Associations of effort-reward imbalance at work and quality of life among workers after stroke: a one-year longitudinal study in Thailand.

Stroke incidence is increasing among working-age population, but the role of psychosocial stress in the workplace in predicting quality of life (QoL) after stroke onset is understudied. This longitudinal study aimed to investigate the relationship between work stress, measured by the effort-reward imbalance (ERI) model, and QoL over one-year period among 103 Thai workers who had experienced a stroke. The study evaluated the effort (E)-reward (R) ratio and over-commitment, the extrinsic and intrinsic components of the ERI model, before discharge; QoL was repeatedly measured at baseline, six months, and 12 months after discharge, respectively, using the Short Form Version 2 (SF-12v2) indicators of physical and mental health composite scores. Generalized estimating equations were used to examine longitudinal relationships between work stress at baseline and QoL over one year by testing the hypotheses that E-R ratio and over-commitment would have direct effects on QoL, and potential moderating effects of over-commitment on E-R ratio and QoL. The results supported the ERI model partially, as over-commitment was significantly associated with poor mental health (coefficient - 8.50; 95% CI: -13.79, -3.20) after adjusting baseline sociodemographic, behavioral, and clinical characteristics, while the E-R ratio was not significantly associated with physical or mental health; the interaction between the E-R ratio and over-commitment was also not significant. These findings suggest that more attention should be paid to workers' personal coping skills and ability to handle work-related problems and prioritize interventions that address over-commitment to promote long-term mental health among workers with stroke.

Genotypic and phenotypic situation of antimicrobial drug resistance of Escherichia coli in water and manure between biogas and non-biogas swine farms in central Thailand.

Currently, Thai livestock is rapidly expanding, especially the production of ruminants, chicken, and swine. The improper use of antibiotics will probably lead to an antimicrobial resistance problem. It has long been suspected that wastewater released from swine farms is a crucial aspect of the spread of antimicrobial resistance to the environment. Biogas systems are wastewater treatment systems commonly used on swine farms; however, little is known about the roles they play in the occurrence and transmission of resistant bacteria between biogas and non-biogas systems. This study collected pooled water, wastewater, and feces samples from five biogas farms and three non-biogas farms in Central Thailand. The samples were isolated to hemolytic E. coli (HEC) and non-hemolytic E. coli (NHEC) to test the drug resistance by using VITEK® 2 Compact (BioMérieux, USA) and detect resistant genes by using the polymerase chain reaction (PCR) technique to correlate the determined phenotypic and genotypic patterns. The results demonstrated that enumeration levels of E. coli ranged from 20.1 to 70.4 (MPN/100 ml), 105 to 107 (cfu/ml), and 105 to 109 (cfu/g), while they were 0-148.7 (MPN/100 ml), 105 to 107 (cfu/ml) and 105 to 109 (cfu/g) for water, wastewater and manure from biogas and non-biogas swine farms, respectively. The amount of E. coli in the sow feces samples was higher than the samples of nursery piglets on biogas farms at a 0.05 significant level (p < 0.05). The antimicrobial resistance indicated the relevant resistance characteristics of E. coli: the highest antimicrobial resistance was for ampicillin (AMP), followed by amoxicillin (AMX), tetracyclines (TET), chloramphenicol (C), and piperacillin (PIP), respectively. Multidrug resistance (MDR) of E. coli was 15 drugs: AMP-AMX-AMC-PIP-CEX-CEV-CPD-XNL-GM-IMP-SXT-C-TE (11.9%) and AMP-AMX-AMC-PIP-CEX-CEV-CPD-XNL-GM-IMP-SXT-C-ENR-MBR-TE (18.55%), which were the most commonly found in biogas and non-biogas swine farms, respectively. The blaTEM, tetA, sul2, and sul3 were dominantly resistant genes isolated from the water from both types of farm; while, blaTEM, aadA1, tetA, dfrA12, sul2, sul3, and cmlA were isolated from feces. The amount of E. coli in the final effluent from biogas swine farms was higher than the non-biogas swine farms; however, it was not significantly different at (p > 0.05). Furthermore, the findings of study found that genotypic characteristic of HEC showed similarity 100%. Thus, it was concluded that the levels of E. coli were accelerated in biogas wastewater treatment systems, and isolated E. coli demonstrated multidrug resistance. Even though E. coli was found in different locations, it showed relevant resistance characteristics. Therefore, regular monitoring of antimicrobial resistance on livestock farms is necessary for efficient management and drug uses on farms.

