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Background/objectives: The diagnosis of encephalitis is a challenging problem due to the heterogeneity of clinical presentations. The objective was to determine the etiology, clinical features, laboratory parameters, radiological findings, and in-hospital outcome of acute encephalitis syndrome (AES) cases in Myanmar., Methods: A prospective descriptive study was conducted at the Neuromedical Ward of Yangon General Hospital from March to August 2023. Eighty-one AES cases were enrolled, and cerebrospinal fluid (CSF) samples were collected. A Qiastat ME Panel was used to detect viral, bacterial, and fungal pathogens., Results: Seventeen out of eighty-one (21%) cases were non-encephalitis with alternative definite diagnosis. Among the remaining 64 encephalitis cases, the exact infectious and immune etiologies were identified in 31 of 64 cases (48.4%); 26 of these (83.9%) were due to infectious causes and 5 (16.1%) were immune encephalitis. Among the infectious causes, six Herpes Simplex Virus-1-, one bacteriologically confirmed and seven probable Mycobacterium tuberculosis -, three Haemophilus influenzae -, two Streptococcus pneumoniae -, one Streptococcus pyogenes -, one Varicella-Zoster Virus (Ramsay Hunt Syndrome with meningoencephalitis)-, and two Cryptococcus neoformans -infected patients and rare causes such as Listeria monocytogenes , Burkholdelria cepacia , Sphingomonas paucimobilis , and Aspergillus were identified. One case was a dual infection with Haemophilus influenzae and Cryptococcus neformans . Abnormal protein levels and CSF pleocytosis were significantly higher among bacterial causes ( p < 0.05). In total, 6.45% (2/31) of encephalitis patients with identified causes and 12.12% (4/33) of those without an identified organism had poor outcome., Conclusions: Herpes encephalitis and tuberculous meningoencepalitis were the commonest. This study highlighted that molecular testing with a multidisciplinary approach is required to ensure the right treatment on time.

Asia remains vulnerable to new and emerging infectious diseases. Understanding how to improve next generation sequencing (NGS) use in pathogen surveillance is an urgent priority for regional health security. Here we developed a pathogen genomic surveillance assessment framework to assess capacity in low-resource settings in South and Southeast Asia. Data collected between June 2022 and March 2023 from 42 institutions in 13 countries showed pathogen genomics capacity exists, but use is limited and under-resourced. All countries had NGS capacity and seven countries had strategic plans integrating pathogen genomics into wider surveillance efforts. Several pathogens were prioritized for human surveillance, but NGS application to environmental and human-animal interface surveillance was limited. Barriers to NGS implementation include reliance on external funding, supply chain challenges, trained personnel shortages and limited quality assurance mechanisms. Coordinated efforts are required to support national planning, address capacity gaps, enhance quality assurance and facilitate data sharing for decision making., (© 2024. The Author(s).)

Dengue is a viral infection caused by the dengue virus (DENV), transmitted to humans through the bite of infected Aedes mosquitoes. About half of the world's population is now at risk of dengue, which represents a global public health concern, especially in tropical and subtropical countries. Early detection of the viral infection is crucial to manage the disease; hence, effective rapid diagnostic tests are essential. In this study, we evaluated the performance between the new Fujifilm Dengue non-structural antigen diagnosis kit (FF NS1 kit) and the SD Bioline NS1 antigen test kit (SD NS1 kit) against the quantitative real-time polymerase chain reaction (qRT-PCR) assays. The 140 acute serum samples collected from the Yangon General Hospital and Yangon Children's Hospital, Myanmar, from 2017 to 2019 were characterised by the three assays. With the qRT-PCR as the standard, the FF NS1 kit and the SD NS1 kit exhibited sensitivity of 94.3% and 88.6%, respectively, and specificity of 100% in both kits. Moreover, the positivity rates of the FF NS1 kit and the SD NS1 kit were 97.5% and 95% in primary infection and 90% and 80% in secondary infection, respectively. Our overall results suggest that the FF NS1 kit is reliable and accurate for detecting DENV infection.

Rotavirus vaccines have substantially decreased rotavirus hospitalizations in countries where they have been implemented. In some high- and middle-income countries, a low-level of increased risk of intussusception, a type of acute bowel obstruction, has been detected following rotavirus vaccination. However, no increased risk of intussusception was found in India, South Africa, or a network of 7 other African countries. We assessed the association between a 2-dose monovalent rotavirus vaccine (Rotarix) and intussusception in 3 early-adopter low-income Asian countries -- Afghanistan, Myanmar, and Pakistan. Children <12 months of age admitted to a sentinel surveillance hospital with Brighton level 1 intussusception were eligible for enrollment. We collected information about each child's vaccination status and used the self-controlled case series method to calculate the relative incidence of intussusception 1-7 days, 8-21 days, and 1-21 days following each dose of vaccine and derived confidence intervals with bootstrapping. Of the 585 children meeting the analytic criteria, the median age at intussusception symptom onset was 24 weeks (IQR: 19-29). Overall, 494 (84 %) children received the first Rotarix dose and 398 (68 %) received the second dose. There was no increased intussusception risk during any of the risk periods following the first (1-7 days: 1.01 (95 %CI: 0.39, 2.60); 8-21 days: 1.37 (95 %CI: 0.81, 2.32); 1-21 days: 1.28 (95 %CI: 0.78, 2.11)) or second (1-7 days: 0.81 (95 %CI: 0.42, 1.54); 8-21 days: 0.77 (95 %CI: 0.53, 1.16); 1-21 days: 0.78 (95 %CI: 0.53, 1.16)) rotavirus vaccine dose. Our findings are consistent with other data showing no increased intussusception risk with rotavirus vaccination in low-income countries and add to the growing body of evidence demonstrating safety of rotavirus vaccines., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Eleanor Burnett reports financial support was provided by GAVI Alliance., (Published by Elsevier Ltd.)

Chikungunya virus (CHIKV) infection is a re-emerging arboviral disease with no approved vaccine, although numerous options are in development. Before vaccine implementation, disease burden, affected age group, and hospitalization rate information should be documented. In 2019, a sizeable outbreak of the East Central South African genotype of CHIKV occurred in Myanmar, and during this period, a cross-sectional study was conducted in two regions, Mandalay and Yangon, to examine the molecular and seropositivity rate of the CHIKV infection. The participants (1124) included dengue-suspected pediatric patients, blood donors, and healthy volunteers, who were assessed using molecular assays (quantitative real-time RT-PCR), serological tests (anti-CHIKV IgM capture and IgG indirect enzyme-linked immunosorbent assays), and neutralization tests. The tests confirmed the following positivity rates: 11.3% (127/1124) for the molecular assay, 12.4% (139/1124) for the anti-CHIKV IgM Ab, 44.5% (500/1124) for the anti-CHIKV IgG Ab, and 46.3% (520/1124) for the CHIKV neutralizing Ab. The highest rate for the molecular test occurred with the dengue-suspected pediatric patients. The seroprevalence rate through natural infection was higher in the healthy volunteers and blood donors than that in the pediatric patients. The results of this study will help stakeholders determine the criteria for choosing appropriate recipients when a CHIKV vaccine is introduced in Myanmar.

