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Abstract Introduction Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and Yellow fever virus (YFV) are mosquito-borne RNA viruses causing major public health threats in major parts of the world. While DENV and ZIKV have been detected in urine samples, data on the presence and stability of flaviviruses in the water environment are limited. Methods In this study, we determined the stability and infectivity of flavivirus in different water environments by utilizing RT-qPCR and plaque assay to explore the feasibility of environmental detection and surveillance of flaviviruses. Results Viral RNA could be detected for up to 49-days, at 4 °C, 25 °C and 37 °C temperatures, and infectious particles could be detected for up to 7 days. While our findings showed that flaviviral RNA has higher stability and better detection rates at lower temperatures, the infectious capacity of flaviviruses was comparatively short at 7 days. Conclusions Our results indicate that flaviviruses retains limited infectivity in general water environments and highlight the feasibility of detection and surveillance in various epidemiologic and environmental settings.

Abstract Vaccine-induced T cells and neutralizing antibodies are essential for protection against SARS-CoV-2. Previously, we demonstrated that an antigen delivery system, pullulan nanogel (PNG), delivers vaccine antigen to lymph node medullary macrophages and thereby enhances the induction of specific CD8+ T cells. In this study, we revealed that medullary macrophage-selective delivery by PNG depends on its binding to a C-type lectin SIGN-R1. In a K18-hACE2 mouse model of SARS-CoV-2 infection, vaccination with a PNG-encapsulated receptor-binding domain of spike protein decreased the viral load and prolonged the survival in the CD8+ T cell- and B cell-dependent manners. T cell receptor repertoire analysis revealed that although the vaccine induced T cells at various frequencies, low-frequency specific T cells mainly promoted virus clearance. Thus, the induction of specific CD8+ T cells that respond quickly to viral infection, even at low frequencies, is important for vaccine efficacy and can be achieved by SIGN-R1+ medullary macrophage-targeted antigen delivery.

The COVID-19 pandemic, driven by the rapid evolution of the SARS-CoV-2 virus, presents ongoing challenges to global public health. SARS-CoV-2 is characterized by rapidly evolving mutations, especially in (but not limited to) the spike protein, complicating predictions about its evolutionary trajectory. These mutations have significantly affected transmissibility, immune evasion, and vaccine efficacy, leading to multiple pandemic waves with over half a billion cases and seven million deaths globally. Despite several strategies, from rapid vaccine development and administration to the design and availability of antivirals, including monoclonal antibodies, already having been employed, the persistent circulation of the virus and the emergence of new variants continue to result in high case numbers and fatalities. In the past four years, immense research efforts have contributed much to our understanding of the viral pathogenesis mechanism, the COVID-19 syndrome, and the host–microbe interactions, leading to the development of effective vaccines, diagnostic tools, and treatments. The focus of this review is to provide a comprehensive analysis of the functional impact of mutations on diagnosis, treatments, and vaccine effectiveness. We further discuss vaccine safety in pregnancy and the implications of hybrid immunity on long-term protection against infection, as well as the latest developments on a pan-coronavirus vaccine and nasal formulations, emphasizing the need for continued surveillance, research, and adaptive public health strategies in response to the ongoing SARS-CoV-2 evolution race.

Objectives: SARS-CoV-2 transmission and epidemic potential is related to the population's immunity levels. As such, assessing different regions’ preexisting immune responses to SARS-CoV-2 is important to understand the transmission potential of emerging SARS-CoV-2 variants. Design: In 975 serum samples from Vietnam (2014 to 2019), anti-SARS-CoV-2 Immunoglobulin G levels were determined by enzyme-linked immunosorbent assay. Plaque reduction neutralization test (PRNT) was performed using Wuhan strain and variants of concern (VOCs). Cross-reactivity was confirmed by analyzing B-cell receptor (BCR) repertoire sequences and identifying BCR repertoire sequences-derived T-cell epitopes. Results: Overall, 20.9% (n = 76/364) and 9.2% (n = 7) demonstrated SARS-CoV-2 neutralizing activity (PRNT50) against the Wuhan and Alpha strain, respectively. Neutralizing activity against Beta, Gamma, and Delta strains was absent (PRNT50

