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Ten samples of tropical lichens collected from Doi Inthanon, Thailand, were explored for the diversity of their bacterial microbiomes through 16S rRNA-based metagenomics analysis. The five predominant lichen-associated bacteria belonged to the phyla Proteobacteria (31.84%), Planctomycetota (17.08%), Actinobacteriota (15.37%), Verrucomicrobiota (12.17%), and Acidobacteriota (7.87%). The diversity analysis metric showed that Heterodermia contained the highest bacterial species richness. Within the lichens, Ramalina conduplicans and Cladonia rappii showed a distinct bacterial community from the other lichen species. The community of lichen-associated actinobacteria was investigated as a potential source of synthesized biologically active compounds. From the total Operational Taxonomic Units (OTUs) found across the ten different lichen samples, 13.21% were identified as actinobacteria, including the rare actinobacterial genera that are not commonly found, such as Pseudonocardia, Kineosporia, Dactylosporangium, Amycolatopsis, Actinoplanes, and Streptosporangium. Evaluation of the pretreatment method (heat, air-drying, phenol, and flooding) and isolation media used for the culture-dependent actinobacterial isolation revealed that the different pretreatments combined with different isolation media were effective in obtaining several species of actinobacteria. However, metagenomics analyses revealed that there were still several strains, including rare actinobacterial species, that were not isolated. This research strongly suggests that lichens appear to be a promising source for obtaining actinobacteria. [ABSTRACT FROM AUTHOR]

One of the main challenges in the world is adaptation to the impacts of climate change, and it is important to understand which impacts will take place in each region. Thailand endures substantial damage from floods every year and needs accurate measurement to mitigate flood disaster predicted under the looming scenario of climate change. The predicted impacts of climate change on flood discharge need to consider land use changes in the future, especially in developing countries because of their vulnerabilities to economic growth. The objective of this study was the quantitative predictions of the impacts of climate change and land use changes on flood discharge, using two simulation models at one watershed in the Song Khwae District, Nan Province, Northern Thailand. This study has three steps: (1) predictions of future land uses (14 scenarios having different proportion of forest cover), (2) calculation of 3.3 and 10-year return period rainfall for the period of 2006-2016 and 2040- 2050, and (3) comparisons of average daily discharge from 3.3 and 10-year return period rainfall in 14 land use patterns. The results showed that although climate change will decrease the average daily discharge from the 3.3 and 10-year return period rainfall, discharge from future rainfall that has a 10-year return period, where land use patterns limit forest areas to less than 45%, will be greater than present levels. These quantitative predictions can lead to cost-benefits performance analysis and contribute to positive adaptation to climate change. [ABSTRACT FROM AUTHOR]

To understand the impact of inter-annual climate change on vegetation-atmosphere mass and energy exchanges, it has become necessary to explore changes in leaf-out onset in response to climatic fluctuations. We examined the response of leaf-out and transpiration onset dates to soil moisture in a teak plantation in northern Thailand based on a 12-year leaf area index and sap flow measurements. The date of leaf-out and transpiration onset varied between years by up to 40 days, and depended on the initial date when the relative extractable water in a soil layer of 0-0.6 m ( Θ) was greater than 0.2 being consistent with our previous results. Our new finding is that the delay in leaf-out and transpiration onset relative to the initial date when Θ > 0.2 increases linearly as the initial date on which Θ > 0.2 becomes earlier. The delay spans about 20 days in years when Θ > 0.2 occurs in March (the late dry season)-much earlier than usual because of heavy pre-monsoon rainfalls-while there is little delay in years when Θ > 0.2 occurs in May. This delay indicates the influence of additional factors on leaf-out onset, which controls the delay in the response of leaf-out to soil moisture increase. The results increased our knowledge about the pattern and extent of the changes in leaf phenology that occur in response to the inter-annual climate variation in tropical regions, where, in particular, such research is needed. [ABSTRACT FROM AUTHOR]

