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Development of large-scale mosquito densovirus production by in vivo methods.

ABSTRACT: BACKGROUND:Mosquito-borne diseases (MBDs) cause a significant proportion of the global infectious disease burden. Vector control remains the primary strategy available to reduce the transmission of MBDs. However, long-term, wide-scale and large-scale traditional chemical pesticide application has caused significant and increased negative effects on ecosystems and broader emerging insecticide resistance in vectors; therefore, the development of a novel alternative approach is urgently needed. Mosquito densoviruses (MDVs) are entomopathogenic viruses that exhibit a narrow host range and multiple transmission patterns, making MDVs a great potential bioinsecticide. However, the application process has been relatively stagnant over the past three decades. The major obstacle has been that viruses must be produced in mosquito cell lines; therefore, the production process is both expensive and time-consuming. METHODS:In our study, two wild-type (wt) MDVs, AaeDV and AalDV-3, and a recombinant rAaeDV-210 were used to infect the Aag2 and C6/36 mosquito cell lines and the 1st-2nd-instar and 3rd-4th-instar larvae of Ae. albopictus, Ae. aegypti and Cx. quinquefasciatus. Viral titers and yields in cells, media, larvae and rearing water and total viral yield were evaluated. Three kinds of virus displayed higher maximum virus titers in vivo than in vitro, and they displayed higher maximum viral yields in rearing water. RESULTS:The three viruses displayed higher total maximum viral yields in C6/36 cells than in Aag2 cells. The three viruses displayed higher total maximum viral yields in Aedes mosquitoes than in Culex mosquitoes. Higher viral yields were produced by 1st-2nd-instar larvae compared to 3rd-4th-instar larvae. The recombinant viruses did not display significantly lower yields than wt viruses in nearly all samples. In summary, by using 100 1st-2nd-instar Aedes mosquito larvae in 200 ml of rearing water, more than 1013 genome equivalents (geq) MDV yield can be obtained. CONCLUSIONS:Considering the lower production cost, this in vivo method has great potential for the large-scale production of MDVs. MDVs exhibit promising prospects and great potential for mosquito control in the future.

SUBMITTER: Sun Y

PROVIDER: S-EPMC6532183 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Development of large-scale mosquito densovirus production by in vivo methods.

Sun Yan Y Dong Yunqiao Y Li Jing J Lai Zetian Z Hao Yanqiang Y Liu Peiwen P Chen Xiaoguang X Gu Jinbao J

Parasites & vectors 20190522 1

<h4>Background</h4>Mosquito-borne diseases (MBDs) cause a significant proportion of the global infectious disease burden. Vector control remains the primary strategy available to reduce the transmission of MBDs. However, long-term, wide-scale and large-scale traditional chemical pesticide application has caused significant and increased negative effects on ecosystems and broader emerging insecticide resistance in vectors; therefore, the development of a novel alternative approach is urgently nee ...[more]

PMID: 31118088

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Project description:BACKGROUND:The analysis of large mosquito samples is expensive and time-consuming, delaying the efficient timing of vector control measurements. Processing a fraction of a sample using a subsampling method can significantly reduce the processing effort. However, a comprehensive evaluation of the reliability of different subsampling methods is missing. METHODS:A total of 23 large mosquito samples (397-4713 specimens per sample) were compared in order to evaluate five subsampling methods for the estimation of the number of specimens and species: area, volume, weight, selection of 200 random specimens and analyses with an image processing software. Each sample was distributed over a grid paper (21.0?×?29.7 cm; 25 grid cells of 4.2?×?5.9 cm) with 200 randomly distributed points. After taking pictures, mosquito specimens closest to each of the 200 points on the paper were selected. All mosquitoes per grid cell were identified by morphology and transferred to scaled tubes to estimate the volume. Finally, the fresh and dry weights were determined. RESULTS:The estimated number of specimens and species did not differ between the area-, volume- and weight-based method. Subsampling 20% of the sample gave an error rate of approximately 12% for the number of specimens, 6% for the proportion of the most abundant species and between 6-40% for the number of species per sample. The error for the estimated number of specimens using the picture processing software ImageJ gave a similar error rate when analyzing 15-20% of the total sample. By using 200 randomly selected specimens it was possible to give a precise estimation of the proportion of the most abundant species (r?=?0.97, P?<?0.001), but the number of species per sample was underestimated by 28% on average. Selecting adjacent grid cells instead of sampling randomly chosen grid cells and using dry weight instead of wet weight did not increase the accuracy of estimates. CONCLUSIONS:Different subsampling methods have various advantages and disadvantages. However, the area-based analysis of 20% of the sample is probably the most suitable approach for most kinds of mosquito studies, giving sufficiently precise estimations of the number of specimens and species, which is slightly less laborious compared to the other methods tested.