Human single-chain variable fragment antibody expressed in E. coli with optimal in vitro cross-neutralizing and no enhancing activity.

Single chain fragment variable (scFv) is a small molecule antibody comprising of only the variable region of heavy and light chain responsible for antigen binding. For dengue disease, the Fc region of antibody molecule was reported to be involved with dengue complication caused by Antibody-dependent enhancement (ADE). We attempted to produce small molecule scFv human monoclonal antibody (HuMAb), which lacking the Fc portion to eliminate the ADE effect of the IgG. This scFv antibody was produced in Escherichia coli. The biologically active form of scFv antibody was successfully generated. 23-1C2D2-scFv showed neutralizing activity similar to the IgG obtained from parental hybridoma, but lacked enhancing activity in all studied concentrations. This antibody was targeted to the 101WXN103 motif of dengue envelop protein domain II, studied by western blot analysis with truncated E protein and random peptide phage display. This scFv is verified as a candidate for further development as therapeutic candidate for DENV infection.

CONSTRUCTION AND EXPRESSION OF H5N1 INFLUENZA VIRUS HEMAGGLUTININ-SPECIFIC scFv-Fc MONOCLONAL ANTIBODIES IN HEK293T CELLS.

Monoclonal antibody (MAb) is a key element in the development of rapid test kits for many infectious diseases. Our group previously developed two antigen-binding fragment (Fab) MAbs, H5Fab-6 and H5Fab-9, specific to hemagglutinin (H5 HA) of influenza A virus H5N1, but these Fabs do not have a constant fragment (Fc) portion with which to bind with gold particles in a strip test. In order to overcome this impediment, we joined a single-chain variable fragment (scFv) with an Fc region to produce a scFv-Fc MAb, which was expressed in mammalian HEK293T cells. Specificity and sensitivity of each generated scFv-Fc MAb for H5 HA was tested using western blotting and dot-enzyme-linked immunosorbent assay (dot-ELISA), respectively. Two scFv-Fcs (designated H5scFvFc-6 and H5scFvFc-9) were constructed and purified to near homogeneity with a yield of 12.87 mg/l and 33.56 mg/l, respectively. Western blotting indicated that both scFv-Fcs reacted as expected with H5 HA with a sensitivity of 60 pg of H5 HA. These scFv-Fc MAbs should prove useful in the development of antibody-based diagnostic tools.

Molecular genetic characterization of rabies virus glycoprotein gene sequences from rabid dogs in Bangkok and neighboring provinces in Thailand, 2013-2014.

Because of its association with dogs, rabies virus (RABV) is still endemic in Thailand, where it is a serious public health problem. The genetic characterization of RABV in Thailand is limited. Therefore, in this study, we investigated the molecular epidemiology and genetic diversity of RABV in the endemic area. Viral RNA from 48 brain specimens from rabid dogs, collected in Bangkok and seven neighboring provinces in 2013-2014, was extracted and sequenced. The complete rabies glycoprotein (G) gene sequences (1575 nt) were aligned, and a phylogenetic analysis was performed using the maximum-likelihood method. All of the Thai rabies virus isolates belonged to lyssavirus genotype 1 and clustered in the same lineage as isolates from South East Asia (SEA) and China. The Thai rabies virus isolates formed two distinct clades, THA-1 and THA-2. Clade THA-1 was the predominant clade and could be divided into two subclades, THA-1A and THA-1B. Clade THA-2 was closely associated with human Thai isolates collected in a previous study. The overall mean rate of evolution based on the G gene was approximately 1.56 × 10(-4) substitutions/site/year. The genetic identities among the isolates from Thailand and other SEA countries were >88.4 % at the nucleotide sequence level and 95 % at the amino acid sequence level. The deduced amino acid sequences of the G proteins of the RABV isolates were compared. A single amino acid change (N194T) in subclade THA-1A distinguished the Thai RABV isolates from other RABV isolates. Our results suggest that these Thai dog RABV isolates share a common ancestor with the RABV isolates circulating in the endemic regions of SEA countries and China. Furthermore, there were strong genetic relationship to RABV from Cambodia, Vietnam and Laos. These data extend our understanding of the relatedness and genetic variation of RABV in Thailand.

