scholarly journals No part gets left behind: Tiled nanopore sequencing of whole ASFV genomes stitched together using Lilo

2021 ◽  
Author(s):  
Amanda Warr ◽  
Caitlin Newman ◽  
Nicky Craig ◽  
Ingrida Vendelė ◽  
Rizalee Pilare ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Low Cost ◽  
Hypervariable Region ◽  
Pcr Amplification ◽  
African Swine Fever ◽  
Pork Production ◽  
Long Time ◽  
Optimal Efficiency ◽  
Public Repositories ◽  
High Level

AbstractAfrican Swine Fever virus (ASFV) is the causative agent of a deadly, panzootic disease, infecting wild and domesticated suid populations. Contained for a long time to the African continent, an outbreak of a particularly infectious variant in Georgia in 2007 initiated the spread of the virus around the globe, severely impacting pork production and local economies. The virus is highly contagious and has a mortality of up to 100% in domestic pigs. It is critical to track the spread of the virus, detect variants associated with pathology, and implement biosecurity measures in the most effective way to limit its spread. Due to its size and other limitations, the 170-190kbp large DNA virus has not been well sequenced with fewer than 200 genome sequences available in public repositories. Here we present an efficient, low-cost method of sequencing ASFV at scale. The method uses tiled PCR amplification of the virus to achieve greater coverage, multiplexability and accuracy on a portable sequencer than achievable using shotgun sequencing. We also present Lilo, a pipeline for assembling tiled amplicon data from viral or microbial genomes without relying on polishing against a reference, allowing for structural variation and hypervariable region assembly other methods fail on. The resulting ASFV genomes are near complete, lacking only parts of the highly repetitive 3’- and 5’telomeric regions, and have a high level of accuracy. Our results will allow sequencing of ASFV at optimal efficiency and high throughput to monitor and act on the spread of the virus.

scholarly journals LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus

2021 ◽  
Author(s):  
Bo YANG ◽  
zhengwang shi ◽  
Yuan Ma ◽  
Lijuan Wang ◽  
Liyan Cao ◽  
...  
Keyword(s):  
Real Time ◽  
Detection System ◽  
African Swine Fever Virus ◽  
Low Cost ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Portable Detection ◽  
Real Time Qpcr

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a system was established in one tube for the detection of the ASFV p72 gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporters and CRISPR-derived RNA (crRNAs) were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples, a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. In the current study, a LAMP coupled with the CRISPR detection method was developed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

scholarly journals Cas12a-based on-site and rapid nucleic acid detection of African swine fever

2019 ◽  
Author(s):  
Jing Bai ◽  
Haosi Lin ◽  
Haojian Li ◽  
Yang Zhou ◽  
Junshan Liu ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Low Cost ◽  
African Swine Fever ◽  
Lateral Flow ◽  
Ease Of Use ◽  
Nucleic Acid Detection ◽  
Lateral Flow Strip ◽  
Trade Offs ◽  
Laboratory Facilities ◽  
Sensitivity Specificity

AbstractThe mortality rate of hemorrhagic African swine fever (ASF), which targets domestic pigs and is caused by African swine fever virus (ASFV), can reach 100%. ASF has been reported in 25 Chinese provinces since August 2018. There is no effective treatment or vaccine for it and the present molecular diagnosis technologies have trade-offs in sensitivity, specificity, cost and speed, and none of them cater perfectly to ASF control. Thus, a technology that overcomes the need for laboratory facilities, is relatively low cost, and rapidly and sensitively detects ASFV would be highly valuable. Here, we describe an RAA-Cas12a-based system that combines recombinase-aided amplification (RAA) and CRISPR/Cas12a for ASFV detection. The fluorescence intensity readout of this system detected ASFV p72 gene levels as low as 10 aM. For on-site ASFV detection, lateral-flow strip readout was introduced for the first time in the RAA-Cas12a based system (named CORDS, Cas12a-based On-site and Rapid Detection System). We used CORDS to detect target DNA highly specifically using the lateral-flow strip readout. CORDS could identify the p72 gene at femtomolar sensitivity in an hour at 37°C, and only requires an incubator. For ease of use, the regents of CORDS was lyophilized to three tubes and remained the same sensitivity when stored at 4 °C for at least 7 days. Thus, CORDS provides a rapid, sensitive and easily operable method for ASFV on-site detection. Lyophilized CORDS can withstand long-term transportation and storage, and is ready for field applications.

