Myosin II mediates Shh signals to shape dental epithelia via control of cell adhesion and movement.

The development of ectodermal organs begins with the formation of a stratified epithelial placode that progressively invaginates into the underlying mesenchyme as the organ takes its shape. Signaling by secreted molecules is critical for epithelial morphogenesis, but how that information leads to cell rearrangement and tissue shape changes remains an open question. Using the mouse dentition as a model, we first establish that non-muscle myosin II is essential for dental epithelial invagination and show that it functions by promoting cell-cell adhesion and persistent convergent cell movements in the suprabasal layer. Shh signaling controls these processes by inducing myosin II activation via AKT. Pharmacological induction of AKT and myosin II can also rescue defects caused by the inhibition of Shh. Together, our results support a model in which the Shh signal is transmitted through myosin II to power effective cellular rearrangement for proper dental epithelial invagination.

Three-dimensional topography of rat trigeminal ganglion neurons using a combination of retrograde labeling and tissue-clearing techniques.

The trigeminal nerve is the sensory afferent of the orofacial regions and divided into three major branches. Cell bodies of the trigeminal nerve lie in the trigeminal ganglion and are surrounded by satellite cells. There is a close interaction between ganglion cells via satellite cells, but the function is not fully understood. In the present study, we clarified the ganglion cells' three-dimensional (3D) localization, which is essential to understand the functions of cell-cell interactions in the trigeminal ganglion. Fast blue was injected into 12 sites of the rat orofacial regions, and ganglion cells were retrogradely labeled. The labeled trigeminal ganglia were cleared by modified 3DISCO, imaged with confocal laser-scanning microscopy, and reconstructed in 3D. Histograms of the major axes of the fast blue-positive somata revealed that the peak major axes of the cells innervating the skin/mucosa were smaller than those of cells innervating the deep structures. Ganglion cells innervating the ophthalmic, maxillary, and mandibular divisions were distributed in the anterodorsal, central, and posterolateral portions of the trigeminal ganglion, respectively, with considerable overlap in the border region. The intermingling in the distribution of ganglion cells within each division was also high, in particular, within the mandibular division. Specifically, intermingling was observed in combinations of tongue and masseter/temporal muscles, maxillary/mandibular molars and masseter/temporal muscles, and tongue and mandibular molars. Double retrograde labeling confirmed that some ganglion cells innervating these combinations were closely apposed. Our data provide essential information for understanding the function of ganglion cell-cell interactions via satellite cells.

Genetic regions affecting the replication and pathogenicity of dengue virus type 2.

Dengue is a mosquito-borne disease that has spread to over 100 countries. Its symptoms vary from the relatively mild acute febrile illness called dengue fever to the much more severe dengue shock syndrome. Dengue is caused by dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. There are four serotypes of DENV, i.e., DENV1 to DENV4, and each serotype is divided into distinct genotypes. Thailand is an endemic area where all four serotypes of DENV co-circulate. Genome sequencing of the DENV2 that was isolated in Thailand in 2016 and 2017 revealed the emergence of the Cosmopolitan genotype and its co-circulation with the Asian-I genotype. However, it was unclear whether different genotypes have different levels of viral replication and pathogenicity. Focus-forming assay (FFA) results showed that clinical isolates of these genotypes differed in focus size and proliferative capacity. Using circular polymerase extension reaction, we generated parental and chimeric viruses with swapped genes between these two DENV2 genotypes, and compared their focus sizes and infectivity titers using FFA. The results showed that the focus size was larger when the structural proteins and/or non-structural NS1-NS2B proteins were derived from the Cosmopolitan virus. The infectious titers were consistent with the focus sizes. Single-round infectious particle assay results confirmed that chimeric viruses with Cosmopolitan type structural proteins, particularly prM/E, had significantly increased luciferase activity. Replicon assay results showed that Cosmopolitan NS1-NS2B proteins had increased reporter gene expression levels. Furthermore, in interferon-receptor knock-out mice, viruses with Cosmopolitan structural and NS1-NS2B proteins had higher titers in the blood, and caused critical disease courses. These results suggested that differences in the sequences within the structural and NS1-NS2B proteins may be responsible for the differences in replication, pathogenicity, and infectivity between the Asian-I and Cosmopolitan viruses.

Developmental process of the modern house shrew's molars: implications for the evolution of the tribosphenic molar in Mesozoic mammals.