Myanmar is an endemic country for arboviruses, and outbreaks occur frequently. A cross-sectional analytical study was conducted during the peak season of the chikungunya virus (CHIKV) outbreak in 2019. A total of 201 patients with acute febrile illness who were admitted to the 550-bedded Mandalay Children Hospital in Myanmar were enrolled in the study, and virus isolation, serological tests, and molecular tests for the dengue virus (DENV) and CHIKV were performed for all samples. Out of 201 patients, 71 (35.3%) were only DENV-infected, 30 (14.9%) were only CHIKV-infected and 59 (29.4%) were coinfected with DENV and CHIKV. The viremia levels of the DENV- and CHIKV- mono-infected groups were significantly higher than those of the group coinfected with DENV and CHIKV. Genotype I of DENV-1, genotypes I and III of DENV-3, genotype I of DENV-4 and the East/Central/South African genotype of CHIKV were co-circulating during the study period. Two novel epistatic mutations of CHIKV (E1:K211E and E2:V264A) were noted. This study highlighted that there were many coinfection cases during the outbreak and that the co-circulation of both viruses in DENV-endemic regions warrants effective monitoring of these emerging pathogens via comprehensive surveillance to facilitate the implementation of effective control measures., Competing Interests: Declaration of competing interest The authors declared that there was no conflict of interests., (Copyright © 2023. Published by Elsevier Masson SAS.)

Background: Sarbecoviruses are a subgenus of Coronaviridae that mostly infect bats with known potential to infect humans (SARS-CoV and SARS-CoV-2). Populations in Southeast Asia, where these viruses are most likely to emerge, have been undersurveyed to date., Methods: We surveyed communities engaged in extractive industries and bat guano harvesting from rural areas in Myanmar. Participants were screened for exposure to sarbecoviruses, and their interactions with wildlife were evaluated to determine the factors associated with exposure to sarbecoviruses., Results: Of 693 people screened between July 2017 and February 2020, 12.1% were seropositive for sarbecoviruses. Individuals were significantly more likely to have been exposed to sarbecoviruses if their main livelihood involved working in extractive industries (logging, hunting, or harvesting of forest products; odds ratio [OR] = 2.71, P = 0.019) or had been hunting/slaughtering bats (OR = 6.09, P = 0.020). Exposure to a range of bat and pangolin sarbecoviruses was identified., Conclusion: Exposure to diverse sarbecoviruses among high-risk human communities provides epidemiologic and immunologic evidence that zoonotic spillover is occurring. These findings inform risk mitigation efforts needed to decrease disease transmission at the bat-human interface, as well as future surveillance efforts warranted to monitor isolated populations for viruses with pandemic potential., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The increasing global emergence of zoonoses warrants improved awareness of activities that predispose vulnerable communities to greater risk of disease. Zoonotic disease outbreaks regularly occur within Myanmar and at its borders partly due to insufficient knowledge of behavioral risks, hindering participatory surveillance and reporting. This study employed a behavioral surveillance strategy among high-risk populations to understand the behavioral risks for zoonotic disease transmission in an effort to identify risk factors for pathogen spillover. To explore behavioral mechanisms of spillover in Myanmar, we aimed to: (1) evaluate the details around animal contact and types of interaction, (2) assess the association between self-reported unusual symptoms (i.e., any illness or sickness that is not known or recognized in the community or diagnosed by medical providers) and animal contact activities and (3) identify the potential risk factors including behavioral practices of self-reported illness. Participants were enrolled at two community sites: Hpa-An and Hmawbi in Southern Myanmar. A behavioral questionnaire was administered to understand participants' animal exposures, behaviors and self-reported illnesses. From these responses, associations between (1) animal contact activities and self-reported unusual illnesses, and (2) potential risk factors and self-reported unusual illness were tested. Contact with poultry seemed to be very frequent (91.1%) and many participants reported raising, handling and having poultry in their houses as well as slaughtering or being scratched/bitten by them, followed by contact with rodents (57.8%) and swine (17.9%). Compared to participants who did not have any unusual symptoms, participants who had unusual symptoms in the past year were more likely to have sold dead animals (OR = 13.6, 95% CI 6.8-27.2), slaughtered (OR = 2.4, 95% CI 1.7-3.3), raised (OR = 3.4, 95% CI 2.3-5.0) or handled animals (OR = 2.1, 95% CI 1.2-3.6), and had eaten sick (OR = 4.4, 95% CI 3.0-6.4) and/or dead animals (OR = 6.0, 95% CI 4.1-8.8) in the same year. Odds of having reported unusual symptoms was higher among those involved in animal production business (OR = 3.4, 95% CI 1.9-6.2) and animal-involved livelihoods (OR = 3.3, 95% CI 1.5-7.2) compared to other livelihoods. The results suggest that there is a high level of interaction between humans, livestock and wild animals in communities we investigated in Myanmar. The study highlights the specific high-risk behaviors as they relate to animal contact and demographic risk factors for zoonotic spillover. Our findings contribute to human behavioral data needed to develop targeted interventions to prevent zoonotic disease transmission at human-animal interfaces., (© 2023. EcoHealth Alliance.)

Nonhuman primates living in proximity to humans increase risks for sylvatic arbovirus transmission. We collected serum samples from nonhuman primates in Hlawga National Park near Yangon, Myanmar, and detected antibodies against chikungunya (33%) and Japanese encephalitis (4%) viruses. Buffer zones between primate and human communities might reduce cross-species arbovirus transmission.