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of neosporosis, recognized as a major cause of bovine abortion around the world. Thailand is a developing agricultural country located in Southeast Asia. Livestock developments particularly in dairy cows of this country have been hampered by low productivity including milk and slow growth rate due to the impact of many pathogens including N. caninum. Currently, there is no effective method for control of neosporosis since there is less information regarding current status of infections. The objective of this study was to investigate the seroprevalence of neosporosis in dairy cows of the northern part of Thailand. During 2006-2007, the sera of 642 cows from 42 small farm holders with the top three highest consensus of dairy farms in the northern provinces, such as Chiang Rai, Chiang Mai and Lumpang were collected and performed tests. Antibodies to N. caninum were assayed by enzyme-linked immunosorbent assay (ELISA) with recombinant N. caninum surface antigen 1 (NcSAG1) and indirect fluorescent antibody test (IFAT). The overall prevalence of N. caninum infection in this study was 46.9% (301/642) by ELISA and 34.3% (220/642) by IFAT. [ABSTRACT FROM AUTHOR]

The article highlights an analysis and comparison of the genetic diversity of the MSA-1 gene among B. bovis isolates in the cattle populations in Thailand, Brazil and Ghana. Topics discussed include the collection of DNA samples, the various testing methods used for the analysis and comparison, and the results of B. bovis detected among Brazilian, Ghana, and Thai cattle populations. Also mentioned is the highly conserved MSA-1 sequences discovered in Thailand.

ABSTRACT The object of this study was to elucidate the seasonal patterns of energy partitioning of net radiation into sensible and latent heat fluxes and carbon dioxide (CO2) flux at a tropical deciduous forest site, i.e. a teak plantation in northern Thailand. In general, latent heat flux was more prominent than sensible heat flux in the wet (growing) season, whereas in the dry (dormant) season, sensible heat was the main form of energy emitted to the atmosphere. More specially, latent heat flux began to increase before leaf-out of teak trees and decreased 1 month earlier than leaf-fall initiation. Daytime net CO2 uptake appeared to increase with increasing amount of leaves but only at the beginning of the wet season; values peaked during the early part of wet season; and declined thereafter. This decline occurred about 3 months earlier than when latent heat flux began to decrease about 4 months earlier than the leaf-fall initiation. These results indicate that the seasonality of water and CO2 exchanges between the atmosphere and the tropical deciduous forest was substantially decoupled during the late wet season. This decoupling of the seasonality of water and carbon exchanges may be particularly important when modelling the ecohydrology of this tropical deciduous forest. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

ABSTRACT In a tropical deciduous forest in northern Thailand, an area affected by the Asian monsoon, we made soil respiration measurements for 4 years to determine their inter-annual variability. We also made continuous measurements of soil moisture, soil temperature, and soil CO2 concentration. Annual soil respiration rates were estimated using two methods: multiple regression analysis and Fick's first law of diffusion. During the study period, between June 2000 and December 2004, the annual rainfall and the length of the wet period showed considerable year-to-year variation, differing by 1162-1435 mm year−1 falling over 146-189 days, respectively. In response to the inter-annual variability in rainfall, annual soil respiration rates estimated by the two models also showed large inter-annual variations for the 4 years (coefficient of variation = 14-17%). The range in the inter-annual variation for soil respiration was comparable with values for net ecosystem exchange reported in other Southeast Asian tropical forests. Historical rainfall records at this site showed further large inter-annual variations that occurred in times other than in the study period, suggesting that long-term monitoring would be required for a better understanding of the C balance at this site. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Although Babesia bovis and Babesia bigemina infections cause economic losses in the cattle industry in northern Thailand, there is inadequate information on Babesia isolates present in the area. Therefore, to determine the prevalence and genetic relationship between Babesia isolates, we screened 200 blood samples of cattle from Chiang Rai, Chiang Mai, and Lumpang provinces of northern Thailand. A nested polymerase chain reaction using primers targeting B. bovis spherical body protein 2 (BboSBP2) and B. bigemina rhoptry-associated protein 1a (BbiRAP-1a) genes revealed a prevalence of 12 and 21 % for B. bovis and B. bigemina, respectively, while that of mixed infections was 6.5 % samples. The prevalences of B. bovis in Chiang Rai, Chiang Mai, and Lumpang were 9.5, 3.7, and 25.5 %, respectively. For B. bigemina, the prevalences were 15.8, 12.9, and 39.2 % in Chiang Rai, Chiang Mai, and Lumpang, respectively. Mixed infections with B. bovis and B. bigemina were 6.3 % in Chiang Rai, 1.9 % in Chiang Mai, and 13.7 % in Lumpang. The identical sequences of either BboSBP2 gene or BbiRAP-1a gene were shared among the Babesia isolates in the three provinces of northern Thailand. Further analysis using the internal transcribed spacer gene revealed at least four genotypes for B. bovis and five genotypes for B. bigemina in northern Thailand, while the sequences present great genetic diversities in the different isolates. Overall, we have demonstrated a high prevalence and polymorphism of Babesia parasites in northern Thailand calling for the need to design effective control programs for bovine babesiosis. [ABSTRACT FROM AUTHOR]

