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.

Development of a real‐time PCR assay for detection of African swine fever virus with an endogenous internal control

2020 ◽  
Vol 67 (6) ◽  
pp. 2446-2454 ◽  
Author(s):  
Yin Wang ◽  
Lizhe Xu ◽  
Lance Noll ◽  
Colin Stoy ◽  
Elizabeth Porter ◽  
...  
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Pcr Assay

scholarly journals Rapid Extraction and Detection of African Swine Fever Virus DNA Based on Isothermal Recombinase Polymerase Amplification Assay

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1731
Author(s):  
Arianna Ceruti ◽  
Rea Maja Kobialka ◽  
Judah Ssekitoleko ◽  
Julius Boniface Okuni ◽  
Sandra Blome ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Isolation ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Recombinase Polymerase Amplification ◽  
Reference Laboratory ◽  
Analytical Sensitivity ◽  
Lysis Buffer

African swine fever virus (ASFV) is the causative agent of a deadly disease in pigs and is spread rapidly across borders. Samples collected from suspected cases must be sent to the reference laboratory for diagnosis using polymerase chain reaction (PCR). In this study, we aimed to develop a simple DNA isolation step and real-time recombinase polymerase amplification (RPA) assay for rapid detection of ASFV. RPA assay based on the p72 encoding B646L gene of ASFV was established. The assays limit of detection and cross-reactivity were investigated. Diagnostic performance was examined using 73 blood and serum samples. Two extraction approaches were tested: silica-column-based extraction method and simple non-purification DNA isolation (lysis buffer and heating, 70 °C for 20 min). All results were compared with well-established real-time PCR. In a field deployment during a disease outbreak event in Uganda, 20 whole blood samples were tested. The assay’s analytical sensitivity was 3.5 DNA copies of molecular standard per µL as determined by probit analysis on eight independent assay runs. The ASFV RPA assay only detected ASFV genotypes. Compared to real-time PCR, RPA diagnostic sensitivity and specificity were 100%. Using the heating/lysis buffer extraction procedure, ASFV-RPA revealed better tolerance to inhibitors than real-time PCR (97% and 38% positivity rate, respectively). In Uganda, infected animals were identified before the appearance of fever. The ASFV-RPA assay is shown to be as sensitive and specific as real-time PCR. Moreover, the combination of the simple extraction protocol allows its use at the point of need to improve control measures.

scholarly journals A seven-gene-deleted African swine fever virus is safe and effective as a live attenuated vaccine in pigs

2020 ◽  
Vol 63 (5) ◽  
pp. 623-634 ◽  
Author(s):  
Weiye Chen ◽  
Dongming Zhao ◽  
Xijun He ◽  
Renqiang Liu ◽  
Zilong Wang ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Live Attenuated Vaccine ◽  
Attenuated Vaccine

Deletion of A137R gene from the pandemic strain of African swine fever virus is attenuated and offers protection against virulent pandemic virus

2021 ◽  
Author(s):  
Douglas P. Gladue ◽  
Elizabeth Ramirez-Medina ◽  
Elizabeth Vuono ◽  
Ediane Silva ◽  
Ayushi Rai ◽  
...  
Keyword(s):  
East Asia ◽  
Central Europe ◽  
Vaccine Candidate ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
Live Attenuated Vaccine ◽  
Attenuated Vaccine ◽  
Highly Virulent

African swine fever virus (ASFV) is causing a devastating pandemic in domestic and wild swine within an extended geographical area from Central Europe to East Asia resulting in economic losses for the regional swine industry. There are no commercial vaccines, therefore disease control relies on identification and culling of infected animals. We report here that the deletion of the ASFV gene A137R from the highly virulent ASFV-Georgia2010 (ASFV-G) isolate induces a significant attenuation of virus virulence in swine. A recombinant virus lacking the A137R gene, ASFV-G-ΔA137R, was developed to assess the role of this gene in ASFV virulence in domestic swine. Animals inoculated intramuscularly with 10 2 HAD 50 of ASFV-G-ΔA137R remained clinically healthy during the 28 day observational period. All animals inoculated with ASFV-G-ΔA137R had medium to high viremia titers and developed a strong virus-specific antibody response. Importantly, all ASFV-G-ΔA137R-inoculated animals were protected when challenged with the virulent parental strain ASFV-G. No evidence of replication of challenge virus was observed in the ASFV-G-ΔA137R-inoculated animals. Therefore, ASFV-G-ΔA137R is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce protection against the highly virulent ASFV Georgia virus that is the cause of the current Eurasian pandemic. Importance: No commercial vaccine is available to prevent African swine fever. The ASF pandemic caused by ASFV Georgia2007 (ASFV-G) is seriously affecting pork production in a contiguous area from Central Europe to East Asia. Here we report the rational development of a potential live attenuated vaccine strain by deleting a virus-specific gene, A137R, from the genome of ASFV-G. The resulting virus presented a completely attenuated phenotype and, importantly, animals infected with this genetically modified virus were protected from developing ASF after challenge with the virulent parental virus. ASFV-G-ΔA137R confers protection even at low doses (10 2 HAD 50 ) demonstrating its potential as a vaccine candidate. Therefore, ASFV-G-ΔA137R is a novel experimental ASF vaccine protecting pigs from the epidemiologically relevant ASFV Georgia isolate.

Development of the test kit for detection of African swine fever virus by Real-Time PCR

2021 ◽  
Vol 24 (11) ◽  
pp. 17-22
Author(s):  
N.I. Salnikov ◽  
◽  
M.E. Senina ◽  
A.S. Preobrazhenskaya ◽  
Z.S. Devrishova ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Test Kit

scholarly journals Research progress on live attenuated vaccine against African swine fever virus

2021 ◽  
pp. 105024
Author(s):  
Le Liu ◽  
Xiangwei Wang ◽  
Ruoqing Mao ◽  
Yahua Zhou ◽  
Juanbin Yin ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Research Progress ◽  
Live Attenuated Vaccine ◽  
Attenuated Vaccine

scholarly journals Evaluation of an Automatic Insulated Isothermal PCR System for Rapid and Reliable Field-Deployable Detection of African Swine Fever Virus

2021 ◽  
Author(s):  
TRAN THI HUYEN NGA ◽  
LE THI NHI-CONG ◽  
PHAM BANG PHUONG ◽  
LUU VAN QUYNH ◽  
Viet-Linh Nguyen
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
High Specificity ◽  
Performance Comparison ◽  
Fever Virus ◽  
Analytical Sensitivity ◽  
Specificity Test ◽  
Insulated Isothermal Pcr

Abstract In the present study, we evaluate an automatic sample-to-answer insulated isothermal PCR system for rapid and reliable field-deployable detection of African swine fever virus in comparison to that of OIE recommended real-time PCR counterparts with samples collected in Vietnam. For analytical sensitivity, the system could detect ASFV up to a dilution of 106 whereas the real time PCR systems could detect up to a dilution of 105 to 106. For specificity test, the system showed high specificity to ASFV in compare to different other types of swine pathogens: PRRSV, FMDV, PCV2, CSFV. The diagnostic performance comparison on 6 different types of samples showed 97.3% to 100% agreement with reference real-time PCR. The results of this study indicated that POCKIT Central-based insulated isothermal PCR system is a rapid, reliable and sample-flexible method for effective detection of ASFV.

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