The dengue virus (DENV) has been endemic in Myanmar since 1970, causing outbreaks every 2-3 years. DENV infection symptoms range from mild fever to lethal hemorrhage. Clinical biomarkers must be identified to facilitate patient risk stratification in the early stages of infection. We analyzed 45 cytokines and other factors in serum samples from the acute phase of DENV infection (within 3-5 days of symptom onset) from 167 patients in Yangon, Myanmar, between 2017 and 2019. All of the patients tested positive for serum DENV nonstructural protein 1 antigen (NS1 Ag); 78.4% and 62.9% were positive for immunoglobulin M (IgM) and G (IgG), respectively; and 18.0%, 19.8%, and 11.9% tested positive for serotypes 1, 3, and 4, respectively. Although the DENV-4 viral load was significantly higher than those of DENV-1 or DENV-3, disease severity was not associated with viral load or serotype. Significant correlations were identified between disease severity and CCL5, SCF, PDGF-BB, IL-10, and TNF-α levels; between NS1 Ag and SCF, CCL5, IFN-α, IL-1α, and IL-22 levels; between thrombocytopenia and IL-2, TNF-α, VEGF-D, and IL-6 levels; and between primary or secondary infection and IL-2, IL-6, IL-31, IL-12p70, and MIP-1β levels. These circulating factors may represent leading signatures in acute DENV infections, reflecting the clinical outcomes in the dengue endemic region, Myanmar.

Background: Rotavirus gastroenteritis (RVGE) is a leading cause of severe diarrhea in children under-five worldwide, with the majority of mortality in lower -income countries. This study aimed to provide baseline information on epidemiology of rotavirus and circulating strains before rotavirus vaccine introduction in Myanmar., Methods: Hospital-based, prospective surveillance was conducted from May 2018 to January 2020 at four sentinel sites; two hospitals in Lower Myanmar, one hospital each in Middle Myanmar and East Myanmar. Children under five years of age hospitalized for acute gastroenteritis were enrolled; demographic and clinical data were collected. Stool samples were screened by ELISA (ProSpecT™ Rotavirus, OXOID-UK) for rotavirus antigen and a subset of ELISA positive samples were genotyped by reverse transcription polymerase chain reaction., Results: Rotavirus was detected in 45.7% (799/1750) of cases enrolled at three sites in May 2018-April 2019 and 42.5% (521/1227) at four sites in May 2019-January 2020. RVGE cases were predominantly male (58.7%; 775/1320) and 92.6% (1223/1320) of RVGE cases occurred in <2 years old. Rotavirus detection was higher in the cold and dry season (November-April). RVGE compared to non-RVGE cases had more frequent vomiting (78.3% Vs 68.1%, p < 0.01), fever (65.8% Vs 61.3%, p = 0.01), severe dehydration (3.6% Vs 2.1%, p < 0.01) and requirement of treatment by IV fluid (58.3% Vs 53.1%, p < 0.01). The most prevalent genotypes identified were G1P[6] (113/359, 31.5%), G1P[8] (94/359, 26.2%) and G2P[4] (33/359, 9.2%)., Conclusions: This study confirms the persistent high prevalence of RVGE among children under-five admitted to hospitals in different parts of Myanmar and the diversity of rotavirus strains over time prior to vaccine introduction. The rotavirus vaccine was introduced nationwide in February 2020 in Myanmar and these data will be important baseline data for post-vaccination monitoring of vaccine impact and circulating strains., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Dengue fever, caused by the mosquito-borne dengue virus (DENV), has been endemic in Myanmar since 1970 and it has become a significant public health burden. It is crucial that circulating DENV strains are identified and monitored, and that their transmission efficiency and association with disease severity is understood. In this study, we analyzed DENV-1, DENV-2, DENV-3, and DENV-4 serotypes in 1235 serum samples collected in Myanmar between 2017 and 2019. Whole-genome sequencing of DENV-1-4 demonstrated that most DENV-1-4 strains had been circulating in Myanmar for several years. We also identified the emergence of DENV-3 genotype-I in 2017 samples, which persisted through 2018 and 2019. The emergence of the strain coincided with a period of increased DENV-3 cases and marked changes in the serotype dynamics. Nevertheless, we detected no significant differences between serum viral loads, disease severity, and infection status of individuals infected with different DENV serotypes during the 3-year study. Our results not only identify the spread of a new DENV-3 genotype into Yangon, Myanmar, but also support the importance of DENV evolution in changing the epidemic dynamics in endemic regions.

Background: Dengue (DEN) is a neglected tropical disease, and surveillance of dengue virus (DENV) serotypes and genotypes is critical for the early detection of outbreaks. Risk factors for outbreaks include the emergence of new genotypes and serotype shifting., Methodology and Principal Findings: To understand the genomic and viral characteristics of DENV-infected patients, we conducted a cross-sectional descriptive study among pediatric patients admitted at the 550-bedded Mandalay Children Hospital during the 2018 DEN endemic season. We conducted virus isolation, serological tests, viremia level measurement, and whole-genome sequencing. Among the 202 serum samples, we detected 85 samples with DENV (46 DENV-1, 10 DENV-3, 26 DENV-4 and three multiple serotype co-infections) via reverse transcription quantitative/real-time PCR (RT-qPCR), and we obtained 49 DENV isolates (31 DENV-1, 10 DENV-3 and 8 DEN-4). We did not detect DENV-2 in this study. The viral genome levels in serum did not differ significantly among virus serotypes, infection status (primary versus secondary) and disease severity. Based on the phylogenetic analysis, we identified DENV-1 genotype-1, DENV-4 genotype-1 and DENV-3 genotype-3 and genotype-1 which was detected for the first time. Next-generation sequencing analysis revealed greater frequencies of nonsynonymous and synonymous mutations per gene in the nonstructural genes. Moreover, mutation rates were also higher among DENV-1., Conclusion/significance: In conclusion, there was an increasing trend of DENV-3 cases during DENV endemic season in 2018 with the first detection of the genotype 1. However, DENV-1 has remained the predominant serotype in this study area since 2013, and we identified stop codon mutations in the DENV-1 genome. This report is the first to feature a complete genome analysis of the strains of DENV-3 and DENV-4 circulating among pediatric patients in Myanmar. This study highlighted the importance of annual surveillance for a better understanding of the molecular epidemiology of DENVs., Competing Interests: No authors have competing interests.

