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.

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.

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.

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.

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.

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.

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.

A Novel Plasmid DNA-Based Foot and Mouth Disease Virus Minigenome for Intracytoplasmic mRNA Production.

Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed.

A new species of Chlamydia isolated from Siamese crocodiles (Crocodylus siamensis).

Chlamydia is a known pathogen in both saltwater and freshwater crocodiles. However, the exact species/strain has not been clearly identified. In this study, we successfully cultivated Siamese crocodile Chlamydia in McCoy cells at a temperature of 30°C. Electron microscopy; phylogeny based on nine conserved taxonomically informative markers, on ompA, or on seven housekeeping genes; and whole-genome sequencing and analysis of the isolate confirmed the identity of the isolate as a new member of the genus Chlamydia, a new species that we name Chlamydia crocodili.

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.

Chimeric flavivirus enables evaluation of antibodies against dengue virus envelope protein in vitro and in vivo.

In a secondary dengue virus (DENV) infection, the presence of non-neutralizing antibodies (Abs), developed during a previous infection with a different DENV serotype, is thought to worsen clinical outcomes by enhancing viral production. This phenomenon is called antibody-dependent enhancement (ADE) of infection, and it has delayed the development of therapeutic Abs and vaccines against DENV, as they must be evaluated for the potential to induce ADE. Unfortunately, limited replication of DENV clinical isolates in vitro and in experimental animals hinders this evaluation process. We have, therefore, constructed a recombinant chimeric flavivirus (DV2ChimV), which carries premembrane (prM) and envelope (E) genes of type 2 DENV (DENV-2) R05-624 clinical (Thai) isolate in a backbone of Japanese encephalitis virus (Nakayama strain). DENV E-protein is the most important viral target, not only for neutralizing Abs, but also for infection-enhancing Abs. In contrast to DENV-2 R05-624, DV2ChimV replicated efficiently in cultured mammalian cells and was lethal in interferon-α/ß-γ-receptor double-knockout mice. With DV2ChimV, we were able to perform neutralization assays, in vitro and in vivo ADE assays, and in vivo protection assays. These results suggest that the chimeric virus is a powerful tool for evaluation of Abs against DENV.

Human Heavy Chain Antibody Genes Elicited in Thai Dengue Patients during DENV2 Secondary Infection.

Dengue is one of the most serious mosquito-borne viral diseases occurring in humans. To combat the complexity of 4 antigenically distinct serotypes, the ideal vaccine for dengue should be able to stimulate cross-neutralizing antibodies. Recently, genetics-based immune responses have been studied to guide vaccine design against several viral pathogens. Despite a recent approval of dengue vaccine, information on genetics-based immune responses against dengue virus (DENV) is still limited. Consequently, we aimed to determine the profiles of immunoglobulin heavy chain genes from DENV2 infected patients. The immunoglobulin heavy chain variable region genes (IGHV) were amplified from peripheral blood mononuclear cells of DENV2 secondary infected patients in the acute, convalescence, and recovery phases. Antibody heavy chain genes were sequenced using next-generation sequencing, and analyzed to identify correlations with neutralizing and enhancing activities of the serum samples. IGHV1-69, 3-23, and 3-30 were frequently discovered in our Thai DENV2 infected patients. Our findings provide new data on the human B cell response during secondary DENV2 infections in Thai dengue patients that offer supportive information for dengue vaccine design and therapeutics development.

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.

Oxidized Carbon Black: Preparation, Characterization and Application in Antibody Delivery across Cell Membrane.

Modulating biomolecular networks in cells with peptides and proteins has become a promising therapeutic strategy and effective biological tools. A simple and effective reagent that can bring functional proteins into cells can increase efficacy and allow more investigations. Here we show that the relatively non-toxic and non-immunogenic oxidized carbon black particles (OCBs) prepared from commercially available carbon black can deliver a 300 kDa protein directly into cells, without an involvement of a cellular endocytosis. Experiments with cell-sized liposomes indicate that OCBs directly interact with phospholipids and induce membrane leakages. Delivery of human monoclonal antibodies (HuMAbs, 150 kDa) with specific affinity towards dengue viruses (DENV) into DENV-infected Vero cells by OCBs results in HuMAbs distribution all over cells' interior and effective viral neutralization. An ability of OCBs to deliver big functional/therapeutic proteins into cells should open doors for more protein drug investigations and new levels of antibody therapies and biological studies.

