A serotype-specific and tiled amplicon multiplex PCR method for whole genome sequencing of dengue virus.

Dengue fever, a mosquito-borne viral disease of significant public health concern in tropical and subtropical regions, is caused by any of the four serotypes of the dengue virus (DENV1-4). Cutting-edge technologies like next-generation sequencing (NGS) are revolutionizing virology, enabling in-depth exploration of DENV's genetic diversity. Here, we present an optimized workflow for full-genome sequencing of DENV 1-4 utilizing tiled amplicon multiplex PCR and Illumina sequencing. Our assay, sequenced on the Illumina MiSeq platform, demonstrates its ability to recover the full-length dengue genome across various viral abundances in clinical specimens with high-quality base coverage. This high quality underscores its suitability for precise examination of intra-host diversity, enriching our understanding of viral evolution and holding potential for improved diagnostic and intervention strategies in regions facing dengue outbreaks.

A genotyping array for the globally invasive vector mosquito, Aedes albopictus.

BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.

Genome evolution of dengue virus serotype 1 under selection by Wolbachia pipientis in Aedes aegypti mosquitoes.

The introgression of antiviral strains of Wolbachia into Aedes aegypti mosquito populations is a public health intervention for the control of dengue. Plausibly, dengue virus (DENV) could evolve to bypass the antiviral effects of Wolbachia and undermine this approach. Here, we established a serial-passage system to investigate the evolution of DENV in Ae. aegypti mosquitoes infected with the wMel strain of Wolbachia. Using this system, we report on virus genetic outcomes after twenty passages of serotype 1 of DENV (DENV-1). An amino acid substitution, E203K, in the DENV-1 envelope protein was more frequently detected in the consensus sequence of virus populations passaged in wMel-infected Ae. aegypti than wild-type counterparts. Positive selection at residue 203 was reproducible; it occurred in passaged virus populations from independent DENV-1-infected patients and also in a second, independent experimental system. In wild-type mosquitoes and human cells, the 203K variant was rapidly replaced by the progenitor sequence. These findings provide proof of concept that wMel-associated selection of virus populations can occur in experimental conditions. Field-based studies are needed to explore whether wMel imparts selective pressure on DENV evolution in locations where wMel is established.

Medication Adherence and Belief about Medication among Vietnamese Patients with Chronic Cardiovascular Diseases within the Context of Implementing Measures to Prevent COVID-19.

BACKGROUND: Long-term adherence is crucial for optimal treatment outcomes in chronic cardiovascular diseases (CVDs), especially throughout the COVID-19 wide-spreading periods, making patients with chronic CVDs vulnerable subjects. AIM: To investigate the relationship between the characteristics, beliefs about prescribed medication, COVID-19 prevention measures, and medication adherence among patients with chronic CVDs. METHODS: This is a cross-sectional study of outpatients with chronic CVDs in Southern Vietnam. The specific parts regarding the Beliefs about Medicines Questionnaires (BMQ-Specific) and the General Medication Adherence Scale (GMAS) were applied to assess the beliefs about and adherence to medication. The implementation measures to prevent COVID-19 in patients were evaluated according to the 5K message (facemask, disinfection, distance, no gathering, and health declaration) of the Vietnam Ministry of Health. A multivariable logistic regression with the Backward elimination (Wald) method was used to identify the associated factors of medication adherence. RESULTS: A slightly higher score in BMQ-Necessity compared to BMQ-Concerns was observed. A total of 40.7% of patients were recorded as having not adhered to their medications. Patients' behavior was most frequently self-reported by explaining their non-adherence (34.7%). Statistical associations were found between rural living place, unemployment status, no or only one measure(s) of COVID-19 prevention application, and medication adherence. CONCLUSION: During the COVID-19 spreading stage, patients generally showed a positive belief about medication when they rated the importance of taking it higher than its side effects. The data analysis suggested that rather than patients' beliefs, the clinicians should consider the patient factors, including living place, employment, and the number of epidemic preventive measures applied for guiding the target patients for improving medication adherence.

Occurrence of Marek's Disease in Backyard Chicken Flocks in Vietnam.

