Attenuated pestiviruses

Abstract

This invention relates to attenuated pestiviruses characterised in that their enzymatic activity residing in glycoprotein E RNS is inactivated, methods of preparing, using and detecting these.

1 - 11 . (canceled)  
     
     
         12 . A nucleic acid coding for a glycoprotein E RNS , wherein the RNase activity residing in said glycoprotein is inactivated by deletions and/or mutations of at least one amino acid of said glycoprotein with the proviso that the amino acids at position 297 and/or 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein are not lysine.  
     
     
         13 . The nucleic acid of  claim 12 , wherein said RNase activity is inactivated by deletions and/or mutations that are located at the amino acids at position 295 to 307 and/or position 338 to 357, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         14 . The nucleic acid of  claim 13 , wherein said RNase activity is inactivated by deletion or mutation of the amino acid at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         15 . The nucleic acid according to  claim 14 , wherein said RNase activity is inactivated by the deletion of the histidine residue at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         16 . A BVDV nucleic acid according to  claim 15 , wherein said RNase activity is inactivated by the deletion of the histidine residue at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other BVDV strains, of said glycoprotein.  
     
     
         17 - 18 . (canceled)  
     
     
         19 . A method for attenuating pestiviruses wherein the RNase activity residing in glycoprotein E RNS  is inactivated.  
     
     
         20 . The method of  claim 19 , wherein said RNase activity is inactivated by deletions and/or mutations of at least one amino acid of said glycoprotein.  
     
     
         21 . The method of  claim 20 , wherein said deletions and/or mutations are located at the amino acids at position 295 to 307 and/or position 338 to 357, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         22 . The method according to  claim 21 , wherein said RNase activity is inactivated by deletion or mutation of the amino acid at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         23 . The method according to  claim 22 , wherein said RNase activity is inactivated by the deletion of the histidine residue at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         24 . A method for producing a specifically attenuated vaccine characterized in that wherein the RNase activity residing in glycoprotein E RNS  is inactivated.  
     
     
         25 . The method of  claim 24 , wherein said RNase activity is inactivated by deletions and/or mutations of at least one amino acid of said glycoprotein.  
     
     
         26 . The method of  claim 25 , wherein said deletions and/or mutations are located at the amino acids at position 295 to 307 and/or position 338 to 357, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         27 . The method according to  claim 26 , wherein said RNase activity is inactivated by deletion or mutation of the amino acid at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         28 . The method according to  claim 27 , wherein said RNase activity is inactivated by the deletion of the histidine residue at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         29 . A method for detectably labeling pestiviruses wherein the RNase activity residing in glycoprotein E RNS  is inactivated.  
     
     
         30 . The method of  claim 29 , wherein said RNase activity is inactivated by deletions and/or mutations of at least one amino acid of said glycoprotein.  
     
     
         31 . The method of  claim 30 , wherein said deletions and/or mutations are located at the amino acids at position 295 to 307 and/or position 338 to 357, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         32 . The method according to  claim 31 , wherein said RNase activity is inactivated by deletion or mutation of the amino acid at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         33 . The method according to  claim 32 , wherein said RNase activity is inactivated by the deletion of the histidine residue at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         34 . (canceled)  
     
     
         35 . A process for the preparation of specifically attenuated pestiviruses characterized in that wherein the RNase activity residing in glycoprotein E RNS  is inactivated.  
     
     
         36 . The process according to  claim 35 , wherein said RNase activity is inactivated by deletions and/or mutations of at least one amino acid of said glycoprotein.  
     
     
         37 . The process according to  claim 36 , wherein said deletions and/or mutations are located at the amino acids at position 295 to 307 and/or position 338 to 357, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         38 . The process according to  claim 37 , wherein said RNase activity is inactivated by deletion or mutation of the amino acid at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         39 . The process according to  claim 38 , wherein said RNase activity is inactivated by the deletion of the histidine residue at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         40 . A process for the preparation of specifically labeled pestiviruses characterized in that wherein the RNase activity residing in glycoprotein E RNS  is inactivated.  
     
     
         41 . The process according to  claim 40 , wherein said RNase activity is inactivated by deletions and/or mutations of at least one amino acid of said glycoprotein.  
     
     
         42 . The process according to  claim 41 , wherein said deletions and/or mutations are located at the amino acids at position 295 to 307 and/or position 338 to 357, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         43 . The process according to  claim 42 , wherein said RNase activity is inactivated by deletion or mutation of the amino acid at position 346. as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         44 . The process according to  claim 43 , wherein said RNase activity is inactivated by the deletion of the histidine residue at position 346, as described in  FIG. 1  for the CSFV Alfort strain in an exemplary manner or corresponding thereto in other strains, of said glycoprotein.  
     
