Infectious bovine viral diarrhea virus

Abstract

The invention belongs to the field of animal health and in particular Bovine Viral Diarrhea Virus (BVDV). The invention provides infectious BVDV clones and methods to produce said BVDV clones. The invention further relates to methods of attenuating said clones, attenuated BVDV clones and vaccines comprising said attenuated clones.

1 . A DNA molecule containing a nucleotide sequence complementary to a BVDV RNA, wherein said RNA, when introduced into susceptible host cells, induces the generation of infectious BVDV particles 
 a) with the capability to induce viraemia and leukopenia in calves for a period of at least 1 day and at least one of the following clinical symptoms of the group comprising diarrhea and/or pyrexia lasting at least one day when infected with a dose of 6×10 6 TCID 50 ; and/or    b) with authentical virulence as compared to a wild-type BVDV isolate from which such DNA molecule has been derived; and/or    c) which are, when BVDV naive calves are infected at a dose of 6×10 6 TCID 50  with such particles, lethal for at least 30% of such calves within a period of 21 days; and/or    d) with a virulence of not less than 90% of BVDV particles comprising an RNA with a sequence complementary to SEQ ID NO. 1; and/or    e) comprising a sequence complementary to SEQ ID NO. 1.    
     
     
         2 . An infectious BVDV clone, capable of serving as a template for transcription into an RNA, wherein said RNA, when introduced into susceptible host cells, induces the generation of infectious BVDV particles 
 a) with the capability to induce viraemia and leukopenia in calves for a period of at least 1 day and at least one of the following clinical symptoms of the group comprising diarrhea and/or pyrexia lasting at least one day when infected with a dose of 6×10 6 TCID 50 ; and/or    b) with authentical virulence as compared to a wild-type BVDV isolate from which such DNA molecule has been derived; and/or    c) which are, when BVDV naive calves aged from 3 to 6 months are infected at a dose of 6×10 6 TCID 50  with such particles, lethal for at least 30% of such calves within a period of 21 days after infection; and/or    d) with a virulence of not less than 90% of BVDV particles comprising an RNA with a sequence complementary to SEQ ID NO. 1; and/or    e) comprising a sequence complementary to SEQ ID NO. 1.    
     
     
         3 . The infectious BVDV clone of  claim 2 , wherein the BVDV clone is a BVDV type 2 clone.  
     
     
         4 . An RNA molecule complementary to the DNA molecule of  claim 1 .  
     
     
         5 . An RNA molecule obtainable by transcription of the DNA molecule of  claim 1 .  
     
     
         6 . A method for the production of an infectious BVDV clone from a wild-type BVDV isolate, said infectious BVDV clone being complementary to a RNA having authentical virulence as compared to said wild-type isolate, comprising the steps of 
 a) isolating viral particles from an infected animal;    b) passaging not more than twice on suitable cell culture cells;    c) preparing RNA from the viral particles;    d) generating full-length complementary DNA after reverse transcription of the RNA; wherein the reverse transcription includes a step at elevated temperatures sufficient to break or reduce secondary structures of the RNA, and the use of a thermostable enzyme for this step, said enzyme being active at these elevated temperatures;    e) incorporating the complementary DNA (cDNA) into a plasmid vector or into a DNA virus capable of directing the transcription of BVDV cDNA into RNA upon infection of suitable cells.    
     
     
         7 . A method of BVDV attenuation of a BVDV strain or clone comprising introducing one or more mutations into the DNA molecule of  claim 1  wherein said mutation or mutations lead to or increase an attenuated phenotype of the recovered BVD virus.  
     
     
         8 . A method of attenuation of a BVDV strain or clone comprising the steps of 
 a) introducing one or more mutations into the DNA molecule of  claim 1;     b) introducing the mutated DNA into susceptible host cells wherein said DNA is transcribed into RNA or introducing an RNA transcribed from said DNA into said cells; and    c) collecting viral particles produced by these cells;    wherein said mutation or mutations results in attenuation.    
     
     
         9 . The method of  claim 7 , wherein the mutation or mutations is a substitution, deletion, insertion, addition, or combination thereof.  
     
     
         10 . The method of  claim 7 , wherein the mutation or mutations is in the glycoprotein E rns  and causes impaired or loss of function of the mutated protein(s).  
     
     
         11 . The method of  claim 10 , wherein the mutation consists of 
 a) deletion of all or part of the glycoprotein Ems; and/or    b) deletion or substitution of histidine at position 300 of SEQ ID NO.1; and/or    c) deletion or substitution of histidine at position 349 of SEQ ID NO. 1.    
     
     
         12 . An attenuated BVDV strain or clone obtained by a method according to  claim 7 .  
     
     
         13 . A vaccine comprising an attenuated BVDV clone or strain according to  claim 12 , optionally in combination with a pharmaceutically acceptable carrier or excipient.  
     
     
         14 . A method of preventing or treating a BVDV infection in an animal comprising administering to the animal an attenuated BVDV clone or strain according to  claim 12 .  
     
     
         15 . A vaccine comprising an attenuated BVD virus type 1, wherein the RNase activity in its protein E rns  is inactivated, combined with an attenuated BVD virus type 2, wherein the RNase activity in its protein E rns  is inactivated, or any other antigenetic group wherein the RNase activity in its protein E rns  is inactivated, and a pharmaceutically acceptable carrier or excipients.  
     
     
         16 . The vaccine according to  claim 15 , wherein the RNase activity in the protein E rns  of the attenuated BVD virus type 1, attenuated BVD virus type 2, and any of such other antigenetic group is inactivated by deletion or substitution at position 295 to 307 and/or position 338 to 357.  
     
     
         17 . The vaccine according to  claim 15 , wherein the RNase activity in the protein E rns  of the attenuated BVD virus type 1, attenuated BVD virus type 2, and any of such other antigenetic group is inactivated by mutation consists of: 
 a) deletion of all or part of the glycoprotein E rns ; and/or    b) deletion or substitution of histidine at position 300 according to SEQ ID NO. 1; and/or    c) deletion or substitution of histidine at position 349 according to SEQ ID NO. 1.    
     
     
         18 . The vaccine according to  claim 15 , wherein the RNase activity in the protein E rns  of the attenuated BVD virus type 1, attenuated BVD virus type 2 and any of such other antigenetic group is inactivated by deletion or substitution of histidine at position 300 and/or deletion or substitution of histidine at position 349.  
     
     
         19 . The vaccine according to  claim 15 , wherein the RNase activity in the protein E rns  of the attenuated BVD virus type 1, attenuated BVD virus type 2 and any of such other antigenetic group is inactivated by deletion or substitution of histidine at position 300.  
     
     
         20 . The vaccine according to  claim 15 , wherein the RNase activity in the protein E rns  of the attenuated BVD virus type 1, attenuated BVD virus type 2 and any of such other antigenetic group is inactivated by deletion of histidine at position 349.