Marek's disease (MD) is a common lymphomatous and neuropathic disease of birds, especially chickens, and has caused significant losses to chicken production as a result of high mortality and morbidity. This study aims to determine the status of MD in backyard flocks in the Mekong Delta of Vietnam and to analyze clinical cases occurring during a year. The study was carried out from August 2018 to July 2019, during which time 16 suspected cases of chicken flocks with MD were observed, 40 chickens were subjected to anatomopathological examination, and PCR was performed for diagnosis of MD and determination of Marek's disease virus (MDV) serotypes. The results showed that all of the examined flocks were confirmed as experiencing MD. Nearly all cases were in an acute form with typical lesions of visceral lymphomas. Besides the presence of Marek's disease virus serotype 1 (MDV-1) from 100.0% of tested chicken flocks (16/16), nononcogenic turkey herpesvirus serotype 3 (MDV-3) was also found from 8 of 16 (50.0%) of examined chicken flocks. Morbidity and mortality at sampling time varied from 1% to 42.11% and 0.6% to 10%, respectively. Chicken flocks with MD vaccination had lower morbidity and mortality. These first findings confirm endemic MD in backyard chicken populations in Vietnam and confirm it continues to be a threat to chickens in backyard flocks and poultry production in general.

Presentación de la enfermedad de Marek en parvadas de pollos de traspatio en Vietnam. La enfermedad de Marek (MD) es una enfermedad linfoproliferativa y neuropática común de las aves, especialmente de los pollos y ha causado pérdidas significativas en la producción de pollos como resultado de la alta mortalidad y morbilidad. Este estudio tiene como objetivo determinar el estado de la enfermedad de Marek en parvadas de traspatio en el delta del Mekong en Vietnam y analizar los casos clínicos que ocurren durante un año. El estudio se llevó a cabo de agosto de 2018 a julio de 2019, tiempo durante el cual se observaron 16 casos sospechosos de parvadas de pollos con enfermedad de Marek, 40 pollos fueron sometidos a examen anatomopatológico y se les realizó PCR para el diagnóstico de enfermedad de Marek y determinación del serotipo del virus. Los resultados confirmaron que todas las parvadas examinadas presentaban enfermedad de Marek y casi todos los casos presentaban una forma aguda con lesiones típicas de linfomas viscerales, especialmente en el hígado. Además de la presencia del serotipo 1 del virus de la enfermedad de Marek (MDV-1) en el 100% de las parvadas de pollos analizadas (16/16), también se encontró el serotipo 3 del herpesvirus del pavo no oncogénico (MDV-3) en 8 de 16 (50.0 %) de las parvadas de pollo examinadas. La morbilidad y la mortalidad en el momento del muestreo variaron del 1% al 42.11% y del 0.6% al 10%, respectivamente. Las parvadas de pollos con vacunación contra la enfermedad de Marek tuvieron menor morbilidad y mortalidad. Estos primeros hallazgos confirman la presencia endémica del virus de la enfermedad de Marek en las poblaciones de pollos de traspatio en Vietnam y confirman que continúa siendo una amenaza para los pollos en parvadas de traspatio y para la producción avícola en general.

Oviposition of Aedes aegypti and Aedes albopictus on Ovitraps in Dry and Rainy Seasons in Southern Vietnam.

Ovitraps were set inside and outside of 15 households in December 2012 (dry season) and August 2013 (rainy season) in 2 communes (An Thanh and Phu Hoa) of Binh Duong Province in southern Vietnam. Eggs laid in the ovitraps were collected after 4 days, dried, and soaked in water. Hatched larvae were transferred to cups and reared to adulthood to identify the species. The rate of positive ovitraps did not differ between December and August for Aedes aegypti, but it was lower in December for Ae. albopictus. The number of eggs laid per ovitrap by Ae. aegypti did not differ between December and August, while that for Ae. albopictus decreased significantly in December in both communes. Moreover, Ae. albopictus laid eggs in ovitraps placed outside the households. Therefore, it is necessary for future studies to investigate whether the major source containers for oviposition had dried in the dry season or Ae. albopictus entered diapause, leading to these observations.

Assessing the vertical transmission potential of dengue virus in field-reared Aedes aegypti using patient-derived blood meals in Ho Chi Minh City, Vietnam.

