and Japanese encephalitis virus (JEV) antibodies using PRNT90. Serum titer above 1:10 was considered positive. Multiple logistic regression was used to assess factors associated with seropositivity. Results Out of all 18 index cases, 9 remained seropositive. The seroprevalence (95% CI) in the two outbreak areas were 43.7% (35.9–51.6%) and 29.7% (23.3–36.0%) in general population, and 24.3% (20.1–28.8%) and 12.8% (9.7–16.5%) in pregnant women. Multivariate analysis showed that seropositivity was independent of the distance gradient from the index’s houses. However, being elderly was associated with seropositivity. DENV1–4 and JEV neutralizing antibodies were present in most ZIKV-positive and negative subsamples. Conclusion Protective herd immunity for ZIKV infection is inadequate, especially among pregnant women in the two post-outbreak areas in southern Thailand.

Background. In Thailand, Japanese encephalitis appears with a higher incidence of the Northern region (0.0/100,000 cases/year) as compared to the central southern region of the country (0.01/100,000/year). Also, all Japanese encephalitis virus (JEV) recently isolated strains in country belong to the genotype I and III.Methods. In order to evaluate the long-term efficacy of the newly developed live attenuated SA 14-14-2 Japanese encephalitis vaccine in Thailand, the immune response in children was studied after a second immunization campaign. Following 2012 immunization campaign, a second JEV immunization vaccine was delivered to children among four provinces of the Northern Region of Thailand. For each province, the recipients were identified accordingly to the two vaccine campaigns time including the children who received the second dose of vaccine: 1/ less than one year before the present study (N=30 by province); 2/ more than one year before the present study (N=70 by province). Altogether, a total of 400 children were enrolled in this project. After receiving the second vaccine dose, blood samples were collected and tested for JEV (Genotypes I and III) neutralizing antibodies following a standard procedure of LLC-MK2 infected cells.Results. All recipient presented neutralizing antibodies cross reacting against the prevalent JEV genotypes I (SM1 JEV strain) and genotype III (SA 14-14-2 and Beijing JEV strains). Most of the children from the first group had a seroconversion rate of 94.2% against homologous (i.e. GIII). Among them, an optimal rate of 100% seroconversion was find at the Phayao Province against both heterologous (GI) and homologous (GIII) JEV strains. Geometric Mean Titer (GMT) of neutralizing antibody against SM1, SA 14-14-2 JEV, and Beijing JEV strains were consistently high, respectively as 239±50, 323±201.2 and 177±36.7. For the second group, seroconversion rate was lower against SM1, SA 14-14-2 and Beijing JEV strains with a rate of 94.3, 90% and 86.8% with respectively a GMT of 154±32, 90±23 and 82±13.6.Conclusion. Although, the immunity appears to decrease within the period of 2.5 years, neutralizing antibody rate are consistent to protect against the JEV infection. Nevertheless, a long-term follow up is suitable to evaluate an eventual third dose opportunity.

Additional file 1: Table 1. Seroprevalence of ZIKV neutralizing antibodies among subgroups of participants in District A and B

Many mosquito transmitted viruses of the genera Alphavirus and Flavivirus are human pathogens of significant concern, and there is currently no specific antiviral for any member of these two genera. This study sought to investigate the broad utility of orlistat (tetrahydrolipstatin) in reducing virus infection for several mosquito borne viruses including flaviviruses (dengue virus (DENV; nine isolates analyzed), Japanese encephalitis virus (JEV; one isolate analyzed) and Zika virus (ZIKV; 2 isolates analyzed)) as well as an alphavirus (chikungunya virus; CHIKV; 2 isolates analyzed). Three different treatment regimens were evaluated, namely pre-treatment (only), post-treatment (only) and pre- and post-treatment, and three factors were evaluated, namely level of infection, virus titer and genome copy number. Results showed that all three treatment modalities were able to significantly reduce virus titer for all viruses investigated, with the exception of three isolates of DENV in the pre-treatment only regimen. Pre- and post-treatment was more effective in reducing the level of infection and genome copy number of all viruses investigated than either pre-treatment or post-treatment alone. Collectively, these results suggest orlistat has potential as a broad-spectrum agent against multiple mosquito transmitted viruses.

