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Article: Deletion of the ASFV dUTPase Gene E165R from the Genome of Highly Virulent African Swine Fever Virus Georgia 2010 Does Not Affect Virus Replication or Virulence in Domestic Pigs

Vuono, Elizabeth A. / Ramirez-Medina, Elizabeth / Pruitt, Sarah / Rai, Ayushi / Espinoza, Nallely / Silva, Ediane / Velazquez-Salinas, Lauro / Gladue, Douglas P. / Borca, Manuel V.

Viruses. 2022 June 28, v. 14, no. 7

2022

Abstract: African swine fever (ASF) is a frequently lethal disease of domestic and wild swine currently producing a pandemic affecting pig production in Eurasia. The causative agent, ASF virus (ASFV) is a structurally complex virus with a large genome harboring ... ...

Abstract	African swine fever (ASF) is a frequently lethal disease of domestic and wild swine currently producing a pandemic affecting pig production in Eurasia. The causative agent, ASF virus (ASFV) is a structurally complex virus with a large genome harboring over 150 genes. One of them, E165R, encodes for a protein belonging to the dUTPase family. The fine structure of the purified protein has been recently analyzed and its dUTPase activity tested. In addition, it has been reported that a BA71 mutant virus, adapted to growth in Vero cells, lacking the E165R gene presented a drastic decreased replication in swine macrophages, its natural target cell. Herein, we report the development of a recombinant virus, ASFV-G-∆E165R, harboring the deletion of the E165R gene from the genome of the highly virulent field isolate ASFV Georgia 2010 (ASFV-G). Interestingly, ASFV-G-∆E165R replicates in primary swine macrophage cultures as efficiently as the parental virus ASFV-G. In addition, ASFV-G-∆E165R also replicates in experimentally inoculated domestic pigs with equal efficacy as ASFV-G and produced a lethal disease almost indistinguishable from that induced by the parental virus. Therefore, results presented here clearly demonstrated that E165R gene is not essential or important for ASFV replication in swine macrophages nor disease production in domestic pigs.
Keywords	African swine fever ; African swine fever virus ; etiological agents ; genes ; macrophages ; mutants ; pandemic ; swine ; swine production ; virulence ; virus replication ; viruses ; Eurasia ; Georgia
Language	English
Dates of publication	2022-0628
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2516098-9
ISSN	1999-4915
ISSN	1999-4915
DOI	10.3390/v14071409
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Evaluation of the Function of ASFV Gene E66L in the Process of Virus Replication and Virulence in Swine.

Ramirez-Medina, Elizabeth / Vuono, Elizabeth A / Rai, Ayushi / Espinoza, Nallely / Valladares, Alyssa / Spinard, Edward / Velazquez-Salinas, Lauro / Gladue, Douglas P / Borca, Manuel V

Viruses

2023 Volume 15, Issue 2

Abstract: African swine fever virus (ASFV) is the etiological agent of an economically important disease of swine currently affecting large areas of Africa, Eurasia and the Caribbean. ASFV has a complex structure harboring a large dsDNA genome which encodes for ... ...

Abstract	African swine fever virus (ASFV) is the etiological agent of an economically important disease of swine currently affecting large areas of Africa, Eurasia and the Caribbean. ASFV has a complex structure harboring a large dsDNA genome which encodes for more than 160 proteins. One of the proteins, E66L, has recently been involved in arresting gene transcription in the infected host cell. Here, we investigate the role of E66L in the processes of virus replication in swine macrophages and disease production in domestic swine. A recombinant ASFV was developed (ASFV-G-∆E66L), from the virulent parental Georgia 2010 isolate (ASFV-G), harboring the deletion of the E66L gene as a tool to assess the role of the gene. ASFV-G-∆E66L showed that the E66L gene is non-essential for ASFV replication in primary swine macrophages when compared with the parental highly virulent field isolate ASFV-G. Additionally, domestic pigs infected with ASFV-G-∆E66L developed a clinical disease undistinguishable from that produced by ASFV-G. Therefore, E66L is not involved in virus replication or virulence in domestic pigs.
MeSH term(s)	Swine ; Animals ; African Swine Fever Virus/genetics ; Virulence ; Sus scrofa ; Virus Replication ; Africa
Language	English
Publishing date	2023-02-18
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v15020566
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Confirming the absence of parental African swine fever virus as a potential contaminant of recombinant live attenuated ASF vaccines.

