Multiple Genome Segments Determine Virulence of Bluetongue Virus Serotype 8

ABSTRACTBluetongue virus (BTV) causes bluetongue, a major hemorrhagic disease of ruminants. In order to investigate the molecular determinants of BTV virulence, we used a BTV8 strain minimally passaged in tissue culture (termed BTV8Lin this study) and a derivative strain passaged extensively in tissue culture (BTV8H) inin vitroandin vivostudies. BTV8Lwas pathogenic in both IFNAR−/−mice and in sheep, while BTV8Hwas attenuated in both species. To identify genetic changes which led to BTV8Hattenuation, we generated 34 reassortants between BTV8Land BTV8H. We found that partial attenuation of BTV8Lin IFNAR−/−mice was achieved by simply replacing genomic segment 2 (Seg2, encoding VP2) or Seg10 (encoding NS3) with the BTV8Hhomologous segments. Fully attenuated viruses required at least two genome segments from BTV8H, including Seg2 with either Seg1 (encoding VP1), Seg6 (encoding VP6 and NS4), or Seg10 (encoding NS3). Conversely, full reversion of virulence of BTV8Hrequired at least five genomic segments of BTV8L. We also demonstrated that BTV8Hacquired an increased affinity for glycosaminoglycan receptors during passaging in cell culture due to mutations in its VP2 protein. Replication of BTV8Hwas relatively poor in interferon (IFN)-competent primary ovine endothelial cells compared to replication of BTV8L, and this phenotype was determined by several viral genomic segments, including Seg4 and Seg9. This study demonstrated that multiple viral proteins contribute to BTV8 virulence. VP2 and NS3 are primary determinants of BTV pathogenesis, but VP1, VP5, VP4, VP6, and VP7 also contribute to virulence.IMPORTANCEBluetongue is one of the major infectious diseases of ruminants, and it is listed as a notifiable disease by the World Organization for Animal Health (OIE). The clinical outcome of BTV infection varies considerably and depends on environmental and host- and virus-specific factors. Over the years, BTV serotypes/strains with various degrees of virulence (including nonpathogenic strains) have been described in different geographical locations. However, no data are available to correlate the BTV genotype to virulence. This study shows that BTV virulence is determined by different viral genomic segments. The data obtained will help to characterize thoroughly the pathogenesis of bluetongue. The possibility to determine the pathogenicity of virus isolates on the basis of their genome sequences will help in the design of control strategies that fit the risk posed by new emerging BTV strains.

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Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets

International Journal of Molecular Sciences ◽

10.3390/ijms22168367 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8367

Author(s):

Hien Lau ◽

Shiri Li ◽

Nicole Corrales ◽

Samuel Rodriguez ◽

Mohammadreza Mohammadi ◽

...

Keyword(s):

Tissue Culture ◽

Oxygen Consumption ◽

Insulin Secretion ◽

Oxygen Consumption Rate ◽

Consumption Rate ◽

In Vitro Development ◽

Porcine Islets ◽

Vitro Development

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

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IN VITRO REPLICATION OF EPIZOOTIC HEMORRHAGIC DISEASE AND BLUETONGUE VIRUSES IN WHITE-TAILED DEER PERIPHERAL BLOOD MONONUCLEAR CELLS AND VIRUS-CELL ASSOCIATION DURING IN VIVO INFECTIONS

Journal of Wildlife Diseases ◽

10.7589/0090-3558-33.3.574 ◽

1997 ◽

Vol 33 (3) ◽

pp. 574-583 ◽

Cited By ~ 9

Author(s):

David E. Stallknecht ◽

Elizabeth W. Howerth ◽

M. Lisa Kellogg ◽

Charlotte F. Quist ◽

Tracy Pisell

Keyword(s):

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Hemorrhagic Disease ◽

Peripheral Blood Mononuclear ◽

In Vitro Replication ◽

Cell Association ◽

Blood Mononuclear Cells

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ELECTRON MICROSCOPY OF PLASMA-CELL TUMORS OF THE MOUSE

The Journal of Cell Biology ◽

10.1083/jcb.9.2.369 ◽

1961 ◽

Vol 9 (2) ◽

pp. 369-381 ◽

Cited By ~ 14

Author(s):

D. F. Parsons ◽

M. A. Bender ◽

E. B. Darden ◽

Guthrie T. Pratt ◽

D. L. Lindsley

Keyword(s):