scholarly journals Comparative Analysis of Full Genome Sequences of African Swine Fever Virus Isolates Taken from Wild Boars in Russia in 2019

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 521
Author(s):  
Ali Mazloum ◽  
Antoinette van Schalkwyk ◽  
Andrey Shotin ◽  
Alexey Igolkin ◽  
Ivan Shevchenko ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Resolving Power ◽  
Full Genome ◽  
Wild Boars ◽  
Sequence Identity ◽  
High Level ◽  
Virus Isolates ◽  
Genotype Ii

In this study, we report on the full genome phylogenetic analysis of four ASFV isolates obtained from wild boars in Russia. These samples originated from two eastern and two western regions of Russia in 2019. Phylogenetic analysis indicated that the isolates were assigned to genotype II and grouped according to their geographical origins. The two eastern isolates shared 99.99% sequence identity with isolates from China, Poland, Belgium, and Moldova, whereas the western isolates had 99.98% sequence identity with isolates from Lithuania and the original Georgia 2007 isolate. Based on the full genome phylogenies, we identified three single locus targets, MGF-360-10L, MGF-505-9R, and I267L, that yielded the same resolving power as the full genomes. The ease of alignment and a high level of variation make these targets a suitable selection as additional molecular markers in future ASFV phylogenetic practices.

scholarly journals How One Pandemic Led To Another: Asfv, the Disruption Contributing To Sars-Cov-2 Emergence in Wuhan

2021 ◽  
Author(s):  
Wei Xia ◽  
Joseph Hughes ◽  
David Robertson ◽  
Xiaowei Jiang
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Pork Production ◽  
Price Increases ◽  
Production And Consumption ◽  
Before And After ◽  
Animal Contact ◽  
Non Local ◽  
Horseshoe Bat ◽  
Ecological Risk Factors

The spillover of a virus from one host species to another requires both molecular and ecological risk factors to align. While extensive research both before and after the emergence of SARS-CoV-2 in 2019 implicates horseshoe bat as the significant reservoir genus for the new coronavirus, it remains unclear why it emerged at this time. One massive disruption to human-animal contact in 2019 is linked to the on-going African swine fever virus (ASFV) pandemic. This began in Georgia in 2007 and was introduced to China in 2018. Pork is the major meat source in the Chinese diet. Severe fluctuations in the pork market prior to December 2019, may have increased the transmission of zoonotic pathogens, including severe acute respiratory syndrome–related coronaviruses, from wildlife to humans, wildlife to livestock and non-local animals to local animals. The major production and consumption regions for pork are geographically separated in China. The dramatic shortage of pork following restrictions of pig movement and culling resulted in price increases, leading to alternative meat consumption and unusual animal and meat movements nationwide such as wildlife and thus greatly increased opportunities for human-sarbecovirus contacts. Pork prices were particularly high in southern provinces (Guangdong, Guangxi, Fujian, Jiangxi, Hunan, and Hubei), where wildlife is farmed on different scales and more frequently consumed. Shandong experienced the biggest losses in pork production (~2 million metric tons), which is also the largest mink farming province. Hence, exposure of SARS-CoV-2 from wildlife or infected animals to humans by contact and consumption are more likely to have taken place in 2019, a year when China was experiencing the worst effects of the ASFV pandemic.

scholarly journals Development of Diagnostic Tests Provides Technical Support for the Control of African Swine Fever

Vaccines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 343
Author(s):  
Zilong Qiu ◽  
Zhaoyao Li ◽  
Quanhui Yan ◽  
Yuwan Li ◽  
Wenjie Xiong ◽  
...  
Keyword(s):  
Early Detection ◽  
African Swine Fever Virus ◽  
Control Strategies ◽  
African Swine Fever ◽  
Laboratory Diagnosis ◽  
Rapid Identification ◽  
Diagnostic Methods ◽  
Clinical Test ◽  
Pork Production ◽  
And Control