Additional file 2: Table 2. Univariable analysis exploring factors associated with ZIKV seropositivity in non-pregnant participants in the two sites combined. (a multi-page table)

Additional file 3: Table 3. Odds ratios for ZIKV seropositivity from the multivariate logistic regression analysis among non-pregnant participants in the two sites combined. (a multi-page table)

Background The study aimed to develop dengue severity score to assess severe manifestations among hospitalized patients with dengue infection. Method Children and adolescents with serologically confirmed dengue infection admitted at Ramathibodi Hospital from 2004 to 2018 and treated by an expert multidisciplinary team were recruited. Medical records were retrospectively reviewed and 14 items, related to clinical parameters and managements during hospitalization, were obtained daily as dengue severity score. Results A total of 191 patients with a mean age of 10.7 years from 2004 to 2013 were recruited. They were classified as dengue fever (35), dengue hemorrhagic fever (DHF) I (53), II (50), III (37) and IV (16). The analysis of 593 daily records revealed the range of daily severity score among patients with DHF grades III (10-20) and IV (31-47) were significantly higher than those of other groups (dengue fever, 5-13; DHF I, 2-10; DHF II, 6-11) with P-values of 0.0001. Using a validity test, a total daily score of ≥12 was an assessment tool for dengue shock syndrome with sensitivity, 86% and specificity, 84%. An additional 51 hospitalized patients with DHF grades II, III and IV with similar ages from 2014 to 2018 were recruited. The number of patients with severe manifestations, having daily score of ≥12, was significantly higher than those without severe manifestations starting from Day -3 to Day +1 of illness. Conclusions Daily dengue severity score of ≥12 was an accurate assessment tool for severe manifestations.

To determine the cardiovascular changes in children with dengue shock syndrome. Echocardiography was performed in 8 children (5 females) with dengue shock syndrome, median age 6.5, 4.2–13.7 yr and weight 34, 12–66 kg. All had massive bleeding with low initial hematocrit in most cases (median 31%), thrombocytopenia (median platelet 37,000/μL), and coagulopathy with massive pleural effusion. Seven (87.5%) developed acute renal failure and hepatic failure. All patients were in either compensate or decompensate shock with alteration of consciousness, tachycardia, poor tissue perfusion, and prolonged capillary refill (>4 s) with mean arterial pressure 65, 39–94 mm Hg. The cardiac dimension was normal to low normal except one had dilated left ventricle. Seven patients had normal left ventricular systolic function (5 with inotrope infusion). One patient had impaired systolic function even with inotrope. All had normal cardiac index (4.14, 3.51–6.37 L/min/m(2)) with increased heart rate (141.5, 110–160/min) but low stroke volume index (30.72, 25.37–42.49 mL/m(2)) and low systemic vascular resistance index (1,072, 223–2,880 dyne/sec/cm(−5)/m(2)). Decreased preload from bleeding and vascular leakage into the third space play an important role in shock in Dengue. However, decreased stroke volume and low systemic vascular resistance may be additional causes of shock.

Recent advances in electron microscopy and tomography have revealed distinct virus-induced endoplasmic reticulum (ER) structures unique for dengue virus (DV) and other flaviviruses in cell culture models, including hepatocytes. These altered ultrastructures serve as sites for viral replication. In this study, we used transmission electron microscopy to investigate whether such structures were present in the liver of fatal dengue hemorrhagic fever (DHF) autopsy cases. In parallel, electron microscopic examination of suckling mouse brains experimentally infected with DV was performed as an in vivo model of acute DV infection. Typical features of ER changes containing abundance of replicative virions were observed in neurons and microglia of DV-infected suckling mouse brains (SMB). This indicated that the in vivo DV infection could induce similar viral replication structures as previously described in the in vitro DV-infected cell model. Nevertheless, liver tissues from autopsy of patients who died of DHF showed scant changes of ER membrane structures and rare particles of virions in hepatocytes, despite overwhelming evidence for the presence of viral antigens and RNA–indicating active virus replication. Instead hepatocytes contained an abundance of steatotic vesicles and structural damages. This lack of structural changes indicative of virus replication in human hepatocytes is discussed.

Objective To identify immunological evidence of Zika virus transmission in Thailand. Methods To undertake a preliminary serosurvey of possible exposure to Zika virus, 21 serum samples from cohort of acute undifferentiated fever patients were examined for immunoreactivity to Zika, Dengue, Japanese encephalitis and Chikungunya envelope antigens by Western blot analysis. Results Twenty of the 21 serum samples showed immunoreactivity to at least one of the antigens, with seven samples showing immunoreactivity to all antigens. Of particular note, two serum samples showed immunoreactivity only to Zika envelope antigen, with no immunoreactivity to other envelope antigens. Conclusions This study presents the first evidence of Zika virus transmission in Thailand, although as yet the relationship between transmission and possible cases of Zika fever in Thailand requires further investigation.