Detection of chikungunya virus antigen by a novel rapid immunochromatographic test.

Chikungunya fever is a mosquito-borne disease of key public health importance in tropical and subtropical countries. Although severe joint pain is the most distinguishing feature of chikungunya fever, diagnosis remains difficult because the symptoms of chikungunya fever are shared by many pathogens, including dengue fever. The present study aimed to develop a new immunochromatographic diagnosis test for the detection of chikungunya virus antigen in serum. Mice were immunized with isolates from patients with Thai chikungunya fever, East/Central/South African genotype, to produce mouse monoclonal antibodies against chikungunya virus. Using these monoclonal antibodies, a new diagnostic test was developed and evaluated for the detection of chikungunya virus. The newly developed diagnostic test reacted with not only the East/Central/South African genotype but also with the Asian and West African genotypes of chikungunya virus. Testing of sera from patients suspected to have chikungunya fever in Thailand (n = 50), Laos (n = 54), Indonesia (n = 2), and Senegal (n = 6) revealed sensitivity, specificity, and real-time PCR (RT-PCR) agreement values of 89.4%, 94.4%, and 91.1%, respectively. In our study using serial samples, a new diagnostic test showed high agreement with the RT-PCR within the first 5 days after onset. A rapid diagnostic test was developed using mouse monoclonal antibodies that react with chikungunya virus envelope proteins. The diagnostic accuracy of our test is clinically acceptable for chikungunya fever in the acute phase.

Antibody germline characterization of cross-neutralizing human IgGs against 4 serotypes of dengue virus.

Dengue virus (DENV), a re-emerging virus, constitutes the largest vector-borne disease virus, with 50-100 million cases reported every year. Although DENV infection induces lifelong immunity against viruses of the same serotypes, the subsequent infection with the heterologous serotypes can cause more severe form of the disease, such as Dengue Haemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS). However, there is neither approved vaccine nor specific drugs available to treat this disease. In this study, previously developed 19 human monoclonal antibodies (HuMAbs) showing strong to moderate cross neutralizing activity were selected. Most of them (13/19) were targeted to domain II of envelop glycoprotein. To understand and clarify the recognition properties, the maturation mechanisms comprising Variable/Diversity/Joining (VDJ) recombination, Variable Heavy (VH)/Variable Light (VL) chain pairing, variability at junctional site, and somatic hypermutation (SHM) of those antibodies were studied and compared with their predecessor germline sequences. IMGT/V-QUEST database was applied to analyze the isolated VH and VL sequences. To confirm the correction of isolated VH/VL, 3 HuMAbs (1A10H7, 1B3B9, 1G7C2) was transiently expressed in HEK293T cell. All three clones of the expressed recombinant IgG (rIgG) showed the same binding and neutralizing activity as same as those from hybridomas. The data obtained in this study will elucidate the properties of those HuMAbs for further genetic modification, and its binding epitopes.

Chikungunya virus was isolated in Thailand, 2010.

