Global research alliance in infectious disease: a collaborative effort to combat infectious diseases through dissemination of portable sequencing.

OBJECTIVE: To disseminate the portable sequencer MinION in developing countries for the main purpose of battling infectious diseases, we found a consortium called Global Research Alliance in Infectious Diseases (GRAID). By holding and inviting researchers both from developed and developing countries, we aim to train the participants with MinION's operations and foster a collaboration in infectious diseases researches. As a real-life example in which resources are limited, we describe here a result from a training course, a metagenomics analysis from two blood samples collected from a routine cattle surveillance in Kulan Progo District, Yogyakarta Province, Indonesia in 2019. RESULTS: One of the samples was successfully sequenced with enough sequencing yield for further analysis. After depleting the reads mapped to host DNA, the remaining reads were shown to map to Theileria orientalis using BLAST and OneCodex. Although the reads were also mapped to Clostridium botulinum, those were found to be artifacts derived from the cow genome. An effort to construct a consensus sequence was successful using a reference-based approach with Pomoxis. Hence, we concluded that the asymptomatic cow might be infected with T. orientalis and showed the usefulness of sequencing technology, specifically the MinION platform, in a developing country.

Insecticidal and Histopathological Effects of Ageratum conyzoides Weed Extracts against Dengue Vector, Aedes aegypti.

Crude extracts and essential oils of A. conyzoides were tested with larva and adult stages of Ae. aegypti mosquitoes to determine their insecticidal properties. The crude extracts and essential oils came from three varieties of A. conyzoides (with white flowers, purple flowers, or white-purple flowers) and from two places on each plant (leaves and flowers), giving six types overall: leaf-white (LW); leaf-purple (LP); leaf white-purple (LW-P); flower-white (FW); flower-purple (FP); and flower white-purple (FW-P). Chemical constituents and components of the essential oils were identified using gas chromatography-mass spectrometry (GC-MS). Electron microscopic and histopathological studies were performed to determine the toxicological effects on mosquitoes in terms of morphological alterations. The six types of crude extracts exhibited no activity against individuals in the larval stages. However, six types of essential oils were effective against adult Ae. aegypti females. The mortality of adult Ae. aegypti females was higher from leaf extracts, particularly LP (median lethal dose, LD50 = 0.84%). The number of chemical constituents identified by GC-MS was high in flowers, especially W-P. Precocene I was the most abundant chemical component among the five types of essential oils, except in LP, in which precocene II was the most abundant. Histopathological alterations in adult Ae. aegypti females included compound eye degeneration, muscular damage with cellular infiltration, gut epithelial degeneration and necrosis, pyknotic nuclei in the malpighian epithelium and ovarian cell degeneration. FW and FP plant types exhibited the highest severity of histopathological alterations in mosquitoes compared with other plants, probably owing to the presence of monoterpene compounds in their tissues. The present study demonstrated LP plant extracts from A. conyzoides could be effective adulticides against adult Ae. aegypti. As natural products are biodegradable and exhibit low toxicity to mammalian and non-target organisms, they are suitable candidates for use in vector control programmes.

Efficacy of Ageratum conyzoides extracts against Giardia duodenalis trophozoites: an experimental study.

