Virus-free cell lines and methods for obtaining same

Abstract

The current teachings are directed to novel virus free cells lines derived from virus-contaminated staring material, such as an organism or a cell line. Methods for obtaining virus free cell lines obtained from virus-contaminated starting material are also provided. Exemplary virus free cell lines include: novel cell lines derived from a Spodoptera frugiperda cell line contaminated with Sf-rhabdovirus, wherein the novel cell lines lack Sf-rhabdovirus; and novel cell lines derived from a Trichoplusia ni cell line contaminated with an alphanodavirus, wherein the novel cell line lacks an alphanodavirus.

What is claimed is: 
     
         1 . A cell line derived from a Sf-rhabdovirus-contaminated  Spodoptera frugiperda  organism or from a Sf-rhabdovirus-contaminated  Spodoptera frugiperda  cell line, the cell line characterized by a lack of virus, wherein the cell line is structurally different from the Sf-rhabdovirus-contaminated  Spodoptera frugiperda  organism or the Sf-rhabdovirus-contaminated  Spodoptera frugiperda  cell line. 
     
     
         2 . The cell line of  claim 1 , wherein the Sf-rhabdovirus-contaminated  Spodoptera frugiperda  cell line comprises Sf-21 or Sf9 cells. 
     
     
         3 . The cell line of  claim 1 , further comprising a cell density, a doubling time, an average cell diameter, a morphology, and a N-glycosylation pattern that is the same or substantially the same as Sf9 cells when the cell line and the Sf9 cells are propagated under the same conditions. 
     
     
         4 . The cell line of  claim 1 , further comprising increasing production of infectious recombinant baculovirus particles compared to production in Sf9 cells under the same conditions. 
     
     
         5 . The cell line of  claim 1  manufactured using a method comprising:
 isolating a cell from a Sf-rhabdovirus-contaminated  Spodoptera frugiperda  organism or from a Sf-rhabdovirus-contaminated  Spodoptera frugiperda  cell line; 
 combining the isolated cell with a cell culture media comprising an antiviral compound to form a first culture composition; 
 incubating the first culture composition under conditions suitable for the cell to grow and divide, thereby generating a multiplicity of cells; 
 removing a portion of the multiplicity of cells or the cell culture media and testing for the presence or absence of a virus; 
 combining at least some of the multiplicity of cells with cell culture media without an antiviral compound to form a second culture composition; and 
 incubating the second culture composition under conditions suitable for the cells to grow and divide, thereby obtaining a virus free cell line. 
 
     
     
         6 . The cell line of  claim 5 , wherein testing comprises (a) RT-PCR, (b) RT-PCR and nested PCR; (c) quantitative RT-PCR; or (d) an antibody-based detection technique. 
     
     
         7 . The cell line of  claim 5 , wherein the antiviral compound is a nucleoside analog, optionally, wherein the nucleoside analog comprises at least one of: ribavirin, 6-azuaridine, vidarabine, acyclovir, 9-/3-D-Arabinofuranosyladenine (Ara-A), cytosine arabinose, adenine arabinoside, and guanine 7-N-oxide (g-7-Ox), optionally wherein the nucleoside analog is 6-azauridine. 
     
     
         8 . The cell line of  claim 5 , wherein the isolating comprises limiting dilution; wherein the antiviral compound is 6-azauridine; and wherein the testing comprises: (a) RT-PCR or (b) RT-PCR and nested PCR. 
     
     
         9 . A method of testing for the presence of Sf-rhabdovirus using the cell line of  claim 1 . 
     
     
         10 . A method of producing at least one selected from the group consisting of a vaccine, a recombinant protein, and a biologic using the cell line of  claim 1 . 
     
     
         11 . The method of  claim 10 , wherein producing utilizes the baculovirus-insect cell system. 
     
     
         12 . The method of  claim 10 , wherein the recombinant protein is a multi-subunit protein complex. 
     
     
         13 . The method of  claim 10 , wherein the vaccine, the recombinant protein, and the biologic are for human or veterinary use.