Crimean-Congo hemorrhagic fever virus (CCHFV) is endemic in Asia, infecting many animal hosts, but CCHFV has not been reported in Myanmar. We conducted a seroepidemiologic survey of logging communities in Myanmar and found CCHFV exposure was common (9.8%) and exposure to wild animal blood and body fluids was associated with seropositivity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a major health concern globally. Genomic epidemiology is an important tool to assess the pandemic of coronavirus disease 2019 (COVID-19). Several mutations have been reported by genome analysis of the SARS-CoV-2. In the present study, we investigated the mutational and phylogenetic analysis of 30 whole-genome sequences for the virus's genomic characteristics in the specimens collected in the early phase of the pandemic (March-June, 2020) and the sudden surge of local transmission (August-September, 2020). The four samples in the early phase of infection were B.6 lineage and located within a clade of the samples collected at the same time in Singapore and Malaysia, while five returnees by rescue flights showed the lineage B. 1.36.1 (three from India), B.1.1 (one from India) and B.1.80 (one from China). However, there was no evidence of local spread from these returnees. Further, all 19 whole-genome sequences collected in the sudden surge of local transmission showed lineage B.1.36. The surge of the second wave on SARS-CoV-2 infection was linked to the single-introduction of a variant (B.1.36) that may result from the strict restriction of international travel and containment efforts. These genomic data provides the useful information to disease control and prevention strategy.

Background: Rotavirus vaccine was planned to be introduced in the National Immunization Program of Myanmar in 2020. Reported potential association of a small increased risk of intussusception after rotavirus vaccination in some countries is a major safety concern and it is mandatory to collect baseline information before vaccine introduction., Methods: Retrospective study reviewed medical records of intussusception cases for past 3 years (2015-2018) and prospective, active study was conducted from August 2018 to January 2020 at three tertiary children hospitals where pediatric surgical facility is present. Brighton Level 1 Criteria was used for confirmation of intussusception among children <2 years of age admitted to surgical wards. Demographic, clinical, diagnostic and treatment practices data were collected and descriptive data analysis was performed., Results: A total of 697 (421 in retrospective and 276 in prospective) confirmed intussusception cases were identified. Majority of intussusception cases (550/697, 78.9%) were observed in the first year of life and most frequent between 5-7 months of age (292/697, 41.9%) with a peak at 6 months (114/697, 16.4%). The most common clinical presentations were vomiting and bloody diarrhea accounting 82.1% and 77.5% respectively. Regarding diagnosis and treatment, 458/697 (65.7%) required surgical intervention either manual reduction or intestinal resection and 34.4% by either air or barium enema. Overall mortality was 0.7% (5/697) and four out of five children died needed intestinal resection. Late arrival to hospital (>3days after onset) is significantly associated with requirement of surgery (61/85, 71.8%), which in turn is significantly associated with longer hospital stay (296/452, 65.5%) (p < 0.05)., Conclusions: Intussusception occurrence is most frequent between 5-7 months age group which is old enough to be vaccinated under the schedule that has now been introduced in Myanmar. More than half of the cases were treated by surgery and late arrival to hospital enhances requirement of surgery and poor outcome. Findings of this baseline surveillance provide important facts for public health officials in balancing risks and benefits of rotavirus vaccine introduction, defining targeted age and dosage scheduling and facilitate monitoring system in post-vaccination., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

Streptococcus pneumoniae causes over one million deaths from lower respiratory infections per annum worldwide. Although mortality is very high in Southeast Asian countries, molecular epidemiological information remains unavailable for some countries. In this study, we report, for the first time, the whole-genome sequences and genetic profiles of pneumococcal strains isolated in Myanmar. We isolated 60 streptococcal strains from 300 children with acute respiratory infection at Yangon Children's Hospital in Myanmar. We obtained whole-genome sequences and identified the species, serotypes, sequence types, antimicrobial resistance (AMR) profiles, virulence factor profiles and pangenome structure using sequencing-based analysis. Average nucleotide identity analysis indicated that 58 strains were S. pneumoniae and the other 2 strains were Streptococcus mitis . The major serotype was 19F (11 strains), followed by 6E (6B genetic variant; 7 strains) and 15 other serotypes; 5 untypable strains were also detected. Multilocus sequence typing analysis revealed 39 different sequence types, including 11 novel ones. In addition, genetic profiling indicated that AMR genes and mutations spread among pneumococcal strains in Myanmar. A minimum inhibitory concentration assay indicated that several pneumococcal strains had acquired azithromycin and tetracycline resistance, whereas no strains were found to be resistant against levofloxacin and high-dose penicillin G. Phylogenetic and pangenome analysis showed various pneumococcal lineages and that the pneumococcal strains contain a rich and mobile gene pool, providing them with the ability to adapt to selective pressures. This molecular epidemiological information can help in tracking global infection and supporting AMR control in addition to public health interventions in Myanmar.

In 2019, an outbreak of chikungunya virus infection occurred in Mandalay, Myanmar, and 3.2% of blood donors and 20.5% of patients who were children were confirmed as being infected. The prevalence rate was up to 6.3% among blood donors. The East Central/South African genotype was predominantly circulating during this outbreak.

Background: Hepatitis B virus (HBV) infections are a severe health concern worldwide. HBV is a DNA virus with a rapid rate of mutation. Based on heterogeneity of the nucleotide sequence, the HBV strains are divided into nine genotypes, each with a characteristic geographical distribution. Identifying and tracking alterations of HBV genotypes is important in epidemiological and transmission studies, and contributes to predicting the risk for development of severe liver disease and response to antiviral treatment. The present study was undertaken to detect HBV genotypes and sub-genotypes in the general population of different states and regions in Myanmar., Methods: In 2015, a total of 5547 adults of the general population, residing in seven states, seven regions and the Nay Pyi Taw Union Territory, were screened for Hepatitis B Surface antigen (HBsAg) by the immunochromatographic test (ICT). Of the 353 HBsAg positive samples, the HBVDNA was identified using polymerase chain reactions (PCR) targeting the DNA sequences encoding the Pre-S region. A total of 153 PCR positive samples were subsequently subjected to genotyping by partial genome sequencing in both directions. The resulting sequences were then edited, aligned, and compared with reference sequences using the National Centre for Biotechnology Information (NCBI) web-based genotyping tool., Results: Three HBV genotypes (HBV genotype B, genotype C and genotype D) were detected in Myanmar, of which genotype HBV genotype C (66.7%) was the most prevalent, followed by HBV genotype D (32%) and HBV genotype B (1.3%). Sub-genotyping revealed a total of 7 variants within the B, C and D genotypes: 2 (B4 and B5) in HBV genotype B, 3 (C1, C5 and C7) in HBV genotype C, and 2 (D3 and D6) in HBV genotype D., Conclusion: HBV genotype C, sub-genotype C1 was predominantly distributed in all states and regions of Myanmar. This study is the first report on the nationwide distribution of HBV genotypes and sub-genotypes in Myanmar. We believe our findings will enable huge support for the hepatitis disease surveillance program, since HBV infection is one of the National Priority Diseases in Myanmar.

