Pathogen lineage-based genome-wide association study identified CD53 as susceptible locus in tuberculosis.

Tuberculosis (TB) is known to be affected by host genetic factors. We reported a specific genetic risk factor through a genome-wide association study (GWAS) that focused on young age onset TB. In this study, we further focused on the heterogeneity of Mycobacterium tuberculosis (M. tb) lineages and assessed its possible interaction with age at onset on host genetic factors. We identified the pathogen lineage in 686 Thai TB cases and GWAS stratified by both infected pathogen lineage information and age at onset revealed a genome-wide significant association of one single-nucleotide polymorphism (SNP) on chromosome 1p13, which was specifically associated with non-Beijing lineage-infected old age onset cases (P=2.54E-08, OR=1.74 (95% CI=1.43-2.12)), when we compared them to the population-matched healthy controls. This SNP locates near the CD53 gene, which encodes a leukocyte surface glycoprotein. Interestingly, the expression of CD53 was also correlated with the patients' active TB status. This is the first report of a pathogen lineage-based genome-wide association study. The results suggested that host genetic risk in TB is depended upon the pathogen genetic background and demonstrate the importance of analyzing the interaction between host and pathogen genomes in TB.

Association of BAK1 single nucleotide polymorphism with a risk for dengue hemorrhagic fever.

BACKGROUND: Dengue hemorrhagic fever (DHF) is a severe life-threatening form of dengue infection. Low platelet count is one of the characteristic clinical manifestations in patients with severe dengue. However, little is known about genetic factors in the host that cause low platelet count in patients with dengue. METHODS: A previous genome-wide association study of hematological and biochemical traits identified single nucleotide polymorphisms (SNPs) associated with low platelet count in healthy subjects. To examine the possible association of these SNPs with DHF, 918 Thai patients with dengue [509 patients with DHF and 409 with dengue fever (DF)] were genotyped for five SNPs: rs5745568 in BAK1, rs6141 in THPO, rs6065 in GP1BA, rs739496 in SH2B3, and rs385893 in RCL1. In addition, rs4804803 in CD209, that has been reported to be associated with dengue infection, was also genotyped to examine if rs4804803 affects the association detected in this study. RESULTS: The allele frequencies of each SNP were compared between the DHF and DF groups. Among the five SNPs, the G allele of rs5745568 in BAK1 was significantly associated with a risk for DHF [P = 0.006 and crude odd ratio (95 % confidence interval) = 1.32 (1.09-1.60)]. The association of this allele with DHF was also significant in a logistic regression analysis adjusted for age, sex, hospital (i.e., geographic region), immune status (i.e., primary or secondary infection), and virus serotype [P = 0.016 and adjusted odd ratio (95 % confidence interval) = 1.29 (1.05-1.58)]. The result was not influenced by rs4804803 [P = 0.0167 and adjusted OR (95 % CI) = 1.29 (1.05-1.58)]. No other SNPs including rs4804803 showed significant association. CONCLUSIONS: The low-level constitutive production of platelets caused by the G allele of rs5745568 seems to increase the risk of bleeding in dengue infection. Our results suggest that BCL-2 homologous antagonist/killer (BAK) protein, encoded by BAK1, plays a crucial role in the pathogenesis of DHF.

Comparison of genetic variation in drug ADME-related genes in Thais with Caucasian, African and Asian HapMap populations.

The objectives of this study are to investigate allele frequencies of drug absorption, distribution, metabolism and elimination (ADME)-related genes in the Thai population and to compare these genes to HapMap populations including Caucasians (CEU), Africans (YRI) and Asians (CHB/JPT). Genetic variations of drug ADME-related genes in 190 Thais were investigated using drug metabolizing enzymes and transporters (DMET) plus genotyping system. We examined 1936 single nucleotide polymorphisms (SNPs) of 225 genes that have documented functional and clinical significances in phase I and phase II drug metabolism enzymes, drug transporters and other genes involved in ADME processes. Distributions of genotyping data from Thai were compared with other HapMap populations including Caucasian, African and Asian populations. The analysis demonstrated 43 SNPs with statistical significance comparing among five populations. However, only 26 SNPs showed statistical significance in pair-wise comparisons between Thai versus CEU and Thai versus CHB/JPT. These 26 SNPs belong to 13 groups of drug ADME-related genes which are CYP2A6, CYP3A5, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, VKORC1, COMT, NAT2, TPMT, UGT1A1 and SLCO1B1. These genes demonstrated clinical significances as previously observed in many studies. The results could explain clinical variability in pharmacokinetics and pharmacodynamics of drugs in Thais based on genetic variations in drug ADME-related gene emphasized in this article.

