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IntroductionCoronavirus disease 2019 (COVID-19) alters the gut microbiome. This study aimed to assess the association between the disease severity of COVID-19 and changes in stool microbes through a seven-month follow-up of stool collection.MethodsWe conducted a multicentre, prospective longitudinal study of 58 COVID-19 patients and 116 uninfected controls. Differences in the gut microbiota were analysed using 16S ribosomal RNA sequencing. The first stool samples were collected at an early convalescent phase of COVID-19, and the second sample was collected at least seven months after COVID-19 infection.Results and discussionAt the order level, Eubacteriales and Bifidobacteriales decreased, while Bacteroidales and Burkholderiales increased in the COVID-19 group compared to the controls. Alpha diversity also decreased in COVID-19 patients compared to controls, with imperfect recovery of the gut microbiome after seven months. The compositional change in the gut microbiome between the early and late convalescent phases was largest in the moderate and severe groups. The severity of COVID-19 was the most influential clinical variable for microbiome composition (Sum of Sqs = 0.686, P = 0.006), and its effect persisted even after partialling out other effects such as antibiotic use and age. Thus, our study indicates a possible interaction between respiratory viral infection and the composition of the gut microbiota community, warranting future mechanistic and prospective longitudinal studies. Additionally, we were able to detect microbiome changes in patients who were re-infected with SARS-CoV-2. Notably, the dominant bacteria in the re-infected group were Lachnospiraceae and Faecalimonas umbilicata, compared to the one-time infected group.

Macrophages play essential roles in maintaining tissue homeostasis and immune defence. However, their extensive infiltration into tumours has been linked to adverse outcomes in multiple human cancers. Within the tumour microenvironment (TME), tumour-associated macrophages (TAMs) promote tumour growth and metastasis, making them prime targets for cancer immunotherapy. Recent single-cell analysis suggest that proliferating TAMs accumulate in human cancers, yet their origins and differentiation pathways remain uncertain. Here, we show that a subpopulation of CD163+ TAMs proliferates in situ within the TME of melanoma, lung cancer, and breast cancer. Consistent with their potential role in suppressing anti-tumour activities of T cells, CD163+ TAMs express a range of potent immunosuppressive molecules, including PD-L1, PD-L2, IL-10, and TGF-β. Other phenotypic markers strongly suggested that these cells originate from CD14+ CCR2+ monocytes, a cell population believed to have minimal capacity for proliferation. However, we demonstrate in vitro that certain myelopoietic cytokines commonly available within the TME induce robust proliferation of human monocytes, especially the combination of interleukin 3 (IL-3) and Macrophage Colony-Stimulating Factor 1 (M-CSF). Monocytic cells cultured with these cytokines efficiently modulate T cell proliferation, and their molecular phenotype recapitulates that of CD163+ TAMs. IL-3-driven proliferation of monocytic cells can be completely blocked by IL-4, associated with the induction of CDKN1A, alongside the upregulation of transcription factors linked to dendritic cell function, such as BATF3 and IRF4. Taken together, our work suggests several novel therapeutic routes to reducing immunosuppressive TAMs in human tumours, from blocking chemokine-mediated recruitment of monocytes to blocking their proliferation.

[This corrects the article DOI: 10.1371/journal.pntd.0001636.].

Urban garden spaces are potentially important habitats for bee conservation. Gardens can host diverse flora, which provide floral resources across foraging seasons for bee species. Recent reviews have focused on the impacts of cityscapes on urban bee assemblages in different green spaces. Urban gardens are distinct from other urban green spaces, and bee communities in urban spaces have been an increasing topic of study over the past few decades. We reviewed 28 urban garden bee studies spanning five decades and 14 countries to compile an original metadataset of bee species' functional traits to understand the conservation value of gardens, identify gaps in bee sampling efforts, and summarize the calls to action included by their authors. Studies of urban garden bees have documented between 674 (conservative count, excluding morphospecies) and 830 (liberal count, including morphospecies) bee species. Urban garden bee communities were taxonomically and functionally diverse, although bee species that were non-eusocial, ground-nesting, generalist foragers, and native were most common in garden habitats. The proportion of parasitic bee species and specialist foragers found in urban gardens was comparable to proportions for global bee taxa. This suggests that gardens contain the hosts and forage needed to support bees with specialized life history requirements, and thus represent high quality habitat for a subset of bee communities. Garden bee research was strongly biased toward the northern hemisphere, which signifies a large gap in our understanding of garden bee communities in other regions. The variety of, and non-standard sampling methods in garden bee research makes it difficult to directly compare results between studies. In addition, both intentional low taxonomic resolution and a lack of collaboration with taxonomists constrains our understanding of bee diversity. Our analyses highlight both successes of past urban garden bee studies, and areas of opportunity for future research as we move into a sixth decade of garden bee research.