Velazquez-Salinas, Lauro / Ramirez-Medina, Elizabeth / Rai, Ayushi / Pruitt, Sarah / Vuono, Elizabeth A / Espinoza, Nallely / Gay, Cyril G / Witte, Steve / Gladue, Douglas P / Borca, Manuel V

Biologicals : journal of the International Association of Biological Standardization

2023 Volume 83, Page(s) 101685

Abstract: African swine fever (ASF) is a devastating disease that is currently producing a panzootic significantly impacting the swine industry worldwide. One of the major challenges for advancing the development of ASF vaccines has been the absence of ... ...

Abstract	African swine fever (ASF) is a devastating disease that is currently producing a panzootic significantly impacting the swine industry worldwide. One of the major challenges for advancing the development of ASF vaccines has been the absence of international standards for ASF vaccine purity, potency, safety, and efficacy. To date, the most effective experimental vaccines have been live attenuated strains of viruses. Most of these promising vaccine candidates have been developed by deleting virus genes involved in the process of viral pathogenesis and disease production. This approach requires genomic modification of a parental virus field strain through a process of homologous recombination followed by purification of the recombinant attenuated virus. In this scenario, it is critical to confirm the absence of any parental virulent virus in the final virus stock used for vaccine production. We present here a protocol to establish the purity of virus stock using the live attenuated vaccine candidates ASFV-G-ΔMGF, ASFV-G-Δ9 GLΔUK and ASFV-G-ΔI177L. Procedures described here includes inoculation in susceptible pigs followed by the assessment of the obtained material by differential qPCRs that allows the identification of vaccine virus from ASFV field isolates. This protocol is proposed as a model to ensure that master seed virus stock used for vaccine production does not contain residual parental virulent virus. Procedures described here includes a passage in susceptible pigs followed by the assessment of the obtained material by differential qPCRs that allows the identification of vaccine virus from ASFV field isolates.
MeSH term(s)	Swine ; Animals ; African Swine Fever Virus/genetics ; African Swine Fever/prevention & control ; Vaccines, Attenuated ; Virulence ; Viral Proteins/genetics ; Vaccines, Synthetic
Chemical Substances	Vaccines, Attenuated ; Viral Proteins ; Vaccines, Synthetic
Language	English
Publishing date	2023-06-03
Publishing country	England
Document type	Journal Article
ZDB-ID	1017370-5
ISSN	1095-8320 ; 1045-1056
ISSN (online)	1095-8320
ISSN	1045-1056
DOI	10.1016/j.biologicals.2023.101685
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Confirming the absence of parental African swine fever virus as a potential contaminant of recombinant live attenuated ASF vaccines

Velazquez-Salinas, Lauro / Ramirez-Medina, Elizabeth / Rai, Ayushi / Pruitt, Sarah / Vuono, Elizabeth A. / Espinoza, Nallely / Gay, Cyril G. / Witte, Steve / Gladue, Douglas P. / Borca, Manuel V.