Here we report on an epidemiological study regarding the molecular prevalence of different genotypes of Theileria orientalis present among domestic cattle and water buffalo populations bred in Thailand. A phylogenetic analysis based on the parasitic gene encoding a major piroplasm surface protein revealed the presence of 5 genotypes (Types 1, 3, 5, 7, and N-3) in cattle and 7 genotypes (Types 1, 3, 4, 5, 7, N-2, and N-3) in water buffaloes. Types 4, 7, and N-3 of T. orientalis were reported for the first time in water buffaloes. The previously reported C and Thai types from Thailand clustered as types 7 and 6, respectively, in the present analysis. Great similarities were observed among nucleotide sequences of isolates of the same genotype from cattle and water buffaloes, and, therefore, water buffaloes were considered to serve as a reservoir for these genotypes of T. orientalis in Thailand. In conclusion, T. orientalis parasites circulating in Thailand are more diverse in their genetic characters than previously anticipated. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a zoonotic protozoan parasite of both medical and veterinary importance worldwide. The parasite can cause severe complications in immunocompromised individuals such as AIDS patients and transplant recipients, where up to 25% of patients will develop toxoplasmic encephalitis. Toxoplasmosis is a zoonosis that causes a public health concern in both developed and developing countries such as Thailand. Livestock development particularly in dairy cows of Thailand have been hampered by low production of milk and slow growth rate because of many pathogens including T. gondii. The objective of this study was to evaluate the serodiagnostic tool to be used for detection of T. gondii infection in dairy cows of Thailand. During 2006–2007, the sera of 700 cows from 55 small farm holders of the highest number of dairy cow population in the northern provinces were collected and analysed. Antibodies to T. gondii were assayed by latex agglutination test (LAT), enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody test (IFAT). The overall prevalence of T. gondii infection in dairy cows was 9.4% (66/700) by LAT and 17% (119/700) by ELISA. Sixty-three seropositive samples by LAT (95.5%) and 107 by ELISA (89.9%) were confirmed by IFAT. These results demonstrated that LAT had the highest specificity for detection of T. gondii infection. [ABSTRACT FROM AUTHOR]

This study aimed to develop a new casemix classification system as an alternative method for the budget allocation of oral healthcare service (OHCS). Initially, the International Statistical of Diseases and Related Health Problem, 10th revision, Thai Modification (ICD-10-TM) related to OHCS was used for developing the software "Grouper". This model was designed to allow the translation of dental procedures into eight-digit codes.Multiple regression analysis was used to analyze the relationship between the factors used for developing the model and the resource consumption. Furthermore, the coefficient of variance, reduction in variance, and relative weight (RW) were applied to test the validity. The results demonstrated that 1,624 OHCS classifications, according to the diagnoses and the procedures performed, showed high homogeneity within groups and heterogeneity between groups. Moreover, the RWof the OHCS could be used to predict and control the production costs. In conclusion, this new OHCS casemix classification has a potential use in a global decision making. [ABSTRACT FROM AUTHOR]

Babesia bovis , an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B . bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 ( msa - 1 ) gene using 162 B . bovis -positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa - 1 gene sequences isolated by PCR amplification were 43.5–100%, and the similarity values among the translated amino acid sequences were 42.8–100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa - 1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B . bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa - 1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9–68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0–39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B . bovis msa - 1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B . bovis in Thailand. [ABSTRACT FROM AUTHOR]

Abstract: Bovine babesiosis is an economically significant disease that affects dairy farming operations in Thailand. In the present study, 1824 blood-DNA samples prepared from cattle bred in 4 different regions of the country (North, Northeast, Central, and South) were screened using a nested PCR for the specific detection of Babesia bovis. While the overall prevalence of B. bovis was 8.8%, the Central region of Thailand was found to be a high-risk area of the country, as the prevalence of the parasite was 15.0%. The positive rate was relatively higher among the animals of 1–5 years of age. The genetic diversity among the B. bovis parasites was also studied based on their MSA-2b gene, and the findings showed that the Thai sequences were dispersed across 8 of 13 total clades observed in the phylogram. Three of these clades were formed only of Thai sequences. Similarity among the deduced MSA-2b amino acid sequences determined in the present study was 68.3–100%. In conclusion, the present study found that all the locations surveyed were infected with B. bovis and that the parasite populations in Thailand were genetically diverse. Our findings highlight the need for further studies in Thailand to generate more information before a sound control strategy could be implemented against B. bovis. [Copyright &y& Elsevier]