This paper describes an automated method and device to conduct the Chair Stand Tests of the Fullerton Functional Test Battery. The Fullerton Functional Test is a suite of physical tests designed to assess the physical fitness of older adults. The Chair Stand Tests, which include the Five Times Sit-to-Stand Test (5xSST) and the 30 Second Sit-to-Stand Test (30CST), are the standard for measuring lower-body strength in older adults. However, these tests are performed manually, which can be labor-intensive and prone to error. We developed a sensor-integrated chair that automatically captures the dynamic weight and distribution on the chair. The collected time series weight–sensor data is automatically uploaded for immediate determination of the sit-to-stand timing and counts, as well as providing a record for future comparison of lower-body strength progression. The automatic test administration can provide significant labor savings for medical personnel and deliver much more accurate data. Data from 10 patients showed good agreement between the manually collected and sensor-collected 30CST data (M = 0.5, SD = 1.58, 95% CI = 1.13). Additional data processing will be able to yield measurements of fatigue and balance and evaluate the mechanisms of failed standing attempts.

Intratumoural administration of unmethylated cytosine-phosphate-guanine motifs (CpG) to stimulate toll-like receptor (TLR)-9 has been shown to induce tumour regression in preclinical studies and some efficacy in the clinic. Because activated natural killer T (NKT) cells can cooperate with pattern-recognition via TLRs to improve adaptive immune responses, we assessed the impact of combining a repeated dosing regimen of intratumoural CpG with a single intratumoural dose of the NKT cell agonist α-galactosylceramide (α-GalCer). The combination was superior to CpG alone at inducing regression of established tumours in several murine tumour models, primarily mediated by CD8+ T cells. An antitumour effect on distant untreated tumours (abscopal effect) was reliant on sustained activity of NKT cells and was associated with infiltration of KLRG1+ NKT cells in tumours and draining lymph nodes at both injected and untreated distant sites. Cytometric analysis pointed to increased exposure to type I interferon (IFN) affecting many immune cell types in the tumour and lymphoid organs. Accordingly, antitumour activity was lost in animals in which dendritic cells (DCs) were incapable of signaling through the type I IFN receptor. Studies in conditional ablation models showed that conventional type 1 DCs and plasmacytoid DCs were required for the response. In tumour models where the combined treatment was less effective, the addition of tumour-antigen derived peptide, preferably conjugated to α-GalCer, significantly enhanced the antitumour response. The combination of TLR ligation, NKT cell agonism, and peptide delivery could therefore be adapted to induce responses to both known and unknown antigens.

The unexpectedly low vaccine efficacy of Dengvaxia®, developed by Sanofi Pasteur, and a higher risk of severe diseases after vaccination among dengue-naive children or children younger than 6 years old, have cast skepticism about the safety of dengue vaccination resulting in the suspension of school-based immunization programs in the Philippines. The absence of immune correlates of protection from dengue virus (DENV) infection hampers the development of other potential DENV vaccines. While tetravalent live-attenuated tetravalent vaccines (LATVs), which mimic natural infection by inducing both cellular and humoral immune responses, are still currently favored, developing a vaccine that provides a balanced immunity to all four DENV serotypes remains a challenge. With the recently advanced understanding of virion structure and B cell immune responses from naturally infected DENV patients, two points of view in developing a next-generation dengue vaccine emerged: one is to induce potent, type-specific neutralizing antibodies (NtAbs) recognizing quaternary structure-dependent epitopes by having four components of vaccine strains replicate equivalently; the other is to induce protective and broadly NtAbs against the four serotypes of DENV with a universal vaccine. This article reviews the studies related to these issues and the current knowledge gap that needs to be filled in.

