Generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection

Abstract

A reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) has been described in which a full-length cDNA, corresponding to the IBV (Beaudette-CK) genome, was inserted into the vaccinia virus genome following in vitro assembly of three contiguous cDNAs [Casais, R., Thiel, V., Siddell, S.G., Cavanagh, D., Britton, P., 2001. Reverse genetics system for the avian coronavirus infectious bronchitis virus. J. Virol. 75, 12359-12369]. The method has subsequently been used to generate a recombinant IBV expressing a chimaeric S gene [Casais, R., Dove, B., Cavanagh, D., Britton, P., 2003. Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism. J. Virol. 77, 9084-9089]. Use of vaccinia virus as a vector for the full-length cDNA of the IBV genome has the advantage that modifications can be made to the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. We describe the use of homologous recombination as a method for modifying the Beaudette full-length cDNA, within the vaccinia virus genome, without the requirement for in vitro assembly of the IBV cDNA. To demonstrate the feasibility of the method we exchanged the ectodomain of the Beaudette spike gene for the corresponding region from IBV M41 and generated two recombinant infectious bronchitis viruses (rIBVs) expressing the chimaeric S protein, validating the method as an alternative way for generating rIBVs.

Fig. 1

Schematic diagram for the construction of the recombination vector for deleting the IBV S gene from the full-length cDNA in the vaccinia virus genome. The recombination vector is based on a GPT expressing plasmid, pGPTNEB193, in which a segment of the IBV genome, corresponding to the IBV S gene and flanking sequences of approximately equal length, was introduced. The majority of the S gene was then removed, leaving only the transmembrane and C-terminal domains within the recombination vector, pGPT-IBV-ΔS. The flanking regions were included to allow homologous recombination events to occur between the IBV sequences in pGPT-IBV-ΔS and the corresponding sequences in the IBV full-length cDNA in the vaccinia virus (vNotI-IBV_FL) genome. The flanking regions were of approximate equal length to allow unbiased homologous recombination events. The various IBV gene sequences and relevant restriction sites are indicated; S represents the spike glycoprotein gene, 3a, 3b, 3c (E) represent the IBV gene 3 products; M represents the integral membrane protein; 5a, 5b represent the IBV gene 5 products; N represents the nucleocapsid protein; 3′ UTR represents the IBV 3′ untranslated region; HδR represents the hepatitis delta ribozyme sequence; T7 term represents the T7 termination sequence; Ecogpt represents the E. coli GPT gene and VV P_7.5 represents the vaccinia virus early/late P_7.5 promoter.

Fig. 2

Schematic diagram for the construction of the recombination vector for inserting the chimaeric S gene into the IBV cDNA lacking an S gene sequence in the vaccinia virus genome. The signal sequence, ectodomain and transmembrane domain of the M41-CK S gene sequence was used to replace the corresponding Beau-CK sequence in pGPT-IBV-StuI-BamHI. The transmembrane domains from the two viruses are identical, therefore the chimaeric S gene has essentially the signal sequence and ectodomain sequences derived from M41-CK and transmembrane and C-terminal domain sequences from Beau-CK in pGPT-M41S. The flanking regions of the chimaeric S gene were as described in the legend for Fig. 1 and were present to allow integration of the chimaeric S gene by homologous recombination events into the IBV cDNA lacking an S gene sequence in vNotI-IBV-ΔS_FL. The various IBV gene sequences are as described in the legend for Fig. 1 and the relevant restriction sites are shown.

Fig. 3

Schematic diagram demonstrating the TDS method for removing the S gene sequence from the full-length IBV cDNA in vNotI-IBV_FL. The diagram represents a potential recombination event between the replicase sequence in one of the flanking regions in pGPT-IBV-ΔS and the corresponding region in the IBV cDNA in vNotI-IBV_FL. The single step recombination event results in the integration of the complete plasmid sequence into the IBV cDNA in the vaccinia virus genome. Resultant recombinant vaccinia viruses are selected in the presence of MPA due to incorporation of the GPT gene under the control of a vaccinia virus promoter. The integrated sequences result in tandem repeat sequences within the IBV cDNA. Such resultant vaccinia viruses are relatively unstable and are only able to maintain the duplicate sequences in the presence of MPA. In the absence of MPA one of the duplicated sequences is lost as a result of spontaneous recombination events. In the absence of MPA, two types of potential recombination events, I and II, are possible resulting in the loss of the GPT gene. Recombination event (I) results in retention of the S gene sequence, such recombinant vaccinia viruses have the same sequence as the original input vaccinia virus; (II) results in the loss of most of the S gene sequence, the desired modification. Both types of recombination events have an equal chance of occurring. The various IBV genes are as described in the legend to Fig. 1 Rep represents the end of the IBV replicase gene, ΔS indicates that the signal sequence and ectodomain are deleted with the transmembrane and C-terminal domain sequences remaining in the partial S gene sequence in the IBV cDNA; 3 and 5 represent the IBV gene 3 and 5 sequences, respectively.

Fig. 4

Schematic diagram demonstrating the TDS method for inserting the chimaeric S gene sequence into the IBV cDNA lacking an S gene sequence in vNotI-IBV-ΔS_FL. The diagram represents two potential recombination events, the first involving the integration of the complete pGPT-M41S plasmid into the IBV cDNA sequence in vNotI-IBV-ΔS_FL with concomitant selection in the presence of MPA. The second type of recombination events occur in the absence of MPA, resulting in the loss of the GPT gene and resulting in recombinant vaccinia viruses with either (I) the original sequence of the input vaccinia virus, with an IBV cDNA sequence lacking the S gene sequence, or (II) insertion of the chimaeric S gene sequence, the desired modification. The various IBV genes are as described in the legend to Fig. 1 B-S indicates that the transmembrane and C-terminal domain are derived from Beau-CK.

Fig. 5

Growth profiles of the rIBVs on (A) CK and (B) Vero cells. The cells were infected with Beau-R, M41-CK, BeauR-M41(S), rIBV-M41S-A and rIBV-M41S-B. Cell medium from the infected cells was analysed, for progeny virus, by plaque titration assay on CK cells 1–48 h post infection.