scholarly journals In Vitro Reassortment between Endemic Bluetongue Viruses Features Global Shifts in Segment Frequencies and Preferred Segment Combinations

2021 ◽  
Vol 9 (2) ◽  
pp. 405
Author(s):  
Jennifer Kopanke ◽  
Justin Lee ◽  
Mark Stenglein ◽  
Christie Mayo
Keyword(s):  
Bluetongue Virus ◽  
In Vitro Propagation ◽  
Severe Disease ◽  
Whole Genome ◽  
Biting Midges ◽  
Genetic Changes ◽  
In Vitro System ◽  
Double Stranded Rna ◽  
The Usa

Bluetongue virus (BTV) is an arthropod-borne pathogen that is associated with sometimes severe disease in both domestic and wild ruminants. Predominantly transmitted by Culicoides spp. biting midges, BTV is composed of a segmented, double-stranded RNA genome. Vector expansion and viral genetic changes, such as reassortment between BTV strains, have been implicated as potential drivers of ongoing BTV expansion into previously BTV-free regions. We used an in vitro system to investigate the extent and flexibility of reassortment that can occur between two BTV strains that are considered enzootic to the USA, BTV-2 and BTV-10. Whole genome sequencing (WGS) was coupled with plaque isolation and a novel, amplicon-based sequencing approach to quantitate the viral genetic diversity generated across multiple generations of in vitro propagation. We found that BTV-2 and BTV-10 were able to reassort across multiple segments, but that a preferred BTV-2 viral backbone emerged in later passages and that certain segments were more likely to be found in reassortant progeny. Our findings indicate that there may be preferred segment combinations that emerge during BTV reassortment. Moreover, our work demonstrates the usefulness of WGS and amplicon-based sequencing approaches to improve understanding of the dynamics of reassortment among segmented viruses such as BTV.

scholarly journals The Genetic Diversification of a Single Bluetongue Virus Strain Using an In Vitro Model of Alternating-Host Transmission

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1038 ◽  
Author(s):  
Jennifer H. Kopanke ◽  
Justin S. Lee ◽  
Mark D. Stenglein ◽  
Christie E. Mayo
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genetic Stability ◽  
Bluetongue Virus ◽  
Genomic Structure ◽  
Field Isolate ◽  
Viral Population ◽  
Whole Genome ◽  
Single Nucleotide Variants

Bluetongue virus (BTV) is an arbovirus that has been associated with dramatic epizootics in both wild and domestic ruminants in recent decades. As a segmented, double-stranded RNA virus, BTV can evolve via several mechanisms due to its genomic structure. However, the effect of BTV’s alternating-host transmission cycle on the virus’s genetic diversification remains poorly understood. Whole genome sequencing approaches offer a platform for investigating the effect of host-alternation across all ten segments of BTV’s genome. To understand the role of alternating hosts in BTV’s genetic diversification, a field isolate was passaged under three different conditions: (i) serial passages in Culicoides sonorensis cells, (ii) serial passages in bovine pulmonary artery endothelial cells, or (iii) alternating passages between insect and bovine cells. Aliquots of virus were sequenced, and single nucleotide variants were identified. Measures of viral population genetics were used to quantify the genetic diversification that occurred. Two consensus variants in segments 5 and 10 occurred in virus from all three conditions. While variants arose across all passages, measures of genetic diversity remained largely similar across cell culture conditions. Despite passage in a relaxed in vitro system, we found that this BTV isolate exhibited genetic stability across passages and conditions. Our findings underscore the valuable role that whole genome sequencing may play in improving understanding of viral evolution and highlight the genetic stability of BTV.

scholarly journals Botrytis cinerea loss and restoration of virulence during in vitro culture follows flux in global DNA methylation