Electron Microscopy ◽

Tissue Culture ◽

Complex Structure ◽

Granular Body ◽

Virus Particles ◽

Culture Cells ◽

Large Numbers ◽

Tumor Agent

The X5563 tumor has been grown in tissue culture. Cells similar to those of the original tumor migrated from the explant and attached to the glass walls of the culture vessels. Electron microscopy showed that large numbers of particles, similar in morphology to virus particles, were associated with these cells after 7 days of culture. The two principal types of particles found in the tumor in vivo appear to be present in vitro. Many more of these particles, however, were larger and showed a more complex structure. Whereas the particles were mainly localized inside endoplasmic reticulum or the Golgi zone in the tumors in vivo, in the tissue culture the majority of the particles were associated with the plasma membrane and were found outside of the cells. The relation of the particles to the granular body is discussed as well as a possible relation to the mammary tumor agent.

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In Vivo Interactions with (Tissue Culture Pretreated) Dermal Sheep Collagen

MRS Proceedings ◽

10.1557/proc-252-117 ◽

1991 ◽

Vol 252 ◽

Cited By ~ 3

Author(s):

P. B. van Wachem ◽

L. H. H. Olde Damink ◽

P. J. Dijkstra ◽

J. Feijen ◽

...

Keyword(s):

Tissue Culture ◽

Cell Death ◽

Marked Reduction ◽

Giant Cells ◽

In Vitro Cytotoxicity ◽

Cell Infiltration ◽

Cytotoxic Effects ◽

Lipid Formation

ABSTRACTPretreatment in tissue culture (TC) was previously found to markedly reduce the in vitro cytotoxicity of two types of crosslinked dermal sheep collagens (DSC's). This in vivo study confirms our in vitro results, in that TC-pretreatment of crosslinked DSC's resulted in the marked reduction or elimination of cytotoxic effects, such as increased cell infiltration, a deviant neutrophil-morphology, lipid formation and cell death. TC-pretreatment affected the crosslinked state of both DSC's in a different way, which could be deduced from the differences in gelatin-formation and presence of giant cells from macrophage- or fibroblast-origin. The results are explained in view of the differences in crosslinking.

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An Overview of Structural Aspects and Health Beneficial Effects of Antioxidant Oligosaccharides

Current Pharmaceutical Design ◽

10.2174/1381612824666180517120642 ◽

2020 ◽

Vol 26 (16) ◽

pp. 1759-1777 ◽

Cited By ~ 1

Author(s):

Tatiane F. Vieira ◽

Rúbia C. G. Corrêa ◽

Rosely A. Peralta ◽

Regina F. Peralta-Muniz-Moreira ◽

Adelar Bracht ◽

...

Keyword(s):

Antioxidant Activity ◽

Antioxidant Activities ◽

Sugar Content ◽

Animal Health ◽

Monosaccharide Composition ◽

Degree Of Polymerization ◽

Chemical Diversity ◽

In Vivo Studies

Background: Non-digestible oligosaccharides are versatile sources of chemical diversity, well known for their prebiotic actions, found naturally in plants or produced by chemical or enzymatic synthesis or by hydrolysis of polysaccharides. Compared to polyphenols or even polysaccharides, the antioxidant potential of oligosaccharides is still unexplored. The aim of the present work was to provide an up-to-date, broad and critical contribution on the topic of antioxidant oligosaccharides. Methods: The search was performed by crossing the words oligosaccharides and antioxidant. Whenever possible, attempts at establishing correlations between chemical structure and antioxidant activity were undertaken. Results: The most representative in vitro and in vivo studies were compiled in two tables. Chitooligosaccharides and xylooligosaccharides and their derivatives were the most studied up to now. The antioxidant activities of oligosaccharides depend on the degree of polymerization and the method used for depolymerization. Other factors influencing the antioxidant strength are solubility, monosaccharide composition, the type of glycosidic linkages of the side chains, molecular weight, reducing sugar content, the presence of phenolic groups such as ferulic acid, and the presence of uronic acid, among others. Modification of the antioxidant capacity of oligosaccharides has been achieved by adding diverse organic groups to their structures, thus increasing also the spectrum of potentially useful molecules. Conclusion: A great amount of high-quality evidence has been accumulating during the last decade in support of a meaningful antioxidant activity of oligosaccharides and derivatives. Ingestion of antioxidant oligosaccharides can be visualized as beneficial to human and animal health.