Objective We examined rates of spontaneous and indicated preterm births (S-PTB and I-PTB, respectively) and clinical risk factors for PTB in adolescents. Study Design This is a population-based, retrospective cohort using 2012 U.S. natality data of nulliparous women who delivered a nonanomalous singleton birth between 20 and 42 weeks' gestation. Maternal age included

Dengue viral (DENV) infection results in a wide spectrum of clinical manifestations from asymptomatic, mild fever to severe hemorrhage diseases upon infection. Severe dengue is the leading cause of pediatric deaths and/or hospitalizations, which are a major public health burden in dengue-endemic or hyperendemic countries. Like other RNA viruses, DENV continues to evolve. Adaptive mutations are obscured by the major consensus sequence (so-called wild-type sequences) and can only be identified once they become the dominant viruses in the virus population, a process that can take months or years. Traditional surveillance systems still rely on Sanger consensus sequencing. However, with the recent advancement of high-throughput next-generation sequencing (NGS) technologies, the genome-wide investigation of virus population within-host and between-hosts becomes achievable. Thus, viral population sequencing by NGS can increase our understanding of the changing epidemiology and evolution of viral genomics at the molecular level. This review focuses on the studies within the recent decade utilizing NGS in different experimental and epidemiological settings to understand how the adaptive evolution of dengue variants shapes the dengue epidemic and disease severity through its transmission. We propose three types of studies that can be pursued in the future to enhance our surveillance for epidemic prediction and better medical management.

Abstract Swine are a critical amplifying host involved in human Japanese encephalitis (JE) outbreaks. Cross-genotypic immunogenicity and sterile protection are important for the current genotype III (GIII) virus-derived vaccines in swine, especially now that emerging genotype I (GI) JE virus (JEV) has replaced GIII virus as the dominant strain. Herein, we aimed to develop a system to generate GI JEV virus-like particles (VLPs) and evaluate the immunogenicity and protection of the GI vaccine candidate in mice and specific pathogen-free swine. A CHO-heparan sulfate-deficient (CHO-HS(-)) cell clone, named 51-10 clone, stably expressing GI-JEV VLP was selected and continually secreted GI VLPs without signs of cell fusion. 51-10 VLPs formed a homogeneously empty-particle morphology and exhibited similar antigenic activity as GI virus. GI VLP-immunized mice showed balanced cross-neutralizing antibody titers against GI to GIV viruses (50% focus-reduction micro-neutralization assay titers 71 to 240) as well as potent protection against GI or GIII virus infection. GI VLP-immunized swine challenged with GI or GIII viruses showed no fever, viremia, or viral RNA in tonsils, lymph nodes, and brains as compared with phosphate buffered saline-immunized swine. We thus conclude GI VLPs can provide sterile protection against GI and GIII viruses in swine.

This paper describes an automated method and device to conduct the Chair Stand Tests of the Fullerton Functional Test Battery. The Fullerton Functional Test is a suite of physical tests designed to assess the physical fitness of older adults. The Chair Stand Tests, which include the Five Times Sit-to-Stand Test (5xSST) and the 30 Second Sit-to-Stand Test (30CST), are the standard for measuring lower-body strength in older adults. However, these tests are performed manually, which can be labor-intensive and prone to error. We developed a sensor-integrated chair that automatically captures the dynamic weight and distribution on the chair. The collected time series weight–sensor data is automatically uploaded for immediate determination of the sit-to-stand timing and counts, as well as providing a record for future comparison of lower-body strength progression. The automatic test administration can provide significant labor savings for medical personnel and deliver much more accurate data. Data from 10 patients showed good agreement between the manually collected and sensor-collected 30CST data (M = 0.5, SD = 1.58, 95% CI = 1.13). Additional data processing will be able to yield measurements of fatigue and balance and evaluate the mechanisms of failed standing attempts. [ABSTRACT FROM AUTHOR]