Association of IL1B -31C/T and IL1RA variable number of an 86-bp tandem repeat with dengue shock syndrome in Thailand.

BACKGROUND: Dengue patients present a range of symptoms: dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). It is not clear whether this variability is due to their genetic background. Here we tested polymorphisms of interleukin 1 beta (IL1B) and interleukin 1 receptor antagonist (IL1RA) genes for association with DSS in the Thai population. METHODS: Polymorphisms of IL1B -31C/T (rs1143627) and IL1RA 86-base-pair tandem repeat were analyzed in 871 patients (DF = 384, DHF = 413, and DSS = 74). RESULTS: IL1B -31C and IL1RA 2/4 genotype were associated with DSS (IL1B -31C: DSS vs DHF: P = .0061, odds ratio [OR, 95% confidence interval {CI}], 3.49 [1.36-8.95]; DSS vs DF: P = .027, OR [95% CI], 2.81 [1.12-7.06]; IL1RA 2/4: DSS vs DHF: P = .017, OR [95% CI], 1.94 [1.12-3.40]; DSS vs DF: P = .024, OR [95% CI], 1.90 [1.07-3.4]). No difference was found between DF and DHF. Logistic regression analysis revealed that IL1B -31C and IL1RA 2/4 genotypes were each independently associated with DSS. CONCLUSIONS: Patients with IL1B -31C carrier, or IL1RA 2/4 genotype carry a risk for DSS, implying that IL1B may play a role in pathogenesis of DSS.

A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses.

Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.

A replication study confirms the association of GWAS-identified SNPs at MICB and PLCE1 in Thai patients with dengue shock syndrome.

BACKGROUND: Dengue shock syndrome (DSS), a severe life-threatening form of dengue infection, mostly occurs in children. A recent genome wide association study (GWAS) identified two SNPs, rs3132468 of major histocompatibility complex class I polypeptide-related sequence B (MICB) and rs3765524 of phospholipase C, epsilon 1 (PLCE1), associated with DSS in Vietnamese children. In this study, to examine whether an identical association is found in a different population, the association of these two SNPs with DSS was assessed in Thai children with dengue. METHODS: The rs3132468 and rs3765524 SNPs were genotyped in 917 Thai children with dengue: 76 patients with DSS and 841 patients with non-DSS. The allele frequencies were compared between DSS and non-DSS groups by one-sided Fisher's exact test. The association of rs3132468 and rs3765524 with the mRNA expression levels of MICB and PLCE1 were assessed in EBV-transformed lymphoblastoid cell lines. RESULTS: The reported DSS-risk alleles were significantly associated with DSS in Thai patients with dengue (one-sided P = 0.0213 and odds ratio [OR] = 1.58 for rs3132468-C and one-sided P = 0.0252 and OR = 1.49 for rs3765524-C). The rs3132468-C allele showed a significant association with lower mRNA level of MICB (P = 0.0267), whereas the rs3765524-C allele did not. These results imply that the MICB molecule may play an important role in the prevention of DSS in dengue infection. CONCLUSIONS: Together with previous association studies, we conclude that rs3132468-C at MICB and rs3765524-C at PLCE1 confer risk of DSS in Southeast Asians.

Incidence and predictors of regimen-modification from first-line antiretroviral therapy in Thailand: a cohort study.