One of the challenges developing a live attenuated tetravalent dengue vaccine (TDV) is to overcome the presumed viral interference that may be preventing the induction of a balanced immune response to all 4 serotypes of the dengue virus (DENV1–4). Our live attenuated TDV candidate was developed from wild-type (wt) parental strains (DENV1/03135, DENV2/99345, DENV3/16562, and DENV4/1036, respectively) using a classical host range mutation strategy: the same strategy used for the approved live attenuated smallpox, polio, and MMR vaccines. Our vaccine candidate is expected to mimic natural dengue virus infection, as it provides all the components of dengue virus, including both structural and nonstructural proteins. Therefore, induction of more solid and comprehensive immune responses against pathogenic dengue viruses is also expected. In this study, we evaluated the neutralizing antibody responses for each serotype induced by a single subcutaneous administration of 6 formulations, which were composed of different combinations of vaccine strains and were all of different dosages. These formulations were tested in dengue-naïve cynomolgus macaques. As a result, regardless of the TDV formulation, all the monkeys immunized with TDVs seroconverted to all the 4 serotypes at day 30. Next, we evaluated protection ability of the selected formulations of TDV candidate, no RNAemia was detected from any of the immunized monkeys upon s.c. challenge with wtDENV. The findings of this non-human primate study indicate that our vaccine candidate is very promising; it can be further evaluated for safety and efficacy in human clinical studies., Microbiology; Virology; Antibody; Immune response; Vaccination; Vaccines; Viral disease; Live attenuated tetravalent dengue vaccine; Protective efficacy; KD-382

Background Japanese encephalitis virus (JEV) is a zoonotic, mosquito-borne flavivirus, distributed across Asia. Infections are mostly mild or asymptomatic, but symptoms include neurological disorders, sequelae, and fatalities. Data to inform control strategies are limited due to incomplete case reporting. Methods We used JEV serological data from a multicountry Asian dengue vaccine study in children aged 2–14 years to describe JEV endemicity, measuring antibodies by plaque reduction neutralization test (PRNT50). Results A total 1479 unvaccinated subjects were included. A minimal estimate of pediatric JEV seroprevalence in dengue-naive individuals was 8.1% in Indonesia, 5.8% in Malaysia, 10.8% in the Philippines, and 30.7% in Vietnam, translating to annual infection risks varying from 0.8% (in Malaysia) to 5.2% (in Vietnam). JEV seroprevalence and annual infection estimates were much higher in children with history of dengue infection, indicating cross-neutralization within the JEV PRNT50 assay. Conclusions These data confirm JEV transmission across predominantly urban areas and support a greater emphasis on JEV case finding, diagnosis, and prevention., Japanese encephalitis virus circulation has been demonstrated in urban areas of Indonesia, Malaysia, the Philippines, and Vietnam. Serological data indicate that up to 5% of children are infected annually.

The complete genome of Zika virus (ZIKV) strain CVD_06-274 was isolated from the serum of an infected patient in Thailand in 2006. Phylogenetic analysis showed that this strain belongs to the Asian lineage and also high titers in Vero cells (RCB 10-87). It has potential for development as an inactivated ZIKV vaccine.

Background To determine the prevalence and associated factors of seroprotection against Japanese encephalitis (JE) virus among HIV-infected adolescents stable on combination antiretroviral treatment (cART). Methods A multicenter seroprevalence study was conducted in Thailand. Perinatally HIV-infected adolescents who aged 11–25 years, had previous evidence of severely immune suppression (CD4 < 15% or < 200 cells/mm3), were currently stable on cART (CD4 > 350 cells/mm3 for > 6 months or CD4 > 200 cells/mm3 with viral suppression [VS; plasma HIV RNA < 50 copies/mL] for > 12 months), and had completed a 3- or 4-dose series of mouse brain-derived inactivated JE vaccine (MBDV) during childhood were enrolled. Adolescents who had clinically or serologically confirmed recent JE virus infections, or received immunosuppressive agents or blood components within 6 months were excluded. Plaque reduction neutralization (PRNT50) assay was conducted to assess neutralizing antibodies to JE virus, and titers of ≥ 10 were considered seroprotective. Logistic regression analysis was performed to identify associated factors of JE seroprotection. Results Of 98 eligible adolescents, 54% were female, a median age was 19 years, and 11% were overweight. Ninety-five percent and 5% of adolescents received 3 and 4 doses of MBDV during childhood, respectively. A median duration since the last dose of MBDV was 16 years. At enrollment, 71% were on NNRTI-based cART regimens, a median cART duration was 13 years. A median current CD4 was 29%, and 89% had VS. Seroprotection against JE virus was identified in 28 (29%) adolescents; of whom, the geometric mean titer (GMT) of neutralizing antibody was 64 (95% CI: 39–106). Proportion of adolescents with JE seroprotection and GMT of neutralizing antibodies to JE virus slightly decreased over time after the last immunization (Figure 1). In a multivariable logistic regression analysis, seroprotection against JE virus was associated with younger age and greater current CD4 count (Table 1). Conclusion The majority of our perinatally HIV-infected adolescents did not maintain seroprotection against JE virus although having completed a series of MBDV during childhood. JE revaccination is an important tool for disease prevention in these adolescents who live in JE endemic areas. Disclosures All authors: No reported disclosures.