BACKGROUND: Giardia duodenalis causes giardiasis in humans, particularly in developing countries. Despite the availability of treatments, resistance to some of the commercial anti-Giardia drugs has been reported in addition to their harmful side effects. Therefore, novel treatments for giardiasis are required. In this study, we aimed to assess the in vitro activity of crude extracts of Ageratum conyzoides against G. duodenalis trophozoites. METHODS: Plants were classified into three groups based on their flower colors: white (W), purple (P), and white-purple (W-P). Plants were separately cut into leaf (L) and flower (F) parts. Changes in internal organelle morphology of trophozoites following exposure to crude extracts were assessed using transmission electron microscopy (TEM). In subsequent experiments, efficacy of the most active essential oils from crude extracts [half maximal inhibitory concentrations (IC50) ≤ 100 µg/mL] against G. duodenalis trophozoites was tested. In vitro anti-Giardia assays using essential oils were performed in the same way as those performed using crude extracts. RESULTS: LW-P and FP extracts showed high activity (IC50 ≤ 100 µg/mL) against G. duodenalis trophozoites, with IC50 ± SD values of 45.67 ± 0.51 and 96.00 ± 0.46 µg/mL, respectively. In subsequent experiments, IC50 ± SD values of LW-P and FP essential oils were 35.00 ± 0.50 and 89.33 ± 0.41 µg/mL, respectively. TEM revealed the degeneration of flagella and ventral discs of G. duodenalis trophozoites following exposure to crude extracts. CONCLUSION: Crude LW-P and FP extracts of A. conyzoides showed the highest activity against G. duodenalis. Exposure to crude extract induced changes in the flagella and ventral discs of G. duodenalis trophozoites, which play important roles in attachment to the surface of mucosal cells. Our results suggest that the tested extracts warrant further research in terms of their efficacy and safety as giardiasis treatment.

Susceptibility of Aedes flavopictus miyarai and Aedes galloisi mosquito species in Japan to dengue type 2 virus

Objective: To evaluate the potential of local mosquitoes to act as vectors for dengue transmission in Japan. Methods: Serotype 2 ThNH28/93 was used to test the dengue susceptibility profiles of Aedes flavopictus miyarai (Ae. f. miyarai), Aedes galloisi (Ae. galloisi) and Aedes albopictus (Ae. albopictus), which were collected in Japan. We used Aedes aegypti from Thailand as a positive control. The mosquitoes were infected with the virus intrathoracically or orally. At 10 or 14 days post infection, the mosquitoes were dissected and total RNA was extracted from their abdomens, thoraxes, heads and legs. Mosquito susceptibility to dengue virus was evaluated using RT-PCR with dengue virus-specific primers. Differences in the infection and mortality rates of the different mosquito species were tested using Fisher's exact probability test. Results: The infection rates for dengue virus administered intrathoracically to Ae. f. miyarai, Ae. galloisi and Aedes aegypti mosquitoes were identical by RT-PCR on Day 10 post infection. All of the body parts we tested were RT-PCR-positive for dengue virus. For the orally admin-istered virus, the infection rates in the different body parts of the Ae. f. miyarai mosquitoes were slightly higher than those of Ae. albopictus mosquitoes, but were similar to the control mosquitoes (P>0.05). The mortality rates for Ae. f. miyarai and Ae. albopictus mosquitoes were similar (P=0.19). Our data indicated that dengue virus was able to replicate and disseminate to secondary infection sites in all of the four mosquito species (Japanese and Thai). Conclusions: Ae. albopictus is a well-known candidate for dengue transmission in Japan. However, our data suggest that Ae. f. miyarai from Ishigaki Island (near Okinawa Island) and Ae. galloisi from Hokkaido (Northern Japan) should also be regarded as potential vectors for dengue transmission in these regions. Further studies on these mosquitoes should be conducted.

Dengue-2 virus carrying capacity of Thai Aedes aegypti strains with different susceptibility to deltamethrin.

Deltamethrin-resistant Aedes aegypti currently threatens the effectiveness of dengue hemorrhagic fever control operations in Thailand. Although a previous study has suggested that insecticide resistance may increase Ae. aegypti susceptibility to dengue-2 virus infection, our experimental data showed no significant association between laboratory-induced deltamethrin-resistance in a Thai Ae. aegypti isolate and its susceptibility to dengue -2 infection.

Investigations on the role of a lysozyme from the malaria vector Anopheles dirus during malaria parasite development.