Chikungunya fever (CHIKF) is an acute febrile illness caused by a mosquito-borne alphavirus, chikungunya virus (CHIKV). This disease re-emerged in Kenya in 2004, and spread to the countries in and around the Indian Ocean. The re-emerging epidemics rapidly spread to regions like India and Southeast Asia, and it was subsequently identified in Europe in 2007, probably as a result of importation of chikungunya cases. On the one hand, chikungunya is one of the neglected diseases and has only attracted strong attention during large outbreaks. In 2008-2009, there was a major outbreak of chikungunya fever in Thailand, resulting in the highest number of infections in any country in the region. However, no update of CHIKV circulating in Thailand has been published since 2009. In this study, we examined the viral growth kinetics and sequences of the structural genes derived from CHIKV clinical isolates obtained from the serum specimens of CHIKF-suspected patients in Central Thailand in 2010. We identified the CHIKV harboring two mutations E1-A226V and E2-I211T, indicating that the East, Central, and South African lineage of CHIKV was continuously circulating as an indigenous population in Thailand.

Screening for lead compounds and herbal extracts with potential anti-influenza viral activity.

Nonstructural protein 1 (NS1) of the highly pathogenic avian influenza virus (H5N1) contains a conserved RNA binding domain (RBD) that inhibits antiviral functions of host-innate immune response. Dimerization of NS1 forms a central groove and binds to double stranded (ds) RNA. This region might serve as a potential drug target. In this study, three dimensional structure model of NS1 RBD protein was constructed and virtual screening was performed to identify lead compounds that bound within and around the central groove. The virtual screening showed that 5 compounds bound within the central groove with binding energy ranging between -16.05 and -17.36 Kcal/mol. Two commercially available compounds, estradiol and veratridine, were selected for using in an in vitro screening assay. The results showed that neither of the compounds could inhibit the association between dsRNA and NS1 RBD protein. In addition, 34 herbal extracts were examined for their inhibitory effects. Five of them were able to inhibit association between NS1 RBD and dsRNA in electrophoresis mobility shift assay. Four herbs, Terminalia belirica, Salacia chinensis, Zingiber montanum and Peltophorum pterocarpum, could reduce > 50% of infectivity of H5N1 in a cell-based assay, and it is worth further studying their potential use as source of antiviral drugs.

Novel mutation detection IN rpoB OF rifampicin-resistant Mycobacterium tuberculosis using pyrosequencing.

Tuberculosis (TB) remains a major global public health problem particularly severe in parts of Asia and Africa, where often it is present in HIV-AIDS patients. Although rifampicin-resistant (RIFr) TB is slow to emerge due to the low rate of mutation of its target leading to RIFE being a marker of TB that is already resistant to other anti-TB drugs, and such cases are prone to treatment failure. More than 95% of rifampicin resistance is associated with mutations in Mycobacterium tuberculosis (MTB) rpoB, with 97% of mutations occurring within the 81 bp rifampicin-resistant determining region (RRDR) of this gene. In this study, we employed pyrosequencing technique to identify mutations in RRDR and 5 codons beyond of 39 MTB strains, comprising of 14 multi-drug resistance TB (MDRTB) and 3 RIF susceptible (RIFs) MTB from the Center of Disease Control (CDC), Ratchaburi Province, and 19 mono RIFr MTB, 1 MDRTB and 2 poly-drug resistant MTB from the Chest Institute, Ministry of Public Health, Thailand. Mu- tations in 8/22 samples from the Chest Institute and 13/14 from CDC were able to be identified. Six point mutations were detected, with Ser531Leu mutation accounting for 13, the silent mutation at Gly536 for 4, deletion of Gly523 for 2, combination of His526Cys and novel Leu533Arg for 1, and a novel Leu538Arg for 1. Mutation analysis of the 81 bp fragment and 5 codons beyond in MTB rpoB using pyrosequencing provides a useful approach in predicting RIFr phenotype allowing early diagnosis and appropriate drug therapy.

Characterization of Vibrio parahaemolyticus isolated from clinical specimens and oysters in Thailand.