Phylogenetically, the tribosphenic molars-prototypes of multi-cusped cheek teeth in marsupial and placental mammals-are derived from the single-cusped conical teeth of reptiles through the addition of cusps. Ontogenetically, mammalian molars are formed through the interface between the dental epithelium and mesenchyme (future enamel-dentin junction), becoming geometrically complex by adding epithelial signaling centers, called enamel knots, which determine future cusp positions. To reevaluate cusp homologies in Mesozoic mammals from an ontogenetic perspective, this study tracked molar development in a living placental mammal species, the house shrew (Suncus murinus), whose molars are morphologically the least derived from tribosphenic prototypes. The development of shrew molars proceeded as if it replayed the evolutionary process of tribosphenic molars. The first formed enamel knots gave rise to the evolutionarily oldest cusps-upper paracone and lower protoconid. The order of formation of other enamel knots and their location in development seemed to trace the order of cusp appearance in evolution. The parallel relationship between ontogeny and phylogeny of mammalian molars, if any, suggests that a change in the timing between developmental events rather than a change in the morphogenetic mechanism itself, should have been a major causal factor for the evolutionary transformation of tooth morphology.

Identification of the Tembusu Virus in Mosquitoes in Northern Thailand.

Among emerging zoonotic pathogens, mosquito-borne viruses (MBVs) circulate between vertebrate animals and mosquitoes and represent a serious threat to humans via spillover from enzootic cycles to the human community. Active surveillance of MBVs in their vectors is therefore essential to better understand and prevent spillover and emergence, especially at the human-animal interface. In this study, we assessed the presence of MBVs using molecular and phylogenetic methods in mosquitoes collected along an ecological gradient ranging from rural urbanized areas to highland forest areas in northern Thailand. We have detected the presence of insect specific flaviviruses in our samples, and the presence of the emerging zoonotic Tembusu virus (TMUV). Reported for the first time in 1955 in Malaysia, TMUV remained for a long time in the shadow of other flaviviruses such as dengue virus or the Japanese encephalitis virus. In this study, we identified two new TMUV strains belonging to cluster 3, which seems to be endemic in rural areas of Thailand and highlighted the genetic specificities of this Thai cluster. Our results show the active circulation of this emerging flavivirus in Thailand and the need for continuous investigation on this poorly known but threatening virus in Asia.

Potential Protective Effect of Dengue NS1 Human Monoclonal Antibodies against Dengue and Zika Virus Infections.

Due to the lack of an effective therapeutic treatment to flavivirus, dengue virus (DENV) nonstructural protein 1 (NS1) has been considered to develop a vaccine owing to its lack of a role in antibody-dependent enhancement (ADE). However, both NS1 and its antibody have shown cross-reactivity to host molecules and have stimulated anti-DENV NS1 antibody-mediated endothelial damage and platelet dysfunction. To overcome the pathogenic events and reactogenicity, human monoclonal antibodies (HuMAbs) against DENV NS1 were generated from DENV-infected patients. Herein, the four DENV NS1-specific HuMAbs revealed the therapeutic effects in viral neutralization, reduction of viral replication, and enhancement of cell cytolysis of DENV and zika virus (ZIKV) via complement pathway. Furthermore, we demonstrate that DENV and ZIKV NS1 trigger endothelial dysfunction, leading to vascular permeability in vitro. Nevertheless, the pathogenic effects from NS1 were impeded by 2 HuMAbs (D25-4D4C3 and D25-2B11E7) and also protected the massive cytokines stimulation (interleukin [IL-]-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-17, eotaxin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, Inducible protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein [MIP]-1 α, MIP-1ß, tumor necrosis factor-α, platelet-derived growth factor, and RANTES). Collectively, our findings suggest that the novel protective NS1 monoclonal antibodies generated from humans has multiple therapeutic benefits against DENV and ZIKV infections.

Development of a rapid assay system for detecting antibody-dependent enhancement of dengue virus infection.

Antibody-dependent enhancement (ADE) is one of the pathogenic mechanisms related to disease severity in dengue virus infection. Conventional assays for detecting ADE activity usually require several days. In this study, we established a rapid assay system to evaluate ADE activity in dengue-seropositive samples using single round infectious particles (SRIPs). Human Fc-gamma receptor-bearing cells (K562 and Mylc cells) were infected with SRIP antigen in the presence of human serum samples to measure ADE activity. Two assay protocols were introduced: (i) rapid assay with 5 h of incubation, and (ii) semi-rapid assay with 24 h of incubation. The rapid assay requires a large quantity of SRIP antigen and gives results in half a day. Although the semi-rapid assay requires slightly more than a day, it can be performed using only a small amount of SRIP. Interestingly, the range of the number of Mylc cells required for the semi-rapid assay was wider than that of K562 cells. Significant correlations were observed between the rapid and semi-rapid assays for both cell types. Although it is difficult to judge which protocol best reflects the current immune status in vivo, both assays could rapidly provide valuable information regarding the risk assessment for severe diseases.