2016 ◽  
Author(s):  
Jimmy Breen ◽  
Luis Mur ◽  
Anushen Sivakumaran ◽  
Aderemi Akinyemi ◽  
Mike Wilkinson ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Pathogenic Fungi ◽  
Global Changes ◽  
Whole Genome ◽  
Genetic Changes ◽  
Whole Genome Bisulfite Sequencing ◽  
Differentially Methylated Genes ◽  
Genome Bisulfite Sequencing

Pathogenic fungi can lose virulence after protracted periods of culture but little is known of the underlying mechanisms. Here we present the first single-base resolution methylome for the plant pathogen B. cinerea and identify differentially methylated genes/genomic regions associated with virulence erosion. Cultures were maintained for eight months with subcultures and virulence testing every month. Methylation-sensitive amplified polymorphisms were performed at monthly intervals to characterise global changes to the pathogen's genome during culture and also on DNA from mycelium inoculated onto Arabidopsis thaliana after eight months in culture. Characterisation of culture-induced epialleles was assessed by whole-genome re-sequencing and whole-genome bisulfite sequencing. Virulence declined with time in culture and recovered after inoculation on A. thaliana. Variation detected by methylation-sensitive amplified polymorphisms followed virulence changes during culture. Whole-genome (bisulfite) sequencing showed marked changes on global and local methylation during culture but no significant genetic changes. We imply that virulence is a non-essential plastic character that is at least partly modified by changing levels of DNA methylation during culture. We hypothesise that changing DNA methylation during culture may be responsible for the high virulence/low virulence transition in B. cinerea and speculate that this may offer fresh opportunities to control pathogen virulence.

The emergence of macrocyclic lactone resistance in the canine heartworm, Dirofilaria immitis.

Parasitology ◽  
2015 ◽  
Vol 142 (10) ◽  
pp. 1249-1259 ◽  
Author(s):  
ADRIAN J. WOLSTENHOLME ◽  
CHRISTOPHER C. EVANS ◽  
PABLO D. JIMENEZ ◽  
ANDREW R. MOORHEAD
Keyword(s):  
Dirofilaria Immitis ◽  
Severe Disease ◽  
Mild Disease ◽  
Wild Canids ◽  
Heartworm Disease ◽  
Macrocyclic Lactone Resistance ◽  
The Face ◽  
The Usa ◽  
Clinical Consequences

SUMMARYPrevention of heartworm disease caused by Dirofilaria immitis in domestic dogs and cats relies on a single drug class, the macrocyclic lactones (MLs). Recently, it has been demonstrated that ML-resistant D. immitis are circulating in the Mississippi Delta region of the USA, but the prevalence and impact of these resistant parasites remains unknown. We review published studies that demonstrated resistance in D.immitis, along with our current understanding of its mechanisms. Efforts to develop in vitro tests for resistance have not yet yielded a suitable assay, so testing infected animals for microfilariae that persist in the face of ML treatment may be the best current option. Since the vast majority of D. immitis populations continue to be drug-sensitive, protected dogs are likely to be infected with only a few parasites and experience relatively mild disease. In cats, infection with small numbers of worms can cause severe disease and so the clinical consequences of drug resistance may be more severe. Since melarsomine dihydrochloride, the drug used to remove adult worms, is not an ML, the ML-resistance should have no impact on our ability to treat diseased animals. A large refugium of heartworms that are not exposed to drugs exists in unprotected dogs and in wild canids, which may limit the development and spread of resistance alleles.

scholarly journals In Vivo and in Vitro Propagation of Selected Cabbage (Brassica oleracea L. var. capitata) Clones

HortScience ◽  
1996 ◽  
Vol 31 (4) ◽  
pp. 628d-628
Author(s):  
José E.B.P. Pinto ◽  
Clovis M. Souza ◽  
W.R. Maluf
Keyword(s):  
Boric Acid ◽  
Vegetative Propagation ◽  
In Vitro Propagation ◽  
Basal Medium ◽  
Inbred Lines ◽  
Indolebutyric Acid ◽  
In Vitro System ◽  
Degree Of Differentiation

