First complete genome sequence of circulating dengue virus serotype 3 in Jeddah, Saudi Arabia.

Here we report the first full-length genome sequence of dengue virus serotype 3 (DENV-3) from a strain isolated from a patient in Jeddah, Saudi Arabia, in 2014. The genome consists of 10 635 bp and shows close similarity to circulating genotype III isolates from Singapore, suggesting possible importation, most probably during religious pilgrimages to Saudi Arabia.

Challenging the Wigglesworthia, Sodalis, Wolbachia symbiosis dogma in tsetse flies: Spiroplasma is present in both laboratory and natural populations.

Profiling of wild and laboratory tsetse populations using 16S rRNA gene amplicon sequencing allowed us to examine whether the "Wigglesworthia-Sodalis-Wolbachia dogma" operates across species and populations. The most abundant taxa, in wild and laboratory populations, were Wigglesworthia (the primary endosymbiont), Sodalis and Wolbachia as previously characterized. The species richness of the microbiota was greater in wild than laboratory populations. Spiroplasma was identified as a new symbiont exclusively in Glossina fuscipes fuscipes and G. tachinoides, members of the palpalis sub-group, and the infection prevalence in several laboratory and natural populations was surveyed. Multi locus sequencing typing (MLST) analysis identified two strains of tsetse-associated Spiroplasma, present in G. f. fuscipes and G. tachinoides. Spiroplasma density in G. f. fuscipes larva guts was significantly higher than in guts from teneral and 15-day old male and female adults. In gonads of teneral and 15-day old insects, Spiroplasma density was higher in testes than ovaries, and was significantly higher density in live versus prematurely deceased females indicating a potentially mutualistic association. Higher Spiroplasma density in testes than in ovaries was also detected by fluorescent in situ hybridization in G. f. fuscipes.

Genetic comparison of Glossina tachinoides populations in three river basins of the Upper West Region of Ghana and implications for tsetse control.

Tsetse flies are the cyclical vectors of African animal trypanosomosis (AAT) and human African trypanosomosis (HAT). In March 2010, the Government of Ghana initiated a large scale integrated tsetse eradication campaign in the Upper West Region (UWR) (≈18,000 km(2)) under the umbrella of the Pan-African Tsetse and Trypanosomosis Eradication Campaign (PATTEC). We investigated the structuring of Glossina tachinoides populations within and between the three main river basins of the target area in the UWR. Out of a total sample of 884 flies, a sub-sample of 266 was genotyped at nine microsatellite loci. The significance of the different hierarchical levels was tested using Yang's parameters estimated with Weir and Cockerham's method. A significant effect of traps within groups (pooling traps no more than 3 km distant from each other), of groups within river basins and of river basins within the whole target area was observed. Isolation by distance between traps was highly significant. A local density of 0.48-0.61 flies/m(2) was estimated and a dispersal distance that approximated 11 m per generation [CI 9, 17]. No significant sex-biased dispersal was detected. Dispersal distances of G. tachinoides in the UWR were relatively low, possibly as a result of the fragmentation of the habitat and the seasonality of the Kulpawn and Sissili rivers. Moreover, very high fly population densities were observed in the sample sites, which potentially reduces dispersal at constant habitat saturation, because the probability that migrants can established is reduced (density dependent dispersal). However, the observed spatial dispersal was deemed sufficient for a G. tachinoides-cleared area to be reinvaded from neighboring populations in adjacent river basins. These data corroborate results from other population genetics studies in West Africa, which indicate that G. tachinoides populations from different river basins cannot be considered isolated.

Prevalence and genetic variation of salivary gland hypertrophy virus in wild populations of the tsetse fly Glossina pallidipes from southern and eastern Africa.

The Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is a rod-shaped, non-occluded double-stranded DNA virus that causes salivary gland hypertrophy (SGH) and reduced fecundity in the tsetse fly G. pallidipes. High GpSGHV prevalence (up to 80%) makes it impossible to mass-rear G. pallidipes colonies for the sterile insect technique (SIT). To evaluate the feasibility of molecular-based GpSGHV management strategies, we investigated the prevalence and genetic diversity of GpSGHV in wild populations of G. pallidipes collected from ten geographical locations in eastern and southern Africa. Virus diversity was examined using a total sequence of 1497 nucleotides (≈ 1% of the GpSGHV genome) from five putative conserved ORFs, p74, pif1, pif2, pif3 and dnapol. Overall, 34.08% of the analyzed flies (n=1972) tested positive by nested PCR. GpSGHV prevalence varied from 2% to 100% from one location to another but phylogenetic and gene genealogy analyses using concatenated sequences of the five putative ORFs revealed low virus diversity. Although no correlation of the virus diversity to geographical locations was detected, the GpSGHV haplotypes could be assigned to one of two distinct clades. The reference (Tororo) haplotype was the most widely distributed, and was shared by 47 individuals in seven of the 11 locations. The Ethiopian haplotypes were restricted to one clade, and showed the highest divergence (with 14-16 single nucleotide mutation steps) from the reference haplotype. The current study suggests that the proposed molecular-based virus management strategies have a good prospect of working throughout eastern and southern Africa due to the low diversity of the GpSGHV strains.

Analysis of the antiviral drugs acyclovir and valacyclovir-hydrochloride in tsetse flies (Glossina pallidipes) using LC-MSMS.

A new simple, sensitive and precise liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of valacyclovir-HCl and acyclovir in tsetse flies (Glossina pallipides). Tsetse flies were extracted by ultrasonication with acidified methanol/acetonitrile, centrifuged and cleaned up by solid phase dispersion using MgSO(4) and MSPD C(18) material. Samples were analysed using a Waters Alliance 2695 series HPLC with a C(18) Gemini analytical column (150 mm x 4.6 mm x 5 microm) and a guard cartridge column connected to a Waters Quattro-Micro triple-quadrupole mass spectrometer. The isocratic mobile phase consisted of methanol:acetonitrile:water (60:30:10, v/v/v) plus formic acid (0.1%) at a flow rate of 0.25 ml/min. The precursor>product ion transition for valacyclovir (m/z 325.1>152) and acyclovir (m/z 226.1>151.9) were monitored in positive electrospray multiple reaction monitoring mode. The method was validated at fortification levels of 0.5, 1 and 2 microg/g. The range of calibration for both drugs was 0.45-4.5 microg/g. The overall accuracy of the method was 92% for valacyclovir and 95% for acyclovir with corresponding within-laboratory reproducibilities of 4.4 and 3.4%, respectively. Mean recoveries were above 80% for both drugs and repeatability ranged from 0.7 to 6.1%. For both drugs the limits of detection and quantification were 0.0625 and 0.2 microg/g, respectively. The method was applied in experiments on the mass rearing of tsetse flies for sterile insect technique (SIT) applications, in which the flies were fed with blood meals containing acyclovir or valcyclovir-HCl prior to analysis to assess effects on Glossina pallidipes Salivary Gland Hypertrophy syndrome.