Biologicals. 2023, p.101685-

2023 , Page(s) 101685–

Abstract	African swine fever (ASF) is a devastating disease that is currently producing a panzootic significantly impacting the swine industry worldwide. One of the major challenges for advancing the development of ASF vaccines has been the absence of international standards for ASF vaccine purity, potency, safety, and efficacy. To date, the most effective experimental vaccines have been live attenuated strains of viruses. Most of these promising vaccine candidates have been developed by deleting virus genes involved in the process of viral pathogenesis and disease production. This approach requires genomic modification of a parental virus field strain through a process of homologous recombination followed by purification of the recombinant attenuated virus. In this scenario, it is critical to confirm the absence of any parental virulent virus in the final virus stock used for vaccine production. We present here a protocol to establish the purity of virus stock using the live attenuated vaccine candidates ASFV-G-ΔMGF, ASFV-G-Δ9GLΔUK and ASFV-G-ΔI177L. Procedures described here includes inoculation in susceptible pigs followed by the assessment of the obtained material by differential qPCRs that allows the identification of vaccine virus from ASFV field isolates. This protocol is proposed as a model to ensure that master seed virus stock used for vaccine production does not contain residual parental virulent virus. Procedures described here includes a passage in susceptible pigs followed by the assessment of the obtained material by differential qPCRs that allows the identification of vaccine virus from ASFV field isolates.
Keywords	African swine fever ; African swine fever virus ; genomics ; homologous recombination ; live vaccines ; pathogenesis ; pork industry ; virulence ; viruses ; Vaccination ; Safety protocol ; Recombinant vaccine ; Contamination
Language	English
Publishing place	Elsevier Ltd
Document type	Article ; Online
Note	Pre-press version
ZDB-ID	1017370-5
ISSN	1095-8320 ; 1045-1056
ISSN (online)	1095-8320
ISSN	1045-1056
DOI	10.1016/j.biologicals.2023.101685
Database	NAL-Catalogue (AGRICOLA)

In stock of ZB MED Cologne/Königswinter

Zs.A 879: Show issues

Location:
Je nach Verfügbarkeit (siehe Angabe bei Bestand)
bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
Jg. 1995 - 2021: Lesesall (1.OG)
ab Jg. 2022: Lesesaal (EG)

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Article ; Online: Evaluation of an ASFV RNA Helicase Gene A859L for Virus Replication and Swine Virulence.

Ramirez-Medina, Elizabeth / Vuono, Elizabeth A / Pruitt, Sarah / Rai, Ayushi / Espinoza, Nallely / Velazquez-Salinas, Lauro / Gladue, Douglas P / Borca, Manuel V

Viruses

2021 Volume 14, Issue 1

Abstract: African swine fever virus (ASFV) is producing a devastating pandemic that, since 2007, has spread to a contiguous geographical area from central Europe to Asia. In July 2021, ASFV was detected in the Dominican Republic, the first report of the disease in ...

Abstract	African swine fever virus (ASFV) is producing a devastating pandemic that, since 2007, has spread to a contiguous geographical area from central Europe to Asia. In July 2021, ASFV was detected in the Dominican Republic, the first report of the disease in the Americas in more than 40 years. ASFV is a large, highly complex virus harboring a large dsDNA genome that encodes for more than 150 genes. The majority of these genes have not been functionally characterized. Bioinformatics analysis predicts that ASFV gene A859L encodes for an RNA helicase, although its function has not yet been experimentally assessed. Here, we evaluated the role of the A859L gene during virus replication in cell cultures and during infection in swine. For that purpose, a recombinant virus (ASFV-G-∆A859L) harboring a deletion of the A859L gene was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. Recombinant ASFV-G-∆A859L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, demonstrating that the A859L gene is non-essential for ASFV replication. Experimental infection of domestic pigs demonstrated that ASFV-G-∆A859L replicates as efficiently and induces a clinical disease indistinguishable from that caused by the parental ASFV-G. These studies conclude that the predicted RNA helicase gene A859L is not involved in the processes of virus replication or disease production in swine.
MeSH term(s)	African Swine Fever/virology ; African Swine Fever Virus/genetics ; African Swine Fever Virus/pathogenicity ; African Swine Fever Virus/physiology ; Animals ; Cells, Cultured ; Gene Deletion ; Genes, Viral ; Macrophages/virology ; RNA Helicases/genetics ; Sus scrofa ; Swine ; Transcription, Genetic ; Viral Proteins/genetics ; Virulence/genetics ; Virus Replication/genetics
Chemical Substances	Viral Proteins ; RNA Helicases (EC 3.6.4.13)
Language	English
Publishing date	2021-12-21
Publishing country	Switzerland
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v14010010
Database	MEDical Literature Analysis and Retrieval System OnLINE

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

Velazquez-Salinas, Lauro / Ramirez-Medina, Elizabeth / Rai, Ayushi / Pruitt, Sarah / Vuono, Elizabeth A / Espinoza, Nallely / Gladue, Douglas P / Borca, Manuel V

Frontiers in veterinary science

2021 Volume 8, Page(s) 768869

Abstract: 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 ... ...

