We have previously demonstrated that the replacement of the S gene from an avirulent strain (Beaudette) of infectious bronchitis virus (IBV) with an S gene from a virulent strain (M41) resulted in a recombinant virus (BeauR-M41(S)) with the in vitro cell tropism of the virulent virus but that was still avirulent. In order to investigate whether any of the other structural or accessory genes played a role in pathogenicity we have now replaced these from the Beaudette strain with those from M41. The recombinant IBV was in effect a chimaeric virus with the replicase gene derived from Beaudette and the rest of the genome from M41. This demonstrated that it is possible to exchange a large region of the IBV genome, approximately 8.4 kb, using our transient dominant selection method. Recovery of a viable recombinant IBV also demonstrated that it is possible to interchange a complete replicase gene as we had in effect replaced the M41 replicase gene with the Beaudette derived gene. Analysis of the chimaeric virus showed that it was avirulent indicating that none of the structural or accessory genes derived from a virulent isolate of IBV were able to restore virulence and that therefore, the loss of virulence associated with the Beaudette strain resides in the replicase gene.

In 2003, the word "coronavirus" spread across the globe, somewhat further than the virus that sparked the panic. In SARS- and Other Coronaviruses: Laboratory Protocols, expert researchers examine thes

Of the many primer combinations that we have investigated for the detection of avian coronaviruses, two have worked better than any of the others: they worked with the largest number of strains/samples of a given coronavirus and the most species of avian coronavirus, and they also produced the most sensitive detection tests. The primer combinations were: oligonucleotide pair 2Bp/4Bm, which is in a region of gene 1 that is moderately conserved among all species of coronavirus (1); and UTR11-/UTR41+, which are in a highly conserved part of the 3' untranslated region of avian coronaviruses related to infectious bronchitis virus (2). The gene 1 primer pair enabled the detection of a new coronavirus in a green-checked Amazon parrot (Amazon viridigenalis Cassin). In this chapter we describe the use of these oligonucleotides in a one-step (single-tube) RT-PCR, and describe the procedure that we used to extract RNA from turkey feces.

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA. We have used it successfully for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA containing the modified IBV cDNA into cells infected with a recombinant fowlpox virus expressing T7 DNA-dependent RNA polymerase.

This chapter covers the genome organization and expression mechanisms of the nidoviruses. Following infection of a susceptible cell by a nidovirus and uncoating of the RNA genome, the first step in a successful replication cycle is the production of the replicase proteins. The nidovirus genomic RNA (gRNA) initially acts as a eukaryotic mRNA for the translation of the replicase proteins. Following synthesis of the replicase proteins, the positive-sense gRNA is copied into negative-sense counterparts which act as templates for the synthesis of new gRNAs. In addition to a negative-sense gRNA, nidoviruses produce a series of negative-sense counterparts of the subgenomic mRNAs (sg mRNAs). The synthesis of both full-length and subgenome-length negative-sense RNAs is initiated at the 3’ end of the gRNA. Synthesis of negative-sense RNAs may terminate at different points along the gRNA template, yielding subgenome-length minus-strand RNAs. Attenuation of minus-strand RNA synthesis occurs at sequences, known as transcription regulatory sequences (TRSs), which are well conserved in a virus type but differ between groups, genera, and families of viruses. The negative-sense sgRNAs act as templates for the synthesis of the positive-sense sgRNAs, which are usually generated in a large excess compared to their negative-sense counterparts. The mechanism for the synthesis of nidovirus sgRNAs is called discontinuous extension of minus-strand RNA.

Infectious bronchitis virus (IBV), the coronavirus of the chicken (Gallus gallus), is one of the foremost causes of economic loss within the poultry industry, affecting the performance of both meat-type and egg-laying birds. The virus replicates not only in the epithelium of upper and lower respiratory tract tissues, but also in many tissues along the alimentary tract and elsewhere e. g. kidney, oviduct and testes. It can be detected in both respiratory and faecal material. There is increasing evidence that IBV can infect species of bird other than the chicken. Interestingly breeds of chicken vary with respect to the severity of infection with IBV, which may be related to the immune response. Probably the major reason for the high profile of IBV is the existence of a very large number of serotypes. Both live and inactivated IB vaccines are used extensively, the latter requiring priming by the former. Their effectiveness is diminished by poor cross-protection. The nature of the protective immune response to IBV is poorly understood. What is known is that the surface spike protein, indeed the amino-terminal S1 half, is sufficient to induce good protective immunity. There is increasing evidence that only a few amino acid differences amongst S proteins are sufficient to have a detrimental impact on cross-protection. Experimental vector IB vaccines and genetically manipulated IBVs-with heterologous spike protein genes-have produced promising results, including in the context of in ovo vaccination.

