scholarly journals Optimizing Release of Nucleic Acids of African Swine Fever Virus and Influenza A Virus from FTA Cards

2021 ◽  
Vol 22 (23) ◽  
pp. 12915
Author(s):  
Ahmed Elnagar ◽  
Timm C. Harder ◽  
Sandra Blome ◽  
Martin Beer ◽  
Bernd Hoffmann
Keyword(s):  
Nucleic Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Universal Method ◽  
Viral Dna ◽  
Dna And Rna ◽  
Fta Cards

FTA cards and related products simplify the collection, transport, and transient storage of biological sample fluids. Here, we have compared the yield and quality of DNA and RNA released from seven different FTA cards using seven releasing/extraction methods with eleven experimental eluates. For the validation, dilution series of African swine fever virus (ASFV) positive EDTA blood and Influenza A virus (IAV) positive allantoic fluid were used. Based on our data, we conclude that direct PCR amplification without the need for additional nucleic acid extraction and purification could be suitable and more convenient for ASFV DNA release from FTA cards. In contrast, IAV RNA loads can be amplified from FTA card punches if a standard extraction procedure including a lysis step is applied. These differences between the amplifiable viral DNA and RNA after releasing and extraction are not influenced by the type of commercial FTA card or the eleven different nucleic acid releasing procedures used for the comparative analyses. In general, different commercial FTA cards were successfully used for the storage and recovery of the ASFV and IAV genetic material suitable for PCR. Nevertheless, the usage of optimized nucleic acid releasing protocols could improve the recovery of the viral genome of both viruses. Here, the application of Chelex® Resin 100 buffer mixed with 1 × Tris EDTA buffer (TE, pH 8.0) or with TED 10 (TE buffer and Dimethylsulfoxid) delivered the best results and can be used as a universal method for releasing viral DNA and RNA from FTA cards.

Genetic variation of african swine fever virus: Variable regions near the ends of the viral DNA

Virology ◽  
1989 ◽  
Vol 173 (1) ◽  
pp. 251-257 ◽  
Author(s):  
Rafael Blasco ◽  
Inmaculada de la Vega ◽  
Fernando Almazan ◽  
Montserrat Aguero ◽  
Eladio Viñuela
Keyword(s):  
Genetic Variation ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Viral Dna ◽  
Variable Regions

scholarly journals A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1129 ◽  
Author(s):  
Ferenc Olasz ◽  
István Mészáros ◽  
Szilvia Marton ◽  
Győző L. Kaján ◽  
Vivien Tamás ◽  
...  
Keyword(s):  
Sample Preparation ◽  
African Swine Fever Virus ◽  
Animal Health ◽  
African Swine Fever ◽  
Fever Virus ◽  
Whole Genome ◽  
Swine Industry ◽  
Viral Dna ◽  
Simple Method ◽  
Generation Sequencing

In the recent years, African swine fever has become the biggest animal health threat to the swine industry. To facilitate quick genetic analysis of its causative agent, the African swine fever virus (ASFV), we developed a simple and efficient method for next generation sequencing of the viral DNA. Execution of the protocol does not demand complicated virus purification steps, enrichment of the virus by ultracentrifugation or of the viral DNA by ASFV-specific PCRs, and minimizes the use of Sanger sequencing. Efficient DNA-se treatment, monitoring of sample preparation by qPCR, and whole genome amplification are the key elements of the method. Through detailed description of sequencing of the first Hungarian ASFV isolate (ASFV_HU_2018), we specify the sensitive steps and supply key reference numbers to assist reproducibility and to facilitate the successful use of the method for other ASFV researchers.

scholarly journals An Isothermal Molecular Point of Care Testing for African Swine Fever Virus Using Recombinase-Aided Amplification and Lateral Flow Assay Without the Need to Extract Nucleic Acids in Blood

2021 ◽  
Vol 11 ◽  
Author(s):  
Yuhang Zhang ◽  
Qingmei Li ◽  
Junqing Guo ◽  
Dongliang Li ◽  
Li Wang ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Point Of Care ◽  
African Swine Fever ◽  
Lateral Flow ◽  
Fever Virus ◽  
Lateral Flow Assay ◽  
Economic Losses ◽  
Point Of Care Testing ◽  
Viral Dna ◽  
Blood Samples