This study was conducted to find the burden of dengue virus (DENV) and Japanese encephalitis virus (JEV) among children under the age of 13, who presented with acute encephalitis syndrome at Mandalay Children Hospital in Myanmar in 2013. Molecular and serological investigations were performed on 123 cerebrospinal fluid (CSF) samples collected from these patients. By neutralization tests and/or virus isolation, four (3.3%) JEV- and one DENV-associated encephalitis cases (0.8%) were confirmed. Antibody titer against JEV Genotype 3 was the highest among the laboratory-confirmed JEV cases. One strain of DENV-1 with Genotype 1 was isolated from the CSF sample of the dengue encephalitis patient; this was similar to the virus circulating in the study area and neighboring countries. This study shows that flaviviruses are important pathogens causing encephalitis in Myanmar. Active disease surveillance, vector control, and vaccination programs should be enforced to reduce the morbidity and mortality caused by flavivirus encephalitis.

Background: In southeast Asia, antibiotic prescription in febrile patients attending primary care is common, and a probable contributor to the high burden of antimicrobial resistance. The objective of this trial was to explore whether C-reactive protein (CRP) testing at point of care could rationalise antibiotic prescription in primary care, comparing two proposed thresholds to classify CRP concentrations as low or high to guide antibiotic treatment., Methods: We did a multicentre, open-label, randomised, controlled trial in participants aged at least 1 year with a documented fever or a chief complaint of fever (regardless of previous antibiotic intake and comorbidities other than malignancies) recruited from six public primary care units in Thailand and three primary care clinics and one outpatient department in Myanmar. Individuals were randomly assigned using a computer-based randomisation system at a ratio of 1:1:1 to either the control group or one of two CRP testing groups, which used thresholds of 20 mg/L (group A) or 40 mg/L CRP (group B) to guide antibiotic prescription. Health-care providers were masked to allocation between the two intervention groups but not to the control group. The primary outcome was the prescription of any antibiotic from day 0 to day 5 and the proportion of patients who were prescribed an antibiotic when CRP concentrations were above and below the 20 mg/L or 40 mg/L thresholds. The primary outcome was analysed in the intention-to-treat and per-protocol populations. The trial is registered with ClinicalTrials.gov, number NCT02758821, and is now completed., Findings: Between June 8, 2016, and Aug 25, 2017, we recruited 2410 patients, of whom 803 patients were randomly assigned to CRP group A, 800 to CRP group B, and 807 to the control group. 598 patients in CRP group A, 593 in CRP group B, and 767 in the control group had follow-up data for both day 5 and day 14 and had been prescribed antibiotics (or not) in accordance with test results (per-protocol population). During the trial, 318 (39%) of 807 patients in the control group were prescribed an antibiotic by day 5, compared with 290 (36%) of 803 patients in CRP group A and 275 (34%) of 800 in CRP group B. The adjusted odds ratio (aOR) of 0·80 (95% CI 0·65-0·98) and risk difference of -5·0 percentage points (95% CI -9·7 to -0·3) between group B and the control group were significant, although lower than anticipated, whereas the reduction in prescribing in group A compared with the control group was not significant (aOR 0·86 [0·70-1·06]; risk difference -3·3 percentage points [-8·0 to 1·4]). Patients with high CRP concentrations in both intervention groups were more likely to be prescribed an antibiotic than in the control group (CRP ≥20 mg/L: group A vs control group, p<0·0001; CRP ≥40 mg/L: group B vs control group, p<0·0001), and those with low CRP concentrations were more likely to have an antibiotic withheld (CRP <20 mg/L: group A vs control group, p<0·0001; CRP <40 mg/L: group B vs control group, p<0·0001). 24 serious adverse events were recorded, consisting of 23 hospital admissions and one death, which occurred in CRP group A. Only one serious adverse event was thought to be possibly related to the study (a hospital admission in CRP group A)., Interpretation: In febrile patients attending primary care, testing for CRP at point of care with a threshold of 40 mg/L resulted in a modest but significant reduction in antibiotic prescribing, with patients with high CRP being more likely to be prescribed an antibiotic, and no evidence of a difference in clinical outcomes. This study extends the evidence base from lower-income settings supporting the use of CRP tests to rationalise antibiotic use in primary care patients with an acute febrile illness. A key limitation of this study is the individual rather than cluster randomised study design which might have resulted in contamination between the study groups, reducing the effect size of the intervention., Funding: Wellcome Trust Institutional Strategic Support Fund grant (105605/Z/14/Z) and Foundation for Innovative New Diagnostics (FIND) funding from the Australian Government., (Copyright © 2019 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: In Asia, rotavirus accounts for approximately 45% of admissions due to acute gastroenteritis in children <5 years, and causes about 145,000 deaths every year. We studied the distribution of rotavirus strains from Myanmar, Sri Lanka, and Nepal during 2009-2015., Methods: Stool samples collected from children <5 years of age hospitalized with acute diarrhea in the three sites and positive for rotavirus antigen by enzyme immunoassay (EIA) were sent to the Christian Medical College, Vellore from 2009 to 2015. G and P typing of rotavirus strains were performed using reverse-transcription polymerase chain reaction (RT-PCR)., Result: Of the 2354 EIA positive samples tested, G12P[8] (36.8%), G1P[8] (30.1%), and G12P[6] (41.3%) were the most common strains isolated from Myanmar, Sri Lanka, and Nepal respectively., Conclusion: There was substantial diversity of rotavirus genotypes, and continued surveillance in developing countries of Asia will help in understanding the epidemiology of rotavirus before and after introduction of vaccines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

An entomological surveillance of arboviruses was conducted in Myanmar in 2014. A total of 8357 Culex mosquito vectors were collected in the Mandalay area and virus isolation was done by using the mosquito cell line C6/36 E2. A total of eighteen strains of Culex flavivirus (CxFV) were isolated from Cx. tritaeniorhynchus, Cx. vishnui and Cx. fuscocephala. Like other insect-specific flaviviruses, CxFV can replicate only in mosquito cells but not in mammalian cells. These CxFV strains that were isolated in Japan from mosquitoes collected in Myanmar were closely related to the Wang Thong virus detected from Cx fusocephalus in Thailand and Cx.theileri flavivirus (CTFV) isolated from Cx. theileri mosquitoes in Portugal and Turkey. They encode a single open reading frame with 3357 amino acid residues. They have the characteristics of flaviviruses and have 95.62% amino acid identity with CTFV. This is the first report of CxFV in Myanmar with the characterized viral genome. This study illustrated that CxFV was circulating among the vectors of human pathogenic arboviruses in Myanmar but the impact of CxFV on other flaviviruses which are endemic in the study area still remains to be explored., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Zika virus (ZIKV), an emerging mosquito-borne flavivirus, causes a dengue-like infection that has recently caught global attention. The infection, which also includes some birth defects, has been documented in the Americas, Pacific Islands, and some parts of Africa and Asia. There are no published reports on local ZIKV transmission in Myanmar. In this study, a total of 462 serum samples from patients and asymptomatic persons were collected in Myanmar from 2004 to 2017. They were analyzed for ZIKV infection by immunoglobulin M capture enzyme-linked immunosorbent assay (ELISA), immunoglobulin G indirect ELISA, neutralization test, real-time polymerase chain reaction (PCR), and conventional PCR. Our study confirmed ZIKV infection in 4.9% of patients with clinical dengue symptoms and in 8.6% of persons who were asymptomatic. This is the first report on ZIKV infection in Myanmar and it suggests the occurrence of ZIKV infection in two geographically distinct sites in this country since at least 2006.