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

Abstract A low-cost SYBR Green-based RT-qPCR method to detect SARS-CoV-2 were developed and validated. Primers targeting a conserved and vital region of the N genes of SARS-CoV-2 were designed. In-silico study was performed to analyse the compatibility of the selected primer pair with Indonesian SARS-CoV-2 genome sequences available from the GISAID database. We determined the linearity of our new assay using serial dilution of SARS-CoV-2 RNA from clinical samples with known virus concentration. The assay was then evaluated using clinically relevant samples in comparison to a commercial TaqMan-based test kit. Finally, we applied the assay in sample pooling strategies for SARS-CoV-2 detection. The SYBR Green-based RT-qPCR method was successfully developed with sufficient sensitivity. There is a very low prevalence of genome variation in the selected N primer binding regions, indicating their high conservation. The validation of the assay using clinical samples demonstrated similar performance to the TaqMan method suggesting the SYBR methods is reliable. The pooling strategy by combining 5 RNA samples for SARS-CoV-2 detection using the SYBR RT-qPCR methods is feasible and provides a high diagnostic yield. However, when dealing with samples having a very low viral load, it may increase the risk of missing positive cases.

Abstract Background Dengue fever, caused by the dengue virus (DENV), is the most common viral infection transmitted by Aedes mosquitoes (mainly Ae. aegypti and Ae. albopictus) worldwide. Aedes aegypti is not currently established in Japan, and Ae. albopictus is the primary vector mosquito for DENV in the country, but knowledge of its viral susceptibility is limited. Therefore, we aimed to clarify the status of DENV susceptibility by comparing the infection and dissemination dynamics of Japanese Ae. albopictus to all known DENV serotypes with those of Ae. aegypti. Methods After propagation of each DENV serotype in Vero cells, the culture supernatants were mixed with defibrinated rabbit blood and adenosine triphosphate, and the mixture was artificially blood-sucked by two colonies of Ae. albopictus from Japan and one colony of Ae. aegypti from a dengue-endemic country (Vietnam). After 14 days of sucking, the mosquito body was divided into two parts (thorax/abdomen and head/wings/legs) and total RNA was extracted from each sample. DENV RNA was detected in these extracted RNA samples using a quantitative RT-PCR method specific for each DENV serotype, and infection and dissemination rates were analyzed. Results The Japanese Ae. albopictus colonies were susceptible to all DENV serotypes. Its infection and dissemination rates were significantly lower than those of Ae. aegypti. However, the number of DENV RNA copies in Ae. albopictus was almost not significantly different from that in Ae. aegypti. Furthermore, Japanese Ae. albopictus differed widely in their susceptibility to each DENV serotype. Conclusions In Japanese Ae. albopictus, once DENV overcame the midgut infection barrier, the efficiency of subsequent propagation and dissemination of the virus in the mosquito body was comparable to that of Ae. aegypti. Based on the results of this study and previous dengue outbreak trends, Ae. albopictus is predicted to be highly compatible with DENV-1, suggesting that this serotype poses a high risk for future epidemics in Japan.

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to nearly 50% of the global population. DENV has been endemic in Nepal since 2006; however, little is known about how DENV is evolving or the prevalence of anti-DENV immunity within the Nepalese population. To begin to address these gaps, we performed a serologic and genetic study of 49 patients from across Nepal who presented at central hospitals during the 2017 dengue season with suspected DENV infection. Of the 49 subjects assessed, 21 (43%) were positive for DENV NS1 antigen; of these; 5 were also anti-DENV IgM+ IgG+; 7 were DENV IgM+ IgG-, 2 were IgM- IgG+, and 7 were IgM-IgG- by specific ELISAs. Seven of the 21 NS1 positive sera were RNA positive by RT-PCR (six DENV2, one DENV3), suggesting that DENV2 was the dominant serotype in our cohort. Whole-genome sequencing of two DENV2 isolates showed similarity with strains circulating in Singapore in 2016, and the envelope genes were also similar to strains circulating in India in 2017. DENV-neutralizing antibodies (nAbs) were present in 31 of 47 sera tested (66%); among these, 20, 24, 26, and 12 sera contained nAbs against DENV1, 2, 3, and 4 serotypes, respectively. Additionally, 27 (58%) samples had nAbs against multiple serotypes (2 or more). Serology analysis suggested that 12 (26%) and 19 (40%) of the 47 subjects were experiencing primary and secondary DENV infections, respectively. Collectively, our results provide evidence for current and/or past exposure to multiple DENV serotypes in our cohort. These data suggest that expanded local surveillance of circulating DENV genotypes and population immunity will be important to effectively manage and mitigate future dengue outbreaks in Nepal.

