Replication Stable and RNase Resistant Chimeras of Pestivirus with Insertion in 3' Nontranslated Region (3'NTR)

Abstract

The invention relates to the field of nucleic acid amplification, particularly to quality control materials for use in viral RNA assays. It specifically relates to the construction of a recombinant Pestivirus by the identification of a region in the 3′NTR of the viral RNA genome where additional sequence elements can be stably inserted. Chimeric Pestivirus with sequence insertions in the 3′ nontranslated region (3′NTR) of the viral RNA genome were stable in replication and capable of forming infectious, RNase resistant virus particles. This chimeric Pestivirus with a 3′NTR insertion can be utilized as a quality control material in analytical assays for RNA targets, including external, internal controls, quantitative standards in PCR and NAT nucleic acid assays.

1 . A chimeric viral RNA sequence comprising: first virus RNA sequence from Pestivirus, and second RNA sequence inserted within a 3′ NTR of the first Pestivirus RNA sequence, wherein the chimeric viral RNA sequence is stable in RNA replication and forms a ribonuclease resistant viral particle. 
     
     
         2 . The chimeric RNA viral sequence of  claim 1 , wherein the first virus RNA sequence is bovine viral diarrhea virus (BVDV). 
     
     
         3 . The chimeric RNA viral sequence of  claim 2 , wherein the first virus RNA sequence is a non-CP7 clone of the bovine viral diarrhea virus (BVDV). 
     
     
         4 . The chimeric RNA viral sequence of  claim 1 , wherein the second RNA sequence is inserted in a variable region of the 3′ NTR of the first Pestivirus RNA sequence. 
     
     
         5 . The chimeric RNA viral sequence of  claim 1 , wherein the second RNA sequence is inserted between a SL STOP  stem-loop structure and a SLII stem-loop structure in the 3′NTR of the first Pestivirus RNA sequence. 
     
     
         6 . The chimeric RNA viral sequence of  claim 5 , wherein the second RNA sequence is inserted between a UGA pos.cons.  box and the SLII stem-loop structure in the 3′V of the 3′NTR. 
     
     
         7 . The chimeric RNA viral sequence of  claim 1 , wherein the second RNA sequence is a portion of an HCV virus. 
     
     
         8 . The chimeric RNA viral sequence of  claim 7 , wherein the portion of the HCV virus is comprised of at least 8 nucleotides. 
     
     
         9 . The chimeric RNA viral sequence of  claim 7 , wherein the second RNA sequence is a 5′NTR region of the HCV virus. 
     
     
         10 . The chimeric RNA viral sequence of  claim 1 , wherein the second RNA sequence is a multiplex sequence comprising sequences from more than one RNA virus. 
     
     
         11 . The chimeric RNA viral sequence of  claim 1 , wherein the second RNA sequence is selected from the group consisting of HCV, HCV genotypes 1-7, HIV, HIV-1, HIV-2, HTLV-1, HTLV-2, hepatitis G, an enterovirus, a respiratory virus and a blood borne virus. 
     
     
         12 . A vector comprising: DNA sequence of a chimeric viral RNA sequence comprising: vector DNA sequence, first DNA sequence derived from a first Pestivirus RNA sequence, and second RNA sequence inserted within a 3′ NTR of the first Pestivirus RNA sequence, wherein the sequence of the chimeric RNA virus derived from the DNA vector is stable in replication, and forms a RNase resistant viral particle. 
     
     
         13 . The chimeric RNA viral sequence of  claim 12 , wherein the second RNA sequence is inserted in a variable region of the 3′ NTR of the first Pestivirus RNA sequence. 
     
     
         14 . The chimeric RNA viral sequence of  claim 12 , wherein the second RNA sequence is inserted between a SL STOP  stem-loop structure and a SLII stem-loop structure in the 3′NTR of the first Pestivirus RNA sequence. 
     
     
         15 . The chimeric RNA viral sequence of  claim 12 , wherein the second RNA sequence is at least a portion of an HCV virus. 
     
     
         16 . The chimeric RNA viral sequence of  claim 15 , wherein the second RNA sequence is at least 8 nucleotides of the HCV virus. 
     
     
         17 . The chimeric RNA viral sequence of  claim 15 , wherein the second RNA sequence is from a 5′ NTR region of the HCV virus. 
     
     
         18 . A culture of cells containing RNA virus comprising: host cell harboring chimeric first and second RNA virus sequences, wherein the first RNA virus is Pestivirus and a second RNA sequence is inserted in a 3′ NTR of the first Pestivirus virus RNA sequence, wherein the chimeric RNA viral sequence is stable in RNA replication, and ribonuclease resistant viral particles comprising the first and second RNA virus sequences when cultured. 
     
     
         19 . The culture of  claim 18 , wherein the second RNA sequence is inserted in a variable region of the 3′ NTR of the first Pestivirus RNA sequence. 
     
     
         20 . The culture of  claim 18 , wherein the second RNA sequence is inserted between a SL STOP  stem-loop structure and a SLII stem-loop structure in the 3′NTR of the first Pestivirus RNA sequence. 
     
     
         21 . The culture of  claim 18 , wherein the second RNA sequence is from an HCV virus. 
     
     
         22 . The culture of  claim 21 , wherein the second RNA sequence is at least 8 nucleotides of the HCV virus. 
     
     
         23 . The culture of  claim 21 , wherein the second RNA sequence is from a 5′ NTR region of the HCV virus. 
     
     
         24 . The culture of  claim 18 , wherein the viral particles are infectious. 
     
     
         25 . The culture of  claim 18 , wherein the viral particles are non-infectious.