scholarly journals DEVELOPMENT OF RECOMBINANT POSITIVE CONTROL FOR AFRICAN SWINE FEVER VIRUS PCR DETECTION

2020 ◽  
Vol 13 (6) ◽  
pp. 58-63
Author(s):  
M. Kit ◽  
Keyword(s):  
African Swine Fever Virus ◽  
Animal Health ◽  
African Swine Fever ◽  
Positive Control ◽  
Fever Virus ◽  
Laboratory Diagnostics ◽  
E Coli ◽  
Pcr Assays ◽  
World Organisation ◽  
Positive Controls

Recombinant plasmids containing target sequences are widely used as positive controls for PCR laboratory diagnostics. The aim of the study was development of recombinant positive control containing a fragment of B646L gene of African swine fever virus. The sequence of interest encodes targets of all the PCR assays for African swine fever laboratory diagnostics recommended by World Organisation for Animal Health. A plasmid containing 1763 bp insertion was cloned in E .coli DH5α strain. After purification, the plasmid ten-fold serial dulutions were used as a positive control while PRC testing. A minimal detectable copy number was 20 copies per reaction for both conventional and real-time PCR assays. The developed plasmid could be used as a safe and effective positive control while ASF laboratory diagnostics by PCR.

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 JOURNEY OF AFRICAN SWINE FEVER VIRUS IN EASTERN EU STATES

2018 ◽  
Vol 6 ◽  
pp. 863-869
Author(s):  
Stelian Baraitareanu ◽  
Dragos Cobzariu ◽  
Mihaela Popp ◽  
Marius Valer Campeanu ◽  
Doina Danes
Keyword(s):  
Czech Republic ◽  
African Swine Fever Virus ◽  
Animal Health ◽  
African Swine Fever ◽  
Fever Virus ◽  
The Black Sea ◽  
Eastern European ◽  
The European Union ◽  
The Czech Republic ◽  
Backyard Pigs

INTRODUCTION: In 2007, African swine fever virus (ASFv) broken its well-known boundaries. This was the reference year for the first report of African swine fever (ASF) in Georgia. Subsequently, the virus reached pigs and boars in Armenia and Russia. From the Caucasus area, ASFv jumped in all directions, between the Black Sea and the Caspian Sea, in relation to the density of backyard pigs and their trade. In the next ten years there have been notifications and registrations of ASFv outbreaks in Russia, Azerbaijan, Ukraine, Belarus, Lithuania, Poland, Estonia, Latvia, Moldova and the Czech Republic. Romania faced the first ASFv outbreak at the end of July 2017, in backyard pigs." in stead "density of backyard pigs and their trade. In the next ten years there have been notifications and registrations of ASFv outbreaks in Russia, Azerbaijan, Ukraine, Belarus, Lithuania, Poland, Estonia, Latvia, Moldova and the Czech Republic. Romania faced the first ASFv outbreak at the end of July 2017, in backyard pigs.OBJECTIVES: The aim of study is to analyse the ways ASFv spread from and into different regions recorded by Eastern European states.METHODS: The immediate notifications on ASFv to the World Organisation for Animal Health (OIE) were analysed from the Eastern-European states between 2007 and 2017. The analysis took into consideration the first occurrence of the disease under scrutiny in the country and the follow-up reports, in relation with the geospatial distribution of the outbreaks.RESULTS: The main route of ASFv introduction into local pig populations indicated by the Member States of the European Union was the trans-boundary circulation of boars. However, the spread of ASFv through both, wild and domestic pigs and also by the human alimentary customs/traditions in the affected areas shouldn’t be ignored. Three cycles of ASFv transmission have been identified and described by the epidemiologists: the domestic cycle, the sylvatic cycle and the tick-pig cycle.CONCLUSION: None of the ways to disseminate the ASFv should be excluded, and the origin of the first outbreaks remains unknown or inconclusive in Eastern EU states.

scholarly journals Effectiveness of Chemical Compounds Used against African Swine Fever Virus in Commercial Available Disinfectants

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 878
Author(s):  
Małgorzata Juszkiewicz ◽  
Marek Walczak ◽  
Natalia Mazur-Panasiuk ◽  
Grzegorz Woźniakowski
Keyword(s):  
Caustic Soda ◽  
Sodium Hypochlorite ◽  
African Swine Fever Virus ◽  
Animal Health ◽  
Virus Titer ◽  
African Swine Fever ◽  
Chemical Compounds ◽  
Fever Virus ◽  
Economic Losses ◽  
Potassium Peroxymonosulfate

