Embryonated eggs were coinfected with two strains of the coronavirus avian infectious bronchitis virus (IBV), IBV-Beaudette and IBV-M41, to investigate whether recombination between the two strains would occur. Virions were isolated from the allantoic fluid of the coinfected eggs and putative hybrid RNAs were detected by polymerase chain reaction (PCR), using strain-specific oligonucleotides. PCR products, of the expected sizes, were obtained as predicted from potential recombination events between the nucleoprotein (N) gene and the 3'-untranslated region of the two IBV genomes. Sequencing confirmed that they corresponded to hybrid RNAs. Virus produced as a result of the mixed infection was treated with an M41-specific neutralizing monoclonal antibody and passaged in Vero cells, in which IBV-Beaudette, but not IBV-M41, replicated. Hybrid RNA was still detectable after three serial passages. Since no IBV-M41 was detectable this confirmed that infectious recombinant genomes had been produced in the embryonated eggs. These findings not only support the circumstantial evidence, from sequencing studies of IBV field strains, that recombination occurs during replication of IBV and contributes to the diversity of IBV, but also show that coronavirus RNA recombination is not limited to mouse hepatitis virus.

The bacteriophage T7 RNA polymerase gene was integrated into the fowlpox virus genome under the control of the vaccinia virus early/late promoter, P7.5-The recombinant fowlpox virus, fpEFLT7pol, stably expressed T7 RNA polymerase in avian and mammalian cells, allowing transient expression of transfected genes under the control of the T7 promoter. The recombinant fowlpox virus expressing T7 RNA polymerase offers an alternative to the widely used vaccinia virus vTF7-3, or the recently developed modified vaccinia virus Ankara (MVA) T7 RNA polymerase recombinant, a highly attenuated strain with restricted host-range. Recombinant fowlpox viruses have the advantage that as no infectious virus are produced from mammalian cells they do not have to be used under stringent microbiological safety conditions. Recombinant vaccinia viruses (rVV) have been produced expressing a variety of bacteriophage DNA-dependent RNA polymerases: T7 RNA polymerase (Fuerst et al., 1986), T3 RNA polymerase (Rodriguez et al., 1990) and SP6 RNA polymerase (Usdin et al., 1993). Cells infected with these rVVs can express biologically active products transiently from genes under the control of the appropriate RNA polymerase promoter. The most utilized system is an rVV expressing T7 RNA polymerase, vTF7-3. Translation of the T7 transcripts was inefficient, as only 5-10 % were capped, but inclusion of an internal ribosome entry signal (IRES) sequence, from encephalomyocarditis virus, at the 5' end of the T7-derived RNA transcripts, resulted in mRNAs that were CAP-independent and efficiently translated (Elroy-Stein et al., 1989).In addition to transient expression, a gene under the control of the T7 RNA polymerase promoter can also be integrated into the genome of a second rVV allowing high level expression of the gene product in cells dually infected with vTF7-3 and the second rVV (Fuerst et al., 1987). The vTF7-3 system has been utilized for the expression of RNA per se, e.g. a defective RNA (D-

A high frequency of recombination has been shown to occur during replication of the coronavirus mouse hepatitis virus (MHV) in vitro as well as in vivo. Although sequencing offield strains of coronavirus infectious bronchitis virus (IBV) has indicated that IBV strains also undergo recombination, there has been no experimental evidence to support this deduction. To investigate whether recombination occurs in IBV, embryonated eggs were coinfected with IBV-Beaudette and IBV-M41. Potential recombinants were detected by strain-specific polymerase chain reaction (PCR) amplifications, using oligonucleotides corresponding to regions in the 3' end of the genome. Sequencing of the peR products confirmed that a number of recombinations had occurred between the two strains.• To whom all correspondence should be sent.

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