ABSTRACT Zoonotic infection by herpes B virus (BV) causes severe neurological disease, which in the absence of appropriate treatment can have a mortality rate of 80%. Long-term administration of acyclovir or ganciclovir is the recommended prophylactic treatment option for BV infections. However, the emergence of acyclovir- and/or ganciclovir-resistant BV may become a major problem. Here, we examined the ability of BV thymidine kinase (TK) from monkeys (mBVTK) and humans (hBVTK) to acquire resistance to acyclovir and ganciclovir. The sequences of mBVTK and hBVTK differ by two amino acids. We constructed and expressed four HSV-1-based recombinant viruses (HSV-1_mBVTK, HSV-1_hBVTK, HSV-1_VZVTK, and HSV_HSV-1TK) in which the HSV-1TK gene was deleted and the TK gene of monkey BV, human BV, varicella-zoster virus, and HSV-1 was inserted, respectively. We then tested the sensitivity of these recombinant viruses to acyclovir, ganciclovir, penciclovir, and brivudine. HSV-1_HSV-1TK and wild-type HSV-1 were more susceptible to ganciclovir, acyclovir, and penciclovir than HSV-1_VZVTK, HSV-1_hBVTK, and HSV-1_mBVTK. Both HSV-1_hBVTK and HSV-1_mBVTK exhibited similar sensitivity profiles against all tested drugs: both were susceptible to acyclovir, ganciclovir, and penciclovir but resistant to brivudine. In addition, we forced HSV-1_hBVTK to replicate under selective pressure from acyclovir or ganciclovir and then examined the ability of the BV TK gene to develop drug resistance. Investigation of the drug-resistant HSV-1_hBVTK gene revealed the absence of amino acid substitutions in BV TK, suggesting that the development of acyclovir- or ganciclovir-resistant BV through mutations in the TK gene is a rare occurrence in this experimental system. IMPORTANCE Zoonotic infection of humans with herpes B virus (BV) causes severe neurological diseases. Acyclovir (ACV) and ganciclovir (GCV), most frequently used as anti-herpes drugs, are recommended for prophylaxis and therapy in human BV infection. In this study, we examined the property of BV thymidine kinase (TK) against anti-herpes drugs using a recombinant herpes simplex virus type 1 (HSV-1) carrying BV TK gene. We found that HSV-1 carrying BV TK was similarly sensitive to GCV as HSV-1 carrying varicella zoster virus TK. In addition, we demonstrated that BV TK was not mutated in the GCV- and ACV-resistant HSV-1 carrying BV TK, suggesting that ACV- or GCV-resistant BV might be rare during treatment with these antiviral drugs. These data can provide a new insight into the properties of BV TK in terms of the development of drug resistance.

Introduction: Arbovirus diseases remain a public health threat in Sri Lanka. Dengue is endemic and two outbreaks of chikungunya infections have been reported. There is limited data on Zika virus (ZIKV) infections in Sri Lanka, and this could be due to a lack of comprehensive ZIKV surveillance. Our aim was to determine the presence of antibodies to dengue, chikungunya, and Zika infections in adults from a suburban population in Sri Lanka. Methods: A total of 149 healthy adult volunteers over 18 years of age (mean age: 43±14 years, males – 43%), with no prior diagnosed arboviral infections and no history of overseas travel, participated in the study. ELISA and neutralization assays were carried out to detect past dengue, chikungunya, or Zika infections. Results: A total of 94.6% (141/149) of the participants demonstrated dengue IgG antibodies, 37.5% (56/149) were positive for chikungunya IgG, and 5.3% (8/149) were positive for anti-ZIKV IgG antibodies. Neutralization assays confirmed ZIKV-specific antibodies in 6.7% (10/149), when 40/149 of the participating population were tested. Conclusion: This clearly demonstrated past ZIKV infections in this population. In addition, this study indicates that >90% of individuals had asymptomatic dengue but no serious symptoms. These results provide a cross-sectional view on the DENV, ZIKV, and CHIKV epidemic status and demonstrate a need for the implementation of enhanced surveillance and more effective measures against the spread of these arbovirus diseases.