BACKGROUND: Antiretroviral therapy markedly reduced mortality in HIV-infected individuals. However, in the previous studies, up to 50% of patients are compelled to modify their regimen in middle and low-income countries where salvage drug is still limited. This cohort study aimed to investigate the incidence and predictors of regimen modification from the first-line antiretroviral regimen in northern Thailand. METHODS: All HIV-infected patients starting antiretroviral therapy (ART) with generic drug (GPOvir®; stavudine, lamivudine and nevirapine) at a governmental hospital in northern Thailand from 2002 to 2007 were recruited. Baseline characteristics and detailed information of regimen modification until the end of 2010 were ascertained from cohort database and medical charts. As a potential genetic predictor of regimen modification, HLA B allele was determined by bead-based array hybridization (WAKFlow® HLA typing kit). We investigated predictors of the regimen modification using Cox's proportional hazard models. RESULTS: Of 979 patients, 914 were eligible for the analysis. The observed events of regimen modification was 377, corresponding to an incidence 13.8/100 person-year-observation (95% CI:12.5-15.3) over 2,728 person years (PY) follow up. The main reasons for regimen modification were adverse effects (73.5%), especially lipodystrophy (63.2%) followed by rash (17.7%). Sixty three patients (17.1%) changed the regimen due to treatment failure. 2% and 19% of patients had HLA-B*35:05 and B*4001, respectively. HLA-B*35:05 was independently associated with rash-related regimen modification (aHR 7.73, 95% CI:3.16-18.9) while female gender was associated with lipodystrophy (aHR 2.11, 95% CI:1.51-2.95). Female gender (aHR 0.54, 95% CI: 0.30-0.96), elder age (aHR 0.56, 95% CI: 0.32-0.99) and having HLA-B*40:01 (aHR 0.29, 95% CI: 0.10-0.82) were protective for treatment failure related modification. CONCLUSION: HLA-B*35:05 and female gender were strong predictors of regimen modification due to rash and lipodystrophy, respectively. Female gender, elder age, and having HLA-B*40:01 had protective effects on treatment failure-related regimen modification. This study provides further information of regimen modification for future tailored ART in Asia.

Lessons learned from influenza A(H1N1)pdm09 pandemic response in Thailand.

In 2009, Thailand experienced rapid spread of the pandemic influenza A(H1N1)pdm09 virus. The national response came under intense public scrutiny as the number of confirmed cases and associated deaths increased. Thus, during July-December 2009, the Ministry of Public Health and the World Health Organization jointly reviewed the response efforts. The review found that the actions taken were largely appropriate and proportionate to need. However, areas needing improvement were surveillance, laboratory capacity, hospital infection control and surge capacity, coordination and monitoring of guidelines for clinical management and nonpharmaceutical interventions, risk communications, and addressing vulnerabilities of non-Thai displaced and migrant populations. The experience in Thailand may be applicable to other countries and settings, and the lessons learned may help strengthen responses to other pandemics or comparable prolonged public health emergencies.

Integrating genome-based informatics to modernize global disease monitoring, information sharing, and response.

The rapid advancement of genome technologies holds great promise for improving the quality and speed of clinical and public health laboratory investigations and for decreasing their cost. The latest generation of genome DNA sequencers can provide highly detailed and robust information on disease-causing microbes, and in the near future these technologies will be suitable for routine use in national, regional, and global public health laboratories. With additional improvements in instrumentation, these next- or third-generation sequencers are likely to replace conventional culture-based and molecular typing methods to provide point-of-care clinical diagnosis and other essential information for quicker and better treatment of patients. Provided there is free-sharing of information by all clinical and public health laboratories, these genomic tools could spawn a global system of linked databases of pathogen genomes that would ensure more efficient detection, prevention, and control of endemic, emerging, and other infectious disease outbreaks worldwide.

Genome-wide association studies of tuberculosis in Asians identify distinct at-risk locus for young tuberculosis.

Tuberculosis (TB) is one of the most devastating chronic infectious diseases, but the role of host genetics in disease development after infection in this disease remains unidentified. Genome-wide association studies (GWASs) in Thais and Japanese were carried out and separately analyzed, attempted replication, then, combined by meta-analysis were not yielding any convincing association evidences; these results suggested that moderate to high effect-size genetic risks are not existed for TB per se. Because of failure in replication attempt of the top 50 single-nucleotide polymorphisms (SNPs) identified form meta-analysis data, we empirically split TB cases into young TB case/control data sets (GWAS-T(young)=137/295 and GWAS-J(young)=60/249) and old TB case/control data sets (GWAS-T(old)=300/295 and GWAS-J(old)=123/685), re-analyzed GWAS based on age-stratified data and replicated the significant findings in two independent replication samples (young TB; Rep-T(young)=155/249, Rep-J(young)=41/462 and old TB; Rep-T(old)=212/187, Rep-J(old)=71/619). GWAS and replication studies conducted in young TB identified at-risk locus in 20q12. Although the locus is located in inter-genic region, the nearest genes (HSPEP1-MAFB) from this locus are promising candidates for TB susceptibility. This locus was also associated with anti-TNF responsiveness, drug with increased susceptibility for TB. Moreover, eight SNPs in an old TB meta-analysis and six SNPs in young TB meta-analysis provided replication evidences but did not survive genome-wide significance.These findings suggest that host genetic risks for TB are affected by age at onset of TB, and this approach may accelerate the identification of the major host factors that affect TB in human populations.