Background: Mosquito-borne diseases are prevalent in Myanmar, with the number of dengue cases showing a significant increase in recent years. Dengue vectors have developed resistance to insecticides and currently used larvicides show only short-term effectiveness. As a result, an alternative larvicide is urgently needed. The present study evaluated the larvicidal effectiveness of long-lasting pyriproxyfen resin discs (SumiLarv®2MR) against dengue virus vectors in schools in Hlaing Thar Yar Township, Yangon., Results: The proportion of Aedes mosquito-infested containers was significantly reduced in the schools applied with the larvicide (OR: 0.24, 95% CI: 0.12-0.48) while there was little reduction noted in the control schools (OR: 0.97, 95% CI: 0.55-1.72). The density of infested containers was also significantly reduced in the intervention schools (Beta: -1.50, 95% CI: -1.98- -1.04), but there was no significant reduction in density in the control schools (Beta: -0.19, 95% CI: -0.53-0.14). The proportion of adult emergence was less than 20% in the treated water collected from the intervention schools for six months, while the proportion was over 90% in the untreated water. In addition, eight-month-old SumiLarv®2MR resin discs were still 100% effective when tested in the laboratory. More than 50% of the discs disappeared from treated containers within two months of intervention., Conclusions: SumiLarv®2MR was effective in reducing Aedes-infested containers at least six months after its application in schools. This new pyriproxyfen formulation has great potential for improving the current dengue vector control program in Myanmar.

Background: The etiology of acute watery diarrhea remains poorly characterized, particularly after rotavirus vaccine introduction., Methods: We performed quantitative polymerase chain reaction for multiple enteropathogens on 878 acute watery diarrheal stools sampled from 14643 episodes captured by surveillance of children <5 years of age during 2013-2014 from 16 countries. We used previously developed models of the association between pathogen quantity and diarrhea to calculate pathogen-specific weighted attributable fractions (AFs)., Results: Rotavirus remained the leading etiology (overall weighted AF, 40.3% [95% confidence interval {CI}, 37.6%-44.3%]), though the AF was substantially lower in the Americas (AF, 12.2 [95% CI, 8.9-15.6]), based on samples from a country with universal rotavirus vaccination. Norovirus GII (AF, 6.2 [95% CI, 2.8-9.2]), Cryptosporidium (AF, 5.8 [95% CI, 4.0-7.6]), Shigella (AF, 4.7 [95% CI, 2.8-6.9]), heat-stable enterotoxin-producing Escherichia coli (ST-ETEC) (AF, 4.2 [95% CI, 2.0-6.1]), and adenovirus 40/41 (AF, 4.2 [95% CI, 2.9-5.5]) were also important. In the Africa Region, the rotavirus AF declined from 54.8% (95% CI, 48.3%-61.5%) in rotavirus vaccine age-ineligible children to 20.0% (95% CI, 12.4%-30.4%) in age-eligible children., Conclusions: Rotavirus remained the leading etiology of acute watery diarrhea despite a clear impact of rotavirus vaccine introduction. Norovirus GII, Cryptosporidium, Shigella, ST-ETEC, and adenovirus 40/41 were also important. Prospective surveillance can help identify priorities for further reducing the burden of diarrhea., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: The spread of artemisinin-resistant Plasmodium falciparum is a global health concern. Myanmar stands at the frontier of artemisinin-resistant P. falciparum. Myanmar also has the highest reported malaria burden in Southeast Asia; it is integral in the World Health Organization's plan to eliminate malaria in Southeast Asia, yet few epidemiological data exist for the general population in Myanmar., Methods: This cross-sectional, probability household survey was conducted in Phyu township, Bago Region (central Myanmar), during the wet season of 2013. Interviewers collected clinical and behavioural data, recorded tympanic temperature and obtained dried blood spots for malaria PCR and serology. Plasmodium falciparum positive samples were tested for genetic mutations in the K13 region that may confer artemisinin resistance. Estimated type-specific malaria PCR prevalence and seroprevalence were calculated, with regression analysis to identify risk factors for seropositivity to P. falciparum. Data were weighted to account for unequal selection probabilities., Results: 1638 participants were sampled (500 households). Weighted PCR prevalence was low (n = 41, 2.5%) and most cases were afebrile (93%). Plasmodium falciparum was the most common species (n = 19. 1.1%) and five (26%) P. falciparum samples harboured K13 mutations. Plasmodium knowlesi was detected in 1.0% (n = 16) and Plasmodium vivax was detected in 0.4% (n = 7). Seroprevalence was 9.4% for P. falciparum and 3.1% for P. vivax. Seroconversion to P. falciparum was 0.003/year in the whole population, but 16-fold higher in men over 23 years old (LR test p = 0.016)., Discussion: This is the first population-based seroprevalence study from central Myanmar. Low overall prevalence was discovered. However, these data suggest endemic transmission continues, probably associated with behavioural risk factors amongst working-age men. Genetic mutations associated with P. falciparum artemisinin resistance, the presence of P. knowlesi and discrete demographic risk groups present opportunities and challenges for malaria control. Responses targeted to working-age men, capable of detecting sub-clinical infections, and considering all species will facilitate malaria elimination in this setting.