Abstract: Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and Babesia bigemina and is characterized by significant morbidity and mortality worldwide. The disease is widespread in the northeastern region of Thailand, where an increasingly large part of the livestock is composed of water buffaloes. The present study was therefore conducted to investigate the epidemiological distribution of B. bovis and B. bigemina in water buffaloes in the northeastern region of Thailand. A total of 305 buffalo blood samples were randomly collected from five provinces and simultaneously analyzed by the nested PCR (nPCR) assay, ELISA, and IFAT techniques. The overall prevalence of B. bovis and B. bigemina was 11.2% and 3.6% by nPCR, 14.7% and 5.9% by ELISA, and 16.8% and 5.6% by IFAT, respectively. The high concordance between the molecular and the serological detection tests revealed the specificity and sensitivity of the diagnostic assays used for the detection of infection as well as the endemic stability status of the parasites in the surveyed areas. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location but not gender. Our data provide valuable information regarding the epidemiology of B. bovis and B. bigemina infection in water buffaloes in the northeastern region of Thailand which will likely be very beneficial for management and control programs of this disease. [Copyright &y& Elsevier]

The diagnostic performance of a cocktail formula consisting of two Babesia (B.) bovis recombinant proteins, including spherical body protein 1 (BbSBP-1) and spherical body protein 4 (BbSBP-4), was evaluated in the present study for the global detection of B. bovis infection in cattle and for the differentiation between B. bovis and B. bigemina infections. The efficacy and the practicality of the rBbSBP-1 and rBbSBP-4 cocktail formula for differentiation between the infection caused by both parasites were assessed using indirect enzyme-linked immunosorbent assay (iELISA) with serum samples collected from cattle experimentally infected by B. bovis (n = 33) or B. bigemina (n = 30). Cocktail antigen exhibited the highest optical density (OD) values with B. bovis –infected sera and the lowest OD values with normal bovine sera or B. bigemina –infected sera in comparison with the single antigen. A total of 581 field serum samples collected from four countries with known B. bovis endemicity: Ghana (n = 154), Egypt (n = 162), Thailand (n = 96), and South Africa (n = 169) were screened also in the current study using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. A cocktail formula (rBbSBP-1 and rBbSBP-4) exhibited the highest concordance rate (89.90%) and kappa value (0.73). The obtained results revealed the reliability of the rBbSBP-1 and rBbSBP-4 cocktail antigen for the detection of specific antibodies to B. bovis in cattle and demonstrated the usefulness of cocktail antigen for differentiation between B. bovis and B. bigemina infections compared with the single antigen in cattle, which will be useful for epidemiological surveys and control of bovine babesiosis. Image 1 • rBbSBP-1 + rBbSBP-4 cocktail antigen efficacy for global detection of B. bovis infection was assessed in this study. • BbSBP-1 + BbSBP-4 cocktail antigen improved the differentiation between B. bovis and B. bigemina infections. • rBbSBP-1 and rBbSBP-4 cocktail antigen has a consistency for the detection of specific antibodies to B. bovis in cattle. [ABSTRACT FROM AUTHOR]

Twenty-two strains of dengue 2 virus, isolated in China, Latin America, New Guinea and Thailand were subjected to phylogenetic analysis. The UPGMA analysis was carried out on each gene region of dengue virus and demonstrated that outcome from most of the gene regions showed similar results except those from NS4B and YUTR with very short nucleotide length. Among ten regions examined, the results from E gene documented the geographical differences of the virus strains most clearly and all the American strains (Mara 4, IQT1797 and S1) were distantly related to the Asian isolates. As for the 16 Thai strains isolated in 1993, they were clustered into three groups and a strain from a DSS patient formed a distinct branch compared to the other two groups. This finding from phylogenetic analysis is consistent with earlier conclusion and support the severity related subtyping of dengue 2 virus based on amino acid changes.