Introduction: Fever is a common symptom of many tropical diseases and in many cases the etiologic agent remains unidentified as a consequence of either the etiologic agent not being part of routine diagnostic screening or as a consequence of false negatives on standard diagnostic tests. Methodology: This study screened a well characterized panel of 274 serum samples collected on day of admission from adult patients with acute undifferentiated fever admitted to a hospital in Nakhon Ratchasima, Thailand by RT-PCR using pan-flavivirus degenerate primers. Results: Subsequent clinical diagnosis was achieved for 38 of the patients, and included 19 cases of dengue fever. RT-PCR screening identified seven positive samples (2.5%) which were revealed by sequence analysis to be dengue virus 1 (2 cases), dengue virus 2 (2 cases) and dengue virus 3 (3 cases). Only 5 out of 19 (26%) serum samples from patients subsequently diagnosed with dengue were positive, but 2 samples which clinically remained undiagnosed were shown to be positive for dengue virus. Sequence analysis suggested that the dengue virus 3 cases occurred as a result of importation of a strain of dengue from India or China. No other flaviviruses were identified. Conclusions: No evidence was found of other flaviviruses besides dengue circulating in this population. Despite improved diagnostic tests, cases of dengue are still evading correct diagnosis.

International audience; Dengue is a mosquito-borne viral disease that afflicts millions of individuals worldwide every year. Infection by any of the 4 dengue virus (DENV) serotypes can result in a spectrum of disease severity. We investigated the impact of variants of interferon-regulated innate immunity genes with a potent antiviral effect on the outcome of DENV infection. We compared the effect of OAS gene family variants on 2 DENV serotypes in cell culture. While both OAS1-p42 and p46 showed antiviral activity against DENV-2, only OAS1-p42 presented anti-DENV-1 activity. Conversely, whereas both OAS3_S381 and R381 variants were able to block DENV-1 infection, the anti-DENV-2 activity observed for OAS3_S381 was largely lost for the R381 variant. By means of an allelic association study of a cohort of 740 patients with dengue, we found a protective effect of OAS3_R381 against shock (odds ratio [OR], 0.37; P < .001). This effect was due to DENV-2 infections (OR, 0.13; P = .007) but was absent for DENV-1, in accordance with the serotype-dependent OAS3 activity found in the functional study. Severe dengue has long been associated with a cytokine storm of unclear origin. This work identifies an early innate immunity process that could lead to the immune overreaction observed in severe dengue and could be triggered by a specific host genotype-pathogen genotype interaction.

Summary: Inactivated mouse-brain-derived Japanese encephalitis vaccine has a worrisome safety profile and the live attenuated vaccine is unsuitable in immunodeficiency. This study aimed to evaluate the immunogenicity and safety of an inactivated chromatographically purified Vero-cell-derived JE vaccine (CVI-JE, Beijing P-3 strain) in children. 152 healthy Thai children, with an average (SD) age of 14.4 (3.8) months, received 3 doses of CVI-JE on days 0, 7–28, and one year. Homologous JE neutralizing antibody titers (NT) were measured. All subjects had seroprotection [geometric mean titer (GMT) 150] 28 days' post 2nd vaccination. The seroprotection rates at 1 year after primary series and and 1 month after the booster were 89.3% (GMT 49) and 100% (GMT 621), respectively. Local and systemic reactions–fever (17.6%), vomiting (8%), and poor appetite (5.3%)–were noted within 28 days' post-vaccination. All these symptoms were self-limited. Conclusions: CVI-JE is safe, immunogenic, and provided high NT.

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.

Background Plaque size is a common feature of viral characterization. Small plaque size is used as a marker of attenuation for live-attenuated vaccine development. Objective To investigate whether the naturally occurring plaque size variation reflects virulence of the variants of chikungunya virus (CHIKV). Methods We selected and purified a variant with small plaque size from the primary isolate. The viral variant was tested for the plaque morphology, in vitro growth kinetics and mouse neurovirulence in comparison with the parental wild type. Results The small plaque size variant showed stable homogenous small plaques after 4 plaque purifications. The small plaque virus grew slower and to the lower titer when compared with wild type virus. After 21 days of infection, mice that received small plaque virus showed 98% survival rate while 74% of mice survived after infected with wild type virus. Conclusion The small plaque size variant of CHIKV can be obtained by plaque purification and the virus displays decreased virulence.