INTRODUCTION: Vibrio parahaemolyticus is a common pathogen that can cause seafood-borne gastroenteritis in humans. We determined the prevalence and characteristics of V. parahaemolyticus isolated from clinical specimens and oysters in Thailand. METHODOLOGY: Isolates of V. parahaemolyticus from clinical specimens (n = 77) and oysters (n = 224) were identified by biochemical testing, polymerase chain reaction (PCR) assays, and serotyping. The toxin genes, antimicrobial resistance, and ß-lactamase production were determined. RESULTS: A total of 301 isolates were confirmed as V. parahaemolyticus by PCR using specific primers for the toxR gene. The majority of clinical isolates carried the tdh+/trh- genotype (82.1%), and one of each isolate was tdh-/trh+ and tdh+/trh+ genotypes. One isolate from oyster contained the tdh gene and another had the trh gene. Twenty-six serotypes were characterized among these isolates, and O3:K6 was the most common (37.7%), followed by OUT:KUT, and O4:K9. In 2010, most clinical and oyster isolates were susceptible to antibiotics, with the exception of ampicillin. In 2012, clinical isolates were not susceptible to cephalothin (52.4%), streptomycin (95.2%), amikacin (66.6%), kanamycin (61.9%), and erythromycin (95.2%), significantly more frequently than in 2010. More than 95% of isolates that were not susceptible to ampicillin produced ß-lactamase enzymes. CONCLUSIONS: We found toxin genes in two oyster isolates, and the clinical isolates that were initially determined to be resistant to several antibiotics. Toxin genes and antimicrobial susceptibility profiles of V. parahaemolyticus from seafood and environment should be continually monitored to determine the spread of toxin and antimicrobial resistance genes.

Nicotiana benthamiana as a potential source for producing anti-dengue virus D54 neutralizing therapeutic antibody.

Dengue virus (DENV), transmitted by mosquitoes, is classified into four serotypes (DENV1-4) and typically causes mild, self-limiting symptoms upon initial infection. However, secondary infection can lead to severe symptoms due to antibody-dependent enhancement (ADE). To address this, anti-DENV antibodies are being developed with the goal of neutralizing infection without ADE activity. Previous attempts using a 54_hG1 antibody from CHO-K1 mammalian cells resulted in ADE induction, increasing viral infection. This study aimed to express the D54 monoclonal antibody in Nicotiana benthamiana. The plant-produced antibody had a similar neutralizing profile to the previous 54_hG1 antibody. Notably, the ADE activities of the plant-derived antibody were successfully eliminated, with no sign of viral induction. These findings suggest that N. benthamiana could be a source of therapeutic DENV antibodies. The method offers several advantages, including lower ADE, cost-effectiveness, simple facility requirements, scalability, and potential industrial-scale production in GMP facilities.

The anthelmintic effect of plumbagin on Schistosoma mansoni.

The anthelmintic effects of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) and praziquantel (PZQ) against adult Schistosoma mansoni in vitro were compared by estimating the relative motility (RM) values, survival indices (SI) and alterations of the tegument of flukes incubated in M-199 medium containing 1, 10 and 100 µg/ml of the drugs, at 1, 3, 6, 12 and 24 h. For the parasites incubated in 10 µg/ml, the RM values of the PB-treated group decreased at a more rapid rate than the PZQ-treated group. When incubated in 100 µg/ml all PB-treated flukes appeared immobile from 1 to 24 h when 91-100% died, while in the PZQ-treated group RM values were higher than that of PB and most flukes were still alive at 1-12 h, and at 24 h only 21% of flukes were killed. Furthermore, male parasites were more affected by PZQ than females as their RM values were significantly less than that of females at all doses. While in PB treatment, males and females showed less difference in response to the drug as their RM values were closer than those treated with PZQ. When observed by SEM, the tegument of untreated S. mansoni displayed no alteration, while in PB treated parasites it exhibited swelling, blebbing, loss of spines, disruption of tubercles and ridges, leading to erosion and lesion, exposure of the basal lamina, and sloughing of the tegument. PZQ induced similar tegumental changes as those observed in PB treatment but at longer incubation time and higher doses. These data indicated that PB had more anthelmintic effect on both sexes of adult S. mansoni than PZQ.