African swine fever (ASF) is a highly contagious and usually deadly porcine infectious disease listed as a notifiable disease by the World Organization for Animal Health (OIE). It has brought huge economic losses worldwide, especially since 2018, the first outbreak in China. As there are still no effective vaccines available to date, diagnosis of ASF is essential for its surveillance and control, especially in areas far from city with limited resources and poor settings. In this study, a sensitive, specific, rapid, and simple molecular point of care testing for African swine fever virus (ASFV) B646L gene in blood samples was established, including treatment of blood samples with simple dilution and boiling for 5 min, isothermal amplification with recombinase-aided amplification (RAA) at 37°C in a water bath for 10 min, and visual readout with lateral flow assay (LFA) at room temperature for 10–15 min. Without the need to extract viral DNA in blood samples, the intact workflow from sampling to final diagnostic decision can be completed with minimal equipment requirement in 30 min. The detection limit of RAA-LFA for synthesized B646L gene-containing plasmid was 10 copies/μl, which was 10-fold more sensitive than OIE-recommended PCR and quantitative PCR. In addition, no positive readout of RAA-LFA was observed in testing classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, pseudorabies virus and porcine circovirus 2, exhibiting good specificity. Evaluation of clinical blood samples of RAA-LFA showed 100% coincident rate with OIE-recommended PCR, in testing both extracted DNAs and treated bloods. We also found that some components in blood samples greatly inhibited PCR performance, but had little effect on RAA. Inhibitory effect can be eliminated when blood was diluted at least 32–64-fold for direct PCR, while only a 2–4 fold dilution of blood was suitable for direct RAA, indicating RAA is a better choice than PCR when blood is used as detecting sample. Taken together, we established an sensitive, specific, rapid, and simple RAA-LFA for ASFV molecular detection without the need to extract viral DNA, providing a good choice for point of care testing of ASF diagnosis in the future.

Evidence for the type of nucleic acid in African swine fever virus

1966 ◽  
Vol 19 (3) ◽  
pp. 289-304 ◽  
Author(s):  
W. Plowright ◽  
F. Brown ◽  
J. Parker
Keyword(s):  
Nucleic Acid ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus

scholarly journals High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system

2019 ◽  
Author(s):  
Qian He ◽  
Dongmei Yu ◽  
Mengdi Bao ◽  
Grant Korensky ◽  
Juhong Chen ◽  
...  
Keyword(s):  
Detection Limit ◽  
Nucleic Acid ◽  
High Throughput ◽  
African Swine Fever Virus ◽  
Point Of Care ◽  
African Swine Fever ◽  
Fever Virus ◽  
Solution Phase ◽  
Nucleic Acid Amplification ◽  
Dna Complex

AbstractHere we report the development of a high throughput, all-solution phase, and isothermal detection system to detect African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with fluorescence-based point-of-care (POC) system we developed for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 hrs. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 hrs of incubation, resulting in an improvement of the detection limit to 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact, multiplexing, and simple ASFV detection, intended for low resource settings.

Detection of African Swine Fever Virus DNA in Blood Samples Stored on FTA Cards from Asymptomatic Pigs in Mbeya Region, Tanzania

2013 ◽  
Vol 62 (1) ◽  
pp. 87-90 ◽  
Author(s):  
U. C. Braae ◽  
M. V. Johansen ◽  
H. A. Ngowi ◽  
T. B. Rasmussen ◽  
J. Nielsen ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Blood Samples ◽  
Fta Cards

scholarly journals Complete genome sequence of an African swine fever virus (ASFV POL/2015/Podlaskie) determined directly from pig erythrocyte-associated nucleic acid

2018 ◽  
Vol 261 ◽  
pp. 14-16 ◽  
Author(s):  
Ann Sofie Olesen ◽  
Louise Lohse ◽  
Marlene Danner Dalgaard ◽  
Grzegorz Woźniakowski ◽  
Graham J. Belsham ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus

scholarly journals DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription

2017 ◽  
Vol 91 (12) ◽  
Author(s):  
Gonçalo Frouco ◽  
Ferdinando B. Freitas ◽  
João Coelho ◽  
Alexandre Leitão ◽  
Carlos Martins ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Topoisomerase Ii ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Dna Supercoiling ◽  
Fever Virus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Replication Sites

ABSTRACT African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (−78.3%), and reduced transcription of a late viral gene (−47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV. IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently available. Remarkably, ASFV is the only known mammalian virus that putatively codes for a histone-like protein (pA104R) that shares extensive sequence homology with bacterial histone-like proteins. In this study, we characterized the DNA-binding properties of pA104R, analyzed the functional importance of two conserved residues, and showed that pA104R and ASFV topoisomerase II cooperate and display DNA-supercoiling activity. Moreover, pA104R is expressed during the late phase of infection and accumulates in viral DNA replication sites, and its downregulation revealed that pA104R is required for viral DNA replication and transcription. These results suggest that pA104R participates in the modulation of viral DNA topology and genome packaging, indicating that A104R deletion mutants may be a good strategy for vaccine development against ASFV.

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