A cDNA encoding a lysozyme was obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) from females of the malaria vector Anopheles dirus A (Diptera: Culicidae). The 623 bp lysozyme (AdLys c-1) cDNA encodes the 120 amino acid mature protein with a predicted molecular mass of 13.4 kDa and theoretical pI of 8.45. Six cysteine residues and a potential calcium binding motif that are present in AdLys c-1 are highly conserved relative to those of c-type lysozymes found in other insects. RT-PCR analysis of the AdLys c-1 transcript revealed its presence at high levels in the salivary glands both in larval and adult stages and in the larval caecum. dsRNA mediated gene knockdown experiments were conducted to examine the potential role of this lysozyme during Plasmodium berghei infection. Silencing of AdLys c-1 resulted in a significant reduction in the number of oocysts as compared to control dsGFP injected mosquitoes.

Efficacy of three insecticides against Anopheles dirus and Anopheles minimus, the major malaria vectors, in Kanchanaburi Province, Thailand.

We conducted this study to determine the insecticide susceptibility of two malaria vectors, Anopheles dirus and Anopheles minimus from Kanchanaburi Province, Thailand. The mosquitoes were collected and reared under laboratory conditions. The test was carried out on unfed F-1 female mosquitoes using a standard WHO testing protocol. The LD50 and LD90 of deltamethrin in both species were tested for by exposing the mosquitoes to various doses of deltamethrin for 1 hour. The lethal time was also tested among mosquitoes by exposing them to deltamethrin (0.05%), permethrin (0.75%) and malathion (5%), for different exposure times, ranging from 0.5 to 15 minutes. Percent knockdown at 60 minutes and mortality at 24 hours were calculated. The resistance ratio (RR) was determined based on the LD50 and LT50 values. LD50 of deltamethrin against An. dirus and An. minimus were 0.00077% and 0.00066%, respectively. LT50 values for deltamethrin (0.05%), permethrin (0.75%) and malathion (5%) against An. dirus and An. minimus were 1.20, 3.16 and 10.07 minutes and 0.48, 1.92 and 5.94 minutes, respectively. The study revealed slightly increased tolerance by both mosquito species, compared with laboratory susceptible strains, based on LD50 values. The two anopheline species had the same patterns of response to the three insecticides, based on LT50 values, although the LT50 values were slightly higher in the An. dirus population. Both An. dirus and An. minimus were fully susceptible to all the insecticides tested, with 100% mortality at 24 hours post-exposure. Deltamethrin was the most effective insecticide, followed by permethrin and malathion.

Field-collected permethrin-resistant Aedes aegypti from central Thailand contain point mutations in the domain IIS6 of the sodium channel gene (KDR).

One of the mechanisms responsible for pyrethroid resistance in mosquitoes is mutations in domain IIS6 of voltage-gated sodium channel gene (kdr). Aedes aegypti larvae were collected from the central provinces of Thailand (Bangkok, Prachin Buri and Ratchaburi) and colonized until they became adults. Partial fragment of kdr of permethrin-resistant mosquitoes were amplified by RT-PCR and sequenced. Among the four nucleotide mutations detected, two mutations resulted in two amino acid substitutions, S(TCC) 989 P(CCC) and V(GTA)1016 G(GGA). Among 94 permethrin-resistant mosquitoes, the SS genotype (SS/VV) was found to predominate (n = 74), followed by SR (SP/VG) (n = 15) and RR (PP/ GG) genotypes (n = 5), with the resistant allele frequency ranging from 0.03 to 0.17. As pyrethroid insecticides are currently being advocated for use in Thailand, investigations of pyrethroid resistance in other regions of the country are needed to prevent potential cross-resistance among different types of insecticides.

Antioxidative systems defense against oxidative stress induced by blood meal in Aedes aegypti.