Variables influencing anti-human immunodeficiency virus type 1 neutralizing human monoclonal antibody (NhMAb) production among infected Thais.

We conducted this study to determine the clinical variables associated with the production of human immunodeficiency virus type 1 (HIV-1) circulating recombinant form (CRF) 01_AE neutralizing human monoclonal antibodies (NhMAbs) using a hybridoma technique. This cross sectional study was performed in 20 asymptomatic HIV-1-infected Thais. Peripheral blood mononuclear cells (PBMCs) were obtained from each study participant and fused with SPYMEG cells. Culture supernatant collected from growing hybridomas was tested for neutralizing activity against HIV-1 CRF01_AE Env-recombinant viruses. Fifty hybridomas expressing anti-HIV-1 NhMAbs with strong neutralizing activity against at least 1 CRF01_AE Env-recombinant virus were found. A positive association between the numbers of hybridomas produced and the CD4 counts of study participants (p = 0.019) was observed. NhMAb-producing hybridomas with strong neutralizing activity were mostly found in participans diagnosed with HIV-1 infection within the previous 1 year. The HIV-1 viral load was not significantly correlated with the numbers of either established hybridomas or clones expressing anti-HIV-1 NhMAbs with strong neutralizing activity. To our knowledge, this is the first study of NhMAb-producing hybridomas obtained from HIV-1 CRF01_AE-infected populations identified by antibody binding to HIV-1 V3 loop peptide enzyme-linked immunosorbent assay (ELISA) or TRUGENE HIV-1 Genotyping Assay (HIV-1 pol sequence). It provides important criterion to slect study participants with high CD4 counts who produce large numbers of hybridoma clones. The results are valuable for further studies related to nurtalizing antibodies production and HIV-1 vaccine development.

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.

Human monoclonal antibodies to neutralize all dengue virus serotypes using lymphocytes from patients at acute phase of the secondary infection.

The global spread of the four dengue virus serotypes (DENV-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, we prepared hybridomas producing human monoclonal antibodies (HuMAbs) against DENV using peripheral blood mononuclear cells (PBMCs) from patients in the acute phase (around 1 week after the onset of illness) or the convalescent phase (around 2weeks after the onset of illness) of secondary infection. Interestingly, a larger number of hybridoma clones was obtained from patients in the acute phase than from those in the convalescent phase. Most HuMAbs from acute-phase infections were cross-reactive with all four DENV serotypes and showed significant neutralization activity to all four DENV serotypes. Thus, secondary DENV infection plays a significant role in stimulating memory cells to transiently increase the number of antibody-secreting plasma cells in patients in the early phase after the secondary infection. These HuMAbs will enable us to better understand the protective and pathogenic effects of DENV infection, which could vary greatly among secondarily-infected individuals.

Mimotope identification using phage displayed random peptide libraries against monoclonal antibodies specific to house dust mite.

Random heptapeptide T7 and random 12mer M13 phage libraries were employed to identify mimotopes binding to monoclonal antibodies (MAb) specific to house dust mite. After selection of bound phage by bio-panning and determination of binding specificity, DNA of selected bound phages was amplified, sequenced and aligned for peptide similarity. Eight mimotopes which were partially matched with Der f 15 allergen were predominant. The amino acid regions, 411-429 and 480-503 of Der f 15 allergen, appeared to be the main epitope clusters. Five mimotopes of MAb B2 and one mimotope of MAb B1 matched with Der p 1 and Der f 2 precursor, respectively.

Characterization of Human Anti-Dengue NS1 Monoclonal Antibodies Derived from Patients Infected with DENV-2.