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

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

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

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

Development of a system to analyze oral frailty associated with Alzheimer's disease using a mouse model.

The rapid aging of the population makes the detection and prevention of frailty increasingly important. Oral frailty has been proposed as a novel frailty phenotype and is defined as a decrease in oral function coexisting with a decline in cognitive and physical functions. Oral frailty has received particular attention in relation to Alzheimer's disease (AD). However, the pathomechanisms of oral frailty related to AD remain unknown. It is assumed that the mesencephalic trigeminal nucleus (Vmes), which controls mastication, is affected by AD pathology, and as a result, masticatory function may be impaired. To investigate this possibility, we included male 3 × Tg-AD mice and their non-transgenic counterpart (NonTg) of 3-4 months of age in the present study. Immunohistochemistry revealed amyloid-ß deposition and excessive tau phosphorylation in the Vmes of 3 × Tg-AD mice. Furthermore, vesicular glutamate transporter 1-immunopositive axon varicosities, which are derived from Vmes neurons, were significantly reduced in the trigeminal motor nucleus of 3 × Tg-AD mice. To investigate whether the AD pathology observed in the Vmes affects masticatory function, we analyzed electromyography of the masseter muscle during feeding. The 3 × Tg-AD mice showed a significant delay in masticatory rhythm compared to NonTg mice. Furthermore, we developed a system to simultaneously record bite force and electromyography of masseter, and devised a new method to estimate bite force during food chewing in mice. Since the muscle activity of the masseter showed a high correlation with bite force, it could be accurately estimated from the muscle activity. The estimated bite force of 3 × Tg-AD mice eating sunflower seeds was predominantly smaller than that of NonTg mice. However, there was no difference in masseter weight or muscle fiber cross-sectional area between the two groups, suggesting that the decreased bite force and delayed mastication rhythm observed in 3 × Tg-AD mice were not due to abnormality of the masseter. In conclusion, the decreased masticatory function observed in 3 × Tg-AD mice was most likely caused by AD pathology in the Vmes. Thus, novel quantitative analyses of masticatory function using the mouse model of AD enabled a comprehensive understanding of oral frailty pathogenesis.

Evolution and development of the mammalian multicuspid teeth.

BACKGROUND: Mammalian premolars and molars (cheek teeth) are teeth with multiple cusps, which are important characteristics of mammals. Endothermic mammals have high basal metabolic rates and must take in much energy by efficient mastication of food using their multicuspid cheek teeth. From the phylogenetic (evolutionary) perspective, the mammalian multicuspid teeth are derived from the reptilian unicuspid teeth with a single cone by adding new cusps around the original cone. Nearly 100 million years of long geological time were required for the unicuspid tooth to transform into a tribosphenic molar, which is the prototype of all molars in modern mammals. From the ontogenetic (developmental) perspective, the shape of the tooth germ becomes complex by adding the secondary enamel knots repeatedly in a short embryonic period. The secondary enamel knots are signaling centers that determine the future cusp positions. HIGHLIGHT: Here we first reviewed the evolutionary process of the tribosphenic molar in the Mesozoic Era (the age of dinosaurs), and cusp homologies in the fossil record. Next, we reviewed the developmental mechanisms controlling the patterning of secondary enamel knots, which determine the final cusp patterns of molars in modern mammals. Finally, we discussed the possible relationship between the two processes from the extremely different time scales. CONCLUSION: A parallel relationship between ontogeny and phylogeny of the mammalian multicuspid teeth was expected.

Retrospective study on the possibility of an SFTS outbreak associated with undiagnosed febrile illness in veterinary professionals and a family with sick dogs in 2003.