African swine fever (ASF) causes huge economic losses and is one of most dangerous diseases of pigs. The disease is known for almost 100 years, an effective vaccine or treatment is still unavailable, only proper biosecurity measures, including disinfection, are being applied, in order to prevent disease outbreaks. Eight active substances, i.e., formaldehyde, sodium hypochlorite, caustic soda, glutaraldehyde, phenol, benzalkonium chloride, potassium peroxymonosulfate and acetic acid, were tested, in order to confirm their effectiveness against African swine fever virus (ASFV). This specific selection was done based on the World Organisation for Animal Health (OIE)’s recommendation and previous disinfectant studies on surfaces. The result of our study shows that most of them inactivate the virus, in recommended concentrations. In order to reduce the cytotoxicity of the four substances, Microspin S-400 HR columns were applied, therefore making it possible to demonstrate four logarithms virus titer reduction. Sodium hypochlorite, glutaraldehyde, caustic soda and potassium peroxymonosulfate showed the best ASFV inactivation rates, achieving titer reductions over 5 logs. Despite microfiltration, the virucidal activity of formaldehyde was not assessable, due to its high cytotoxicity. Our results showed that cleaning is particularly important, because removal of the soiling provides improved effectiveness of the tested chemical compounds.

scholarly journals Structure of African Swine Fever Virus and Associated Molecular Mechanisms Underlying Infection and Immunosuppression: A Review

2021 ◽  
Vol 12 ◽  
Author(s):  
Yue Wang ◽  
Weifang Kang ◽  
Wenping Yang ◽  
Jing Zhang ◽  
Dan Li ◽  
...  
Keyword(s):  
Immune Response ◽  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
African Swine Fever Virus ◽  
Animal Health ◽  
African Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
Cell Immune Response ◽  
Effective Vaccine

African swine fever (ASF) is an acute, highly contagious, and deadly infectious disease. The mortality rate of the most acute and acute ASF infection is almost 100%. The World Organization for Animal Health [Office International des épizooties (OIE)] lists it as a legally reported animal disease and China lists it as class I animal epidemic. Since the first diagnosed ASF case in China on August 3, 2018, it has caused huge economic losses to animal husbandry. ASF is caused by the African swine fever virus (ASFV), which is the only member of Asfarviridae family. ASFV is and the only insect-borne DNA virus belonging to the Nucleocytoplasmic Large DNA Viruses (NCLDV) family with an icosahedral structure and an envelope. Till date, there are still no effective vaccines or antiviral drugs for the prevention or treatment of ASF. The complex viral genome and its sophisticated ability to regulate the host immune response may be the reason for the difficulty in developing an effective vaccine. This review summarizes the recent findings on ASFV structure, the molecular mechanism of ASFV infection and immunosuppression, and ASFV-encoded proteins to provide comprehensive proteomic information for basic research on ASFV. In addition, it also analyzes the results of previous studies and speculations on the molecular mechanism of ASFV infection, which aids the study of the mechanism of clinical pathological phenomena, and provides a possible direction for an intensive study of ASFV infection mechanism. By summarizing the findings on molecular mechanism of ASFV- regulated host cell immune response, this review provides orientations and ideas for fundamental research on ASFV and provides a theoretical basis for the development of protective vaccines against ASFV.

scholarly journals Linear epitopes in African swine fever virus p72 recognized by monoclonal antibodies prepared against baculovirus-expressed antigen

2018 ◽  
Vol 30 (3) ◽  
pp. 406-412 ◽  
Author(s):  
Mallory E. Heimerman ◽  
Maria V. Murgia ◽  
Ping Wu ◽  
Andre D. Lowe ◽  
Wei Jia ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Major Capsid Protein ◽  
Vaccine Development ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
Fever Virus ◽  
E Coli ◽  
Important Target ◽  
Linear Epitopes