Genetic characterization of measles viruses that circulated in Thailand from 1998 to 2008.

During the period between 1998 and 2008, 48 representative measles viruses (MeVs) circulating in Thailand were subjected to genetic characterization. Three genotypes, G2, D5, and D9 were detected. The results suggested that measles genotype D5, which has been circulating since at least 1998, is the endemic genotype in Thailand. Genotype G2 was detected between 1998 and 2001. In addition, almost all of the MeVs detected throughout the country in 2008 were genotype D9. This is the first report of genotype D9 in Thailand. This report provides important baseline data about measles genotypes in Thailand and this information will be needed to help verify measles elimination in Thailand.

Immune response in diarrheal patients and asymptomatic carrier with CS6-producing enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of diarrhea in children and travelers in developing countries. ETEC colonization factors (CFs) are virulence determinants considered as protective antigens and major targets for vaccine development against ETEC infections. One of the most prevalent CFs, coli surface antigen 6 (CS6), a non-fimbrial polymeric protein consisting of two major subunits, CssA and CssB, is produced by approximately 25-35% of ETEC worldwide. We could isolate only CS6-producing ETEC strains from two diarrheal patients and one asymptomatic carrier, but we could not detect CssA- or CssB-specific antibodies in the feces and blood of two patients convalescing from natural ETEC infection and of an asymptomatic carrier using western blotting. Therefore, in order to protect against infection with CS6-producing ETEC, protective levels of CS6 immunity should be incorporated in any future vaccines against ETEC.

Neutralization titers against influenza A (H3N2) and influenza B viruses among a non-vaccinated population from Thailand.

Influenza A and B viruses are viral respiratory pathogens that can cause severe infections among birds and mammals. Neutralization assays using human sera are useful to evaluate the risk of circulating viruses to humans. In this study, 359 serum samples from healthy Thai volunteers, who had not been vaccinated against influenza for at least five years, were investigated by microneutralization (MN) assays against influenza A H3N2 and influenza B viruses in 2009. There was no significant difference in neutralization activities against 2006 and 2008 isolates of influenza A H3N2 viruses. However, neutralization titers to influenza B viruses among 2008 isolates were quite low. The results indicate the non-vaccinated study population had some neutralizing antibodies against influenza A H3N2 but not against influenza B viruses.

The serotype-independent but concentration-dependent enhancing antibodies among Thai dengue patients.

Antibody-dependent enhancement of infection (ADE) is central to explaining the development of severe disease at the end of post-dengue virus infection. Non-neutralizing anti-dengue antibodies bound to the dengue virion enhances the virus entrance into the target cells via the Fc receptor. The titer of enhancing antibodies in dengue patients is not determined during dengue virus infection. Sensitive flow cytometry detecting dengue virus-infected K562 cells was used to quantitate enhancing activity among Thai DF and DHF patients against four serotypes and the patient's dengue isolate. The titer was defined as the reciprocal of the final dilution that loses enhancing activity. The serum of Thai patients confirmed to have dengue infection were found to have high titers of enhancing antibodies and increased gradually through the convalescent phase of infection. The enhancing antibody titers were not different among the four serotypes or from the infecting isolate. The anti-dengue antibodies from dengue patients can enhance dengue virus infections in a concentration-dependent, serotype-independent manner.

Evidence for subclinical avian influenza virus infections among rural Thai villagers.

