Cdna construct of salmonidae alphavirus

Abstract

The invention concerns recombinant DNA's comprising c NDA of genomic RNA of a Salmonidae alphavirus preceded by a spacer sequence, under the control of a suitable promoter. Said recombinant DNA's are useful for obtaining expression vectors, producing recombinant Salmonidae alphavirus, and for obtaining vaccines.

1 . A method for preparing a Salmonidae alphavirus RNA replicon, comprising introducing a recombinant DNA in to a host cell and culturing said host cell, wherein the recombinant DNA is derived from a genome of a Salmonidae alphavirus and comprises:
 a transcription promoter and, downstream of said promoter and under transcriptional control thereof;   a spacer sequence of at least 5 nucleotides; and   a cDNA of a genomic RNA of a Salmonidae alphavirus,   and the spacer sequence is defined by general formula (I):   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 29) 
                 
                     
                   5′ X 1 CTGANGARX 2 B 2 X′ 2 YGAAAX 3 B 3 X′ 3 TH 3′ (I) 
                 
             
                
                
               
            
           
         
         in which A, T, G, and C have their usual meaning; H represents C, T or A; R represents A or G; Y represents C or T; N represents A, T, G or C; X 1  represents an oligonucleotide of at least 3 nucleotides, preferably from 6 to 10 nucleotides, of sequence complementary to that of the 5′ end of the genome of said alphavirus; X 2  represents an oligonucleotide of at least 3 nucleotides, preferably from 3 to 5 nucleotides, of any sequence; B 2  represents an oligonucleotide of 4 or 5 nucleotides, of any sequence; X′ 2  represents an oligonucleotide complementary to X 2 ; X 3  represents an oligonucleotide of at least 2 nucleotides, preferably of 6 to 10 nucleotides, of any sequence; B 3  represents an oligonucleotide of 4 to 5 nucleotides, of any sequence; X′ 3  represents an oligonucleotide complementary to X 3 . 
       
     
     
         2 . A Salmonidae alphavirus RNA replicon obtained by the method according to  claim 1 . 
     
     
         3 . A method for preparing a recombinant Salmonidae alphavirus, comprising introducing a recombinant DNA or an RNA replicon into a host cell in which all structural proteins of said alphavirus that are required for encapsidation are expressed, and culturing said host cell wherein the recombinant DNA is derived from a genome of a Salmonidae alphavirus and comprises:
 a transcription promoter and, downstream of said promoter and under transcriptional control thereof;   a spacer sequence of at least 5 nucleotides; and   a cDNA of a genomic RNA of a Salmonidae alphavirus,   and the spacer sequence is defined by general formula (I):   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 29) 
                 
                     
                   5′ X 1 CTGANGARX 2 B 2 X′ 2 YGAAAX 3 B 3 X′ 3 TH 3′ (I) 
                 
             
                
                
               
            
           
         
         in which A, T, G, and C have their usual meaning; H represents C, T or A; R represents A or G; Y represents C or T; N represents A, T, G or C; X 1  represents an oligonucleotide of at least 3 nucleotides, preferably from 6 to 10 nucleotides, of sequence complementary to that of the 5′ end of the genome of said alphavirus; X 2  represents an oligonucleotide of at least 3 nucleotides, preferably from 3 to 5 nucleotides, of any sequence; B 2  represents an oligonucleotide of 4 or 5 nucleotides, of any sequence; X′ 2  represents an oligonucleotide complementary to X 2 ; X 3  represents an oligonucleotide of at least 2 nucleotides, preferably of 6 to 10 nucleotides, of any sequence; B 3  represents an oligonucleotide of 4 to 5 nucleotides, of any sequence; X′ 3  represents an oligonucleotide complementary to X 3 , or 
         introducing the RNA replicon according to  claim 5  into a host cell in which all structural proteins of said alphavirus that are required for encapsidation are expressed, and culturing said host cell. 
       
     
     
         4 . The method according to  claim 3 , wherein all genetic information for the expression of said structural proteins is carried by said recombinant DNA or said RNA replicon. 
     
     
         5 . The method according to  claim 3 , wherein all or part of genetic information for the expression of said structural proteins is provided in trans by the host cell. 
     
     
         6 . A recombinant Salmonidae alphavirus obtained by a method according to  claim 3 . 
     
     
         7 . A method for obtaining a vaccine, incorporating a recombinant Salmonidae alphavirus according to  claim 6  into the vaccine. 
     
     
         8 . A vaccine comprising a Salmonidae alphavirus RNA replicon according to  claim 2 . 
     
     
         9 . A vaccine comprising a recombinant Salmonidae alphavirus according to  claim 6 .