Flavivirus infections, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), present significant global public health challenges. For successful vaccine design, the assessment of neutralizing antibody activity requires reliable and robust methodologies for determining antibody titers. Although the plaque reduction neutralization test (PRNT) is commonly acknowledged as the gold standard, it has limitations in terms of time and cost, and its usage may be limited in resource-limited settings. To address these challenges, we introduced the micro-neutralization test (MNT) as a simplified alternative to the PRNT. The MNT employs a 96-well plate format, conducts microscale neutralization assays, and assesses cell viability by dissolving cells to create a uniform color solution, which is measured with a spectrometer. In this study, we evaluated the utility of the MNT by contrasting the end-point titers of the MNT and PRNT using 4 monoclonal antibodies, 15 non-human primate serum samples, and 2 therapeutic drug candidates across flaviviruses. The results demonstrated a strong correlation between the MNT and PRNT titers, affirming the robustness and reproducibility of the MNT for evaluating control measures against flaviviruses. This research contributes valuable insights toward the development of a cost-effective antibody titer testing approach that is particularly suitable for resource-limited settings.

Abstract Background Dengue virus (DENV) is a member of insect vector-borne viruses, and it causes dengue fever. Southeast Asia is the epi-center of dengue fever in the world. The characterization of the virus is essential to identify the transmission and evolution of DENV. Objectives In 2017, there was an outbreak of Dengue virus type 1 (DENV1) in northern Vietnam and the neighboring countries. To identify the genetic character of the outbreak virus in the area, we conducted whole-genome sequencing analysis on the samples positive for the DENV1 along with real-time PCR. Study design In total, 1026 blood samples were collected from patients with suspected dengue fever in Ha Nam and Hai Duong province, nearby areas of the capital of Vietnam. After screening by real-time PCR, 40 of DENV1 positive samples were subjected to whole-genome sequencing, and 28 complete coding sequences were obtained. Results All 28 sequences were genotype I of DENV1, which is dominant in the southeast and East Asian countries. The phylogenetic analysis of the E region showed that they fell into a single cluster with the reported sequences from Vietnam between 2009 and 2016, in which the isolates from other countries are very rare. Our results suggested that the 2017 outbreak in the area was caused by locally circulating viruses.

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

Abstract Background Between 2016 and 2019, 265 cases of Zika virus (ZIKV) infection were reported in Vietnam, predominantly in southern Vietnam. In 2016, a case of ZIKV-associated microcephaly was confirmed in the Central Highlands, and several members of the infant’s family were confirmed to be infected with ZIKV. The study aims to determine the level of immunity to ZIKV in the general population of the ZIKV epidemic region. Methods A total of 879 serum samples were collected from 801 participants between January 2017 and July 2018, during and after the ZIKV epidemic in Vietnam. The samples were tested for anti-ZIKV immunoglobulin M (IgM) and immunoglobulin G (IgG), and anti-dengue virus (DENV) IgG antibodies using enzyme-linked immunosorbent assays (ELISA). Plaque-reduction neutralization test (PRNT) for ZIKV was performed on all samples, and for DENV on the samples that ZIKV neutralizing antibody positive. Results A total of 83 (10.3%) participants had anti-ZIKV IgM. Of the 83, 6 were confirmed to be ZIKV antibodies positive using PRNT and anti-ZIKV IgG ELISA. Of the 718 participants who were anti-ZIKV IgM negative, a further 3 cases were confirmed as positive for antibodies against ZIKV. Of the 9 participants with ZIKV infection, 5 lived in the same village as the infant with ZIKV-associated microcephaly and the other 4 lived in 2 neighboring communes. Repeat samples were collected from the 83 ZIKV IgM positive participants 1.5 years after the first collection. No new cases of ZIKV infection were detected. In addition, 2 of 3 participants with anti-ZIKV NS1 IgG demonstrated a 4- to 8-fold increase in ZIKV neutralizing antibody titer. Conclusions ZIKV was present in the area around Krong Buk, with the rate of ZIKV-specific antibodies was 1.1% in the community since at least 2016. While the low levels of circulation together with low seroprevalence suggests a limited outbreak in the region, the results also reflect on low levels of protective immunity to Zika within the population. These results provide a better understanding of the current ZIKV epidemic status in the region and demonstrate a need for implementation of more effective ZIKV infection control measures.