Abstract	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.
Language	English
Publishing date	2021-10-27
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2834243-4
ISSN	2297-1769
ISSN	2297-1769
DOI	10.3389/fvets.2021.768869
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Evaluation of the Function of the ASFV

Vuono, Elizabeth A / Ramirez-Medina, Elizabeth / Pruitt, Sarah / Rai, Ayushi / Espinoza, Nallely / Velazquez-Salinas, Lauro / Gladue, Douglas P / Borca, Manuel V

Viruses

2021 Volume 13, Issue 6

Abstract: African swine fever virus (ASFV) causes a devastating disease of swine that has caused outbreaks in Central Europe since 2007, spreading into Asia in 2018. ASFV is a large, structurally complex virus with a large dsDNA genome encoding for more than 160 ... ...

Abstract	African swine fever virus (ASFV) causes a devastating disease of swine that has caused outbreaks in Central Europe since 2007, spreading into Asia in 2018. ASFV is a large, structurally complex virus with a large dsDNA genome encoding for more than 160 genes, most of them still uncharacterized. p22, encoded by the ASFV gene KP177R, is an early transcribed, structural virus protein located in the ASFV particle. Although its exact function is unknown, p22 has recently been identified as an interacting partner of several host proteins. Here, we describe the development of a recombinant ASFV (ASFV-G-∆KP177R) lacking the KP177R gene as a tool to evaluate the role of p22 in virus replication and virulence in swine. The recombinant ASFV-G-∆KP177R demonstrated that the KP177R gene is non-essential for ASFV replication in primary swine macrophages, with virus yields similar to those of the parental, highly virulent field isolate Georgia2010 (ASFV-G). In addition, experimental infection of domestic pigs with ASFV-G-∆KP177R produced a clinical disease similar to that caused by the parental ASFV-G. Therefore, and surprisingly, p22 does not seem to be involved in virus replication or virulence in swine.
MeSH term(s)	African Swine Fever/virology ; African Swine Fever Virus/genetics ; African Swine Fever Virus/pathogenicity ; Amino Acid Sequence ; Animals ; Cells, Cultured ; Conserved Sequence ; Gene Deletion ; Macrophages/virology ; Mutation ; Swine ; Viral Load ; Viral Structural Proteins/genetics ; Virulence ; Virulence Factors/genetics ; Virus Replication
Chemical Substances	Viral Structural Proteins ; Virulence Factors
Language	English
Publishing date	2021-05-26
Publishing country	Switzerland
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v13060986
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Evaluation of the Function of the ASFV KP177R Gene, Encoding for Structural Protein p22, in the Process of Virus Replication and in Swine Virulence

Vuono, Elizabeth A. / Ramirez-Medina, Elizabeth / Pruitt, Sarah / Rai, Ayushi / Espinoza, Nallely / Velazquez-Salinas, Lauro / Gladue, Douglas P. / Borca, Manuel V.

Viruses. 2021 May 26, v. 13, no. 6

2021

Abstract	African swine fever virus (ASFV) causes a devastating disease of swine that has caused outbreaks in Central Europe since 2007, spreading into Asia in 2018. ASFV is a large, structurally complex virus with a large dsDNA genome encoding for more than 160 genes, most of them still uncharacterized. p22, encoded by the ASFV gene KP177R, is an early transcribed, structural virus protein located in the ASFV particle. Although its exact function is unknown, p22 has recently been identified as an interacting partner of several host proteins. Here, we describe the development of a recombinant ASFV (ASFV-G-∆KP177R) lacking the KP177R gene as a tool to evaluate the role of p22 in virus replication and virulence in swine. The recombinant ASFV-G-∆KP177R demonstrated that the KP177R gene is non-essential for ASFV replication in primary swine macrophages, with virus yields similar to those of the parental, highly virulent field isolate Georgia2010 (ASFV-G). In addition, experimental infection of domestic pigs with ASFV-G-∆KP177R produced a clinical disease similar to that caused by the parental ASFV-G. Therefore, and surprisingly, p22 does not seem to be involved in virus replication or virulence in swine.
Keywords	African swine fever virus ; DNA ; genes ; macrophages ; structural proteins ; swine ; viral proteins ; virulence ; virus replication ; viruses ; Asia ; Central European region
Language	English
Dates of publication	2021-0526
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2516098-9
ISSN	1999-4915
ISSN	1999-4915
DOI	10.3390/v13060986
Database	NAL-Catalogue (AGRICOLA)

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Article: Evaluation of an ASFV RNA Helicase Gene A859L for Virus Replication and Swine Virulence

Ramirez-Medina, Elizabeth / Vuono, Elizabeth A. / Pruitt, Sarah / Rai, Ayushi / Espinoza, Nallely / Velazquez-Salinas, Lauro / Gladue, Douglas P. / Borca, Manuel V.