Generation and characterization of cross neutralizing human monoclonal antibody against 4 serotypes of dengue virus without enhancing activity.

BACKGROUND: Dengue disease is a leading cause of illness and death in the tropics and subtropics. Most severe cases occur among patients secondarily infected with a different dengue virus (DENV) serotype compared with that from the first infection, resulting in antibody-dependent enhancement activity (ADE). Our previous study generated the neutralizing human monoclonal antibody, D23-1B3B9 (B3B9), targeting the first domain II of E protein, which showed strong neutralizing activity (NT) against all four DENV serotypes. However, at sub-neutralizing concentrations, it showed ADE activity in vitro. METHODS: In this study, we constructed a new expression plasmid using the existing IgG heavy chain plasmid as a template for Fc modification at position N297Q by site-directed mutagenesis. The resulting plasmid was then co-transfected with a light chain plasmid to produce full recombinant IgG (rIgG) in mammalian cells (N297Q-B3B9). This rIgG was characterized for neutralizing and enhancing activity by using different FcγR bearing cells. To produce sufficient quantities of B3B9 rIgG for further characterization, CHO-K1 cells stably secreting N297Q-B3B9 rIgG were then established. RESULTS: The generated N297Q-B3B9 rIgG which targets the conserved N-terminal fusion loop of DENV envelope protein showed the same cross-neutralizing activity to all four DENV serotypes as those of wild type rIgG. In both FcγRI- and RII-bearing THP-1 cells and FcγRII-bearing K562 cells, N297Q-B3B9 rIgG lacked ADE activity against all DENV serotypes at sub-neutralizing concentrations. Fortunately, the N297Q-B3B9 rIgG secreted from stable cells showed the same patterns of NT and ADE activities as those of the N297Q-B3B9 rIgG obtained from transient expression against DENV2. Thus, the CHO-K1 stably expressing N297Q-B3B9 HuMAb can be developed as high producer stable cells and used to produce sufficient amounts of antibody for further characterization as a promising dengue therapeutic candidate. DISCUSSION: Human monoclonal antibody, targeted to fusion loop of envelope domainII (EDII), was generated and showed cross-neutralizing activity to 4 serotypes of DENV, but did not cause any viral enhancement activity in vitro. This HuMAb could be further developed as therapeutic candidates.

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.

Migration, health, and socioenvironmental safety net among children of Dhaka, Bangladesh.

This study quantifies the diarrhea burden among migrant children under age 5 (who have migrated due to environmental degradation) in Dhaka. We used a multifactor socioepidemiological as well as environmental approach with pretested questionnaires and observations. It was found that 52% of the children were affected by diarrhea. Disability-adjusted life years (DALYs) lost was reduced manifold with the increase of mothers' behavioral determinants. Health losses were 1,718 fold with significant coefficient (ß) in the migrant group. DALYs lost were significantly associated with socioenvironmental factors such as mother's illiteracy (ß = .18; p < .001), no hand wash before eating (ß = .08; p = .004), and no hand wash after defecation (ß = .10; p < .001). This puts emphasis clearly on the awareness at household level, especially of mothers and children under age 5 in Dhaka, Bangladesh, in formulating migration-related policies.

Genomic studies of envelope gene sequences from mosquito and human samples from Bangkok, Thailand.

Dengue virus (DENV) is an RNA virus showing a high degree of genetic variation as a consequence of its proofreading inability. This variation plays an important role in virus evolution and pathogenesis. Although levels of within-host genetic variation are similar following equilibrium, variation among different hosts is frequently different. To identify dengue quasispecies present among two hosts, we collected patient samples from six acute DENV cases and two pools of Aedes aegypti mosquitoes and analyzed the genetic variation of regions of the viral envelope gene. Among human and mosquito samples, we found three major clusters originating from two subpopulations. Although several shared lineages were observed in the two hosts, only one lineage showing evidence of neutral selection was observed among two hosts. Taken together, our data provide evidence for the existence of a DENV quasispecies, with less genetic variation observed in mosquitoes than humans and with circulating lineages found in both host types.

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.