T-cell recognition of antigen epitopes is a crucial step for the induction of adaptive immune responses, and the identification of such T-cell epitopes is, therefore, important for understanding diverse immune responses and controlling T-cell immunity. A number of bioinformatic tools exist that predict T-cell epitopes; however, many of these methods highly rely on evaluating conventional peptide presentation by major histocompatibility complex (MHC) molecules, but they ignore epitope sequences recognized by T-cell receptor (TCR). Immunogenic determinant idiotopes are present on the variable regions of immunoglobulin molecules expressed on and secreted by B-cells. In idiotope-driven T-cell/B-cell collaboration, B-cells present the idiotopes on MHC molecules for recognition by idiotope-specific T-cells. According to the idiotype network theory formulated by Niels Jerne, such idiotopes found on anti-idiotypic antibodies exhibit molecular mimicry of antigens. Here, by combining these concepts and defining the patterns of TCR-recognized epitope motifs (TREMs), we developed a T-cell epitope prediction method that identifies T-cell epitopes derived from antigen proteins by analyzing B-cell receptor (BCR) sequences. This method allowed us to identify T-cell epitopes that contain the same TREM patterns between BCR and viral antigen sequences in two different infectious diseases caused by dengue virus and SARS-CoV-2 infection. The identified epitopes were among the T-cell epitopes detected in previous studies, and T-cell stimulatory immunogenicity was confirmed. Thus, our data support this method as a powerful tool for the discovery of T-cell epitopes from BCR sequences.

The first wave of COVID-19 epidemic began in late January in Malaysia and ended with a very small final size. The second wave of infections broke out in late February and grew rapidly in the first 3 weeks. Authorities in the country responded quickly with a series of control strategies collectively known as the Movement Control Order (MCO) with different levels of intensity matching the progression of the epidemic. We examined the characteristics of the second wave and discussed the key control strategies implemented in the country. In the second wave, the epidemic doubled in size every 3.8 days (95% confidence interval [CI]: 3.3, 4.5) in the first month and decayed slowly after that with a halving time of approximately 3 weeks. The time-varying reproduction number Rt peaked at 3.1 (95% credible interval: 2.7, 3.5) in the 3rd week, declined sharply thereafter and stayed below 1 in the last 3 weeks of April, indicating low transmissibility approximately 3 weeks after the MCO. Experience of the country suggests that adaptive triggering of distancing policies combined with a population-wide movement control measure can be effective in suppressing transmission and preventing a rebound.

We analyzed 2 clusters of 12 patients in Vietnam with severe acute respiratory syndrome coronavirus 2 infection during January–February 2020. Analysis indicated virus transmission from a traveler from China. One asymptomatic patient demonstrated virus shedding, indicating potential virus transmission in the absence of clinical signs and symptoms.

Currently, there are no specific therapeutics for flavivirus infections, including dengue virus (DENV) and Zika virus (ZIKV). In this study, we evaluated extracts from the plants Hedyotis diffusa (HD) and Artemisia capillaris (AC) to determine the antiviral activity against DENV, ZIKV, and Japanese encephalitis virus (JEV). HD and AC demonstrated inhibitory activity against JEV, ZIKV, and DENV replication and reduced viral RNA levels in a dose–responsive manner, with non-cytotoxic concentration ranging from 0.1 to 10 mg/mL. HD and AC had low cytotoxicity to Vero cells, with CC50 values of 33.7 ± 1.6 and 30.3 ± 1.7 mg/mL (mean ± SD), respectively. The anti-flavivirus activity of HD and AC was also consistent in human cell lines, including human glioblastoma (T98G), human chronic myeloid leukemia (K562), and human embryonic kidney (HEK-293T) cells. Viral-infected, HD-treated cells demonstrated downregulation of cytokines including CCR1, CCL26, CCL15, CCL5, IL21, and IL17C. In contrast, CCR1, CCL26, and AIMP1 were elevated following AC treatment in viral-infected cells. Overall, HD and AC plant extracts demonstrated flavivirus replication inhibitory activity, and together with immunoregulatory cytokine signatures, these results suggest that HD and AC possess bioactive compounds that may further be refined as promising candidates for clinical applications.