Protein p72 is the major capsid protein of African swine fever virus (ASFV) and is an important target for test and vaccine development. Monoclonal antibodies (mAbs) were prepared against a recombinant antigenic fragment, from amino acid (aa) 20–303, expressed in baculovirus. A total of 29 mAbs were recovered and tested by immunofluorescent antibody (IFA) staining on ASFV Lisbon-infected Vero cells. Six antibodies were IFA-positive and selected for further characterization. Epitope mapping was performed against overlapping polypeptides expressed in E. coli and oligopeptides. Based on oligopeptide recognition, the mAbs were divided into 4 groups: mAb 85 (aa 165–171); mAbs 65-3 and 6H9-1 (aa 265–280); mAbs 8F7-3 and 23 (aa 280–294); and mAb 4A4 (aa 290–303). All mAbs were located within a highly conserved region in p72. This panel of antibodies provides the opportunity to develop new assays for the detection of ASFV antibody and antigen.

scholarly journals Development Real-Time PCR Assays to Genetically Differentiate Vaccinated Pigs From Infected Pigs With the Eurasian Strain of African Swine Fever Virus

2021 ◽  
Vol 8 ◽  
Author(s):  
Lauro Velazquez-Salinas ◽  
Elizabeth Ramirez-Medina ◽  
Ayushi Rai ◽  
Sarah Pruitt ◽  
Elizabeth A. Vuono ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
African Swine Fever Virus ◽  
Vaccination Campaign ◽  
African Swine Fever ◽  
Fever Virus ◽  
Live Attenuated Vaccine ◽  
Vaccine Candidates ◽  
Attenuated Vaccine ◽  
Pcr Assays

Currently, African swine fever virus (ASFV) represents one of the most important economic threats for the global pork industry. Recently, significant advances have been made in the development of potential vaccine candidates to protect pigs against this virus. We have previously developed attenuated vaccine candidates by deleting critical viral genes associated with virulence. Here, we present the development of the accompanying genetic tests to discriminate between infected and vaccinated animals (DIVA), a necessity during an ASFV vaccination campaign. We describe here the development of three independent real-time polymerase chain reaction (qPCR) assays that detect the presence of MGF-360-12L, UK, and I177L genes, which were previously deleted from the highly virulent Georgia strain of ASFV to produce the three recombinant live attenuated vaccine candidates. When compared with the diagnostic reference qPCR that detects the p72 gene, all assays demonstrated comparable levels of sensitivity, specificity, and efficiency of amplification to detect presence/absence of the ASFV Georgia 2007/1 strain (prototype virus of the Eurasian lineage) from a panel of blood samples from naïve, vaccinated, and infected pigs. Collectively, the results of this study demonstrate the potential of these real-time PCR assays to be used as genetic DIVA tests, supporting vaccination campaigns associated with the use of ASFV-ΔMGF, ASFV-G-Δ9GL/ΔUK, and ASFV-ΔI177L or cell culture adapted ASFV-ΔI177LΔLVR live attenuated vaccines in the field.

scholarly journals ANALYSIS OF MONITORING STUDIES ON AFRICAN SWINE FEVER VIRUS DNA DETECTION CARRIED OUT IN 2017

2018 ◽  
pp. 21-25
Author(s):  
D. N. Fedoseyeva ◽  
Ye. V. Aronova ◽  
A. A. Varentsova ◽  
A. A. Yelsukova ◽  
Ali Mazloum ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Animal Health ◽  
African Swine Fever ◽  
Virus Genome ◽  
Fever Virus ◽  
Uniform Sampling ◽  
Development Organization ◽  
Wild Fauna ◽  
Federal Districts ◽  
First Time

The paper describes the results of testing of biomaterial from domestic pigs and wild boars by real-time PCR used for African swine fever virus genome detection, carried out in the FGBI “Federal Centre for Animal Health” (Vladimir). In 2017 8,500 samples from 44 subjects of the Russian Federation were tested within the framework of the state laboratory monitoring. African swine fever virus genome was detected in 504 samples. In 2017 ASF outbreaks were registered in the Urals and Siberian Federal Districts of the RF for the first time. The conducted research and persistent ASF infection in the territory of the RF have demonstrated the need for further surveillance in the populations of susceptible animals. Development, organization and implementation of the program for ASF spread surveillance in wild fauna remains a high priority. It is necessary to create and implement sampling schedules with uniform sampling of biomaterial and submission of the collected samples to the research laboratories for timely ASF outbreak containment at the regional level.

scholarly journals Immunizing Mice using Recombinant Truncated p72 Protein of African Swine Fever Virus and Establishment of an Indirect ELISA

2021 ◽  
Vol 25 (03) ◽  
pp. 609-614
Author(s):  
Jinliang Wang
Keyword(s):  
Prokaryotic Expression ◽  
Indirect Elisa ◽  
Serological Test ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
E Coli ◽  
Average Value ◽  
Coated Material ◽  
Prokaryotic Expression Vector