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Effect of China berry crude extracts on broad mites in vivo and in vitro

Journal of Biotechnology Research Center ◽

10.24126/jobrc.2008.2.1.19 ◽

2008 ◽

Vol 2 (1) ◽

pp. 12-18

Author(s):

Assma Gatta ◽

Luaay K. Al – ani ◽

Nabeel Al - ani

Keyword(s):

Biological Control ◽

Tissue Culture ◽

Melia Azedarach ◽

Concentration Increase ◽

Water Extracts ◽

Crude Extracts ◽

Number Increase ◽

Ethanol Extracts

Tissue culture were established from leaf and stem of china berry (Melia azedarach ) tree . Using MS media the best regulator to form callus were 6mg/l BAP, all other concentrations did not give callus . The crude extracts from leaves and callus established from leaves were extracted with water and ethanol with different concentrations. In ethanol extracts the least concentration 0.0001 half of the treated parasites were killed in 24 hours while the number increase as the concentration increase . However in callus the ethanol extracts were much higher about 8.5 were killed in the above concentration . In water extracts the least concentration 0.0001 killed half of the treated parasites in 24 hours .This number was increased 8 or 9 in 48 and 72 hours respectively . These results give us preliminary idea about the biological control of this dangerous parasite.

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183PREGNANCY RATE FOLLOWING TRANSFER OF IN VITRO- AND IN VIVO-PRODUCED BOVINE EMBRYOS TO LH-TREATED RECIPIENTS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab183 ◽

2004 ◽

Vol 16 (2) ◽

pp. 213 ◽

Cited By ~ 2

Author(s):

J. Small ◽

M. Colazo ◽

D. Ambrose ◽

R. Mapletoft ◽

J. Reeb ◽

...

Keyword(s):

Pregnancy Rate ◽

Animal Health ◽

Beef Cows ◽

Water Bath ◽

Bovine Embryos ◽

Chi Square ◽

Embryo Survival ◽

Frozen Embryos

The objective was to evaluate the effect of pLH treatment on pregnancy rates in recipients receiving in vivo- or in vitro-produced bovine embryos. Heifers (n=37) and lactating (n=28) and non-lactating (n=150) beef cows were treated at random stages of the cycle with 100μg GnRH i.m. (Cystorelin, Merial Canada Inc., Victoriaville, Quebec, Canada) on Day −9, 500μg cloprostenol i.m. (PGF; Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day —2 and GnRH on Day 0 (66h post-PGF; without estrus detection). Cattle were placed at random, by class, into three groups: no further treatment (Control; n=71), or 12.5mg pLH (Lutropin-V, Bioniche Animal Health, Belleville, Ontario, Canada) on Day 5 (n=72) or on Day 7 (n=72) after the second GnRH. On Day 7, cattle with a CL >10mm in diameter (determined ultrasonically) received in vivo-produced, fresh (Simmental) or frozen (Holstein), or in vitro-produced frozen (Holstein) embryos (embryo type balanced among groups). Embryos were cryopreserved in 10% ethylene glycol; in vivo-produced frozen embryos were thawed 5 to 10s in air, 15s in a water-bath at 30°C and then “direct-transferred” nonsurgically. In vitro-produced frozen embryos (donated by IND Lifetech Inc., Delta, British Columbia, Canada) were thawed in a water-bath at 27°C for 10s and placed in ViGro Holding Plus medium (AB Technology, Pullman, WA, USA) at room temperature, evaluated and then transferred nonsurgically. Pregnancy was determined by ultrasonography on Day 35. Data were analyzed with CATMOD, chi-square and GLM procedures (SAS Institute, Cary, NC, USA.). Twenty cattle (9.3%) did not receive embryos; five heifers had cervical problems, and five heifers and 10 cows did not have a CL >10mm. Overall, 7.1% of the recipients had two CL on the day of embryo transfer. There was no effect (P>0.05) of treatment, embryo type (or interaction) or class of recipient on pregnancy rate (overall, 44.1%, 86/195; Table 1). Similarly, mean (±SD) CL diameter and luteal area did not differ (P>0.05) among groups or between pregnant and open recipients (overall, 22.0±3.4mm and 352.0±108.7mm, respectively). However, recipients with a CL diameter ≥18mm tended (P<0.1) to have a higher pregnancy rate (45.8 vs 25.0%). In a subset of 40 recipients examined ultrasonically on Day 12, 50% of those treated on Day 5 and 70% of those treated with pLH on Day 7 had two CL. In summary, overall pregnancy rate in GnRH-synchronized recipients receiving in vitro- or in vivo-produced embryos by nonsurgical transfer was 44.1%. Embryo survival to Day 35 was not affected by type of embryo or treatment with pLH 5 or 7 days after ovulation. Table 1 Pregnancy rate in recipients on Day 35 based on pLH treatment and embryo-type