Hybrid cabbage cultivars can be produced via seed-propagated self-incompatible (SI) inbred lines, or, alternatively, via vegetative propagation of SI clones. Cabbage clones differ in their ability to undergo vegetative propagation, a fact that appears to be related to the degree of differentiation of the axillary buds inside the head. A procedure for in vivo and in vitro propagation is described for cabbage clones known for difficulty in undergoing vegetative propagation. Cuttings from clonal families 800 (easy-to-propagate) and 007 (difficult to propagate) were immersed in indolebutyric acid (IBA—0, 5, 25, and 125 mg·L–1) + boric acid (100 mg·L–1) + sucrose (20 g·L–1) for 15 hours and maintained in glasshouses. Induction of roots was more effective with 125 mg·L–1 IBA supplemented with boric acid and sucrose. This treatment showed the highest frequency of rooting and the largest number of roots per cutting. The in vitro system of propagation was performed on the basal medium of Murashige and Skoog (MS), to which triadizuron (TDZ), benzyladeninepurine (BAP), and kinetin (Kin) were added in different combinations. TDZ was more effective than BAP or Kin in the promotion of shoot regeneration.

scholarly journals Multiple Genome Segments Determine Virulence of Bluetongue Virus Serotype 8

2015 ◽  
Vol 89 (10) ◽  
pp. 5238-5249 ◽  
Author(s):  
Anna Janowicz ◽  
Marco Caporale ◽  
Andrew Shaw ◽  
Salvatore Gulletta ◽  
Luigina Di Gialleonardo ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Bluetongue Virus ◽  
Control Strategies ◽  
Animal Health ◽  
Hemorrhagic Disease ◽  
Genetic Changes ◽  
Virus Serotype ◽  
World Organization

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.

scholarly journals Field Performance of Conventional vs. in Vitro Propagules of Plantain (Musa spp., AAB Group)

HortScience ◽  
1996 ◽  
Vol 31 (5) ◽  
pp. 862-865 ◽  
Author(s):  
Dirk R. Vuylsteke ◽  
Rodomiro Ortiz
Keyword(s):  
Tissue Culture ◽  
Phenotypic Variation ◽  
In Vitro Propagation ◽  
Seed Set ◽  
Severe Disease ◽  
Field Performance ◽  
Musa Spp ◽  
Planting Material

In vitro-propagated plants of plantain (Musa spp., AAB group) did not manifest consistently superior horticultural performance compared to conventional propagules. Tissue culture plants grew vigorously and taller than sucker-propagated plants, but higher yield was not obtained, probably because of severe disease and suboptimal husbandry input. Phenotypic variation was higher in tissue culture plants, although this increase was not always statistically significant. There were no other detrimental effects of in vitro propagation on field performance. Botanical seed set rates for the two types of propagules were similar. The advantages of tissue-culture-derived plants as improved planting material would be most relevant for establishing field nurseries for further clean, conventional propagation of newly bred or selected genotypes.

scholarly journals Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in Arabidopsis

2021 ◽  
Vol 118 (34) ◽  
pp. e2102885118
Author(s):  
Manabu Yoshikawa ◽  
Yong-Woon Han ◽  
Hirofumi Fujii ◽  
Shu Aizawa ◽  
Tatsuya Nishino ◽  
...  
Keyword(s):  
Recognition System ◽  
Small Interfering Rnas ◽  
In Vitro System ◽  
Double Stranded Rna ◽  
Cleavage Fragment ◽  
Secondary Sirnas ◽  
Secondary Sirna ◽  
Interfering Rna ◽  
Target Rna

