Location: Foreign Animal Disease Research
Project Number: 3022-32000-063-004-I
Project Type: Interagency Reimbursable Agreement
Start Date: Aug 9, 2018
End Date: Sep 30, 2022
Objective:
Commercial production of an African swine fever (ASF) vaccine requires the ability of the vaccine to replicate in a mammalian cell line. Currently a stable cell line that supports replication of African swine fever virus (ASFV) is unknown. For basic research studies ASFV is able to replicate in freshly harvested primary swine macrophages. These cells do not replicate, and have to be harvested fresh each day. Therefore, primary swine macrophages are not suitable for vaccine production. Identification of a stable cell line that supports ASFV vaccine growth is necessary for large scale commercial vaccine production, and for large scale diagnostics of suspected cases of ASFV. The objective of this research project is to identify a stable cell line which is capable of supporting stable ASFV growth without genome changes for field isolates and recombinant derivatives.
Specific objectives include:
1. Identify stable swine cell lines permissive for ASFV. Including: initial screening of available stable cell lines, adaptation of ASFV to cell lines, immortalization of primary swine macrophages to create stable cell lines and evaluation of different ASFV isolates for ability to replicate in selected cell lines.
2. Determination of genome stability Next generation sequence (NGS) of the full ASFV genome sequence will be conducted to identify potential anomalies within the viral genome and evaluate its genome stability after viral growth and adaption to stable cell cultures. Resulting viruses with changes in the genome will be tested for the ability of ASFV to replicate in macrophages (Adapted ASFVs that do not grow in swine macrophages historically have lost the ability to cause disease in swine, and were not able to produce an immune response as a vaccine). Resulting viruses will then be tested for the ability to cause disease in swine.
Approach:
1. Initial screening of swine cell lines, either commercially available or previously made in other laboratories, will be conducted to assess their ability to grow ASFV. To date we have only tested primary swine macrophages for the ability to grow ASFV. In addition, porcine Aortic Endothelial Cells (PAOEC) will be tested as the genetics of these cells is similar to macrophages. After initial screening of cell lines, serial passageing of ASFV in the selected cell line will occur to adapt the virus to grow in the particular cell line. Primary swine macrophages will be immortalized, cloned and tested for their ability to support ASFV replication. Cell lines which support ASFV growth will be evaluated for their susceptibility to different ASFV field and vaccine strains such as Georgia/2007, Malawi and Pretoria.
2. Adaptation of ASFV to cell cultures results in potential changes to the viral genome. It will therefore, be necessary to determine the extent of these potential changes and if these changes alter the ability for ASFV to cause disease. Adapted viruses will be plaque purified and their full length sequence obtained by next generation sequencing. In addition, the ability of these adapted strains to grow in primary swine macrophages as well as their ability to infect swine will be determined. Next generation sequencing of the full genome will be conducted to identify any potential viral amino acid deletion/mutations that may occur in the viral genome. An evaluation will be conducted on the genomic stability with during successive passages in susceptible cell lines.
A study to determine the ability of selected adapted ASFV strains to grow in swine macrophage will be conducted. Growth in swine macrophages is required for an ASFV to either behave as potential vaccine candidate as well as to cause disease in swine. This can be used to help determine which adapted viruses should be tested in swine. An in vivo trial will be performed to determine the ability of the adapted ASFV strains to induce virulence and/or its use as vaccine candidate.
3. Gene cell expression profiling will be conducted to determine the patterns associated with increased virus replication. Gene expression profiles between susceptible and non-susceptible swine cell lines will be compared to determine which pathways may be turned on or off in cell lines that are susceptible or non-susceptible to ASFV. This will be accomplished either by NGS RNAseq or Microarray. Modify cellular pathways that have been determined to be different between susceptible and non-susceptible cell lines, will be modified using CRISPR/Cas9 to either downregulate the pathway by introducing gene deletions or upregulate key components by introducing constitutively active promoters for specific genes. The pathways targeted will depend on what pathways are identified above that are likely to increase the ability of ASFV to grow in cell culture.