African swine fever (ASF) is a serious infectious pestilence characterized by bleeding in domestic pigs. Therefore, it is necessary to develop effective methods to diagnose this virus, serological detection of specific antibodies against ASFV infection is important for successful clinical diagnosis. In this study, E. coli was used to express the truncated P72 (tP72) gene cloned into the prokaryotic expression vector pET28a (+). Rosetta (DE3). An indirect ELISA assay which against African swine fever virus (ASFV) was established by using purification of recombinant tP72 protein as coated material for detection antibodies. Most effective in exhibiting positive result was observed when the coated material at a concentration of 3.625 μg/mL, serum was diluted to 1:160 and the concentration of HRP-conjugated secondary antibody was 1:2000. Our results showed that the method displayed an excellent specificity (100%) and better sensitivity (1:1600) during serological test based on the criterion of an average value plus three standard deviations. © 2021 Friends Science Publishers

scholarly journals Genotyping of African Swine Fever Virus (ASFV) Isolates in Romania with the First Report of Genotype II in Symptomatic Pigs

2021 ◽  
Vol 8 (12) ◽  
pp. 290
Author(s):  
Andrei Ungur ◽  
Cristina Daniela Cazan ◽  
Luciana Cătălina Panait ◽  
Marian Taulescu ◽  
Oana Maria Balmoș ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Animal Health ◽  
African Swine Fever ◽  
Fever Virus ◽  
Swine Industry ◽  
First Report ◽  
The World ◽  
First Time ◽  
Genotype Ii

The World Organisation for Animal Health has listed African swine fever as the most important deadly disease in domestic swine around the world. The virus was recently brought from South-East Africa to Georgia in 2007, and it has since expanded to Russia, Eastern Europe, China, and Southeast Asia, having a devastating impact on the global swine industry and economy. In this study, we report for the first time the molecular characterization of nine African swine fever virus (ASFV) isolates obtained from domestic pigs in Mureş County, Romania. All nine Romanian samples clustered within p72 genotype II and showed 100% identity with all compared isolates from Georgia, Armenia, Russia, Azerbaijan, Ukraine, Belarus, Lithuania, and Poland. This is the first report of ASFV genotype II in the country.

scholarly journals Regulated Emergence of the African swine fever virus: B646L (p72) Gene Based Bayesian Coalescent Analysis

2021 ◽  
Vol 11 (4) ◽  
Author(s):  
Venkataravana Prabhakarareddy Anapalli ◽  
SR Santosh Kumar ◽  
Suresh Kuralayanapalya Puttahonnappa ◽  
S Patil Sharanagouda ◽  
Indrabalan Uma Bharathi ◽  
...  
Keyword(s):  
Evolutionary Rate ◽  
African Swine Fever Virus ◽  
Animal Health ◽  
Evolutionary Process ◽  
African Swine Fever ◽  
Fever Virus ◽  
Virus Infections ◽  
Geographical Regions ◽  
Evolutionary Epidemiology ◽  
High Death

African swine fever virus (ASFV) belongs to the genus of virus of the Asfaviridae family. ASFV infection causes hemorrhage and high death rate hence increased loss to the swine community. It is a complex infectious disease of swine, which constitutes devastating impacts on animal health and the economy of the pig farmers. It has been confirmed that virus infections has been spreading in swine population for many years. In this study, the evolutionary epidemiology analysis of ASF virus from the geographical regions Africa, Europe, and Asia, respectively were retrieved from GenBank for the analysis. The nucleotide gene sequences of the viral protein p72 encoded by B646L gene published during 1960-2020 was taken in to study. The Bayesian skyline model with uncorrelated randomized clock model was employed to reconstruct the evolutionary history of the virus, to identify virus population demographics. Results of the analysis suggested that ASFV exhibited a high evolutionary rate, as the divergence caused reduction in the population in the recent years. The B646L gene of ASFV had an evolutionary rate of 4.13 X 10-6 substitution/site/year and the tMRCA as 3.15 x 105 with 95 percent HPD range in years (2.4 x 104 to 1.23 x 106) was obtained. In conclusion, the evolutionary study of ASFV with p72 protein from the ASFV of the B646L genes indicated that they evolved at a faster rate and plays a major role in the evolutionary process. Further, this study may help in designing or developing vaccines to control the spread of the disease.

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