The stability and infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in liquid samples are of great concern to virus transmission via common beverages and sewage water. Here, we investigated the stability of SARS-CoV-2 in 32 liquids including common beverages, bodily fluids, and commonly used viral transport media. Our results showed that the infectious virus could be recovered up to 77-days from common beverages including milk and water. Viral RNA could be detected at high levels in all samples up to 28-days, indicating that while viral RNA demonstrates higher stability than infectivity, viral RNA levels do not reflect the infectious capability of SARS-CoV-2. These results indicate that SARS-CoV-2 is highly stable in optimal conditions and a sufficient control measure is needed in reducing the risk of exposure and controlling and preventing future outbreaks.

The dengue virus (DENV) has been endemic in Myanmar since 1970, causing outbreaks every 2–3 years. DENV infection symptoms range from mild fever to lethal hemorrhage. Clinical biomarkers must be identified to facilitate patient risk stratification in the early stages of infection. We analyzed 45 cytokines and other factors in serum samples from the acute phase of DENV infection (within 3–5 days of symptom onset) from 167 patients in Yangon, Myanmar, between 2017 and 2019. All of the patients tested positive for serum DENV nonstructural protein 1 antigen (NS1 Ag); 78.4% and 62.9% were positive for immunoglobulin M (IgM) and G (IgG), respectively; and 18.0%, 19.8%, and 11.9% tested positive for serotypes 1, 3, and 4, respectively. Although the DENV-4 viral load was significantly higher than those of DENV-1 or DENV-3, disease severity was not associated with viral load or serotype. Significant correlations were identified between disease severity and CCL5, SCF, PDGF-BB, IL-10, and TNF-α levels; between NS1 Ag and SCF, CCL5, IFN-α, IL-1α, and IL-22 levels; between thrombocytopenia and IL-2, TNF-α, VEGF-D, and IL-6 levels; and between primary or secondary infection and IL-2, IL-6, IL-31, IL-12p70, and MIP-1β levels. These circulating factors may represent leading signatures in acute DENV infections, reflecting the clinical outcomes in the dengue endemic region, Myanmar.

Abstract 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.

Abstract 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.

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

Owing to genotype-specific neutralizing antibodies, analyzing differences in the immunogenic variation among dengue virus (DENV) genotypes is central to effective vaccine development. Herein, we characterized the viral kinetics and antibody response induced by DENV type 2 Asian I (AI) and Asian/American (AA) genotypes using marmosets (Callithrix jacchus) as models. Two groups of marmosets were inoculated with AI and AA genotypes, and serial plasma samples were collected. Viremia levels were determined using quantitative reverse transcription-PCR, plaque assays, and antigen enzyme-linked immunosorbent assay (ELISA). Anti-DENV immunoglobulin M and G antibodies, neutralizing antibody titer, and antibody-dependent enhancement (ADE) activity were determined using ELISA, plaque reduction neutralization test, and ADE assay, respectively. The AI genotype induced viremia for a longer duration, but the AA genotype induced higher levels of viremia. After four months, the neutralizing antibody titer induced by the AA genotype remained high, but that induced by the AI genotype waned. ADE activity toward Cosmopolitan genotypes was detected in marmosets inoculated with the AI genotype. These findings indicate discrepancies between heterologous genotypes that influence neutralizing antibodies and viremia in marmosets, a critical issue in vaccine development.

Reinfection cases have been reported in some countries with clinical symptoms ranging from mild to severe. In addition to clinical diagnosis, virus genome sequence from the first and second infection has to be confirmed to either belong to separate clades or had significant mutations for the confirmation of SARS-CoV-2 reinfection. While phylogenetic analysis with paired specimens offers the strongest evidence for reinfection, there remains concerns on the definition of SARS-CoV-2 reinfection, for reasons including accessibility to paired-samples and technical challenges in phylogenetic analysis. In light of the emergence of new SARS-CoV-2 variants that are associated with increased transmissibility and immune-escape further understanding of COVID-19 protective immunity, real-time surveillance directed at identifying COVID-19 transmission patterns, transmissibility of emerging variants and clinical implications of reinfection would be important in addressing the challenges in definition of COVID-19 reinfection and understanding the true disease burden.