Background/Purposes: Early diagnosis of dengue virus (DENV) infection to monitor the potential progression to hemorrhagic fever can influence the timely management of dengue-associated severe illness. Nonstructural protein 1 (NS1) antigen detection in acute serum specimens has been widely accepted as an early diagnostic assay for dengue infection; however, lower sensitivity of the NS1 antigen-capture enzyme-linked immunosorbent assay (Ag-ELISA) in secondary dengue viral infection has been reported. Methods: In this study, we developed two forms of Ag-ELISA capable of detecting E-Ag containing virion and virus-like particles, and secreted NS1 (sNS1) antigens, respectively. The temporal kinetics of viral RNA, sNS1, and E-Ag were evaluated based on the in vitro infection experiment. Meanwhile, a panel of 62 DENV-2 infected patients' sera was tested. Results: The sensitivity was 3.042 ng/mL and 3.840 ng/mL for sNS1 and E, respectively. The temporal kinetics of the appearance of viral RNA, E, NS1, and infectious virus in virus-infected tissue culture media suggested that viral RNAs and NS1 antigens could be detected earlier than E-Ag and infectious virus. Furthermore, a panel of 62 sera from patients infected by DENV Serotype 2 was tested. Treating clinical specimens with the dissociation buffer increased the detectable level of E from 13% to 92% and NS1 antigens from 40% to 85%. Conclusion: Inclusion of a low-pH glycine buffer treatment step in the commercially available Ag-ELISA is crucial for clinical diagnosis and E-containing viral particles could be a valuable target for acute DENV diagnosis, similar to NS1 detection.

Circulation of Zika virus (ZIKV) was first identified in the Western hemisphere in late 2014. Primarily transmitted through mosquito bite, ZIKV can also be transmitted through sex and from mother to fetus, and maternal ZIKV infection has been associated with fetal malformations. We assessed immunodeficient AG129 mice for their capacity to shed ZIKV in semen and to infect female mice via sexual transmission. Infectious virus was detected in semen between 7 and 21 days post-inoculation, and ZIKV RNA was detected in semen through 58 days post-inoculation. During mating, 73% of infected males transmitted ZIKV to uninfected females, and 50% of females became infected, with evidence of fetal infection in resulting pregnancies. Semen from vasectomized mice contained significantly lower levels of infectious virus, though sexual transmission still occurred. This model provides a platform for studying the kinetics of ZIKV sexual transmission and prolonged RNA shedding also observed in human semen.

Genotype I (GI) virus has replaced genotype III (GIII) virus as the dominant Japanese encephalitis virus (JEV) in the epidemic area of Asia. The mechanism underlying the genotype replacement remains unclear. Therefore, we focused our current study on investigating the roles of mosquito vector and amplifying host(s) in JEV genotype replacement by comparing the replication ability of GI and GIII viruses. GI and GIII viruses had similar infection rates and replicated to similar viral titers after blood meal feedings in Culex tritaeniorhynchus. However, GI virus yielded a higher viral titer in amplifying host-derived cells, especially at an elevated temperature, and produced an earlier and higher viremia in experimentally inoculated pigs, ducklings, and young chickens. Subsequently we identified the amplification advantage of viral genetic determinants from GI viruses by utilizing chimeric and recombinant JEVs (rJEVs). Compared to the recombinant GIII virus (rGIII virus), we observed that both the recombinant GI virus and the chimeric rJEVs encoding GI virus-derived NS1-3 genes supported higher replication ability in amplifying hosts. The replication advantage of the chimeric rJEVs was lost after introduction of a single substitution from a GIII viral mutation (NS2B-L99V, NS3-S78A, or NS3-D177E). In addition, the gain-of-function assay further elucidated that rGIII virus encoding GI virus NS2B-V99L/NS3-A78S/E177E substitutions re-gained the enhanced replication ability. Thus, we conclude that the replication advantage of GI virus in pigs and poultry is the result of three critical NS2B/NS3 substitutions. This may lead to more efficient transmission of GI virus than GIII virus in the amplifying host-mosquito cycle.