The rotavirus-induced diarrhea of human and animal neonates is a major public health concern worldwide. Until recently, no effective therapy is available to specifically inactivate the rotavirion particles within the gut. Passive immunotherapy by oral administration of chicken egg yolk antibody (IgY) has emerged of late as a fresh alternative strategy to control infectious diseases of the alimentary tract and has been applied in the treatment of diarrhea due to rotavirus infection. The purpose of this concise review is to evaluate evidence on the properties and performance of anti-rotavirus immunoglobulin Y (IgY) for prevention and treatment of rotavirus diarrhea in human and animal neonates. A survey of relevant anti-rotavirus IgY basic studies and clinical trials among neonatal animals (since 1994-2015) and humans (since 1982-2015) have been reviewed and briefly summarized. Our analysis of a number of rotavirus investigations involving animal and human clinical trials revealed that anti-rotavirus IgY significantly reduced the severity of clinical manifestation of diarrhea among IgY-treated subjects relative to a corresponding control or placebo group. The accumulated information as a whole depicts oral IgY to be a safe and efficacious option for treatment of rotavirus diarrhea in neonates. There is however a clear need for more randomized, placebo controlled and double-blind trials with bigger sample size to further solidify and confirm claims of efficacy and safety in controlling diarrhea caused by rotavirus infection especially among human infants with health issues such as low birth weights or compromised immunity in whom it is most needed., Competing Interests: Competing Interests The authors declare that they have no competing interests.

Human rotavirus samples from 54 children with acute gastroenteritis in Myanmar in 2011 were subjected to reverse transcription-PCR to determine their G and P types. On G typing, G2 (24/54; 44.4%) was found to be the most prevalent, followed by G12 (17/54; 31.5%) and G1 (1/54; 1.9%). Mixed cases with G2 and G12 were found in 12 of the 54 (22.2%) samples. On P typing, P[4] was found to be the most predominant (29/54; 53.7%), followed by P[8] (17/54; 31.5%) and P[6] (4/54; 7.4%). Mixed cases with P[4] and P[8] were detected in 4 of 54 (7.4%) samples. Thus, occurrence of G2 and unusual G12 in high proportions was characteristic of human rotaviruses in Myanmar in this study setting.

The four genetically divergent dengue virus (DENV) types are traditionally classified as serotypes. Antigenic and genetic differences among the DENV types influence disease outcome, vaccine-induced protection, epidemic magnitude, and viral evolution. We characterized antigenic diversity in the DENV types by antigenic maps constructed from neutralizing antibody titers obtained from African green monkeys and after human vaccination and natural infections. Genetically, geographically, and temporally, diverse DENV isolates clustered loosely by type, but we found that many are as similar antigenically to a virus of a different type as to some viruses of the same type. Primary infection antisera did not neutralize all viruses of the same DENV type any better than other types did up to 2 years after infection and did not show improved neutralization to homologous type isolates. That the canonical DENV types are not antigenically homogeneous has implications for vaccination and research on the dynamics of immunity, disease, and the evolution of DENV., (Copyright © 2015, American Association for the Advancement of Science.)

G12 rotaviruses are emerging rotavirus strains causing severe diarrhea in infants and young children worldwide. However, the whole genomes of only a few G12 strains have been fully sequenced and analyzed. In this study, we sequenced and characterized the complete genomes of six G12 strains (RVA/Human-tc/MMR/A14/2011/G12P[8], RVA/Human-tc/MMR/A23/2011/G12P[6], RVA/Human-tc/MMR/A25/2011/G12P[8], RVA/Human-tc/MMR/P02/2011/G12P[8], RVA/Human-tc/MMR/P39/2011/G12P[8], and RVA/Human-tc/MMR/P43/2011/G12P[8]) detected in six stool samples from children with acute gastroenteritis in Myanmar. On whole genomic analysis, all six Myanmarese G12 strains were found to have a Wa-like genetic backbone: G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strains A14, A25, P02, P39, and P43, and G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strain A23. Phylogenetic analysis showed that most genes of the six strains examined in this study were genetically related to globally circulating human G1, G3, G9, and G12 strains. Of note is that the NSP4 gene of strain A23 exhibited the closest relationship with the cognate genes of human-like bovine strains as well as human strains, suggesting the occurrence of reassortment between human and bovine strains. Furthermore, strains A14, A25, P02, P39, and P43 were very closely related to one another in all the 11 gene segments, indicating derivation of the five strains from a common origin. On the other hand, strain A23 consistently formed distinct clusters as to all the 11 gene segments, indicating a distinct origin of strain A23 from that of strains A14, A25, P02, P39, and P43. To our knowledge, this is the first report on whole genome-based characterization of G12 strains that have emerged in Myanmar. Our observations will provide important insights into the evolutionary dynamics of spreading G12 rotaviruses in Asia.

Background: Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar., Methodology/principal Findings: We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study., Conclusions/significance: Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti.

This study aims to evaluate the effect of hyperimmune immunoglobulin Y (IgY) against human rotavirus (HRV) among pediatric patients receiving standard supportive treatment for rotavirus-associated diarrhea mostly with an enteric non-cholera co-pathogen in a hospital setting. Two natural HRV reassortant clinical strains ATCC VR 2273 and ATCC VR 2274 were used as mixed immunizing antigens in poultry hens to generate anti-HRV IgY (Rotamix IgY). The Rotamix IgY was used in laboratory and clinical studies against control or placebo IgY. The control or placebo IgY was prepared using tissue culture medium from mock-infected MA104 cell line as antigen for poultry immunization. In vitro, Rotamix IgY exhibited multi-serotypic cross neutralization activities along with synergistic effects against major global serotypes G1, G2, G3, G4 and other human or animal rotavirus strains when compared with mono-specific IgY. Suckling mice (ICR strain) pre-treated orally once with Rotamix IgY and then challenged with rotavirus 3h later showed a significant dose-dependent reduction in frequency (p<0.05) and duration (p<0.05) of diarrhea compared to placebo IgY-treated mice. Out of 114 children aged between 3 and 14 months and with diarrhea upon admission in a Myanmar hospital, 54 dehydrated and rotavirus-positive children were randomized into Rotamix IgY group and placebo IgY group. Of these, only 52 children had complete data with n=26 children per study group. Ninety-two percent of patients in each of these groups were positive for co-infecting enteric non-cholera pathogen and all patients received standard supportive therapy for diarrhea. The patients were monitored for volume and duration of oral rehydration fluid (ORF) and intravenous fluid (IVF) intake, daily stool frequency and overall duration of diarrhea, and frequency and duration of rotavirus shedding. Compared to placebo IgY group, the Rotamix IgY group had statistically significant reduction in mean ORF intake (p=0.004), mean duration of intravenous fluid administration (p=0.03), mean duration of diarrhea from day of admission (p<0.01) and mean duration of rotavirus clearance from stool from day of admission (p=0.05). Overall, our novel approach using oral Rotamix IgY for rotavirus-infected children mostly with non-cholera enteric pathogen co-infection appears to be a promising, safe and effective adjunct to management of acute diarrhea in pediatric patients., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.