Comparative nucleotide sequencing was carried out on dengue type 2 virus (DEN-2) strains isolated from patients in Northeast Thailand during the epidemic season in 1993. The patients exhibited different clinical manifestations ranging from dengue fever (DF) to dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS). The results classified 19 DEN-2 strains into 3 subtypes according to nonsynonymous amino acid replacements. The strain isolated from a DSS patient eliciting secondary serological response belonged to subtype I, whereas 13 strains isolated from DHF patients with secondary response and 2 strains from DF patients with primary response belonged to subtype II. On the other hand, 3 strains isolated from DF cases evoking either primary or secondary response belonged to subtype III. These results suggest that subtype III virus infection could result in clinically milder manifestation irrespective of the serological response compared with subtype I or II viruses. The RNA secondary structure predicted for the 3' noncoding region showed 4 different structures (A, B, C, and D). The result also indicates that different subtypes of DEN-2 serotypes are circulating in a single epidemic in Thailand.

To characterize the dengue epidemic that recently occurred in Malaysia, we sequenced cDNAs from nine 1993-1994 dengue virus type-3 (DEN-3) isolates in Malaysia (DEN-3 was the most common type in Malaysia during this period). Nucleic acid sequences (720 nucleotides in length) from the nine isolates, encompassing the precursor of membrane protein (preM) and membrane (M) protein genes and part of the envelope (E) protein gene were aligned with various reference DEN-3 sequences to generate a neighbor-joining phylogenetic tree. According to the constructed tree, the nine Malaysian isolates were grouped into subtype II, which comprises Thai isolates from 1962 to 1987. Five earlier DEN-3 virus Malaysian isolates from 1974 to 1981 belonged to subtype I. The present data indicate that the recent dengue epidemic in Malaysia was due to the introduction of DEN-3 viruses previously endemic to Thailand.

A virus isolate, ThCAr105/92, from a pool of mosquitos, Culex tritaeniorhynchus, collected in Chiang Mai, Thailand in 1992, appeared to be a member of the genus Flavivirus of the family Flaviviridae, based on the reverse transcription polymerase chain reaction (RT-PCR) using flavivirus cross-reacting primer pairs, electron microscopic examination, and serological tests. However, RT-PCR using Japanese encephalitis (JE) virus-specific primers showed that the isolate was different from JE virus. Sucrose density gradient sedimentation of the virus replicated in C6/36 cells indicated that the virus is relatively unstable in the infected culture fluids at 37 degrees C. Antibody prepared against this virus and a virus seed for the isolate were tested by cross neutralization against a panel of flaviviruses and the results showed that the new isolate was a distinct subtype of Tembusu virus.

Dengue virus infection has been counted among emerging and re-emerging diseases because of (1) the increasing number of patients, (2) the expansion of epidemic areas, and (3) the appearance of severe clinical manifestation of dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS), which is often fatal if not properly treated. In the meantime, there are no effective dengue control measures: a dengue vaccine is still under development and vector control does not provide a long-lasting effect. In order to obtain direct evidence for the virulent virus theory concerning the pathogenesis of DHF/DSS, type 2 dengue virus strains isolated from patients with different clinical severities in the same epidemic area in northeast Thailand, during the same season, were comparatively sequenced. The result revealed a DF strain specific amino acid substitution from I to R in the PrM, and a DSS strain specific amino acid substitution from D to G in the NS1 gene regions, which could significantly alter the nature of these proteins. Moreover, DF strain specific nucleotide substitutions in the 3' noncoding region were predicted to alter its secondary structure. These amino acid and nucleotide substitutions in other strains isolated in different epidemic areas during other seasons, together with their biological significance, remain to be confirmed. In order to innovate dengue vector control, field tests were carried out in dengue epidemic areas in Vietnam to examine the efficacy of Olyset Net screen, which is a wide-mesh net made of polyethylene thread impregnated with permethrin. The results show that Olyset Net (1) reduced the number of principal dengue vector species, Aedes aegypti, (2) interrupted the silent transmission of dengue viruses and (3) was highly appreciated by the local people as a convenient and comfortable vector control method. This encouraging evaluation of the Olyset Net screen should be confirmed further by other tests under different settings.