Purifying selection during dengue viral infection has been suggested as the driving force of viral evolution and the higher complexity of the intra-host quasi-species is thought to offer an adaptive advantage for arboviruses as they cycle between arthropod and vertebrate hosts. However, very few studies have been performed to investigate the viral genetic changes within (intra-host) and between (inter-host) humans in a spatio-temporal scale. Viruses of different serotypes from various countries imported to Taiwan cause annual outbreaks. During 2001-2003, two consecutive outbreaks were caused by dengue virus serotype 2 (DENV-2) and resulted in a larger-scale epidemic with more severe dengue cases in the following year. Phylogenetic analyses showed that the viruses from both events were similar and related to the 2001 DENV-2 isolate from the Philippines. We comprehensively analyzed viral sequences from representative dengue patients and identified three consensus genetic variants, group Ia, Ib and II, with different spatio-temporal population dynamics. The phylodynamic analysis suggested group Ib variants, characterized by lower genetic diversity, transmission rate, and intra-host variant numbers, might play the role of maintenance variants. The residential locations among the patients infected by group Ib variants were in the outer rim of case clusters throughout the 2001-2003 period whereas group Ia and II variants were located in the centers of case clusters, suggesting that group Ib viruses might serve as "sheltered overwintering" variants in an undefined ecological niche. Further deep sequencing of the viral envelope (E) gene directly from individual patient serum samples confirmed the emergence of variants belonging to three quasi-species (group Ia, Ib, and II) and the ancestral role of the viral variants in the latter phase of the 2001 outbreak contributed to the later, larger-scale epidemic beginning in 2002. These findings enhanced our understanding of increasing epidemic severity over time in the same epidemic area. It also highlights the importance of combining phylodynamic and deep sequencing analysis as surveillance tools for detecting dynamic changes in viral variants, particularly searching for and monitoring any specific viral subpopulation. Such subpopulations might have selection advantages in both fitness and transmissibility leading to increased epidemic severity.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein VLP derived from DENV serotype-2 were engineered becoming highly matured (mD2VLP) and showed variable size distribution with diameter of ~31 nm forming the major population under cryo-electron microscopy examination. Furthermore, mD2VLP particles of 31 nm diameter possess a T = 1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Mice vaccinated with mD2VLP generated higher cross-reactive (CR) neutralization antibodies (NtAbs) and were fully protected against all 4 serotypes of DENV. Our results highlight the potential of ‘epitope-resurfaced’ mature-form D2VLPs in inducing quaternary structure-recognizing broad CR NtAbs to guide future dengue vaccine design.

Objective To differentiate exposure to the newly introduced chikungunya virus from exposure to endemic dengue virus and other pathogens in Haiti. Methods We used a multiplex bead assay to detect [...]

In mid-2015, Salvador, Brazil, reported an outbreak of Guillain-Barré syndrome (GBS), coinciding with the introduction and spread of Zika virus (ZIKV). We found that GBS incidence during April-July 2015 among those ≥12 years of age was 5.6 cases/100,000 population/year and increased markedly with increasing age to 14.7 among those ≥60 years of age. We conducted interviews with 41 case-patients and 85 neighborhood controls and found no differences in demographics or exposures prior to GBS-symptom onset. A higher proportion of case-patients (83%) compared to controls (21%) reported an antecedent illness (OR 18.1, CI 6.9-47.5), most commonly characterized by rash, headache, fever, and myalgias, within a median of 8 days prior to GBS onset. Our investigation confirmed an outbreak of GBS, particularly in older adults, that was strongly associated with Zika-like illness and geo-temporally associated with ZIKV transmission, suggesting that ZIKV may result in severe neurologic complications.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) acts through tumor vascular disruption and cytokine production and is the first of its class to enter phase 3 trials. We characterized leukocytes and cytokines in murine Colon 38 tumors before and after DMXAA treatment. Tumor mass declined 50% 24 hours after DMXAA administration, but the leukocyte count per gram of tumor increased threefold owing to a large influx of Ly6G+CD11b+F4/80−. cells with the morphology of neutrophils. However, B and T lymphocytes, natural killer cells, and macrophages in the tumor all decreased in numbers. Seven chemokines were substantially induced in the tumor, spleen, and serum 4 hours after DMXAA administration. Using cultured spleen cell subpopulations, CD11b+ cells (largely monocytes and macrophages) were shown to be the primary producers of tumor necrosis factor á, interleukin 6 (IL-6), and macrophage inflammatory 1α (MIP-1α). CD49b+ natural killer cells produced IP-10, whereas CD45R+ B lymphocytes produced regulated upon activation normal T cell express sequence. T lymphocytes were not major producers of cytokines in the response to DMXAA. Murine peripheral blood leukocytes (PBLs) produced a similar panel of cytokines in culture to that detected in mouse serum after DMXAA treatment. Cytokines in human PBL cultures were subsequently measured with the aim of identifying potential serum markers of the human response to DMXAA. IP-10 (P < .001), monocyte chemoattractant protein 1 (P < .001), and sCD40L (P < .01) were decreased, whereas IL-8 (P < .001) and MIP-1α (P = .03) were increased in DMXAA-treated compared with untreated PBL cultures from a group of 12 donors.

Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H2O2, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H2O2, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines.

The four serotypes of dengue virus (DENV1-4) pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE). Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs) against DENV4. Using plaque reduction neutralization test (PRNT), we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP) mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine.

Domestic dogs and cats were infected by mosquito bite and evaluated as hosts for West Nile virus (WNV). Viremia of low magnitude and short duration developed in four dogs but they did not display signs of disease. Four cats became viremic, with peak titers ranging from 103.0 to 104.0 PFU/mL. Three of the cats showed mild, non-neurologic signs of disease. WNV was not isolated from saliva of either dogs or cats during the period of viremia. An additional group of four cats were exposed to WNV orally, through ingestion of infected mice. Two cats consumed an infected mouse on three consecutive days, and two cats ate a single infected mouse. Viremia developed in all of these cats with a magnitude and duration similar to that seen in cats infected by mosquito bite, but none of the four showed clinical signs. These results suggest that dogs and cats are readily infected by WNV. The high efficiency of oral transmission observed with cats suggests that infected prey animals may serve as an important source of infection to carnivores. Neither species is likely to function as an epidemiologically important amplifying host, although the peak viremia observed in cats may be high enough to infect mosquitoes at low efficiency.

The lymphatic sinuses in human lymph nodes (LNs) are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs). A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs); in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs) in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209.

Nucleotide sequence analysis was performed on 14 dengue virus isolates (13 dengue-2 viruses and 1 dengue-3 virus) recovered from febrile soldiers in Somalia in 1993. The dengue-2 viruses were most closely related to dengue-2 virus recovered in Somalia in 1984. However, differences in nucleotide sequence (0.35% to 1.35%) were evident among the 1993 isolates. These differences were closely associated with the geographic location of the infection as well as with different times of infection at the same location. Genetic difference between strains was not associated with differences in clinical features. Molecular analysis of dengue viruses is a useful adjunct to epidemiologic investigation of their distribution over distance and time.

West Nile virus (WNV) poses a serious threat to public health, especially to the elderly and the immuno-compromised. Neither vaccines nor treatments ate available for humans. Active hexose correlated compound (AHCC) is ah extract of Lentinula edodes of the Basidiomycetefamily of fungi rich in [alpha]-glucans. In this study, we evaluated the effect of AHCC on host susceptibility in the murine model of WNV infection. Mice orally administered with AHCC (600 mg/kg) every other day for 1 wk before and at d 1 and 3 postinfection were assessed using viremia levels, survival rate, and protective immunity. AHCC administration in young (6- to 8-wk-old) mice attenuated viremia and mortality following lethal WNV infection. WNV-specific IgM and IgG production and [gamma][delta] T cell expansion were also enhanced in these mice. Aged (21- to 22-mo-old) mice were more susceptible to WNV infection than young mice, partially due to the dysfunction of [gamma][delta] T cell subsets. AHCC administration in aged mice enhanced the protective V[gamma][1.sup.+] T cell response as well as WNV-specific IgG but not IgM antibodies production. AHCC administration in aged mice attenuated viremia levels but led to no difference in mortality rate. Overall, our data suggests that AHCC enhances protective host immune responses against WNV infection in young and aged mice. Dietary supplementation with AHCC may be potentially immunotherapeutic for WNV-susceptible populations.