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Use of Tannin-Containing Plants as Antimicrobials Influencing the Animal Health

The Iraqi Journal of Veterinary Medicine ◽

10.30539/ijvm.v45i2.1258 ◽

2022 ◽

Vol 45 (2) ◽

pp. 33-40

Author(s):

Mohammed M Dakheel ◽

Afnan A Al-Mnaser ◽

Jessica Quijada ◽

Martin J Woodward ◽

Caroline Rymer

Keyword(s):

Nutritional Quality ◽

Condensed Tannins ◽

Animal Health ◽

Degree Of Polymerization ◽

Gram Negative Bacteria ◽

Animal Feeding ◽

The Subject

The antimicrobial effects of diverse tannin-containing plants, particularly condensed tannins (CTs) produced from various plants, are the subject of this study. CT components can be determined using CT-specific procedures such the HCl-Butanol Acetone assay, Thiolysis reaction, and HPLC/MS analysis. These methods indicate CT contents, including mean degree of polymerization, the procyanidins and prodelphinidins ratio (PC/PD%), the isomers of trans- and cis-, and CT concentration. Tannin-containing plants possess antibacterial action, which can be attributed to their protein linkage technique, and tannin-type variations, particularly CTs extract and their PC/PD%. The effects of CT components on the development of Gram-positive and Gram-negative bacteria have been documented for their relative PC/PD%; this is regarded to be a key predictor of tannin characteristics in terms of antimicrobials. In conclusion, tannins, more specific CT compositions, have significant impacts on in vivo trials of animal productions and utilization of metabolites and fermentation in vitro experiments. These findings need further investigations to fully understand how CT-types act on animal feeding in terms of enhanced nutritional quality of animal diets, which may have implications for human and animal health.

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Chimeric RNA:DNA Donorguide Improves HDR in vitro and in vivo

10.1101/2021.05.28.446234 ◽

2021 ◽

Author(s):

Brandon W Simone ◽

Han B Lee ◽

Camden L Daby ◽

Santiago Restrepo-Castillo ◽

Hirotaka Ata ◽

...

Keyword(s):

Cell Types ◽

Repair Process ◽

Strand Break ◽

Genetic Alterations ◽

Precise Integration ◽

Genetic Changes ◽

Area Of Interest ◽

Temporal Localization

Introducing small genetic changes to study specific mutations or reverting clinical mutations to wild type has been an area of interest in precision genomics for several years. In fact, it has been found that nearly 90% of all human pathogenic mutations are caused by small genetic variations, and the methods to efficiently and precisely correct these errors are critically important. One common way to make these small DNA changes is to provide a single stranded DNA (ssDNA) donor containing the desired alteration together with a targeted double-strand break (DSB) at the genomic target. The donor is typically flanked by regions of homology that are often ~30-100bp in length to leverage the homology directed repair (HDR) pathway. Coupling a ssDNA donor with a CRISPR-Cas9 to produce a targeted DSB is one of the most streamlined approaches to introduce small changes. However, in many cell types this approach results in a low rate of incorporation of the desired alteration and has undesired imprecise repair at the 5' or 3' junction due to artifacts of the DNA repair process. We herein report a technology that couples the spatial temporal localization of an ssDNA repair template and leverages the nucleic acid components of the CRISPR-Cas9 system. We show that by direct fusion of an ssDNA template to the trans activating RNA (tracrRNA) to generate an RNA-DNA chimera, termed Donorguide, we recover precise integration of genetic alterations, with both increased integration rates and decreased imprecision at the 5' or 3' junctions relative to an ssODN donor in vitro in HEK293T cells as well as in vivo in zebrafish. Further, we show that this technology can be used to enhance gene conversion with other gene editing tools such as TALENs.

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