scholarly journals Development of High-growth African Swine Fever Virus (ASFV) in MA-104 Cells

2021 ◽  
Author(s):  
Duy Tien Do ◽  
Toan Tat Nguyen
Keyword(s):  
Tissue Sample ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
High Growth ◽  
Fever Virus ◽  
Blood Samples ◽  
Vaccine Candidates ◽  
Ct Value ◽  
Tissue Homogenates ◽  
Blood Specimens

Abstract The goal of this study was to identify a candidate commercial cell line for the replication of African swine fever virus (ASFV) by comparing several available cell lines with various medium factors. In the sensitivity test of cells, MA104 and MARC-145 had strong potential for ASFV replication. Next, MA104 cells were used to compare the adaptation of ASFV obtained from tissue homogenates and blood samples in various infectious media. At the 10th passage, the ASFV obtained from the blood sample had a significantly higher viral load than that obtained from the tissue sample (P = 0.00), exhibiting a mean Ct value = 20.39 ± 1.99 compared with 25.36 ± 2.11. For blood samples, ASFV grew on infectious medium B more robustly than on infectious medium A (P = 0.006), corresponding to a Ct value = 19.58 ± 2.10 versus 21.20 ± 1.47. ASFV originating from blood specimens continued to multiply gradually and peaked in the 15th passage, exhibiting a Ct value = 14.36 ± 0.22 in infectious medium B and a Ct value = 15.42 ± 0.14 in infectious medium A. However, there was no difference (P = 0.062) in ASFV growth between infectious media A and B when ASFV was cultured from tissue homogenates. In this study, a model was developed to enhance ASFV replication through adaptation to MA104 cells and the lack of mutation in serial culture passages may serve to maintain the immunogenicity of ASFV isolates when they are developed as vaccine candidates.

scholarly journals Development of Optimized Protocol for Culturing African Swine Fever Virus (ASFV) Field Isolates in MA104 Cells

2021 ◽  
Author(s):  
Hyeok-il Kwon ◽  
DUY tien DO ◽  
Hung Van Vo ◽  
Seung-Chul Lee ◽  
Min-Ho kim ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Vaccine Development ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Hemorrhagic Disease ◽  
Blood Samples ◽  
Ct Value ◽  
Tissue Homogenates

Abstract I. Background: ASFV causes a highly contagious hemorrhagic disease with a high mortality rates in domestic pigs. The virus has been isolated across various cell lines, but identifying a cell line to develop an effective commercial vaccine has been challenging which a major obstacle to effective vaccine development is identifying a commercial cell line that is suitable for high-yield viral replication.II. Methods and Results: The goal of this study was to identify a candidate commercial cell line for the replication of African swine fever virus (ASFV) by comparing several available cell lines with various medium factors. In the sensitivity test of cells, MA104 and MARC-145 had strong potential for ASFV replication. Next, MA104 cells were used to compare the adaptation of ASFV obtained from tissue homogenates and blood samples in various infectious media. At the 10th passage, the ASFV obtained from the blood sample had a significantly higher viral load than that obtained from the tissue sample (P = 0.000), exhibiting a mean Ct value = 20.39 ± 1.99 compared with 25.36 ± 2.11. For blood samples, ASFV grew on infectious medium B more robustly than on infectious medium A (P = 0.006), corresponding to a Ct value = 19.58 ± 2.10 versus 21.20 ± 1.47. ASFV originating from blood specimens continued to multiply gradually and peaked in the 15th passage, exhibiting a Ct value = 14.36 ± 0.22 in infectious medium B and a Ct value = 15.42 ± 0.14 in infectious medium A. However, there was no difference (P = 0.062) in ASFV growth between infectious media A and B when ASFV was cultured from tissue homogenates. III. Conclusions: A model was developed to enhance ASFV replication through adaptation to MA104 cells and the lack of mutation in serial culture passages may serve to maintain the immunogenicity of ASFV isolates when they are developed as vaccine candidates.

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.