The release of iron from hemoglobin via the digestion of a blood meal in female mosquitoes can potentially induce oxidative damage and even death. These mosquitoes need an effective antioxidant to prevent this. We carried out this study to determine the antioxidant activities of ferritin, glutathione peroxidase (GPx), glutathione S-transferase (GST) and catalase, and glutathione (GSH). These enzymes had their greatest activity among 4 day old virgin female mosquitoes. Using a single blood feed model, groups of female mosquitoes were tested at 4, 7 and 20 days post-emergence. They were allowed to feed on a hamster for 1 hour. The engorged mosquitoes were collected at 48 and 72 hours after their blood meal. There were no changes in GSH, GPx, GST or catalase levels, but ferritin levels increased markedly (about 2-3 fold) by 48 hours post blood-feed in all mosquito age groups. On repeated blood-feed experiments, mosquitoes aged 4 days were blood fed, once every 3 days and were collected 48 hours after their most recent blood meal. A significant decrease in GSH and GPx activity and a further increase in ferritin, were detected. Ferritin levels were 0.19+/-0.03 and 0.14+/-0.02 ng/microg protein in the repeat and single blood-feed groups, respectively. These results suggest ferritin is an inducible, sensitive defense system protecting against oxidative stress caused by iron derived from blood meals in Aedes aegypti mosquitoes.

Competitive suppression between chikungunya and dengue virus in Aedes albopictus c6/36 cell line.

Aedes albopictus C6/36 cell line was used to evaluate dengue virus serotype-3 (DENV-3) and chikungunya virus (CHIKV) co-infection. Virus infection was determined using a one-step duplex reverse transcriptase polymerase chain reaction (D- RT-PCR). D-RT-PCR was positive for both viruses when equal multiplicity of infection (MOI) was utilized. Co-infection with different titers between a higher CHIKV titer (MOI = 1.0) than DENV-3 titer (MOI = 0.1) showed similar results with that of equal titer. However, co-infection with a lower CHIKV titer (MOI = 0.1) than DENV-3 titer (MOI = 1.0) revealed positive results for only dengue virus infection, suggesting DENV competitive suppression of CHIKV. This competitive suppression occurred in mixed-infection samples but not in individually double infection (super infection) samples which produced both dengue and chikungunya virus progenies. Both virus replications depend on the amount of virus titer rather than serial infection. These findings have provided information regarding pathogen-pathogen interaction in host cell, which could be used to predict outbreaks, and to develop virus detection and vector control.

Insecticide susceptibility of the dengue vector, Aedes aegypti (L.) in Metropolitan Bangkok.

Mosquito larvae were collected from the houses of dengue infected patients in Bangkok, Thailand from 55 sites (36 out of the 50 districts of Metropolitan Bangkok). Aedes aegypti larvae were tested against temephos using WHO bioassay techniques. Adult mosquitoes were tested for susceptibility to permethrin, deltamethrin, cyfluthrin, malathion and DDT using WHO diagnostic doses. Most of the larvae tested were susceptible to temephos. Only few specimens were resistant to temephos. Most adult mosquitoes were highly susceptible to malathion. Deltamethrin resistance was seen in 6 districts of Bangkok. Variable levels of susceptibility were seen with cyfluthrin. Most of the specimens showed resistance to permethrin and all specimens were resistant to DDT.

Comparative identification of protein profiles and major allergens of saliva, salivary gland and whole body extracts of mosquito species in Thailand.

Allergic reactions to mosquito bites, such as generalized urticaria or severe local reactions are common problems worldwide. The diverse sources of allergen prepared from different mosquito body parts usage are a major obstacle to obtaining safe and effective tests and immunotherapy for mosquito bite allergy. Thus, the reactions are often not recognized and allergen immunotherapy is seldom used for severe reaction to mosquito bites. In a search for appropriate allergen sources, the protein profiles of saliva, salivary glands and whole body extracts were comparatively analyzed from 4 common mosquito species of Thailand and/or South East Asia; viz. Culex quinquefasciatus, Aedes aegypti, Aedes albopictus and a zoophilic strain, Anopheles minimus. The major allergens in the extracts which elicited specific IgE responses in the pooled sera of subjects allergic to mosquito bites were identified. It was concluded that mosquito saliva was the best source of allergens. Additionally, both species-specific and species-shared allergens of the 4 mosquito species were identified. The major saliva allergens having MWs of 36, 32 and 22 kDa were identified. The identificstion of major allergens should facilitate the production of specific recombinant allergens and contribute to improvement in the diagnosis and specific immunotherapy of Thai mosquito bite allergy patients.