Project description:Hematopoietic stem and progenitor cells (HSPCs) have been cultured using a wide variety of cytokines to promote differentiation into megakaryocytic cells (Mks), the precursors to platelets. Greater Mk DNA content, or ploidy, has been correlated with increased platelet release. Gradients of pH, pO2, and signaling factors regulate megakaryopoiesis in the bone marrow niche. In this study, we demonstrate that a 3-phase culture process with increasing pH and pO2 and different cytokine cocktails greatly increases megakaryocyte production. CD34(+) HSPCs were first cultured at 5% O2 and pH 7.2 with a cytokine cocktail previously shown to promote Mk progenitor production. At day 5, cells were shifted to 20% O2 and pH 7.4 and maintained in 1 of 17 cytokine cocktails identified using a 2(4) factorial design of experiments method to evaluate the effects of interleukin (IL)-3, IL-6, IL-9, and high- or low-dose stem cell factor (SCF), in conjunction with thrombopoietin (Tpo) and IL-11, on expansion of mature Mks from progenitors. The combination of Tpo, high-dose SCF, IL-3, IL-9, and IL-11 best promoted Mk expansion. IL-3 greatly increased total cell fold expansion, but this was partially offset by lower Mk purity. IL-9 promoted CD41 and CD42b expression. High-dose (100?ng/mL) SCF increased Mk production and ploidy. Different commercial media and IL-3 sources substantially impacted differentiation, and X-VIVO 10 serum-free media best supported mature Mk expansion. Shifting from pH 7.4 to pH 7.6 at day 7 increased Mk production by 30%. Treatment with nicotinamide at day 7 or day 8 more than doubled the fraction of high-ploidy (>4N) Mks. Ultimately, the 3-phase culture system gave rise to 44.5±8.1?Mks and 8.5±3.1 high-ploidy Mks per input HSPC. Further optimization was required to improve platelet production. Using Iscove's modified Dulbecco's medium (IMDM)+20% BSA, insulin and transferin (BIT) 9500 Serum Substitute greatly improved the frequency and quality of Mk proplatelet extensions without affecting Mk expansion, commitment, or polyploidization in the 3-phase process. Mks cultured in IMDM+20% BIT 9500 gave rise to platelets with functional activity similar to that of fresh platelets from normal donors, as evidenced by basal tubulin distribution and the expression of surface markers and spreading in response to platelet agonists.

Project description:BackgroundNannochloropsis is a marine microalga that has been extensively studied. The major carotenoid produced by this group of microalgae is violaxanthin, which exhibits anti-inflammatory, anti-photoaging, and antiproliferative activities. Therefore, it has a wide range of potential applications. However, large-scale production of this pigment has not been much studied, thereby limiting its industrial application.ResultsTo develop a novel strain producing high amount of violaxanthin, various Nannochloropsis species were isolated from seawater samples and their violaxanthin production potential were compared. Of the strains tested, N. oceanica WS-1 exhibited the highest violaxanthin productivity; to further enhance the violaxanthin yield of WS-1, we performed gamma-ray-mediated random mutagenesis followed by colorimetric screening. As a result, Mutant M1 was selected because of its significant higher violaxanthin content and biomass productivity than WS-1 (5.21 ± 0.33 mg g- 1 and 0.2101 g L- 1 d- 1, respectively). Subsequently, we employed a 10 L-scale bioreactor to confirm the large-scale production potential of M1, and the results indicated a 43.54 % increase in violaxanthin production compared with WS-1. In addition, comparative transcriptomic analysis performed under normal light condition identified possible mechanisms associated with remediating photo-inhibitory damage and other key responses in M1, which seemed to at least partially explain enhanced violaxanthin content and delayed growth.ConclusionsNannochloropsis oceanica mutant (M1) with enhanced violaxanthin content was developed and its physiological characteristics were investigated. In addition, enhanced production of violaxanthin was demonstrated in the large-scale cultivation. Key transcriptomic responses that are seemingly associated with different physiological responses of M1 were elucidated under normal light condition, the details of which would guide ongoing efforts to further maximize the industrial potential of violaxanthin producing strains.

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Development of large-scale mosquito densovirus production by in vivo methods.

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Development of large-scale mosquito densovirus production by in vivo methods.

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