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 . A live vaccine comprising a pestivirus, wherein the RNase activity residing in glycoprotein E RNS  is inactivated.  
     
     
         2 . The vaccine of  claim 1 , wherein said RNase activity is inactivated by deletions and/or mutations of at least one amino acid of said glycoprotein.  
     
     
         3 . The vaccine according to  claim 2 , 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.  
     
     
         4 . The vaccine according to anyone of  claims 1  to  3 , 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.  
     
     
         5 . The vaccine according to anyone of  claims 1  to  4 , 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.  
     
     
         6 . A vaccine according to anyone of  claims 1  to  5  comprising a BVDV pestivirus, 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.  
     
     
         7 . A pestivirus, wherein the RNase activity residing in glycoprotein E RNS  is inactivated by deletions and/or mutations ot 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.  
     
     
         8 . The pestivirus of  claim 7 , wherein said RNase activity is inactivated by deletions and/or mutations 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.  
     
     
         9 . The pestivirus of  claim 7  or  8 , 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.  
     
     
         10 . The pestivirus according to anyone of  claims 7  to  9 , 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.  
     
     
         11 . A BVDV pestivirus according to anyone of  claims 7  to  10 , 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.  
     
     
         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 12  or  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 anyone of  claims 12  to  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 anyone of  claims 12  to  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 . Use of nucleic acids according to anyone of  claims 12  to  16  for preparing nucleotide- and/or vector-vaccines.  
     
     
         18 . A pharmaceutical composition comprising a vaccine according to anyone of  claims 1  to  6 , and/or a pestivirus according to anyone of  claims 7  to  11 , and/or a nucleotide sequence according to anyone of  claims 12  to  16 .  
     
     
         19 . A method for attenuating pestiviruses characterized in that 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 19  or  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 anyone of  claims 19  to  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 anyone of  claims 19  to  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 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 24  or  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 anyone of  claims 24  to  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 anyone of  claims 24  to  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 characterized in that 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 29  or  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 anyone of  claims 29  to  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 anyone of  claims 29  to  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 . A method for the prophylaxis and treatment of pestivirus infections in animals characterized in that a vaccine according to anyone of  claims 1  to  6  or a pharmaceutical composition according to  claim 18  is applied to an animal in need of such prophylaxis or treatment.  
     
     
         35 . A process for the preparation of specifically attenuated pestiviruses characterized in that 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 35  or  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 anyone of  claims 35  to  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 anyone of  claims 36  to  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 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 40  or  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 anyone of  claims 40  to  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 anyone of  claims 40  to  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 . Use of a vaccine of anyone of  claims 1  to  6  for the prophylaxis and treatment of pestivirus infections in animals.  
     
     
         46 . Use of a pharmaceutical composition of  claim 18  for the prophylaxis and treatment of pestivirus infections in animals.  
     
     
         47 . Use of a pestivirus of anyone of  claims 7  to  11  and/or a nucleotide sequence according to anyone of  claims 12  to  16  for the preparation of a vaccine or a pharmaceutical composition.  
     
     
         48 . A method for distinguishing pestivirus-infected animals from animals vaccinated with a specifically attenuated pestivirus, wherein said specifically attenuated pestivirus is attenuated according to a method of anyone of  claims 19  to  23 , comprising the following steps: 
 Obtaining a sample from an animal of interest suspected of pestivirus infection or a vaccinated animal;  
 Identifying the nucleotide sequence of a pestivirus within said sample;  
 Correlating the deletions and/or mutations of the E RNS  nucleotide sequence as present in the vaccine with a vaccinated animal and correlating the absence of said deletions and/or mutations with a pestivirus infection of said animal.  
 
     
     
         49 . The method of  claim 48 , comprising the following steps: 
 Obtaining a sample from an animal of interest suspected of pestivirus infection or a vaccinated animal;    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 a method according to anyone of  claims 19  to  23 , whereby said monoclonal or polyclonal antibodies do not bind to unmodified E RNS  glycoproteins;    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 under the proviso that the presence of pestiviral material in said animal and/or said sample is established otherwise.    
     
     
         50 . The method of  claim 49 , comprising the following steps: 
 Obtaining a sample from an animal of interest suspected of pestivirus infection or a vaccinated animal;    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 a method according to anyone of  claims 19  to  23 , whereby said monoclonal or polyclonal antibodies do not bind to modified Ems glycoproteins;    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 an vaccinated animal under the proviso that the presence of pestiviral material in said animal and/or said sample is established otherwise.    
     
     
         51 . The method of  claim 48 , comprising the following steps: 
 Obtaining a sample from an animal of interest suspected of pestivirus infection or a vaccinated animal;    Determining the absence or presence of RNase activity of a glycoprotein E RNS  within said sample;    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.    
     
     
         52 . The method of  claim 48 , comprising the following steps: 
 Obtaining a sample of polyclonal antibodies from an animal of interest suspected of pestivirus infection or a vaccinated animal;    Identifying any specific binding of said polyclonal antibodies to unmodified glycoprotein E RNS  or glycoprotein E RNS  as modified according to the invention.    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 according to the invention with a vaccinated.