United States of America, As Represented by the Secretary of Agriculture

University of Connecticut

Universidad Del Pais Vasco/euskal Herriko Univertsitatea (upv-ehu)

Douglas P Gladue

Manuel V Borca

Lauren G Holinka-Patterson

Vivian O'Donnell

Guillermo R Risatti

Jose Nieva

Ayushi Rai

Vivian K O'DONNELL

Vivian K O'donnell

Provided herein are details on the construction of a recombinant African Swine Fever Virus (ASFV) live attenuated vaccine for prevention of ASF caused by various strains of ASFV, such as the highly virulent Georgia 2007 isolate ('ASFV-G'). An exemplary vaccine comprises the ASFV-G&#x394;I1771 modified virus, a recombinant ASFV-G modified by deleting a portion of the I177L ORF rendering the I177L gene nonfunctional.

Development of a novel live attenuated African Swine Fever vaccine based in the deletion of gene I177L

Provided herein are details on the construction of a recombinant African Swine Fever Virus (ASFV) live attenuated vaccine for prevention of ASF caused by various strains of ASFV, such as the highly virulent Georgia 2007 isolate ('ASFV-G'). An exemplary vaccine comprises the ASFV-G&#x394;A137R modified virus, a recombinant ASFV-G modified by deleting a portion of the A137R ORF rendering the A137R gene nonfunctional.

Development of a novel live attenuated African swine fever vaccine based in the deletion of gene A137R

Methods are provided herein utilizing a stable cell line capable of efficient infection by African swine fever virus (ASFV) and also provides for the detection of the presence of virus in samples applied to the cells. Detection of the virus by means such as red blood cell rosetting is a surprising result given that the cell line is derived from African green monkeys. This cell line provides a marked improvement over the currently available testing strategies.

Continuous stable cell line for identification of infectious African swine fever virus in clinical samples

Provided herein are details on the construction of a recombinant African Swine Fever Virus (ASFV) live attenuated vaccine for prevention of ASF caused by various strains of ASFV, such as the highly virulent Georgia 2007 isolate ('ASFV-G'). An exemplary vaccine comprises a deletion of multiple genes allowing for industrial-scale growth in stable cell lines.

Genomic deletion in African swine fever vaccine allowing efficient growth in stable cell lines

The role of a specific E2 region containing a putative fusion peptide (FP) sequence was evaluated. FPs critically contribute to the interaction between proteins and the membrane system of the host cell. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how amino acid substitutions within this region of E2 may affect replication of BICv in cell cultures and affect virus virulence in swine. Interestingly, mutated virus FPi.c was completely attenuated when inoculated intranasally at a dose of 10TCID50 in swine. Importantly, animals infected with FPi.c virus were protected against the virulent challenge with Brescia virus at 3 and 28 days after vaccination. Protection was evidenced by absence of clinical signs related with CSF as well as the absence of viremia produced by the challenge virulent virus.

Live attenuated classical swine fever vaccine based in genetic manipulation of a putative fusion peptide area in the virus structural glycoprotein E2

African swine fever virus (ASFV) is the etiological agent of a contagious, often lethal viral disease of domestic pigs. The control of African Swine Fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFVs; however, these vaccines are only successful when protecting against homologous viruses. We have constructed a recombinant &#x394;9GL/&#x394;UK virus derived from the highly virulent ASFV Georgia 2007 (ASFV-G) isolate by deleting the specific virulence-associated 9GL (B119L) and the UK (DP96R) genes. In vivo, ASFV-G &#x394;9GL/&#x394;UK administered intramuscularly to swine even at relatively high doses (10HAD) does not induce disease. Importantly, animals infected with 10or 10HADare solidly protected against the presentation of clinical disease when challenged at 28 days post infection with the virulent parental strain Georgia 2007.

Rationally developed african swine fever attenuated virus strain protects against challenge with parental virus georgia 2007 isolate

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for swine breeding. Control of ASF has been hampered by the unavailability of vaccines. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against the challenge with parental viruses. Here we report the construction of a recombinant &#x394;9GL virus derived from the highly virulent ASFV Georgia 2007 (ASFV-G) isolate. In vivo, ASFV-G &#x394;9GL administered intramuscularly (IM) to swine at relatively high doses (10HAD) retains a virulent phenotype practically indistinguishable from the parental virus. Conversely, at low IM doses (10or 10HAD), ASFV-G &#x394;9GL does not induce disease. Importantly, animals infected with 10HADare protected against the presentation of clinical disease when challenge at 28 days post infection with the virulent parental strain Georgia 2007.

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Guilford, CT, United States of America

Douglas P Gladue