Permissive cells and uses thereof
The invention relates generally to the field of virology. More particularly, the present invention relates to methods for determining the permissiveness of a cell for a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The invention further provides methods and compositions related to the generation of host cells permissive for a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, in particular for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). Methods of using said cells thus identified or thus generated, in preparing a culture of a virus that is a member of the family Arteriviridae or Coronaviridae or Asfarviridae, as well as the use of said virus for the purpose of vaccine production or diagnosis, are also provide by the present invention.
1. A method for preparing Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the method comprising:
determining CD163 expression in a cell line;
determining sialoadhesin expression in the cell line;
identifying a cell line having both CD163 and sialoadhesin expression as a permissive cell line for PRRSV;
infecting the identified cell line with PRRSV; and
harvesting PRRSV from the cell line.
2. A method for preparing Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the method comprising:
treating a cell to yield an expression of both CD163 and sialoadhesin;
determining CD163 expression in the cell;
determining sialoadhesin expression in the cell;
identifying a cell having both CD163 and sialoadhesin expression as a permissive cell for PRRSV;
infecting the identified cell with PRRSV; and
harvesting PRRSV from the cell line.
3. The method according to claim 2 , wherein the expression of CD163 and sialoadhesin is determined at the nucleic acid level.
4. The method according to claim 3 , wherein the expression of CD163 and sialoadhesin is determined at the mRNA level.
5. The method according to claim 3 , further comprising isolating nucleic acid or protein from the cell.
6. The method according to claim 4 , further comprising isolating mRNA from the cell.
7. The method according to claim 3 , further comprising contacting the cell or an extract thereof with an antibody for CD163 or sialoadhesin.
8. The method according to claim 1 , wherein the expression of CD163 and sialoadhesin is determined at the protein level.
9. The method of claim 8 , wherein the protein level is determined using an immunological method.
10. The method according to claim 2 , wherein the CD163 protein is at least 70% identical to porcine CD163 encoded by the gene characterized by SEQ ID NO:1, including biologically active fragments thereof containing at least one Scavenger Receptor Cysteine Rich (SRCR) domain.
11. The method according to claim 2 , wherein the sialoadhesin protein is at least 70% identical to porcine sialoadhesin encoded by the gene characterized by SEQ ID NO:9, including biologically active fragments thereof containing at least the N-terminal domain.
12. The method according to claim 2 , further comprising the step of inactivating the virus harvested from the cell culture.
13. The method according to claim 2 , wherein treating a cell comprises the introduction of exogenous nucleic acid into the cell.
14. The method according to claim 13 , wherein the exogenous nucleic acid encodes CD163 and/or sialoadhesin.