Rotavirus positive stool specimens collected from children less than 5-year-old admitted to Yangon Children's Hospital for diarrhoea were analysed for their G and P genotypes. In 2004, rotavirus was detected in 57% of 1004 samples tested with G3 and P[8] being the most common genotypes identified. Corresponding figures in 2005, were 55% of 1175 samples positive for rotavirus with G3 and P[4] genotypes being most common. The most common G and P combinations were G3P[8] and G1P[8], with some unusual combinations (G1P[4], G1P[6] and G3P[4]) also being identified. In Myanmar, ongoing rotavirus surveillance to understand the distribution of G and P genotypes will be important for monitoring the impact of rotavirus vaccines following their introduction into the infant immunization schedule.

Dengue and dengue hemorrhagic fever re-emerged in Bangladesh in 2000 and 2001 and nearly all viruses isolated were dengue type 3. Phylogenetic analyses of the envelope genes of examples of these viruses indicated that they were most closely related to recently emerged dengue type 3 viruses from neighboring Thailand and Myanmar but distinct from those from India and Sri Lanka. Since this strain of dengue virus type 3 had not been associated with unusual patterns of disease in Thailand or Myanmar, it suggested that the outbreak in Bangladesh was due to local factors after the introduction of viruses from countries to the east rather than to the evolution of an unusually virulent strain of virus in Bangladesh.

In 2001, dengue virus type 1 (DENV-1) populations in humans and mosquitoes from Myanmar acquired a stop-codon mutation in the surface envelope (E) protein gene. Within a year, this stop-codon strain had spread to all individuals sampled. The presence of truncated E protein species within individual viral populations, along with a general relaxation in selective constraint, indicated that the stop-codon strain represents a defective lineage of DENV-1. We propose that such long-term transmission of defective RNA viruses in nature was achieved through complementation by coinfection of host cells with functional viruses.

Outbreaks of dengue due to dengue virus type 1 (DENV-1) occurred almost simultaneously in 2001 in Myanmar and at multiple sites almost 10,000 km away in the Pacific. Phylogenetic analyses of the E protein genes of DENV-1 strains recovered from Asia and the Pacific revealed three major viral genotypes (I, II, and III) with distinct clades within each. The majority of strains from the Pacific and Myanmar, and a number of other Asian strains fell into genotype I. Genotype II comprised a smaller set of Asian and Pacific strains, while genotype III contained viruses from diverse geographical localities. These analyses suggested that the continuing outbreak of dengue in the Pacific has been due to multiple, direct, introductions of dengue viruses from a variety of locations in Asia followed by local transmission. There was no evidence that the introduction of these viruses into the Pacific was associated with any adaptive changes in the E protein of the viruses.

In 2001, Myanmar (Burma) had its largest outbreak of dengue-15,361 reported cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), including 192 deaths. That year, 95% of dengue viruses isolated from patients were serotype 1 viruses belonging to two lineages that had diverged from an earlier, now extinct, lineage sometime before 1998. The ratio of DHF to DSS cases in 2001 was not significantly different from that in 2000, when 1,816 cases of DHF/DSS were reported and dengue 1 also was the most frequently isolated serotype. However, the 2001 ratio was significantly higher than that in 1998 (also an outbreak year) and in 1999, when all four serotypes were detected and serotypes 1, 2, and 3 were recovered in similar numbers. The large number of clinical cases in 2001 may have been due, in part, to a preponderance of infections with dengue 1 viruses.

Envelope (E) protein genes sampled from populations of dengue 2 (DEN-2) virus in individual Aedes aegypti mosquitoes and in serum from dengue patients were copied to cDNA, cloned, and sequenced. The nucleotide sequences of the E genes in more than 70% of the clones differed from the consensus sequence for the corresponding virus population at up to 11 sites, and 24 of the 94 clones contained at least one stop codon. Virus populations recovered up to 2 years apart yielded clones with similar polymorphisms in the E gene. For one mosquito, the clones obtained fell into two genotypes. One group of sequences was closely related to those of viruses recovered from dengue patients in the same locality (Yangon, Myanmar) since 1995 and were classified as Asian 1 genotype. The second group were Cosmopolitan genotype viruses which were also circulating in Yangon in 2000 and which were related to DEN-2 viruses sampled from southern China in 1999. Finally, one clone was identified as a recombinant genome composed of portions of these two "parental" genotypes. This is the first report of recombinant and parental dengue viruses in a single host.

Strains of dengue 3 (DEN-3) virus circulating in Thailand prior to 1992 appear to have disappeared from that location and to have been replaced by two new lineages which have evolved locally, rather than being introduced. Similar DEN-3 virus extinctions may have occurred previously in Thailand in 1962 and 1973. Although no causal relationship could be shown, this strain replacement event was accompanied by DEN-3 replacing DEN-2 as the serotype recovered most frequently from patients in Thailand. Although this implies a change in selection pressure, we found no evidence for positive natural selection at the level of either the E protein or the E protein gene. Further, the extinction of the pre-1992 strains and the appearance of the new lineages occurred during an interepidemic period, suggesting that a genetic bottleneck, rather than selection, might have been important in the emergence of these two new strains of virus. The pre-1992 DEN-3 virus lineage could still be found in 1998, to the west, in Myanmar. The ratio of nonsynonymous-to-synonymous nucleotide changes within a DEN-3 virus population from a single patient was less than the ratio among the consensus sequences of DEN-3 viruses from different patients, suggesting that many of the nonsynonymous nucleotide changes which occurred naturally in the E protein were deleterious and removed by purifying selection.

The effect of temperature and relative humidity on dengue virus propagation in the mosquito as one of the possible contributing factors to dengue hemorrhagic fever (DHF) outbreaks was studied. Ae. aegypti mosquitos were reared under standard conditions and inoculated intrathoracically with dengue virus. Virus propagation in the mosquitos was determined at the temperature and relative humidity of all 3 seasons of Yangon and for the simulated temperature and relative humidity of Singapore. The virus propagation was detected by direct fluorescent antibody technique (DFAT) with mosquito head squash and the virus titer was determined by plaque forming unit test (PFUT) in baby hamster kidney-21 cells. The results show that the infected mosquitos kept under the conditions of the rainy season and under the simulated conditions of Singapore had a significantly higher virus titer (p=<0.05) when compared with the other 2 seasons of Yangon. So it is thought that the temperature and relative humidity of the rainy season of Yangon and that of Singapore favors dengue virus propagation in the mosquito and is one of the contributing factors to the occurence of DHF outbreaks.