Infectious clones

Abstract

Methods of preparing a DNA comprising sequences derived from the genomic RNA (gRNA) of a coronavirus are provided, comprising cloning under the expression of a promoter a coronavirus interfering defective genome into a bacterial artificial chromosome (BAC) and reinserting into said genome the deleted sequences within the defective genome, wherein said sequences have a homology of at least 60% to the natural sequence of the virus, wherein said sequences code for an RNA-dependent RNA polymerase and at least one structural or non-structural protein, and wherein a fragment of said DNA is capable of being transcribed into RNA and assembeld to a virion. Also provided are infective clones, recombinant viral vectors, and vaccines.

1 .- 74 . (canceled) 
     
     
         75 . A method of preparing a DNA comprising sequences derived from the genomic RNA (gRNA) of a coronavirus comprising:
 cloning under the expression of a promoter a coronavirus interfering defective genome into a bacterial artificial chromosome (BAC); and   re-inserting into said genome the deleted sequences within the defective genome   
       wherein said sequences have a homology of at least 60% to the natural sequence of the virus, wherein said sequences code for an RNA-dependent RNA polymerase and at least one structural or non-structural protein, and wherein a fragment of said DNA is capable of being transcribed into RNA and assembled to a virion. 
     
     
         76 . The method according to  claim 75 , wherein sequences within the viral genome which are toxic for the bacteria into which it is to be cloned are identified before re-insertion into said DNA. 
     
     
         77 . The method according to  claim 76 , wherein the toxic sequences within the viral genome are the last sequences to be re-inserted when completing the genome. 
     
     
         78 . The method according to  claim 75 , wherein an infective clone is obtained which comprises a full-length copy of complementary DNA (cDNA) to the gRNA of a coronavirus cloned under a transcriptional regulatory sequence, said method further comprising:
 constructing the full-length cDNA from the gRNA of a coronarivus; and   joining the transcriptional-regulatory elements to said full-length cDNA.   
     
     
         79 . The method according to  claim 78 , wherein construction of the full-length cDNA of the gRNA of a coronavirus comprises:
 cloning an interfering defective genome derived from said coronavirus under a promoter of expression in a BAC;   completing the deletions of said interfering defective genome by regenerating the deleted sequences with respect to the infective gRNA;   identifying the sequences in the coronavirus which are toxic for the bacteria in which it is going to be cloned;   removing said toxic sequences; and   inserting said toxic sequences just before effecting the transfection in eukaryotic cells   
       to obtain the cDNA clone corresponding to the gRNA of the coronavirus. 
     
     
         80 . An infective clone comprising a full-length copy of complementary DNA (cDNA) to the genomic RNA (gRNA) of a coronavirus cloned under a transcription-regulatory sequence, wherein said infective cDNA is cloned in a bacterial artificial chromosome (BAC). 
     
     
         81 . The infective clone according to  claim 80 , wherein said coronavirus is an isolate of the porcine transmissible gastroenteritis virus (TGEV). 
     
     
         82 . The infective clone according to  claim 80 , wherein the promoter employed is the immediately early (IE) promoter of expression of cytomegalovirus (CMV). 
     
     
         83 . The infective clone according to  claim 80 , wherein said full-length cDNA is flanked at the 3′ end by a poly(A) tail, the ribozyme of the hepatitis delta virus (HDV), and the termination and polyadenylation sequences of bovine growth hormone (BGH). 
     
     
         84 . The infective clone according to  claim 80 , wherein said infective cDNA is obtainable from the BAC of  E. coli  deposited under CECT 5265 at the Spanish Collection of Type Cultures. 
     
     
         85 .  E. coli  deposited under CECT 5265 at the Spanish Collection of Type Cultures. 
     
     
         86 . A recombinant viral vector comprising an infective clone according to  claim 80  modified to contain a heterologous nucleic acid inserted into said infective clone under conditions that allow said heterologous nucleic acid to be expressed. 
     
     
         87 . The recombinant viral vector according to  claim 86 , wherein said heterologous nucleic acid is selected between a gene and a gene fragment that codes a gene product of interest. 
     
     
         88 . A vaccine for protecting an animal against the infection caused by an infectious agent comprising at least one recombinant viral vector according to  claim 86 , wherein said viral vector expresses either at least one antigen suitable for inducing an immune response against said infectious agent or an antibody that provides protection against said infectious agent. 
     
     
         89 . The vaccine according to  claim 88 , further comprising a pharmaceutically-acceptable excipient. 
     
     
         90 . The vaccine according to  claim 88 , wherein said viral vector expresses at least one antigen capable of inducing a systemic immune response and/or an immune response in mucous membranes against different infectious agents that propagate in respiratory or intestinal mucous membranes. 
     
     
         91 . The vaccine according to  claim 88 , wherein the vaccine is a multivalent vaccine for protection against the infection caused by more than one infectious agent. 
     
     
         92 . The multivalent vaccine according to  claim 91 , further comprising independent recombinant viral vectors for mixing and joint inoculation, wherein said recombinant viral vectors each express at least one different antigen or a different antibody.