Infectious bronchitis coronavirus (IBV) is the cause of the single most economically costly infectious disease of domestic fowl in the UK--and probably so in many countries that have a developed poultry industry. A major reason for its continued dominance is its existence as many serotypes, determined by the surface spike protein (S), cross-protection being poor. Although controlled to some degree by live and inactivated vaccines, a new generation of IB vaccines is called for. Reverse genetic or 'infectious clone' systems, which allow the manipulation of the IBV genome, are key to this development. New vaccines would ideally be: genetically stable (i.e. maintain a stable attenuated phenotype); administered in ovo; and be flexible with respect to the source of the spike protein gene. Rational attenuation of IBV requires the identification of genes that are simultaneously not essential for replication and whose absence would reduce pathogenicity. Being able to modify a 'core' vaccine strain to make it applicable to a prevailing serotype requires a procedure for doing so, and the demonstration that 'spike-swapping' is sufficient to induce good immunity. We have demonstrated that four small IBV proteins, encoded by genes 3 and 5, are not essential for replication; failure to produce these proteins had little detrimental affect on the titre of virus produced. Our current molecularly cloned IBV, strain Beaudette, is non-pathogenic, so we do not know what effect the absence of these proteins would have on pathogenicity. That said, plaque size and composition of various gene 3/5 recombinant IBVs in cell culture, and reduced output and ciliostasis in tracheal organ cultures, shows that they are less aggressive than the wild-type Beaudette. Consequently these genes remain targets for rational attenuation. We have recently obtained evidence that one or more of the 15 proteins encoded by gene 1 are also determinants of pathogenicity. Hence gene 1 is also a target for rational attenuation. Replacing the S protein gene of Beaudette with that from the pathogenic M41 strain resulted in a recombinant virus that was still non-pathogenic but which did induce protection against challenge with M41. We have since made other 'spike-swapped' recombinants, including ones with chimaera S genes. Uniquely, our molecular clone of Beaudette is benign when administered to 18-day-old embryos, even at high doses, and induces immunity after this route of vaccination. Taken together, our results point to the creation of a new generation of IB vaccines, based on rational modification of the genome, as being a realisable objective.

The avian coronavirus infectious bronchitis virus (IBV) expresses four nonstructural, non-gene 1 proteins (3a, 3b, 5a and 5b) which have been shown to be dispensable for virus replication in cell culture. These IBV accessory proteins have no sequence homology to any of the accessory proteins of the group I and II coronaviruses but are highly conserved among the group III coronaviruses. Characterisation of naturally occurring strains of IBV which do not express two or more of the accessory proteins and of genetically modified recombinant IBVs has demonstrated that these accessory proteins contribute at most a minor role to the pathogenicity of the virus. To understand the relevance of these proteins for IBV the subcellular location of the 3a, 5a and 5b proteins have been characterised along with the identification of potential protein-protein interactions for the 3a protein. The subcellular location and protein-protein interactions of the 3b protein were attempted but specific problems were encountered. Indirect immunofluorescence with confocal microscopy of IBV-infected chick kidney cells was used to study the subcellular location of the accessory proteins. The 3a protein displayed a punctate, cytoplasmic distribution pattern which colocalised with virally-induced double-stranded RNA. A diffuse, cytoplasmic distribution was observed for the 5b protein which produced limited colocalisation with an IBV structural protein. Expression of a FLAG-tagged 5a protein in transfected Vero cells resulted in a punctate, cytoplasmic pattern. The protein interactions of the 3a protein were identified using FLAG-tag pull-down experiments with tandem mass spectrometry. Six cellular proteins were identified as interacting with the FLAG/3a protein within transfected Vero cells, three of which, GCN1, PP2A and Exportin-1, may interact with native 3a protein in IBV-infected cells. The 3a protein could sequester the viral dsRNA to hide it from the innate immune system and the potential interactions with three cellular proteins indicate that the IBV 3a protein may contribute to attenuation of host cell translation, induce cell cycle arrest and/or attenuate the nuclear export of a specific subset of mRNAs.

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.

The available detection methods for avian pneumoviruses (turkey rhinotracheitis virus; genus Metapneumovirus ) in turkeys, domestic fowl and other species are reviewed. The advantages and disadvantages of virus isolation techniques, virus or genome (polymerase chain reaction) detection and serology are discussed. Some of the problems likely to be encountered are considered, including the detection of yet to be discovered subtypes, as are the factors that are likely to influence the outcome of the work

The universality of seven pairs of oligonucleotides for detection of the coronavirus infectious bronchitis virus (IBV) by reverse-transcription polymerase chain reaction (RT-PCR) was examined using 41 isolates of IBV collected over five decades from Europe, Japan and the USA. Oligonucleotides specific for sequences within the S2 region of the spike (S) gene (Lin et al., 1991a) and nucleocapsid (N) gene (Zwaagstra et al., 1992) gave the appropriate products with all 41 isolates. Oligonu-cleotide pair UTR1 - UTR2 +, corresponding to sequences within the 3 untranslated region (UTR) of the genome, also gave the predicted product with all the isolates. Oligonucleotide pair UTR3 - /UTR4 + was internal to oligonucleotides UTR1 - /UTR2 + and was used in a nested-set arrangement for greater specificity and sensitivity, giving the correct product with the 39 isolates examined. Oligonucleotide pair S1Unil - /S1Uni2 + was used to produce a 1.6 kb cDNA, corresponding to most of the S1 region of the S gene, with 24/24 isolates tested. This oligonucleotide pair was less suited than the others for routine detection of IBV but is recommended for the amplification of the S1 region of the S gene of new isolates for subsequent analysis. Other oligonucleotide pairs, yielding cDNA corresponding to the variable region of the IBV genome where genes 3 and 4 (M) overlap, were selected to be largely specific for Massachusetts serotype isolates, in the context of European strains. RT-PCR analysis using these oligonucleotide pairs showed that a number of field isolate preparations also contained a small amount of Massachusetts serotype virus, probably of vaccine origin and indicative of low level persistent infection. These results suggest that any strain of IBV is likely to be detectable by RT-PCR with at least one of our primer pairs.