BACKGROUND: Dengue viruses (DENV) can be transmitted from an adult female Aedes aegypti mosquito through the germ line to the progeny; however, there is uncertainty if this occurs at a frequency that is epidemiologically significant. We measured vertical transmission of DENV from field-reared Ae. aegypti to their F1 progeny after feeding upon blood from dengue patients. We also examined the transmission potential of F1 females. METHODS: We examined the frequency of vertical transmission in field-reared mosquitoes, who fed upon blood from acutely viremic dengue patients, and the capacity for vertically infected females to subsequently transmit virus horizontally, in two sets of experiments: (i) compared vertical transmission frequency of field-reared Ae. aegypti and Ae. albopictus, in individual progeny; and (ii) in pooled progeny derived from field- and laboratory-reared Ae. aegypti. RESULTS: Of 41 DENV-infected and isofemaled females who laid eggs, only a single female (2.43%) transmitted virus to one of the F1 progeny, but this F1 female did not have detectable virus in the saliva when 14 days-old. We complemented this initial study by testing for vertical transmission in another 460 field-reared females and > 900 laboratory-reared counterparts but failed to provide any further evidence of vertical virus transmission. CONCLUSIONS: In summary, these results using field-reared mosquitoes and viremic blood from dengue cases suggest that vertical transmission is uncommon. Field-based studies that build on these observations are needed to better define the contribution of vertical DENV transmission to dengue epidemiology.

Synthesis, structure and in vitro cytotoxicity testing of some 2-aroylbenzofuran-3-ols.

Five 2-aroyl-5-bromobenzo[b]furan-3-ol compounds (two of which are new) and four new 2-aroyl-5-iodobenzo[b]furan-3-ol compounds were synthesized starting from salicylic acid. The compounds were characterized by mass spectrometry and 1H NMR and 13C NMR spectroscopy. Single-crystal X-ray diffraction studies of four compounds, namely, (5-bromo-3-hydroxybenzofuran-2-yl)(4-fluorophenyl)methanone, C15H8BrFO3, (5-bromo-3-hydroxybenzofuran-2-yl)(4-chlorophenyl)methanone, C15H8BrClO3, (5-bromo-3-hydroxybenzofuran-2-yl)(4-bromophenyl)methanone, C15H8Br2O3, and (4-bromophenyl)(3-hydroxy-5-iodobenzofuran-2-yl)methanone, C15H8BrIO3, were also carried out. The compounds were tested for their in vitro cytotoxicity on the four human cancer cell lines KB, Hep-G2, Lu-1 and MCF7. Six compounds show good inhibiting abilities on Hep-G2 cells, with IC50 values of 1.39-8.03 µM.

Multiple Wolbachia strains provide comparative levels of protection against dengue virus infection in Aedes aegypti.

The insect bacterium Wolbachia pipientis is being introgressed into Aedes aegypti populations as an intervention against the transmission of medically important arboviruses. Here we compare Ae. aegypti mosquitoes infected with wMelCS or wAlbB to the widely used wMel Wolbachia strain on an Australian nuclear genetic background for their susceptibility to infection by dengue virus (DENV) genotypes spanning all four serotypes. All Wolbachia-infected mosquitoes were more resistant to intrathoracic DENV challenge than their wildtype counterparts. Blocking of DENV replication was greatest by wMelCS. Conversely, wAlbB-infected mosquitoes were more susceptible to whole body infection than wMel and wMelCS. We extended these findings via mosquito oral feeding experiments, using viremic blood from 36 acute, hospitalised dengue cases in Vietnam, additionally including wMel and wildtype mosquitoes on a Vietnamese nuclear genetic background. As above, wAlbB was less effective at blocking DENV replication in the abdomen compared to wMel and wMelCS. The transmission potential of all Wolbachia-infected mosquito lines (measured by the presence/absence of infectious DENV in mosquito saliva) after 14 days, was significantly reduced compared to their wildtype counterparts, and lowest for wMelCS and wAlbB. These data support the use of wAlbB and wMelCS strains for introgression field trials and the biocontrol of DENV transmission. Furthermore, despite observing significant differences in transmission potential between wildtype mosquitoes from Australia and Vietnam, no difference was observed between wMel-infected mosquitoes from each background suggesting that Wolbachia may override any underlying variation in DENV transmission potential.

DNA-HPV transition rate and related factors in HPV-infected women in Can Tho city, Vietnam.