     
         45 - 52 . (canceled)  
     
     
         53 . A vaccine comprising the nucleic acid according to  claim 12 .  
     
     
         54 . A method for the prophylaxis or treatment of a pestivirus infection in an animal comprising administering a vaccine of claim  1  to an animal in need of such prophylaxis or treatment.  
     
     
         55 . A method for the prophylaxis or treatment of a pestivirus infection in an animal comprising administering the pharmaceutical composition of claim  18  to an animal in need of such prophylaxis or treatment.  
     
     
         56 . A method for distinguishing pestivirus-infected animals from animals vaccinated with a specifically attenuated pestivirus, wherein said specifically attenuated pestivirus is attenuated according to the method of  claim 19 , comprising: 
 (a) obtaining a sample from an animal suspected of pestivirus infection or from an animal vaccinated with a specifically attenuated pestivirus;    (b) identifying the nucleotide sequence of a pestivirus within said sample; and    (c) correlating the presence of deletions and/or mutations of the E RNS  nucleotide sequence with a vaccinated animal and correlating the absence of said deletions and/or mutations with a pestivirus infection of said animal.    
     
     
         57 . A method for distinguishing pestivirus-infected animals from animals vaccinated with a specifically attenuated pestivirus, wherein said specifically attenuated pestivirus is attenuated according to the method of  claim 19 , comprising: 
 (a) obtaining a sample from an animal suspected of pestivirus infection or from an animal vaccinated with a specifically attenuated pestivirus;    (b) identifying a modified E RNS  glycoprotein of an attenuated pestivirus by the specific binding of monoclonal or polyclonal antibodies to E RNS  glycoproteins present in said sample, said glycoproteins being modified by deletions and/or mutations of at least one amino acid, whereby said monoclonal or polyclonal antibodies do not bind to unmodified E RNS  glycoproteins; and    (c) correlating the specific binding of said monoclonal or polyclonal antibodies with a vaccinated animal and correlating the absence of antibody binding to a pestivirus infection of said animal with the proviso that the presence of pestiviral material in said animal and/or said sample is established otherwise.    
     
     
         58 . A method for distinguishing pestivirus-infected animals from animals vaccinated with a specifically attenuated pestivirus, wherein said specifically attenuated pestivirus is attenuated according to the method of  claim 19 , comprising: 
 (a) obtaining a sample from an animal suspected of pestivirus infection or from an animal vaccinated with a specifically attenuated pestivirus;    (b) identifying an unmodified E RNS  glycoprotein of a pestivirus by the specific binding of monoclonal or polyclonal antibodies to E RNS  glycoproteins present in said sample, said glycoproteins not being modified by deletions and/or mutations of at least one amino acid, whereby said monoclonal or polyclonal antibodies do not bind to modified E RNS  glycoproteins; and    (c) correlating the specific binding of said monoclonal or polyclonal antibodies with a pestivirus infection in said animal and correlating the absence of antibody binding to a vaccinated animal with the proviso that the presence of pestiviral material in said animal and/or said sample is established otherwise.    
     
     
         59 . A method for distinguishing pestivirus-infected animals from animals vaccinated with a specifically attenuated pestivirus, wherein said specifically attenuated pestivirus is attenuated according to the method of  claim 19 , comprising: 
 (a) obtaining a sample from an animal suspected of pestivirus infection or from an animal vaccinated with a specifically attenuated pestivirus;    (b) determining the absence or presence of RNase activity of a glycoprotein E RNS  within said sample; and    (c) correlating the absence of RNase activity of glycoprotein E RNS  with a vaccinated animal and correlating the presence of said activity with a pestivirus infection of said animal.    
     
     
         60 . A method for distinguishing pestivirus-infected animals from animals vaccinated with a specifically attenuated pestivirus, wherein said specifically attenuated pestivirus is attenuated according to the method of  claim 19 , comprising: 
 (a) obtaining a sample of polyclonal antibodies from an animal suspected of pestivirus infection or from an animal vaccinated with a specifically attenuated pestivirus;    (b) identifying any specific binding of said polyclonal antibodies to unmodified glycoprotein E RNS  or glycoprotein E RNS  as modified by deletions and/or mutations of at least one amino acid; and    (c) correlating the binding of said polyclonal antibodies to unmodified glycoprotein E RNS  with a pestivirus infection and correlating the binding of said polyclonal antibodies to glycoprotein E RNS  as modified by deletions and/or mutations of at least one amino acid with a vaccinated animal.