Small interfering RNAs (siRNAs) are often amplified from transcripts cleaved by RNA-induced silencing complexes (RISCs) containing a small RNA (sRNA) and an Argonaute protein. Amplified siRNAs, termed secondary siRNAs, are important for reinforcement of target repression. In plants, target cleavage by RISCs containing 22-nucleotide (nt) sRNA and Argonaute 1 (AGO1) triggers siRNA amplification. In this pathway, the cleavage fragment is converted into double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase 6 (RDR6), and the dsRNA is processed into siRNAs by Dicer-like proteins. Because nonspecific RDR6 recruitment causes nontarget siRNA production, it is critical that RDR6 is specifically recruited to the target RNA that serves as a template for dsRNA formation. Previous studies showed that Suppressor of Gene Silencing 3 (SGS3) binds and stabilizes 22-nt sRNA–containing AGO1 RISCs associated with cleaved target, but how RDR6 is recruited to targets cleaved by 22-nt sRNA–containing AGO1 RISCs remains unknown. Here, using cell-free extracts prepared from suspension-cultured Arabidopsis thaliana cells, we established an in vitro system for secondary siRNA production in which 22-nt siRNA–containing AGO1-RISCs but not 21-nt siRNA–containing AGO1-RISCs induce secondary siRNA production. In this system, addition of recombinant Silencing Defective 5 (SDE5) protein remarkably enhances secondary siRNA production. We show that RDR6 is recruited to a cleavage fragment by 22-nt siRNA–containing AGO1-RISCs in coordination with SGS3 and SDE5. The SGS3–SDE5–RDR6 multicomponent recognition system and the poly(A) tail inhibition may contribute to securing specificity of siRNA amplification.

In Vitro Assay of Platelet Adhesiveness with Washed and Tanned Human Red Cells

1968 ◽  
Vol 20 (03/04) ◽  
pp. 384-396 ◽  
Author(s):  
G Zbinden ◽  
S Tomlin
Keyword(s):  
Platelet Adhesion ◽  
Sodium Citrate ◽  
Blood Platelets ◽  
In Vitro Assay ◽  
Red Cells ◽  
Platelet Adhesiveness ◽  
Vitro Assay ◽  
In Vitro System ◽  
Human Red Cells

SummaryAn in vitro system is described in which adhesion of blood platelets to washed and tannic acid-treated red cells was assayed quantitatively by microscopic observation. ADP, epinephrine and TAME produced a reversible increase in platelet adhesiveness which was antagonized by AMP. With Evans blue, polyanetholsulfonate, phthalanilide NSC 38280, thrombin and heparin at concentrations above 1-4 u/ml the increase was irreversible. The ADP-induced increase in adhesiveness was inhibited by sodium citrate, EDTA, AMP, ATP and N-ethylmaleimide. EDTA, AMP and the SH-blocker N-ethylmaleimide also reduced spontaneous platelet adhesion to red cells. No significant effects were observed with adenosine, phenprocoumon, 5-HT, phthalanilide NSC 57155, various estrogens, progestogens and fatty acids, acetylsalicylic acid and similarly acting agents, hydroxylamine, glucose and KCN. The method may be useful for the screening of thrombogenic and antithrombotic properties of drugs.

The Mesothelial Cell as a Non-Thrombogenic Surface

1984 ◽  
Vol 52 (02) ◽  
pp. 102-104 ◽  
Author(s):  
L J Nicholson ◽  
J M F Clarke ◽  
R M Pittilo ◽  
S J Machin ◽  
N Woolf
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Blood Flow ◽  
Cell Surface ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Plastic Film ◽  
In Vitro System ◽  
Scanning Electron

SummaryA technique for harvesting mesothelial cells is described. This entails collagenase digestion of omentum after which the cells can be cultured. The technique has been developed using the rat, but has also been successfully applied to human tissue. Cultured rat mesothelial cells obtained in this way have been examined by scanning electron microscopy. Rat mesothelial cells grown on plastic film have been exposed to blood in an in vitro system using a Baumgartner chamber and have been demonstrated to support blood flow. No adhering platelets were observed on the mesothelial cell surface. Fibroblasts similarily exposed to blood as a control were washed off the plastic.

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