OBJECTIVES: To determine DNA-HPV transition rates and related factors in HPV-infected women 18-69 years of age in Can Tho City from 2013 to 2018. METHODS: Both a retrospective and a prospective cohort study were done. Interviews, gynaecological examinations and HPV testing by PCR (cervical fluid) were used to collect data. The results were recorded and compared with those of HPV in 2013 to assess the development of HPV over time. Transition was defined as conversion to HPV-positive state in 2018 from a negative state in 2013. No transition was defined as clearance of HPV when the positive 2013 result was negative in 2018 or when the result remained negative or positive in 2013 and 2018. Factors related to the change were analysed. RESULTS: Among a sample size of 204 cases, the average age of participants was 48.9 ± 10.4 years. Women >45 comprised 63.2% of participants; 82.8% lived with their husbands, 6.4% were divorced, and 2.9% lived apart from their husbands due to work. After 5 years of observation, 16.2% of DNA-HPV cases had converted to HPV-positive state and 66.2% of DNA-HPV cases had cleared to HPV-negative state. Factors related to conversion to HPV-positive state were age ≤ 45 years (3.14 times higher risk of transition than in the >45 age group (95% CI: 1.12-8.8)); change of sexual partner (OR = 3.75 (95% CI: 1.15-12.2)); change of sexual partner by husband (OR = 3.69 (95% CI: 1.20-11.3); sexually transmitted diseases (OR = 5.19 (95% CI: 1.09-24.8)); and a history of vacuum aspiration or dilation and evacuation abortion (OR = 1.4 (95% CI: 0.29-6.4)). CONCLUSIONS: 16.2% of women with DNA-HPV transition converted to HPV-positive state. Changes in sexual habits increase the risk of developing HPV positivity.

TAUX DE TRANSITION ADN-VPH ET FACTEURS CONNEXES CHEZ LES FEMMES INFECTÉES PAR LE VPH À CAN THO CITY, VIETNAM: OBJECTIFS: Déterminer les taux de transition ADN-VPH et les facteurs connexes chez les femmes infectées par le VPH, âgées de 18 à 69 ans à Can Tho City de 2013 à 2018. MÉTHODES: Une étude de cohorte rétrospective et une prospective ont été effectuées. Des entretiens, des examens gynécologiques et des tests pour le VPH par PCR (liquide cervical) ont été utilisés pour collecter des données. Les résultats ont été enregistrés et comparés à ceux du VPH en 2013 afin d'évaluer l'évolution du VPH au fil du temps. La transition a été définie comme étant la conversion d'un état négatif au VPH en 2013 à un état positif en 2018. Aucune transition n'a été définie comme une élimination du VPH lorsque le résultat positif de 2013 était négatif en 2018 ou lorsque le résultat est resté négatif ou positif en 2013 et 2018. Les facteurs liés au changement ont été analysés. RÉSULTATS: Sur un échantillon de 204 cas, l'âge moyen des participantes était de 48,9 ± 10,4 ans. Les femmes > 45 ans représentaient 63,2% des participantes; 82,8% vivaient avec leur mari, 6,4% étaient divorcées et 2,9% vivaient séparées de leur mari à cause de leur travail. Après 5 ans d'observation, 16,2% des cas d'ADN-VPH étaient passés à l'état VPH positif et 66,2% des cas d'ADN-VPH avaient tout éliminé et étaient passés à l'état HPV négatif. Les facteurs liés à la conversion à l'état positif au VPH étaient les suivants: âge ≤ 45 ans (risque de transition de 3,14 fois supérieur à celui du groupe d'âge > 45 ans (IC95%: 1,12 à 8,8)), changement de partenaire sexuel (OR = 3,75 (IC95%: 1,15-12,2)), changement de partenaire sexuel par le mari (OR = 3,69 (IC95%: 1,20-11,3), maladies sexuellement transmissibles (OR = 5,19 (IC95%: 1,09-24,8)) et antécédents d'aspiration ou de dilatation et d'évacuation d'avortement (OR = 1,4 (IC95%: 0,29 à 6,4)). CONCLUSIONS: 16,2% des femmes présentant une transition ADN-VPH sont devenues positives au VPH. Les changements d'habitudes sexuelles augmentent le risque de développer une positivité au VPH.

French invasive Asian tiger mosquito populations harbor reduced bacterial microbiota and genetic diversity compared to Vietnamese autochthonous relatives.

The Asian tiger mosquito Aedes albopictus is one of the most significant pathogen vectors of the twenty-first century. Originating from Asia, it has invaded a wide range of eco-climatic regions worldwide. The insect-associated microbiota is now recognized to play a significant role in host biology. While genetic diversity bottlenecks are known to result from biological invasions, the resulting shifts in host-associated microbiota diversity has not been thoroughly investigated. To address this subject, we compared four autochthonous Ae. albopictus populations in Vietnam, the native area of Ae. albopictus, and three populations recently introduced to Metropolitan France, with the aim of documenting whether these populations display differences in host genotype and bacterial microbiota. Population-level genetic diversity (microsatellite markers and COI haplotype) and bacterial diversity (16S rDNA metabarcoding) were compared between field-caught mosquitoes. Bacterial microbiota from the whole insect bodies were largely dominated by Wolbachia pipientis. Targeted analysis of the gut microbiota revealed a greater bacterial diversity in which a fraction was common between French and Vietnamese populations. The genus Dysgonomonas was the most prevalent and abundant across all studied populations. Overall genetic diversities of both hosts and bacterial microbiota were significantly reduced in recently established populations of France compared to the autochthonous populations of Vietnam. These results open up many important avenues of investigation in order to link the process of geographical invasion to shifts in commensal and symbiotic microbiome communities, as such shifts may have dramatic impacts on the biology and/or vector competence of invading hematophagous insects.