African swine fever is a highly contagious global disease caused by the African swine fever virus. Since African swine fever (ASF) was introduced to Georgia in 2007, it has spread to many Eurasian countries at an extremely fast speed. It has recently spread to China and other major pig-producing countries in southeast Asia, threatening global pork production and food security. As there is no available vaccine at present, prevention and control must be carried out based on early detection and strict biosecurity measures. Early detection should be based on the rapid identification of the disease on the spot, followed by laboratory diagnosis, which is essential for disease control. In this review, we introduced the prevalence, transmission routes, eradication control strategies, and diagnostic methods of ASF. We reviewed the various methods of diagnosing ASF, focusing on their technical characteristics and clinical test results. Finally, we give some prospects for improving the diagnosis strategy in the future.

scholarly journals A CRISPR/Cas12a Based Universal Lateral Flow Biosensor for the Sensitive and Specific Detection of African Swine-Fever Viruses in Whole Blood

Biosensors ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 203
Author(s):  
Jinghua Wu ◽  
Omar Mukama ◽  
Wei Wu ◽  
Zhiyuan Li ◽  
Jean De Dieu Habimana ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Simultaneous Detection ◽  
Pcr Amplification ◽  
African Swine Fever ◽  
Food Samples ◽  
Lateral Flow ◽  
Economic Threat ◽  
Polymerase Chain ◽  
High Fluorescence ◽  
Safety Applications

Cross-border pathogens such as the African swine fever virus (ASFV) still pose a socio-economic threat. Cheaper, faster, and accurate diagnostics are imperative for healthcare and food safety applications. Currently, the discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has paved the way for the diagnostics based on Cas13 and Cas12/14 that exhibit collateral cleavage of target and single-stranded DNA (ssDNA) reporter. The reporter is fluorescently labeled to report the presence of a target. These methods are powerful; however, fluorescence-based approaches require expensive apparatuses, complicate results readout, and exhibit high-fluorescence background. Here, we present a new CRISPR–Cas-based approach that combines polymerase chain reaction (PCR) amplification, Cas12a, and a probe-based lateral flow biosensor (LFB) for the simultaneous detection of seven types of ASFV. In the presence of ASFVs, the LFB responded to reporter trans-cleavage by naked eyes and achieved a sensitivity of 2.5 × 10−15 M within 2 h, and unambiguously identified ASFV from swine blood. This system uses less time for PCR pre-amplification and requires cheaper devices; thus, it can be applied to virus monitoring and food samples detection.

scholarly journals LAMP assay coupled with CRISPR/Cas12a system for portable detection of African swine fever virus

2021 ◽  
Author(s):  
Bo YANG ◽  
zhengwang shi ◽  
Yuan Ma ◽  
Lijuan Wang ◽  
Liyan Cao ◽  
...  
Keyword(s):  
Real Time ◽  
Detection System ◽  
African Swine Fever Virus ◽  
Low Cost ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Portable Detection ◽  
Real Time Qpcr

African swine fever (ASF) is one of the most severe infectious diseases of pigs. In this study, a LAMP assay coupled with the CRISPR Cas12a system was established in one tube for the detection of the ASFV p72 gene. The single-strand DNA-fluorophore-quencher (ssDNA-FQ) reporter and CRISPR-derived RNA (crRNAs) were screened and selected for the CRISPR detection system. In combination with LAMP amplification assay, the detection limit for the LAMP-CRISPR assay can reach 7 copies/μl of p72 gene per reaction. Furthermore, this method displays no cross-reactivity with other porcine DNA or RNA viruses. The performance of the LAMP-CRISPR assay was compared with real-time qPCR tests for clinical samples, a good consistency between the LAMP-CRISPR assay and real-time qPCR was observed. The method shed a light on the convenient, portable, low cost, highly sensitive and specific detection of ASFV, demonstrating a great application potential for monitoring on-site ASFV in the field.

scholarly journals Deletion of the H240R Gene of African Swine Fever Virus Decreases Infectious Progeny Virus Production due to Aberrant Virion Morphogenesis and Enhances the Inflammatory Cytokines Expression in Porcine Macrophages

2021 ◽  
Author(s):  
Pingping Zhou ◽  
Lian-Feng Li ◽  
Kehui Zhang ◽  
Bing Wang ◽  
Lijie Tang ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Inflammatory Cytokines ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Progeny Virus ◽  
Hemorrhagic Disease ◽  
High Level ◽  
Icosahedral Capsid ◽  
Infectious Progeny Virus