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 Molecular Detection of Torque Teno Sus Virus and Coinfection with African Swine Fever Virus in Blood Samples of Pigs from Some Slaughterhouses in Nigeria

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Pam D. Luka ◽  
Joseph Erume ◽  
Bitrus Yakubu ◽  
Olajide A. Owolodun ◽  
David Shamaki ◽  
...  
Keyword(s):  
Molecular Detection ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Comparative Sequence Analysis ◽  
Fever Virus ◽  
Nucleic Acid Detection ◽  
Viral Nucleic Acid ◽  
Blood Samples ◽  
Domestic Pigs ◽  
Torque Teno Sus Virus

Torque teno sus virus 1 (TTSuV1a/TTSuV1b) infection is present in pig herds worldwide. This study investigated the prevalence of TTSuV1a/TTSuV1b infections in domestic pigs from some slaughterhouses in Nigeria as well as coinfection with African swine fever virus (ASFV) and described the phylogeny in relation to global strains. One hundred and eighty-one (181) blood samples from four slaughterhouses were used for the study and viral nucleic acid detection was carried out by PCR. Comparative sequence analysis was carried out to infer phylogeny. The overall prevalence of TTSuV1a/b was 17.7%. Prevalence of individual genotypes was 10.5% and 7.2% for TTSuV1a and TTSuV1b, respectively. Coinfection of ASFV/TTSuV1a/b was 7.7% while that of TTSuV1a and TTSuV1b was 1.7%. ASFV alone was detected in 11.91% of the total samples. The Nigerian TTSuV1a and TTSuV1b shared a sequence identity of 91–100% and 95–100%, respectively, among each other. The ASFV sequences were 100% identical to members of genotype 1. This is the first report on the presence of TTSuV1a/b in domestic pigs in Nigeria and coinfection with ASFV. Although the prevalence of TTSuV1a/b in Nigeria was low, we recommend further studies to establish the trend and possible role in the pathogenesis of ASFV.

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 Natural Oil Blend Formulation (NOBF) as an anti- African Swine Fever Virus (ASFV) agent in Primary Porcine Alveolar Macrophases (PAM) cells of Swine

2020 ◽  
Author(s):  
Haig Yousef Babikian ◽  
Rajeev Kumar Jha ◽  
Quang Lam Truong ◽  
Lan Thi Nguyen ◽  
Hoa Thi Nguyen ◽  
...  
Keyword(s):  
Pinus Sylvestris ◽  
Eucalyptus Globulus ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Ct Value ◽  
Natural Oil ◽  
Oil Blend ◽  
Lavandula Latifolia

ABSTRACTAfrican swine fever is one of the severe pathogens of swine. It has a significant impact on production and on economics. So far, there are no known remedies, such as vaccines or drugs, reported. The natural oil blend formulation (NOBF) successfully tested against the African swine fever virus (ASFV) in in vitro conditions. The natural oil blend formulation (NOBF) combines Eucalyptus globulus, Pinus sylvestris, and Lavandula latifolia. We used a two-fold serial dilution to test the NOBF formulation dose. The in vitro trial results demonstrated that NOBF up to dilution 13 or 0.000625 ml deactivates the lethal dose 105HAD50 of ASFV. There was no hemadsorption (Rosetta formation) up to dilution 12 or 0.00125 ml of NOBF. The Ct value of the NOBF group at 96 hours post-infection was the same as the initial value or lower (25), whereas the Ct value of positive controls increased several folds (17.84). The in vitro trial demonstrated that NOBF can deactivate the African swine fever virus.HIGHLIGHTSThe natural oil blend formulation (NOBF) was formulated using three natural oils, i.e., Eucalyptus globulus, Pinus sylvestris, and Lavandula latifolia.The in vitro trial was conducted using porcine alveolar macrophages (PAMs) and further passaged in the PAMs; the stock used in the present study was that obtained after the 15th passage.The natural oil blend formulation (NOBF) showed protection against ASF virus up to a dilution of 13 or 0.000625 of a dilution of 16 or 0.000078 ml that was tried.The real-time PCR analysis showed that the virus did not replicate in the NOBF group, which implies that either ASFV growth was inhibited in the presence of NOBF or that it was inactivated.The in vitro trial outcome showed that NOBF has anti-ASFV properties.

Genetic markers of the African swine fever virus

2020 ◽  
Vol 23 (04) ◽  
pp. 21-26
Author(s):  
A.K. Sibgatullova ◽  
◽  
M.E. Vlasov ◽  
I.A. Titov ◽  
◽  
...  
Keyword(s):  
Genetic Markers ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus
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