Community-based control of Aedes aegypti by using Mesocyclops in southern Vietnam.

We previously reported a new community-based mosquito control strategy that resulted in elimination of Aedes aegypti (Linn.) in 40 of 46 communes in northern and central Vietnam, and with annual recurrent total costs (direct and indirect) of only $0.28-$0.89 international dollars per person. This control strategy was extended to four provinces in southern Vietnam in Long An and Hau Giang (2004-2007) and to Long An, Ben Tre, and Vinh Long (2005-2010). In a total of 14 communes with 124,743 residents, the mean ± SD of adult female Ae. aegypti was reduced from 0.93 ± 0.62 to 0.06 ± 0.09, and the reduction of immature Ae. aegypti averaged 98.8%. By the final survey, no adults could be collected in 6 of 14 communes, and one commune, Binh Thanh, also had no immature forms. Although the community-based programs also involved community education and clean-up campaigns, the prevalence of Mesocyclops in large water storage containers > 50 liters increased from 12.77 ± 8.39 to 75.69 ± 9.17% over periods of 15-45 months. At the conclusion of the study, no confirmed dengue cases were detected in four of the five communes for which diagnostic serologic analysis was performed. The rate of progress was faster in communes that were added in stages to the program but the reason for this finding was unclear. At the completion of the formal project, sustainability funds were set up to provide each commune with the financial means to ensure that community-based dengue control activities continued.

Effect of water supply system installation on distribution of water storage containers and abundance of Aedes aegypti immatures in urban premises of Ho Chi Minh City, Viet Nam.

To assess the effect of installation of a water supply system on the distribution of water storage containers and abundance of Aedes aegypti immatures, we conducted two successive entomological surveys in October 2007 and July 2008 in an urban neighbourhood of Ho Chi Minh City, Viet Nam. In all, 850 water-holding containers, including plastic buckets, water jars, concrete basins, flower vases, ant-traps, outdoor discards, used tyres and plant saucers were observed in 122 premises. Of these, 44 premises already had a water supply system installed at the time of the first survey (i.e. control premises). In the remaining 78 premises, a water supply system was installed 3–6 months before the second survey (i.e. intervention premises). There was a drastic reduction in the number of water storage containers, except concrete basins, between the first and second surveys. We also observed that the distribution of water storage containers in the control and intervention premises was similar. This was probably because the construction of a water supply system before the second survey increased the reliability of the system not only for the residents in intervention premises but also in the control premises (i.e. increased water pressure in the water supply system). The number of plastic buckets and water jars was reduced in the second survey; however, these containers were still an important source of Ae. aegypti pupae. For effective control of dengue infection, it is necessary to conduct entomological monitoring considering the seasonal changes in vector abundance and dengue transmission threshold after installing a water supply system

Second trimester abortion in Viet Nam: changing to recommended methods and improving service delivery.

In Viet Nam, abortion has been legal up to 22 weeks of pregnancy since the 1960s. There are about one million induced abortions every year. First trimester abortion is provided at central, provincial, district and commune level, while second trimester abortion is provided only at central and provincial level. For second trimester abortion, dilatation and evacuation (D&E) has been introduced at some central and provincial hospitals, and medical abortion protocols have been included in the draft National Standards and Guidelines currently being updated. However, Kovac's, an unsafe method, is still often used at many provincial hospitals. While access to first trimester abortion services is not difficult, there are still many barriers to second trimester abortion, especially for young, unmarried women. In order to prevent unwanted pregnancies, increase access to safe abortion and improve quality of care, the Vietnamese Ministry of Health is working with others to establish national policies and developing effective models for women-friendly comprehensive abortion care, including post-abortion family planning. This paper, based on published information, interviews and observations by the second author of service delivery in 2006-2008, provides an overview of second trimester abortion services in Viet Nam and ongoing plans for improving them.