In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication

Abstract

Molecular clones of infectious bronchitis virus (IBV), derived from the Vero cell adapted Beaudette strain, were constructed, using an in vitro assembly method. In vitro transcribed RNA from a cDNA template that had been constructed from seven cDNA fragments, encompassing the entire genome of IBV, was electroporated into BHK-21 cells. The cells were overlaid onto the susceptible Vero cells and viable virus was recovered from the molecular clone. The molecularly cloned IBV (MIBV) demonstrated growth kinetics, and plaque size and morphology that resembled the parental Beaudette strain IBV. The recombinant virus was further manipulated to express enhanced green fluorescent protein (EGFP) by replacing an open reading frame (ORF) of the group-specific gene, ORF 5a, with the EGFP ORF. The rescued recombinant virus, expressing EGFP (GIBV), replicated to lower viral titers and formed smaller plaques compared to the parental virus and the MIBV. After six passages of GIBV, a minority of plaques were observed that had reverted to the larger plaque size and virus from these plaques no longer expressed EGFP. Direct sequencing of RT-PCR products derived from cells infected with the plaque-purified virus, which had lost expression of EGFP, confirmed loss of the EGFP ORF. The loss of EGFP expression (Delta5a IBV) was also accompanied by reversion to growth kinetics resembling the standard virus and intact recombinant virus. This study demonstrates that the 5a ORF is not essential for viral multiplication in Vero cells.

Fig. 1

SapI and BsmBI restriction enzyme sites were used for in vitro assembly of the cDNA for the full-length IBV genome. (A) The 5′ end of the A fragment was amplified with primers that incorporated the T7 RNA polymerase promoter and similarly, a 19 nucleotide polyadenylated tail was inserted at the 3′ end of E. Gray boxes represent ORF of the IBV genome. Open boxes represent genome fragments amplified by RT-PCR and black boxes represent 5′ and 3′ untranslated regions. (B) DNA amplicons used for in vitro ligation. Each amplicon was digested with the appropriate restriction enzyme and separated from the plasmid vectors using agarose gel electrophoresis. Lane M represents the 1 kb DNA ladder; lane 1, fragment A prepared by XhoI digestion, CIP treatment and then SapI restriction enzyme digestion; lane 2, fragment B1 prepared by BsmBI and SapI; lane 3, fragment B2 prepared by digestion with BsmBI and SapI restriction enzyme; lane 4, fragment C1 prepared by BsmBI and SapI digestion; lane 5, fragment C2 prepared by BsmBI digestion; lane 6, fragment D prepared by BsmBI digestion; lane 7, fragment E1 prepared by EcoRI digestion followed by BsmBI digestion, and lane 8, the E2 fragment. (C) In vitro orderly ligation of the generated cDNA fragments. The inserts were ligated stepwise as follows. Lane M represents the 1-kb DNA extension ladder; lane 1, ligation products of A and B1; lane 2, ligation products of B2 and C1; lane 3, purified ligation product of C2 and D; lane 4, purified ligation product of E1 and E2; lane 5, ligation products of lanes 1 and 2; lane 6, ligation products of lanes 3 and 4; lane 7, the final ligation product of lane 5 and 6 (shown by the arrow to the right).

Fig. 2

MIBV generated similar sized plaques as the wild type IBV. Vero cells were infected with each virus and then overlaid with media containing 0.8% agarose. Three days after infection, agarose was removed and cells were stained with crystal violet. The MIBV plaques are shown in panel A and standard cell adapted Beaudette IBV, in panel B.

Fig. 3

Comparison of the replication kinetics of standard cell-adapted and recombinant IBV. Standard IBV and recombinant IBV, MIBV, GIBV, and Δ5a IBV were used to infect Vero cells and triplicate samples of the infected cells were harvested every 4 h for 24 h. Total virus was harvested by freezing and thawing cells three times and the viral titer was measured by counting plaque forming units/ml at each time point.

Fig. 4

Construction of EGFP/Δ5a amplicon. (A) The 5a ORF was replaced with the EGFP ORF. Open boxes represent ORF of the IBV genome. Black lines represent PCR products. Arrows indicate the restriction enzyme sites inserted into the PCR products. (B) PCR products containing EGFP in place of ORF 5a. M represents the 1-kb DNA molecular size marker; lane 1, PCR product of EGFP, denoted as F in (A); lane 2, PCR product of M ORF and junction of 5′ of ORF 5a, denoted as G in (A); lane 3; PCR product encompassing 3′ end of 5a ORF and downstream of the ORF through 3′ UTR of IBV, denoted as H in (A). (C) Underlined are IBV sequences and bold are EGFP sequences.

Fig. 5

Expression of EGFP in Vero cells infected with GIBV. (A) GIBV infection produced syncytia typical of the CPE from Vero cells infected with the standard IBV. (B) Expression of EGFP was observed by UV microscopy. Expression of EGFP could be observed prior to the typical IBV-induced CPE.

Fig. 6

EGFP deletion revertant of GIBV showed comparable plaques to standard IBV or MIBV. After six serial passages of GIBV, heterogeneous-sized plaques were observed. The arrow indicates typical GIBV plaques with the smaller size compared to MIBV and the arrow head indicates a larger plaque from the GIBV stock that was comparable to the standard cell adapted IBV, and plaques purified from the larger plaques in A are shown in panel B.

Fig. 7

GIBV phenotypic revertant had lost the EGFP ORF. The absence of the EGFP ORF was confirmed by RT-PCR and nucleotide sequencing of the region corresponding to nucleotides 25,162 to 25,613 of the standard cell adapted IBV. Compared to GIBV, the GIBV revertant produced a smaller RT-PCR product, smaller than the E amplicon, suggesting that the revertant had lost the EGFP and 5a ORFs. (A) RT-PCR and PCR products of the region corresponding to nucleotides 25,162 to 25,613 of GIBV, the phenotypic 5aGFP deletion revertant and the control, E amplicon. The M lanes represent the DNA molecular size marker; lane 1, RT-PCR product of GIBV expressing EGFP; lane 2, the RT-PCR product of the phenotypic revertant that had lost expression of EGFP, and 3, as a control, the PCR product from the E fragment. (B) Alignment of the nucleotide sequences of the region spanning the 5a/EGFP ORF of Δ5aGFP viruses identified four distinct types of EGFP deletions. Italicized nucleotides and the positions starred below the sequence alignment represent conserved sequences. Every mutant lost the EGFP start codon but maintained the 5b start codon (underlined).