Viruses. 2021 Dec. 21, v. 14, no. 1

2021

Abstract	African swine fever virus (ASFV) is producing a devastating pandemic that, since 2007, has spread to a contiguous geographical area from central Europe to Asia. In July 2021, ASFV was detected in the Dominican Republic, the first report of the disease in the Americas in more than 40 years. ASFV is a large, highly complex virus harboring a large dsDNA genome that encodes for more than 150 genes. The majority of these genes have not been functionally characterized. Bioinformatics analysis predicts that ASFV gene A859L encodes for an RNA helicase, although its function has not yet been experimentally assessed. Here, we evaluated the role of the A859L gene during virus replication in cell cultures and during infection in swine. For that purpose, a recombinant virus (ASFV-G-∆A859L) harboring a deletion of the A859L gene was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. Recombinant ASFV-G-∆A859L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, demonstrating that the A859L gene is non-essential for ASFV replication. Experimental infection of domestic pigs demonstrated that ASFV-G-∆A859L replicates as efficiently and induces a clinical disease indistinguishable from that caused by the parental ASFV-G. These studies conclude that the predicted RNA helicase gene A859L is not involved in the processes of virus replication or disease production in swine.
Keywords	African swine fever virus ; DNA ; RNA helicases ; bioinformatics ; genes ; macrophages ; pandemic ; swine ; virulence ; virus replication ; viruses ; Central European region ; Dominican Republic ; Georgia
Language	English
Dates of publication	2021-1221
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2516098-9
ISSN	1999-4915
ISSN	1999-4915
DOI	10.3390/v14010010
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Deletion of the EP296R Gene from the Genome of Highly Virulent African Swine Fever Virus Georgia 2010 Does Not Affect Virus Replication or Virulence in Domestic Pigs.

Vuono, Elizabeth A / Ramirez-Medina, Elizabeth / Pruitt, Sarah / Rai, Ayushi / Espinoza, Nallely / Spinard, Edward / Valladares, Alyssa / Silva, Ediane / Velazquez-Salinas, Lauro / Borca, Manuel V / Gladue, Douglas P

Viruses

2022 Volume 14, Issue 8

Abstract: African swine fever virus (ASFV) causes a lethal disease (ASF) in domestic pigs, African swine fever (ASF). ASF is currently producing a pandemic affecting pig production across Eurasia, leading to a shortage of food accessibility. ASFV is structurally ... ...

Abstract	African swine fever virus (ASFV) causes a lethal disease (ASF) in domestic pigs, African swine fever (ASF). ASF is currently producing a pandemic affecting pig production across Eurasia, leading to a shortage of food accessibility. ASFV is structurally complex, harboring a large genome encoding over 150 genes. One of them, EP296R, has been shown to encode for an endonuclease that is necessary for the efficient replication of the virus in swine macrophages, the natural ASFV target cell. Here, we report the development of a recombinant virus, ASFV-G-∆EP296R, harboring the deletion of the EP296R gene from the genome of the highly virulent field isolate ASFV Georgia 2010 (ASFV-G). The recombinant ASFV-G-∆EP296R replicates in primary swine macrophages with similar kinetics as the parental virus ASFV-G. Pigs experimentally infected by the intramuscular route with 10
MeSH term(s)	African Swine Fever ; African Swine Fever Virus ; Animals ; Gene Deletion ; Sus scrofa ; Swine ; Virulence/genetics ; Virus Replication
Language	English
Publishing date	2022-07-30
Publishing country	Switzerland
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v14081682
Database	MEDical Literature Analysis and Retrieval System OnLINE

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