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus that causes African swine fever, a lethal hemorrhagic disease that currently threatens the pig industry. Recent studies have identified the viral structural proteins of infectious ASFV particles. However, the functional roles of several ASFV structural proteins remain largely unknown. Here, we characterized the function of the ASFV structural protein H240R (pH240R) in virus morphogenesis. pH240R was identified as a capsid protein using immunoelectron microscopy and interacted with the major capsid protein p72 by pulldown assays. Using a recombinant ASFV, ASFV-ΔH240R, with the H240R gene deletion from the wild-type ASFV (ASFV-WT) genome, we revealed that the infectious progeny virus titers were reduced by approximately 2.0 logs compared with ASFV-WT. Furthermore, we demonstrated that the growth defect was due to the generation of non-infectious particles with a high particle-to-infectious titer ratio in ASFV-ΔH240R-infected porcine primary alveolar macrophages (PAMs) than those of ASFV WT. Importantly, we found that pH240R did not affect virus-cell binding, endocytosis or egress but ASFV assembly; non-infectious virions containing large aberrant tubular and bilobulate structures, occupied nearly 98% of all virions were observed in ASFV-ΔH240R-infected PAMs by electron microscopy. Notably, we demonstrated that ASFV-ΔH240R infection induced high-level inflammatory cytokines expression in PAMs. Collectively, we show for the first time that pH240R is essential for ASFV icosahedral capsid formation and infectious particle production. Also, these results highlight the importance of pH240R in ASFV morphogenesis and provide a novel target for the development of ASF vaccines and antivirals. IMPORTANCE African swine fever is a lethal hemorrhagic disease of global concern that is caused by African swine fever virus (ASFV). Despite extensive research, there exist relevant gaps in knowledge of the fundamental biology of the viral life cycle. In this study, we identified pH240R as a capsid protein that interacts with the major capsid protein p72. Furthermore, we showed that pH240R was required for the efficient production of infectious progeny virus as indicated by the H240R- deleted ASFV mutant (ASFV-ΔH240R). More specifically, pH240R directs the morphogenesis of ASFV toward the icosahedral capsid in the process of assembly. In addition, ASFV-ΔH240R infection induced high-level inflammatory cytokines expression in porcine primary alveolar macrophages. Our results elucidate the role of pH240R in the process of ASFV assembly, which may instruct future research on effective vaccines or antiviral strategies.

scholarly journals Genotypic Characteristics of African Swine Fever Virus Maintained in Various Outbreaks in Vietnam During 2019-2021

2021 ◽  
Author(s):  
Nam Minh Nguyen ◽  
Tram Thi Ngoc Ngo ◽  
Duyen Thi Minh Nguyen ◽  
Hai Ngoc Nguyen ◽  
Trang Thi Phuong Nguyen ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
African Swine Fever Virus ◽  
Clinical Signs ◽  
Hypervariable Region ◽  
African Swine Fever ◽  
Amino Acid Sequences ◽  
Fever Virus ◽  
Southern Vietnam ◽  
Novel Variant ◽  
Genotypic Characteristics

Abstract This study aimed to identify potential genetic diversity among African swine fever virus (ASFV) strains circulating in central and southern Vietnam. Thirty ASFV strains were collected from domestic pigs and convalescent pigs with ASFV-infected clinical signs from 19 different provinces of central and southern Vietnam during 2019–2021. A portion of the B646L (p72) gene and the entire E183L (p54), CP204L (p30), and B602L (CVR) genes were amplified, purified, and sequenced. Web-based BLAST and MEGA-X software were used for sequence analysis. Analysis of the partial B646L (p72) gene, the full-length E183L (p54) and CP204L (p30) genes, and the central hypervariable region (CVR) of the B602L gene sequence showed that all 30 ASFV isolates belonged to genotype II and were 100% identical to the previously identified strains in Vietnam and China. Analysis of the p72, p54, and p30 regions did not indicate any change in the nucleotide and amino acid sequences among these strains in 3 years of research. No novel variant was found in the CVR within the B602L gene. Analysis of the CVR showed that these ASFV strains belong to subgroup XXXII. The results of this study revealed that these ASFVs shared high homology with ASFV isolates detected previously in northern Vietnam and China. Taken together, the results of this study and a previous study in Vietnam showed high stability and no genetic diversity in the ASFV genome.

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