BACKGROUND: Regions of Thailand reported sporadic outbreaks of A/H5N1 highly pathogenic avian influenza (HPAI) among poultry between 2004 and 2008. Kamphaeng Phet Province, in north-central Thailand had over 50 HPAI poultry outbreaks in 2004 alone, and 1 confirmed and 2 likely other human HPAI infections between 2004 and 2006. METHODS: In 2008, we enrolled a cohort of 800 rural Thai adults living in 8 sites within Kamphaeng Phet Province in a prospective study of zoonotic influenza transmission. We studied participants' sera with serologic assays against 16 avian, 2 swine, and 8 human influenza viruses. RESULTS: Among participants (mean age 49.6 years and 58% female) 65% reported lifetime poultry exposure of at least 30 consecutive minutes. Enrollees had elevated antibodies by microneutralization assay against 3 avian viruses: A/Hong Kong/1073/1999(H9N2), A/Thailand/676/2005(H5N1), and A/Thailand/384/2006(H5N1). Bivariate risk factor modeling demonstrated that male gender, lack of an indoor water source, and tobacco use were associated with elevated titers against avian H9N2 virus. Multivariate modeling suggested that increasing age, lack of an indoor water source, and chronic breathing problems were associated with infection with 1 or both HPAI H5N1 strains. Poultry exposure was not associated with positive serologic findings. CONCLUSIONS: These data suggest that people in rural central Thailand may have experienced subclinical avian influenza infections as a result of yet unidentified environmental exposures. Lack of an indoor water source may play a role in transmission.

Genotypic profile of Streptococcus suis serotype 2 and clinical features of infection in humans, Thailand.

To examine associations between clinical features of Streptococcus suis serotype 2 infections in humans in Thailand and genotypic profiles of isolates, we conducted a retrospective study during 2006-2008. Of 165 patients for whom bacterial cultures of blood, cerebrospinal fluid, or both were positive for S. suis serotype 2, the major multilocus sequence types (STs) found were ST1 (62.4%) and ST104 (25.5%); the latter is unique to Thailand. Clinical features were examined for 158 patients. Infections were sporadic; case-fatality rate for adults was 9.5%, primarily in northern Thailand. Disease incidence peaked during the rainy season. Disease was classified as meningitis (58.9%) or nonmeningitis (41.1%, and included sepsis [35.4%] and others [5.7%]). Although ST1 strains were significantly associated with the meningitis category (p<0.0001), ST104 strains were significantly associated with the nonmeningitis category (p<0.0001). The ST1 and ST104 strains are capable of causing sepsis, but only the ST1 strains commonly cause meningitis.

Two N-linked glycosylation sites in the V2 and C2 regions of human immunodeficiency virus type 1 CRF01_AE envelope glycoprotein gp120 regulate viral neutralization susceptibility to the human monoclonal antibody specific for the CD4 binding domain.

A recombinant human monoclonal antibody, IgG1 b12 (b12), recognizes a conformational epitope on human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) gp120 that overlaps the CD4 binding domain. Although b12 is able to broadly neutralize HIV-1 subtype B, C, and D viruses, many HIV-1 CRF01_AE viruses are resistant to b12-mediated neutralization. In this report, we examined the molecular mechanisms underlying the low neutralization susceptibility of CRF01_AE viruses to b12, using recently established CRF01_AE Env recombinant viruses. Our results showed that two potential N-linked glycosylation (PNLG) sites in the V2 and C2 regions of Env gp120 played an important role in regulating the susceptibility of CRF01_AE Env to b12. The locations of these PNLG sites correspond to amino acid positions 186 and 197 in HXB2 Env gp120; thus, they are designated N186 and N197 in this study. Removal of N186 significantly conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones, 65CC4 and 107CC2, while the introduction of N186 reduced the b12 susceptibility of a susceptible CRF01_AE Env clone, 65CC1. In addition, removal of both N186 and N197 conferred the b12 susceptibility of 3 resistant CRF01_AE Env clones, 45PB1, 62PL1, and 101PL1, whereas the removal of either N186 or N197 was not sufficient to confer the b12 susceptibility of these CRF01_AE Env clones. Finally, removal of N197 conferred the b12 susceptibility of 2 resistant CRF01_AE Env clones lacking N186, 55PL1 and 102CC2. Taken together, we propose that two PNLG sites, N186 and N197, in Env gp120 are important determinants of the b12 resistance of CRF01_AE viruses.

Insecticide resistance in bedbugs in Thailand and laboratory evaluation of insecticides for the control of Cimex hemipterus and Cimex lectularius (Hemiptera: Cimicidae).