Bionomic status of Anopheles epiroticus Linton & Harbach, a coastal malaria vector, in Rayong Province, Thailand.

A longitudinal entomological survey was conducted to provide in-depth information on An. epiroticus and determine whether ecological and entomological factors could influence malaria transmission in Rayong Province, Thailand. The mosquitoes were collected monthly from May 2007 to April 2008 by human landing catch technique from 6:00-12:00 PM for 2 consecutive nights, at 3 collection sites. A total of 3,048 mosquitoes within 5 species were captured: An. epiroticus, Culex quinquefasciatus Say, Cx. sitiens Wiedemann, Aedes aegypti (L.) and Ae. albopictus Skuse. PCR was used for molecular identification of An. sundaicus complex, by determination of COI, ITS2, and D3 genes. The target mosquitoes were An. epiroticus, which was the predominant species, accounting for 43.8% of specimens collected. The biting cycle pattern increased during 6:00-8:00 PM and reached a maximum of 6.6 bites/person/hour by 12:00 PM. The mosquitoes varied in population density throughout the year. The highest biting rate was 37.6 bites/person/ half night in September and the lowest (10.2 bites/person/half night) in January. Nested PCR and real-time PCR techniques were used to detect the malaria parasite in An. epiroticus adult females. Nine of 926 (0.97%) mosquitoes tested were malaria parasite positive: 6 P. falciparum and 3 P. vivax. The infective mosquitoes were found in the dry and early rainy seasons. The overall annual entomological inoculation rate (EIR) in the village was 76.6. The overall parity rate was 74%. A total of 38 cement tanks were used to characterize the nature of the breeding places of An. epiroticus. An. epiroticus larvae coexisted with Aedes and Culex larvae; the maximum larval density was more than 140 larvae per dip in May. Breeding places included fresh, brackish and salt water, typically with full sunlight and mats of green algae on the water surface. The salinity of the water ranged from 0.5 to 119.4 g/l, with a narrow pH range of 8.2-8.7. Dissolved oxygen was highest in November (6.27 mg/l) and lowest in March (3.46 mg/l). The water temperature varied between 24.6 and 32.8 degrees C.

Climate associated size and shape changes in Aedes aegypti (Diptera: Culicidae) populations from Thailand.

In spite of the adult body size variability of Aedes aegypti (Linnaeus) and its likely association with life history and vectorial capacity, the causes of size variation itself have been only partially identified. In particular, possible important factors such as climatic variation have not received much attention. The objective of this 2-year study was to describe from field collections the relationship of Ae. aegypti metric properties with available climatic data. The study took place in a dengue hyperendemic area of Thailand. Fourth instar larvae (L(4)) and pupae were collected from the same breeding places allowing the comparisons between seven successive collections, four in 2007 and three in 2008. Climatic data were relative humidity (RH) and temperature (T). They were considered for the periods covering either the pre-imaginal development or, assuming heritability of size, the previous generation. The pre-imaginal period was further subdivided into embryonic and larval phases of development. Size was estimated by traditional and geometric techniques, the latter based on 18 landmarks collected at the intersections of veins also allowing estimation of shape. The shape variation of the wing followed similar patterns as for size and was shown to be a passive allometric change. No significant correlation of size or shape could be disclosed with T. In contrast, significant correlation with RH was found during two periods of examination: (i) the period affecting the generation previous to the time of collection, suggesting possible selective mechanisms on genitors, and (ii) the one occurring during pre-imaginal development. The subdivision of the latter into embryonic and larval phases allowed to evidence a possible selecting effect on embryonic development. The selection would act through the resistance to water loss which is known to depend on the relative surface of the cuticle. In conclusion, our data highlight the importance of the emerged period of Ae. aegypti eggs as a critical time for the size of future adults, and point to the relative humidity as the likely selecting factor.

Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome.

Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNA(val) promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes.

Residual effects of Mossmann 100 (permethrin 10% EC) impregnated bed nets and its impact on malaria vectors and incidence of malaria.

The objective of this field trial was to assess the residual effectiveness of permethrin 10% treated mosquito nets on malaria control compared with untreated nets. The study was carried out between July and December 2007 in the Pong Nam Ron District of Chantaburi Province, Thailand. Mosquito population densities were assessed using the landing catch method. Mosquitoes were collected between 6:00 PM and 12:00 PM. Residual effectiveness of the treated nets was assessed using standard WHO bioassay tests carried out monthly using Anopheles dirus mosquitoes reared in the insectary of the Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Thailand. The results showed the population densities of Anopheles spp, including the malaria vector Anopheles minimus, were unaffected in the study area where mosquito nets treated with Mossmann 100 (permethrin 10% EC) at 300 mg/m2 were used. WHO bioassay tests showed the nets treated with Mossmann 100 remained biologically effective against An. dirus for up to six months. Indigenous cases of malaria were reduced by 27.7% at the site where the nets treated with Mossman 100 (permethrin 10%EC) were used but no changes in malaria cases at the control site were seen.

Novel virus discovery in field-collected mosquito larvae using an improved system for rapid determination of viral RNA sequences (RDV ver4.0).

In this study, we improved a method for rapid determination of viral RNA sequences (RDV) to overcome the limitations of previous versions. The RDV ver4.0 method can detect RNA sequences with at least 1,000 copies as starting material. A novel virus, which was isolated from field-collected Aedes aegypti larvae in the Phasi Charoen district of Thailand using C6/36 cells, was identified using the RDV ver4.0 protocol. The virus was named Phasi Charoen virus (PhaV). We used a high-throughput pyrosequencing approach to obtain more information about the genome sequence of PhaV. Analysis of a phylogenic tree based on amino acid sequences strongly suggested that PhaV belongs to the family Bunyaviridae.

Residual effect of 10% bifenthrin wp on mosquitoes, and community acceptance, in eastern Thailand.

This study was conducted from May to October 2008 in two villages in Chanthaburi Province: village No. 2 Tup Sai Canton (control) and village No.12 Pong Nam Ron (treatment area). Indoor residual spraying, using 10% bifenthrin WP (Bitecthrin WP) was conducted at a concentration of 25 mg/m2 with 87.3% spray coverage of the houses in the treated area. Monthly entomological studies showed that in the control area, Anopheles minimus density was significantly higher than the treatment area. A WHO cone bioassay test showed the residual effect against laboratory-bred, An. dirus persisted for up to 6 months. Community acceptability was good and most preferred insecticide spraying. 10% bifenthrin WP applied six-monthly can be used as an indoor residual spray for malaria control.

Intramolecular integration assay validates integrase phi C31 and R4 potential in a variety of insect cells.

Phage phi C31 and R4 integrases are site-specific and unidirectional serine recombinases. We have analyzed the ability of these integrases to mediate intramolecular integration between their attB and attP sites in 7 important insect cell lines as a means of predicting their relative mobility in the corresponding insect species. Both integrases exhibit significantly higher frequencies in Drosophila S2 cells than in the other insect cell lines examined, but do work well in all of the species tested. Our results, coupled with previous results of the activity of phi C31 integrase in D. melanogaster and Aedes aegypti, suggest the family of serine catalyzed integrases will be useful site-specific integration tools for functional genome analysis and genetic engineering in a wide range of insect species.

Rapid determination of viral RNA sequences in mosquitoes collected in the field.

A method for rapid determination of viral RNA sequences (RDV) was applied to homogenates of Aedes aegypti collected in Thailand in an area in which dengue fever (dengue hemorrhagic fever) is endemic, using the mosquito cell line C6/36. Nucleic acid sequences of dengue virus type 4 and cell fusing agent virus were detected. This RDV method has the potential to become a standard method for detection of both known and newly emerging, unknown mosquito-borne viruses.