Mouse antibodies specific to dengue NS1 have been widely investigated for their cross-reactivity with several human biomolecules. This is the first study demonstrating the cross-reactivity of human monoclonal antibodies (HuMAbs) specific to dengue NS1 isolated from patients infected with dengue virus serotype-2 (DENV-2). Nine anti-NS1 HuMAbs, which were mainly derived from patients in convalescent-phase after secondary infection of DENV-2, were characterized. Their cross-reactivity with plasminogen, thrombin, and endothelial cells was investigated, following which plasmin-formation assays were performed. All anti-NS1 HuMAbs exhibited cross-reactivity with human plasminogen (Plg), but not with thrombin or endothelial cells. Moreover, all HuMAbs exhibiting cross-reactivity with Plg converted Plg to plasmin in the plasmin-formation assay. These results suggest the implications and drawbacks of using anti-NS1 antibodies in immunotherapy.

Spread of a Novel Indian Ocean Lineage Carrying E1-K211E/E2-V264A of Chikungunya Virus East/Central/South African Genotype across the Indian Subcontinent, Southeast Asia, and Eastern Africa.

The Indian Ocean Lineage (IOL) of the chikungunya virus (CHIKV) East/Central/South African (ECSA) genotype, which originated in Kenya, spread to the Indian ocean and the Indian subcontinent, and then expanded through Southeast Asia in the previous decade. It carried an adaptive mutation E1-A226V, which enhances CHIKV replication in Aedes albopictus. However, the IOL CHIKV of the most recent outbreaks during 2016-2020 in India, Pakistan, Bangladesh, the Maldives, Myanmar, Thailand, and Kenya lacked E1-A226V but carried E1-K211E and E2-V264A. Recent CHIKV genome sequences of the Maldives and Thailand were determined, and their phylogenetic relationships were further investigated together with IOL sequences reported in 2004-2020 in the database. The results showed that the ancestral IOLs diverged to a sub-lineage E1-K211E/E2-V264A, probably in India around 2008, and caused sporadic outbreaks in India during 2010-2015 and in Kenya in 2016. The massive expansion of this new sub-lineage occurred after the acquisition of E1-I317V in other neighboring and remote regions in 2014-2020. Additionally, the phylogenetic tree indicated that independent clades formed according to the geographical regions and introduction timing. The present results using all available partial or full sequences of the recent CHIKVs emphasized the dynamics of the IOL sub-lineages in the Indian subcontinent, Southeast Asia, and Eastern Africa.

A replication competent luciferase-secreting DENV2 reporter for sero-epidemiological surveillance of neutralizing and enhancing antibodies.

Dengue virus (DENV) specific neutralizing and enhancing antibodies play crucial roles in dengue disease prevention and pathogenesis. DENV reporters are gaining popularity in the evaluation of these antibodies; their accessibility and acceptance may improve with more efficient production systems and indications of their antigenic equivalence to the wild-type virus. This study aimed to generate a replication competent luciferase-secreting DENV reporter (LucDENV2) and evaluate its feasibility in neutralizing and infection-enhancing antibody assays in comparison with wild-type DENV2, strain 16681, and a luciferase-secreting, single-round infectious DENV2 reporter (LucSIP). LucDENV2 replicated to similarly high levels as that of the parent 16681 virus in a commonly used mosquito cell line. LucDENV2 was neutralized in an antibody concentration-dependent manner by a monoclonal antibody specific to the flavivirus fusion loop and two antibodies specific to the E domain III, which closely resembled the neutralization patterns employing the LucSIP and wild-type DENV2. Parallel analysis of LucDENV2 and wild-type DENV2 revealed good agreement between the luciferase-based and focus-based neutralization and enhancement assays in a 96-well microplate format when employed against a set of clinical sera, suggesting comparable antigenic properties of LucDENV2 with those of the parent virus. The high-titer, replication competent, luciferase-secreting DENV reporter presented here should be a useful tool for fast and reliable quantitation of neutralizing and infection-enhancing antibodies in populations living in DENV-endemic areas.