Bedbugs are found in many countries around the world, and in some regions they are resistant to numerous insecticides. This study surveyed bedbugs in Thailand and determined their resistance to insecticides. The surveys were carried out in six provinces that attract large numbers of foreign tourists: Bangkok, Chonburi, Chiang Mai, Ubon Ratchathani, Phuket, and Krabi. Bedbugs were collected from hotels and colonized in the laboratory to evaluate their resistance to insecticides. Cimex hemipterus (F.) was found in some hotels in Bangkok, Chonburi, Phuket, and Krabi, whereas Cimex lectularius L. was found only in hotels in Chiang Mai. No bedbugs were found in Ubon Ratchathani. The colonized bedbugs showed resistance to groups of insecticides, including organochlorines (dichlorodiphenyl trichloroethane, dieldrin), carbamates (bendiocarb, propoxur), organophosphates (malathion, fenitrothion), and pyrethroids (cyfluthrin, deltamethrin, permethrin, lambda-cyhalothrin, etofenprox) in tests using World Health Organization insecticide-impregnated papers. The new insecticides imidacloprid (neonicotinoid group), chlorfenapyr (pyrrole group), and fipronil (phenylpyrazole group) were effective against the bedbugs; however, organophosphate (diazinon), carbamates (fenobucarb, propoxur), and pyrethroids (bifenthrin, cypermethrin, esfenvalerate, etofenprox) were ineffective. Aerosols containing various pyrethroid insecticides with two to four different active ingredients were effective against the bedbugs. The results obtained from this study suggested that both species of bedbugs in Thailand have developed marked resistance to various groups of insecticides, especially those in the pyrethroid group, which are the most common insecticides used for pest control. Therefore, an integrated pest management should be implemented for managing bedbugs in Thailand.

Temporal and spatial distribution, sex ratio and fecundity of the eye fly siphunculina funicola (Diptera: Chloropidae) at aggregation sites during diurnal and nocturnal periods.

The present study was aimed to determine the distribution and abundance of the eye fly Siphunculina funicola (de Meijere) in Thailand and to investigate the sex ratio and fecundity of eye flies from aggregation sites collected during the day-time and night-time. The flies were collected from several provinces in central Thailand and Phuket in the south. Observations were regarding the relative abundance of eye flies in different regions and seasons. During 2007 and 2008, large populations of eye flies were noted at resting sites in central Thailand with both day and night collections. Males flies outnumbered female flies. Smaller populations were seen in Chumphon and Surat Thani Provinces with increasing numbers in Krabi and Phuket Provinces in the south. The gravid rate was nil in the few females collected in Chomphon and Surat Thani but were 3.9% and 36.3% in Krabi and Phuket, respectively. The gravid rates were higher during the dry season or during dry spells than during wet and rainy periods, suggesting egg retention by the females when oviposition sites (presumably soil) were dry. Numerous day and night collections were made in Chon Buri Province. In most collections males predominated but there was no differences in the numbers of flies collected during the two time periods. There was a slightly greater percentage of females (still lower than males) during the night collections. During the dry and hot season, due to lack of optimum oviposition sites because of dryness, the eggs were retained in the females. A series of day time collections at the end of April 2008 and in February-March 2009 had higher numbers of gravid females. Day time collections in May 2008 (start of the rainy season) showed a moderate number of gravid females, but the gravid rates were low during the rainy season, indicating higher oviposition activity by females.

Characterization of H5N1 influenza viruses isolated from humans in vitro.

Since December 1997, highly pathogenic avian influenza A H5N1 viruses have swept through poultry populations across Asian countries and been transmitted into African and European countries. We characterized 6 avian influenza H5N1 viruses isolated from humans in 2004 in Thailand. A highly pathogenic (HP) KAN353 strain showed faster replication and higher virulence in embryonated eggs compared to other strains, especially compared to the low pathogenic (LP) SP83 strain. HP KAN353 also showed strong cytopathogenicity compared to SP83 in Madin-Darby canine kidney cells. Interestingly, LP SP83 induced smaller plaques compared to other strains, especially HP KAN353. PB2 amino acid 627E may contribute to low virulence, whereas either PB2 amino acid 627 K or the combination of 627E/701N seems to be associated with high virulence. The in vitro assays used in this study may provide the basis for assessing the pathogenesis of influenza H5N1 viruses in vivo.