Objective There is a need for a systematic method to implement the World Health Organization's Clinical Progression Scale (WHO-CPS), an ordinal clinical severity score for coronavirus disease 2019 patients, to electronic health record (EHR) data. We discuss our process of developing guiding principles mapping EHR data to WHO-CPS scores across multiple institutions. Materials and Methods Using WHO-CPS as a guideline, we developed the technical blueprint to map EHR data to ordinal clinical severity scores. We applied our approach to data from 2 medical centers. Results Our method was able to classify clinical severity for 100% of patient days for 2756 patient encounters across 2 institutions. Discussion Implementing new clinical scales can be challenging; strong understanding of health system data architecture was integral to meet the clinical intentions of the WHO-CPS. Conclusion We describe a detailed blueprint for how to apply the WHO-CPS scale to patient data from the EHR. [ABSTRACT FROM AUTHOR]

Dengue viruses (DENV) are the most important mosquito transmitted viral pathogens infecting humans. DENV infection produces a spectrum of disease, most commonly causing a self-limiting flu-like illness known as dengue fever; yet with increased frequency, manifesting as life-threatening dengue hemorrhagic fever (DHF). Waning cross-protective immunity from any of the four dengue serotypes may enhance subsequent infection with another heterologous serotype to increase the probability of DHF. Decades of effort to develop dengue vaccines are reaching the finishing line with multiple candidates in clinical trials. Nevertheless, concerns remain that imbalanced immunity, due to the prolonged prime-boost schedules currently used in clinical trials, could leave some vaccinees temporarily unprotected or with increased susceptibility to enhanced disease. Here we develop a DENV serotype 1 (DENV-1) DNA vaccine with the immunodominant cross-reactive B cell epitopes associated with immune enhancement removed. We compare wild-type (WT) with this cross-reactivity reduced (CRR) vaccine and demonstrate that both vaccines are equally protective against lethal homologous DENV-1 challenge. Under conditions mimicking natural exposure prior to acquiring protective immunity, WT vaccinated mice enhanced a normally sub-lethal heterologous DENV-2 infection resulting in DHF-like disease and 95% mortality in AG129 mice. However, CRR vaccinated mice exhibited redirected serotype-specific and protective immunity, and significantly reduced morbidity and mortality not differing from naïve mice. Thus, we demonstrate in an in vivo DENV disease model, that non-protective vaccine-induced immunity can prime vaccinees for enhanced DHF-like disease and that CRR DNA immunization significantly reduces this potential vaccine safety concern. The sculpting of immune memory by the modified vaccine and resulting redirection of humoral immunity provide insight into DENV vaccine induced immune responses.

BACKGROUND: Genotype I (GI) Japanese encephalitis virus (JEV) that replaced GIII virus has become the dominant circulating virus in Asia. Currently, all registered live and inactivated JEV vaccines are derived from genotype III viruses. In Taiwan, the compulsory JEV vaccination policy recommends that children receives four doses of formalin-inactivated Nakayama (GIII) JEV vaccine. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the influence of genotype replacement on the post-vaccination viral neutralizing ability by GIII and GI viruses, the small panel of vaccinated-children serum specimens was assembled, and the reciprocal 50% plaque-reduction neutralizing antibody titers (PRNT(50)) were measured against Nakayama vaccine strain, CJN GIII human brain isolate and TC2009-1 GI mosquito isolate. The seropositivity rate (PRNT(50) ≥ 1:10) and geometric mean titers (GMT) against the TC2009-1 virus were the lowest among the three viruses. The protective threshold against the CJN and TC2009-1 viruses could only be achieved when the GMT against Nakayama virus was ≥ 1:20 or ≥ 1:80, respectively. Using undiluted vaccinees' sera, the enhancement of JEV infection in K562 cells was observed in some low or non-neutralizing serum specimens. CONCLUSIONS/SIGNIFICANCE: Our preliminary study has shown that neutralizing antibodies, elicited by the mouse brain-derived and formalin-inactivated JEV Nakayama vaccine among a limited number of vaccinees, have reduced neutralizing capacity against circulating GI virus, but more detailed studies are needed to address the potential impact on the future vaccine policy.

BACKGROUND: Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control. METHODOLOGY/PRINCIPAL FINDINGS: We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials. CONCLUSIONS/SIGNIFICANCE: We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.