INTRODUCTION: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-born disease and its animal-to-human transmission has come to attention recently. During our sero-survey of SFTS virus (SFTSV) among veterinary professionals in 2018, a veterinarian and his assistant working in an animal hospital were tested positive by enzyme-linked immunosorbent assay (ELISA). An additional survey implied a cluster of SFTS cases in which four more people, a family who brought two sick dogs to the animal hospital in 2003, were involved. This study aimed at assessing the possibility of animal-to-human transmission of SFTSV in this cluster. METHODS: Retrospective interviews were performed with the owner family of the dogs and their clinical records were obtained from each hospital. SFTSV-IgG were tested by ELISA and virus neutralization test using the sera collected from them in 2018. RESULTS: The interviews revealed that a total of six people, the two veterinary professionals and the owner family who took care of the sick dogs, suffered from SFTS-like symptoms in the same period of time in 2003. All patients did not have tick bite before the onset and all suspected causative agents were excluded by laboratory tests. The serological tests in this study revealed the four owner family members were all positive for SFTSV antibodies. CONCLUSIONS: Considering the extremely low seroprevalence of SFTSV antibodies among inhabitants of the region, the existence of SFTSV antibodies in all these six people presents a possibility that they were involved in an SFTS outbreak originated in the sick dogs in 2003.

Thoracic Empyema Caused by Campylobacter rectus.

Campylobacter rectus is a campylobacterium considered to be a primary periodontal pathogen. Thus, C. rectus has rarely been isolated from extraoral specimens, especially in the thoracic region. We herein report a case of thoracic empyema in which Campylobacter infection was suspected after Gram staining of the pleural effusion, and C. rectus was isolated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Fusobacterium nucleatum was also detected. Molecular identification was performed using polymerase chain reaction amplification and a sequencing analysis of the 16S rRNA gene. Estimation of the causative bacteria using Gram staining led to the proper culture and identification of the causative bacteria.

The potential of COVID-19 patients' sera to cause antibody-dependent enhancement of infection and IL-6 production.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many vaccine trials have been initiated. An important goal of vaccination is the development of neutralizing antibody (Ab) against SARS-CoV-2. However, the possible induction of antibody-dependent enhancement (ADE) of infection, which is known for other coronaviruses and dengue virus infections, is a particular concern in vaccine development. Here, we demonstrated that human iPS cell-derived, immortalized, and ACE2- and TMPRSS2-expressing myeloid cell lines are useful as host cells for SARS-CoV-2 infection. The established cell lines were cloned and screened based on their function in terms of susceptibility to SARS-CoV-2-infection or IL-6 productivity. Using the resulting K-ML2 (AT) clone 35 for SARS-CoV-2-infection or its subclone 35-40 for IL-6 productivity, it was possible to evaluate the potential of sera from severe COVID-19 patients to cause ADE and to stimulate IL-6 production upon infection with SARS-CoV-2.

Development of a Dengue Virus Serotype-Specific Non-Structural Protein 1 Capture Immunochromatography Method.

Four serotypes of dengue virus (DENV), type 1 to 4 (DENV-1 to DENV-4), exhibit approximately 25-40% of the difference in the encoded amino acid residues of viral proteins. Reverse transcription of RNA extracted from specimens followed by PCR amplification is the current standard method of DENV serotype determination. However, since this method is time-consuming, rapid detection systems are desirable. We established several mouse monoclonal antibodies directed against DENV non-structural protein 1 and integrated them into rapid DENV detection systems. We successfully developed serotype-specific immunochromatography systems for all four DENV serotypes. Each system can detect 104 copies/mL in 15 min using laboratory and clinical isolates of DENV. No cross-reaction between DENV serotypes was observed in these DENV isolates. We also confirmed that there was no cross-reaction with chikungunya, Japanese encephalitis, Sindbis, and Zika viruses. Evaluation of these systems using serum from DENV-infected individuals indicated a serotype specificity of almost 100%. These assay systems could accelerate both DENV infection diagnosis and epidemiologic studies in DENV-endemic areas.

Tissue-resident M2 macrophages directly contact primary sensory neurons in the sensory ganglia after nerve injury.