Hyperimmune antisera against four Japanese encephalitis (JE) virus strains, ThCMAr4492 and ThCMAr6793 from Thailand and Nakayama and JaGAr01 from Japan, were used to analyze the antigenic relationships among 12 Thai strains belonging to genotype 1, and two Japanese strains and one Chinese strain belonging to genotype 3. The antiserum for ThCMAr6793 significantly neutralized nine of the 12 Thai strains, none of which was significantly neutralized by antisera for the Nakayama and JaGAr01 strains. The antiserum for ThCMAr4492 neutralized only its homologous strain; therefore, ThCMAr4492 was antigenically different from all other strains. Two Thai strains (Subin and KE-093/83) were significantly less neutralized by all four of the antisera tested. In the deduced amino-acid sequence of the E protein, the 12 Thai strains revealed 100 to 98.2% identity among them and 90.0 to 98.8% identity with the published strains, respectively. Among significant amino-acid substitutions, three residues at positions E-222, E-327 and E-366 were shared by all of the Thai strains, whereas residues at E-89, E-123, E-131, E-178, E-293, E-351 and E-373 seemed to be strain-specific. The amino acids at positions E-178, E-327, E-351, E-373 and E-366 are found either in the peptides with functional T-helper cell epitopes or in the ectodomain of the E protein of other flaviviruses. These amino acids may therefore be responsible for determining the antigenic heterogeneity of these strains.

We have determined the 5' and 3' non-coding regions (NCR) of four dengue-2 viruses isolated from dengue patients with different clinical severities in Nakhon Phanom, Northeastern Thailand in 1993. The results were compared to prototype dengue-2 strains and were found to have highest homology with the New Guinea C strain. The sequence of the 5' NCR was completely conserved among all 4 isolates and were identical to the sequence of the New Guinea C strain. Homology of the 3' NCR sequence of the four isolates with the prototype strain ranged from 97.3 to 97.8%. Isolate ThNH-p11/93 from a mild dengue fever case showed the highest divergence from the prototype strain and the rest of the isolates from severe hemorrhagic cases, (1.11%). This includes a change in triad 297-299 nucleotides from the 3' terminus. Computer predicted secondary structures showed that isolate ThNHp-11/93 had significant structural differences from the other three isolates at this region.

Two strains of Japanese encephalitis (JE) virus were isolated from a pool of Culex tritaeniorhynchus captured in 1992 and another pool of Cx. vishnui captured in 1993, in Chiang Mai Area, Northern Thailand. These two strains, ThCMAr44/92 and ThCMAr67/93, could not be identified either as Nakayama or JaGAr01 subtype by the hemagglutination-inhibition (HI) and the neutralization (N) tests using immune sera raised against these standard JE virus strains. Reverse transcription-polymerase chain reaction showed the presence of JE-specific conserved sequences in these strains. Sequencing of 240 nucleotides in their PrM gene region identified that these two strains belong to the genotype 1 of JE virus. Nucleotide and encoded amino acid sequences of their envelope glycoprotein gene revealed 98.8 and 99.8% identity, respectively. These two strains shared 77.8 to 87.7% homology in the nucleotide sequence and 90.0 to 98.8% homology in the amino acid sequence with other reported JE strains. Five strain-specific amino acid changes were noted in ThCMAr44/92 strain, while one in ThCMAr67/93. In addition, four common amino acid changes were found in both strains. Thus, the findings indicated that these two strains were structurally different from each other as well as different from all the reported strains which was in agreement with the serological tests by hemagglutination-inhibition and neutralization.

We determined the 240-nucleotide sequences of the E/NS1 gene junction of four dengue-2 viruses by the primer extension dideoxy chain termination method. These viruses were isolated from dengue patients with different clinical severities in Nakhon Phanom, Northeastern Thailand in 1993. The results were compared with the 52 published dengue-2 sequences of the same gene region. Sequence divergence of four new isolates varied from 4.17% to 5.42% compared with dengue-2 prototype New Guinea C strain whereas it varied from 5.42% to 6.67% and from 6.67% to 7.09% when compared with Jamaica 1409 strain and PR159/S1 strain, respectively. All nucleotide substitutions were found at the 3rd position of the codons which were silent mutations. All 56 isolates studied were classified into five genotypic groups by constructing the dendrogram. The results indicated that four new isolates from Northeastern Thailand belong to genotype II of dengue virus serotype 2, and were most closely related to prototype New Guinea C strain. We also observed the variation in nucleotide and amino acid sequences among clusters of isolates (Thailand-1980, Malaysia-1989 and Thailand-1993) which were obtained from the dengue patients with different clinical severities. The significance of these genetic differences have been discussed in terms of the possible correlation between genetic variability and virulence.

