FIP is one of the most serious diseases in cats. Despite its importance, however, little is known about the molecular biology of this infection. A major obstacle has been the lack of a suitable reverse genetic system for its causative agent, FIPV. As we describe here, we have now established such a system based on targeted RNA recombination. Central to our approach was the generation of an interspecies chimeric virus, mFIPV, that carries the ectodomain of a spike protein which is derived from another coronavirus, MHV. The consequent change in host cell tropism allowed the selection of this virus by its growth in murine cells. The strategy essentially involves the use of mFIPV as the recipient virus in a reverse recombination event that results in the restoration of the spike gene, allowing the recombinant viruses to be selected in turn by their growth in feline cells. Thus, we prepared wt-rFIPV, which appeared to be indistinguishable from the parental virus—FIPV strain 79-1146—not only by its in vitro growth features but also with respect to its lethal phenotype in the natural host, the cat. Finally, to demonstrate the feasibility of our strategy for reverse genetic purposes, we introduced a mutation into the FIPV genome that effectively impaired the expression of the nonstructural 7b gene. After the groundbreaking development of the prototypic MHV system, the present work establishes the second targeted RNA recombination system for coronaviruses.
The synthetic donor RNA that we designed for our recombination system was modeled after the highly effective defective interfering (DI) RNAs that were used as donors in the MHV paradigm. Here it was estimated that frequencies of recombination increase by some orders of magnitude by using a coreplicating RNA rather than a subgenomic RNA (29
). DI RNAs are quite ubiquitous among nidoviruses, their occurrence having been demonstrated for all groups of coronaviruses (including TGEV [31
] and MHV [27
]), for BCV (4
), for IBV (41
), and for the Berne torovirus (45
), as well as for the equine arterivirus (32
). Remarkably, however, no naturally occurring DI has thus far been observed for FIPV. Although we did not confirm whether our synthetic donor RNA was actually replicated in (m)FIPV-infected cells, our recombinant virus yields support this assumption. We routinely observed 102
recombinants per 5 × 106
infected or transfected cells. Our analyses with the donor RNA carrying a lethal mutation in the N gene also showed that a substantial fraction (~4%) of our recombinant viruses had apparently been generated by double crossovers. Clearly, the FIPV system is recombinogenic as had already been made clear by the emergence of serotype II feline coronaviruses through the recombination of feline and canine coronaviruses (18
). Generally, the particular mode of replication and the huge genome size of coronaviruses are thought to favor polymerase template switching, thereby resulting in a remarkably high recombination rate (for a review, see reference 24
The power of the reverse genetics strategy that we established for FIPV lies in the ease and efficiency with which recombinant viruses are selected from the pool of progeny viruses resulting from a recombination trial. The distinctive receptor specificities of coronaviruses allowed the design of a selection principle based on the switch of host cell specificity that accompanies the exchange of different spike ectodomain sequences. Coronavirus spikes are trimeric assemblies (11
) that are incorporated into viral particles by interactions with the M protein (10
). Sequences in both the transmembrane and the endodomain of the spike protein are responsible for this interaction (B. J. Bosch and P. J. M. Rottier, unpublished data). It appeared that the swapping of ectodomains between different coronavirus S proteins was tolerated without an apparent loss of biological features (17
). Thus, chimeric spikes with FIPV- and MHV-derived ectodomains were incorporated into otherwise MHV- and FIPV-based virus-like particles, respectively (17
). These observations were subsequently instrumental for the generation of the chimeric viruses fMHV (22
) and mFIPV that form the basis of the respective reverse genetics systems for these viruses.
Although the genetic exchange did not affect the replication of the recombinant mFIPV to a great extent, it did result in a slower formation of syncytia and the inability to produce plaques in LR7 monolayer cultures. The latter might be explained by the strongly reduced cleavage efficiency of the chimeric S protein compared to the MHV S protein (17
). Inhibition of this cleavage does not affect MHV's specific infectivity, but it clearly reduces the cell-to-cell fusion caused by the spike protein that gives rise to syncytia and plaque formation (K. Stadler, G. Godeke, and P. J. M. Rottier, unpublished data).
The disruption of the initiation codon of ORF 7b abolished the expression of this gene product. The 7b polypeptide is a soluble glycosylated protein, antibodies against which are induced in infected cats (49
). It is a secretory glycoprotein that presumably functions extracellularly. Deletions in the 7b gene readily occur upon propagation of FIPV in tissue culture cells. Sequence observations suggest that deletions in ORF 7b are associated with loss of virulence (19
). Our reverse genetics system will now allow us to investigate the functions of this and other gene products in great detail. The demonstration that the wild-type FIPV strain 79-1146 that we recreated in our recombination system had fully maintained its phenotype is obviously an essential basis for such experiments.
Targeted RNA recombination offers a very convenient general strategy for the genetic manipulation of coronaviruses. The approach had so far been applied only to MHV, except for one case in which Sanchez et al. (44
) demonstrated a role for the TGEV spike protein in pathogenesis. The MHV recombination system has already shown its usefulness for the study of aspects as diverse as virus assembly (8
), transcription and replication (14
), and tropism and virulence (22
) and for the development of coronaviruses as vectors (14
; C. A. M. de Haan, B. J. Haijema, H. Volders, and P. J. M. Rottier, unpublished data). When compared to the different full-size infectious cDNA clones that were recently described for TGEV (1
), HuCV (46
), IBV (2
), and MHV (52
), targeted RNA recombination systems based on interspecies chimeric viruses such as mFIPV and fMHV obviously have attractive technical advantages for the engineering of coronavirus genomes. The most important of these advantages are the easier manipulation of the smaller, subgenomic cDNA donor constructs used, the high efficiency of homologous recombination, and the extremely simple selection of the recombinant viruses. There are, however, clear limitations. One is the inherent inability to study lethal mutations due to the requirement for passaging, a limitation not met with infectious cDNA clones which in principle allow such mutations to be analyzed in a single round of infection. Another limitation is associated with the limited genomic segment, i.e., the domain downstream of the ORF 1b gene, that is accessible for manipulation by the targeted recombination systems, as opposed to the entire virus genome that can be covered by infectious cDNA clones. Although there is no solution to the first problem, the second limitation is not as dramatic as it seems since it still allows the manipulation of all but one of the coronaviral genes, including all intergenic regions and the 3′ noncoding region.