BACKGROUND: Macrophages in the peripheral nervous system are key players in the repair of nerve tissue and the development of neuropathic pain due to peripheral nerve injury. However, there is a lack of information on the origin and morphological features of macrophages in sensory ganglia after peripheral nerve injury, unlike those in the brain and spinal cord. We analyzed the origin and morphological features of sensory ganglionic macrophages after nerve ligation or transection using wild-type mice and mice with bone-marrow cell transplants. METHODS: After protecting the head of C57BL/6J mice with lead caps, they were irradiated and transplanted with bone-marrow-derived cells from GFP transgenic mice. The infraorbital nerve of a branch of the trigeminal nerve of wild-type mice was ligated or the infraorbital nerve of GFP-positive bone-marrow-cell-transplanted mice was transected. After immunostaining the trigeminal ganglion, the structures of the ganglionic macrophages, neurons, and satellite glial cells were analyzed using two-dimensional or three-dimensional images. RESULTS: The number of damaged neurons in the trigeminal ganglion increased from day 1 after infraorbital nerve ligation. Ganglionic macrophages proliferated from days 3 to 5. Furthermore, the numbers of macrophages increased from days 3 to 15. Bone-marrow-derived macrophages increased on day 7 after the infraorbital nerve was transected in the trigeminal ganglion of GFP-positive bone-marrow-cell-transplanted mice but most of the ganglionic macrophages were composed of tissue-resident cells. On day 7 after infraorbital nerve ligation, ganglionic macrophages increased in volume, extended their processes between the neurons and satellite glial cells, and contacted these neurons. Most of the ganglionic macrophages showed an M2 phenotype when contact was observed, and little neuronal cell death occurred. CONCLUSION: Most of the macrophages that appear after a nerve injury are tissue-resident, and these make direct contact with damaged neurons that act in a tissue-protective manner in the M2 phenotype. These results imply that tissue-resident macrophages signal to neurons directly through physical contact.

Glycan engineering of the SARS-CoV-2 receptor-binding domain elicits cross-neutralizing antibodies for SARS-related viruses.

Broadly protective vaccines against SARS-related coronaviruses that may cause future outbreaks are urgently needed. The SARS-CoV-2 spike receptor-binding domain (RBD) comprises two regions, the core-RBD and the receptor-binding motif (RBM); the former is structurally conserved between SARS-CoV-2 and SARS-CoV. Here, in order to elicit humoral responses to the more conserved core-RBD, we introduced N-linked glycans onto RBM surfaces of the SARS-CoV-2 RBD and used them as immunogens in a mouse model. We found that glycan addition elicited higher proportions of the core-RBD-specific germinal center (GC) B cells and antibody responses, thereby manifesting significant neutralizing activity for SARS-CoV, SARS-CoV-2, and the bat WIV1-CoV. These results have implications for the design of SARS-like virus vaccines.

Seroprevalence of Flavivirus Neutralizing Antibodies in Thailand by High-Throughput Neutralization Assay: Endemic Circulation of Zika Virus before 2012.

Thailand is a hyperendemic country for flavivirus infections in Southeast Asia. Although the reporting system for flavivirus surveillance in Thailand is well established, syndromic surveillance tends to underestimate the true epidemiological status of flaviviruses due to the majority of infections being asymptomatic. To accurately understand the prevalence of flaviviruses in endemic regions, we performed neutralization tests against multiple flaviviruses using 147 serum samples from healthy donors collected from four distinct regions in Thailand. Single-round infectious particles (SRIP) for six flaviviruses, dengue virus types 1 to 4 (DENV-1 to -4), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), were used as antigens for developing a safe, high-throughput neutralization assay. Titers of neutralizing antibodies (NAbs) against the six flaviviruses revealed that DENV-1 and DENV-2, followed by ZIKV were the predominant circulating flaviviruses in a total of four regions, whereas the prevalence of NAbs against JEV varied among regions. Although the seroprevalence of ZIKV was low relative to that of DENV-1 and DENV-2, the findings strongly suggested that ZIKV has been circulating at a sustained level in Thailand since before 2012. These findings not only demonstrated the application of an SRIP-neutralization test in a serological study, but also elucidated the circulation and distribution trends of different flaviviruses in Thailand. IMPORTANCE Neutralization tests are the most reliable assay for flavivirus antibody detection; however, these assays are not suitable for high-throughput processing due to their time-consuming and labor-intensive nature. In this study, we developed single-round infectious particles (SRIPs) with a luciferase gene for dengue virus types 1 to 4, Japanese encephalitis virus, and Zika virus for use in a safe, high-throughput neutralization assay. We performed neutralization tests against multiple flaviviruses using 147 serum samples that were collected from healthy donors residing in four distinct regions of Thailand in 2011 to 2012. The assay was useful for surveys of flavivirus seroprevalence. The data revealed that dengue virus type 1 (DENV-1) and DENV-2 were the predominant circulating flaviviruses in Thailand and that Zika virus has been circulating at a sustained level in Thailand since before 2012.

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

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