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.

Mosquito-borne flavivirus infections, including dengue virus and Zika virus, are major public health threats globally. While the plaque reduction neutralization test (PRNT) is considered the gold standard for determining neutralizing antibody levels to flaviviruses, the assay is time-consuming and laborious. This study, therefore, aimed to develop an enzyme-linked immunosorbent assay (ELISA)-based microneutralization test (EMNT) for the detection of neutralizing antibodies to mosquito-borne flaviviruses. The inhibition of viral growth due to neutralizing antibodies was determined colorimetrically by using EMNT. Given the significance of Fcγ-receptors (FcγR) in antibody-mediated neutralization and antibody-dependent enhancement (ADE) of flavivirus infection, non-FcγR and FcγR-expressing cell lines were used in the EMNT to allow the detection of the sum of neutralizing and immune-enhancing antibody activity as the neutralizing titer. Using anti-flavivirus monoclonal antibodies and clinical samples, the utility of EMNT was evaluated by comparing the end-point titers of the EMNT and the PRNT. The correlation between EMNT and PRNT titers was strong, indicating that EMNT was robust and reproducible. The new EMNT assay combines the biological functional assessment of virus neutralization activity and the technical advantages of ELISA and, is simple, reliable, practical, and could be automated for high-throughput implementation in flavivirus surveillance studies and vaccine trials.

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.

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.

Common marmosets (Callithrix jacchus) developed high levels of viremia, clinical signs including fever, weight loss, a decrease in activity and hematuria upon inoculation with dengue virus (DENV). Presence of DENV genome in urine samples and pathological changes in kidneys were examined in the present study. Levels of DENV genome were determined in 228 urine samples from 20 primary DENV-inoculated marmosets and in 56 urine samples from four secondary DENV-inoculated marmosets. DENV genome was detected in 75% (15/20) of marmosets after primary DENV infection. No DENV genome was detected in urine samples from the marmosets with secondary infection with homologous DENV (0%, 0/4). Two marmosets demonstrated hematuria. Pathological analysis of the kidneys demonstrated non-suppressive interstitial nephritis with renal tubular regeneration. DENV antigen-positive cells were detected in kidneys. In human dengue virus infections, some patients present renal symptoms. The results indicate that marmosets recapitulate some aspects of the involvement of kidneys in human DENV infection, and suggest that marmosets are potentially useful for the studies of the pathogenesis of DENV infection, including kidneys.

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 Côte d’Ivoire. Phylogenetic analysis of the envelope gene showed that 2 genetically related virus isolates belonged to dengue virus type 3 genotype III.

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.

Introduction: Arbovirus diseases remain a public health threat in Sri Lanka. Dengue is endemic and two outbreaks of chikungunya infections have been reported. There is limited data on Zika virus (ZIKV) infections in Sri Lanka, and this could be due to a lack of comprehensive ZIKV surveillance. Our aim was to determine the presence of antibodies to dengue, chikungunya, and Zika infections in adults from a suburban population in Sri Lanka. Methods: A total of 149 healthy adult volunteers over 18 years of age (mean age: 43±14 years, males – 43%), with no prior diagnosed arboviral infections and no history of overseas travel, participated in the study. ELISA and neutralization assays were carried out to detect past dengue, chikungunya, or Zika infections. Results: A total of 94.6% (141/149) of the participants demonstrated dengue IgG antibodies, 37.5% (56/149) were positive for chikungunya IgG, and 5.3% (8/149) were positive for anti-ZIKV IgG antibodies. Neutralization assays confirmed ZIKV-specific antibodies in 6.7% (10/149), when 40/149 of the participating population were tested. Conclusion: This clearly demonstrated past ZIKV infections in this population. In addition, this study indicates that >90% of individuals had asymptomatic dengue but no serious symptoms. These results provide a cross-sectional view on the DENV, ZIKV, and CHIKV epidemic status and demonstrate a need for the implementation of enhanced surveillance and more effective measures against the spread of these arbovirus diseases. [ABSTRACT FROM AUTHOR]