Dengue virus (DENV) infection is considered one of the most important mosquito‐borne diseases. It causes a spectrum of illness that could be due to qualitative and/or quantitative difference(s) of the natural killer (NK) cell responses during acute DENV infection. This view prompted us to perform a detailed phenotypic comparative characterization of NK cell subsets from DENV‐infected patients with dengue fever (DF), patients with dengue haemorrhagic fever (DHF) and healthy controls. The activation/differentiation molecules, CD69 and CD57 and a variety of tissue homing molecules were analysed on the CD56(hi) CD16(−) and CD56(lo) CD16(+) NK cells. Although there was no increase in the frequency of the total NK cells during DENV infection compared with the healthy individuals, there was a significant increase in the frequency of the CD56(hi) CD16(−) subset and the frequency of CD69 expression by both NK cell subsets during the febrile phase of infection. We also found an increase in the frequencies of cells expressing CD69 and CD57 in the CD56(lo) CD16(+) subset compared with those in the CD56(hi) CD16(−) subset. Moreover, although the CD56(lo) CD16(+) subset contained a high frequency of cells expressing skin‐homing markers, the CD56(hi) CD16(−) subset contained a high frequency of cells expressing bone marrow and lymph node trafficking markers. Interestingly, no differences of these NK cell subsets were noted in samples from patients with DF versus those with DHF. These findings suggest that activation and differentiation and the patterns of tissue homing molecules of the two major NK cell subsets are different and that these might play a critical role in the immune response against acute DENV infection.

Real-time PCR validation of siRNA mediated gene silencing of fatty acid synthase (FASN) gene. HEK293T/17 cells were not treated (mock) or treated with a siRNA control (GFP) or treated with one of four siRNAs directed to FASN (FASN1 to FASN 4). On days 1 to 5 post transfection the level of FASN transcript was determined by real time PCR. Normalization expression data relative to actin is shown. Bars show mean +/−SD. (a) 1 day post transfection, (b) 2 days post transfection, (c) 3 days post transfection, (d) 4 days post transfection and (e) 5 days post transfection. Bars show mean +/−SD (*; p value

List of gene specific primers (PDF 56 kb)

Dengue is the most significant arthropod borne viral disease worldwide, and infection with the dengue virus causes a wide range of symptoms in humans, including bone marrow suppression. While the target cells of the virus remain poorly characterized, cells of the myeloid lineage have been shown to be important mediators of the disease. This study sought to determine whether erythroid precursor cells were susceptible to dengue virus infection, and whether erythroid cells from thalassemia trait carriers showed any protection against infection. Infection with a laboratory adapted high passage DENV-2 resulted in high levels of infection during certain stages of differentiation, and cells derived from thalassemia trait carriers showed significantly reduced susceptibility to dengue virus infection. Infection with low passage isolates resulted in only scattered cells showing evidence of infection, but high bystander apoptosis that was reduced by both a caspase 8 inhibitor and anti-tumor necrosis factor 1 receptor antibodies.

Biological markers are normally used to evaluate the candidate of live-attenuated dengue vaccines. D3V 16562 Vero 23 and D3V 16562 Vero 33 which were derivatives of D3V 16562, parental strain, showed the similar biological data. We used molecular techniques and computational tools to evaluate these derivatives. The nucleotide and amino acid sequences of the derivatives were compared to their parent. The secondary structures of untranslated regions and B-cell epitopes were predicted. The results showed that nucleotide substitutions mostly occurred in NS5 and NS5 of V2 was unusual because of amino acid change at 3349 (tryptophan →stop codon). The nucleotide substitutions in 5'UTR, prM, E, NS1, NS2A, NS3, and 3'UTR were 4, 1, 2, 2, 1, 3, and 2, respectively. The secondary structure of 5'UTR of V2 was different from P and V1. The secondary structure of 3'UTR of V2 was similar to P and certainly distinct from V1. Furthermore, B-cell epitopes prediction revealed that there were 21 epitopes of envelope and the interesting epitope was at position 297-309 because it was in domain III in which the neutralizing antibody is induced. For this study, the attenuation of derivatives was caused by the nucleotide substitutions in 5'UTR, 3'UTR, and NS5 regions. The genotypic data and B-cell epitope make the derivatives attractive for the chimeric and peptide DENV vaccine development.