The nucleotide (nt) sequence of the envelope glycoprotein (E) gene of dengue virus type 2 was determined by the primer-extension dideoxy chain-termination method for 3 dengue virus type 2 (D2) strains which had been isolated from patients with dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), in Maha Sarakham, Northeast Thailand, in 1986-1987. Their nt sequences were essentially the same except for a single silent nt replacement in each DHF and DSS strain compared with DF strain. Therefore, these 3 strains possessed identical deduced amino acid (AA) sequences in their E protein. The result indicated that the primary structure of the E protein of D2 virus is not related to the clinical severity of the infected patients. Eleven nt replacements which resulted in 4 amino acid replacements were found to be unique to these 3 Northeast Thai strains. Sequence similarity showed that the 3 Northeast Thai strains were closest to the DSS isolate (H) followed by the DHF isolate (D) identified in Bangkok in 1980.

Genetic variation in Japanese encephalitis (JE) virus isolates from Japan and Thailand was examined by oligonucleotide fingerprints of the 42S genome RNA. Japanese isolates were rather similar to each other, as were the recent isolates from Thailand. However, recent Thai isolates were significantly different from recent Japanese isolates. From these results it was presumed that mutations and selections of the JE virus genome would have progressed independently in these geographically distant areas.

As part of a virological and epidemiological survey of encephalitis in the Chiang Mai area, the neutralizing (N) antibody levels of healthy persons to Japanese encephalitis (JE) and dengue (DEN) type 1-4 viruses were examined. A total of 985 blood samples was collected by the filter paper method from subjects of nine age groups in five districts, four (Pasang, Sarapee, Doi Saket and Mae Taeng) in the Chiang Mai Valley and one (Fang) in another valley separated by several ranges of mountains from the Chiang Mai Valley. From analyses of the results of N tests on the specimens, the following conclusions were drawn about the prevalences of JE and DEN viruses in the Chiang Mai area: (1) In the Chiang Mai Valley, the percentage incidences of N antibodies to JE and DEN viruses increased with age and by the age of 15, two thirds or more of the residents had been infected with JE and all DEN viruses except DEN type 2 virus, which showed the lowest prevalence. (2) In the Fang district, the percentage incidence of N antibody to JE virus increased with age, but those to DEN viruses did not, indicating much lower prevalences in the past of all four serotypes of DEN viruses in this district than in the Chiang Mai Valley. (3) At present, most infants in the Chiang Mai area, including the Fang district, seem to be exposed to DEN viruses first and later to JE virus.

Immunoglobulin M (IgM) antibody titers in paired sera from 19 encephalitis and 44 dengue hemorrhagic fever (DHF) patients in Thailand and 42 Japanese encephalitis (JE) patients in Japan were measured by the antibody capture ELISA and applied to distinguish JE virus infection from dengue virus infection. Titer distribution and the ratio of the titers against JE and dengue antigens led to the following diagnostic criteria. The specimens can be considered as positive with JE when IgM-ELISA titer showed over 200 against JE and 4-fold or more higher than titers against any types of dengue antigens. The specimens can be considered as positive with dengue infection when IgM ELISA titer showed over 200 against one of the 4 types of dengue antigens and 4-fold or more higher than against JE antigen. Based on these criteria, 41 of 42 patients in Japan and 11 of 19 encephalitis patients in Thailand could be diagnosed as having JE virus infection while 2 of 19 encephalitis patients in Thailand and 26 of 44 DHF patients in Thailand could be diagnosed as having dengue virus infections.

Infection by JE virus still constitutes major cause of encephalitis in Chiang Mai Area, although some cases of possible dengue encephalopathy were observed. In spite of many apparent encephalitis cases, infection of vector mosquitoes by JE virus was not demonstrated. Virus isolation from hospitalized patients showed that the principal type of dengue virus circulating in Chiang Mai in 1982 was type 1 virus. Seroepidemiological survey on healthy humans indicated that the northern part of Chiang Mai Province in the region of the Maekong Valley has not yet been invaded so much by dengue viruses, compared with the Chiang Mai Valley, where dengue infection apparently became more prevalent than 12 years ago. The survey also indicated that the spread of JE virus in the study area was not uniform. Survey on vertebrates showed that anti-JE antibodies were highly prevalent among swine, horses, mules, sheep, and dogs. On the other hand, antibody prevalence was low in monkeys, ducks, and sparrows, and was negative among chickens and lizards. IgM-ELISA appeared to help differential diagnosis on JE from dengue even when the HI test did not give positive results.