Coronaviruses from pheasants (Phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys.

Reverse-transcriptase polymerase chain reactions (RT-PCRs) were used to examine RNA extracted from mouth/nasal swabs from pheasants exhibiting signs of respiratory disease. The oligonucleotides used were based on sequences of infectious bronchitis virus (IBV), the coronavirus of domestic fowl. A RT-PCR for the highly conserved region II of the 3' untranslated region of the IBV genome detected a coronavirus in swabs from 18/21 estates. Sequence identity with the corresponding region of IBVs and coronaviruses from turkeys was > 95%. A RT-PCR for part of the S1 region of the spike protein gene was positive with 13/21 of the samples. Sequence analysis of the RT-PCR products derived from nine of the pheasant viruses revealed that some of the viruses differed from each other by approximately 24%, similar to the degree of difference exhibited by different serotypes of IBV. Further analysis of the genome of one of the viruses revealed that it contained genes 3 and 5 that are typical of IBV but absent in both the transmissible gastroenteritis virus and murine hepatitis virus groups of mammalian coronaviruses. The nucleotide sequences of genes 3 and 5 of the pheasant virus had a similar degree of identity (approximately 90%) with those of coronaviruses from turkeys and chickens, as is observed when different serotypes of IBV are compared. This work: (a) confirms that coronaviruses are present in pheasants (indeed, commonly present in pheasants with respiratory disease); (b) demonstrates that their genomes are IBV-like in their organization; and (c) shows that there is sequence heterogeneity within the group of pheasant coronaviruses, especially within the spike protein gene. Furthermore, the gene sequences of the pheasant viruses differed from those of IBV to similar extents as the sequence of one serotype of IBV differs from another. On the genetic evidence to date, there is a remarkably high degree of genetic similarity between the coronaviruses of chickens, turkeys and pheasants.

Infectious bronchitis virus vaccine interferes with the replication of avian pneumovirus vaccine in domestic fowl.

Experiments were performed in chickens to ascertain whether application of infectious bronchitis (IB) H120 vaccine had an effect on the replication of an attenuated avian pneumovirus (APV) strain, using as indicators virus detection, humoral antibody responses and clinical protection against in vivo APV challenge. A preliminary experiment demonstrated that pharyngeal swabs were as efficient for recovery of APV as were buccal cavity swabs, and that either site was superior to swabbing the nasal cavity. APV was detected to a similar extent by both a reverse transcriptase-polymerase chain reaction (RT-PCR) and virus isolation; therefore, RT-PCR was used in subsequent experiments. In chickens vaccinated with APV alone, APV was detected by RT-PCR in most birds for 1 week after vaccination. When IB vaccine had been applied 1 week earlier, APV detection was delayed and much reduced. This interference by IBV resulted in a lower APV antibody response to vaccination. Following challenge with virulent APV, birds that had been vaccinated with APV alone were fully protected both clinically and virologically. Chickens that had received both vaccines were still protected clinically, but challenge virus could be detected in some pharyngeal swabs 4 days after challenge. In contrast, the APV vaccine had no effect on either the antibody response to the IB vaccine or the level of protection against IB challenge. It is concluded that IB vaccination interferes with the replication of APV, resulting in a reduction in the antibody response but with no adverse effect on the induction of protective immunity.

Detection of a coronavirus from turkey poults in Europe genetically related to infectious bronchitis virus of chickens.

Intestinal contents of 13-day-old turkey poults in Great Britain were analysed as the birds showed stunting, unevenness and lameness, with 4% mortality. At post mortem examination, the main gross features were fluid caecal and intestinal contents. Histological examination of tissues was largely unremarkable, apart from some sections that showed crypt dilation and flattened epithelia. Negative contrast electron microscopy of caecal contents revealed virus particles, which in size and morphology had the appearance of a coronavirus. RNA was extracted (turkey/UK/412/00) and used in a number of reverse transcription-polymerase chain reactions (RT-PCRs) with the oligonucleotides based on sequences derived from avian infectious bronchitis virus (IBV), a coronavirus of domestic fowl. The RT-PCRs confirmed that turkey/UK/412/00 was a coronavirus and, moreover, showed that it had the same partial gene order (S-E-M-5-N-3' untranslated region) as IBV. This gene order is unlike that of any known mammalian coronavirus, which does not have a gene analogous to the gene 5 of IBV.The gene 5 of the turkey virus had two open reading frames, 5a and 5b, as in IBV and the coronaviruses isolated from turkeys in North America. The turkey/UK/412/00 also resembled IBV, but not mammalian coronaviruses, in having three open reading frames in the gene encoding E protein (gene 3). The percentage differences between the nucleotide sequences of genes 3 and 5 and the 3' untranslated region of turkey/UK/412/00 when compared with those of IBVs were similar to the differences observed when different strains of IBV were compared with each other. No sequences unique to the turkey isolates were identified. These results demonstrate, for the first time, that a coronavirus was associated with disease in turkeys outside of North America and that it is a Group 3 coronavirus, like IBV.

Longitudinal field studies of infectious bronchitis virus and avian pneumovirus in broilers using type-specific polymerase chain reactions.

In longitudinal studies, 13 flocks were swabbed twice each week for the life of the flock (up to 46 days). The swabs were analyzed by type-specific reverse transcriptase polymerase chain reactions. Massachusetts type vaccinal infectious bronchitis virus (IBVs), applied at the hatchery, were usually maximal during the first week, as expected and, notably, remained detectable for 3 to 4 weeks, occasionally longer. IBV of the 793/B type (also known as 4/91 and CR88) was detected in 11/13 flocks (85%). The time of first detection of 793/B varied over several weeks and was sometimes within the first week in low amounts, which increased gradually. In some flocks, detection of 793/B remained intermittent. IBV types D274 and D1466 were each detected once, in the same flock, for short periods, in low amounts, and in the presence of higher amounts of 793/B. In swabs from a further 30 broiler flocks, plus those already mentioned, there was an incidence for 793/B, D274 and D1466 of 79, 10 and 2%, respectively. Avian pneumovirus (APV) (avian or turkey rhinotracheitis virus) vaccines, applied at the hatchery or later, were either not detected or were detected only after a delay of 1 to 3 weeks. In five flocks that received no APV-A vaccine and two flocks that received only APV type A vaccine, field infection by APV type B was detected but only during the last week or so of life. In six flocks that had received an APV-B vaccine, no field APV-B, differentiated from vaccinal APV-B by restriction enzyme analysis, was detected. In swabs from 30 other flocks, the great majority of which had not been vaccinated against APV, the incidence of APV types B and A was 50 and 3%, respectively. The results show (a) that vaccinal IBV can be detected for several weeks, (b) the dominance of the IBV 793/B type and

Does IBV change slowly despite the capacity of the spike protein to vary greatly?

We have sequenced that part of the spike protein (S) gene which encodes the aminoterminal and most variable quarter (hypervariable region, HVR) of the S1 subunit of 28 isolates of the 793/B (also known as CR88 and 4/91) serotype of infectious bronchitis virus (IBV) and the whole of S1 for nine of them. The isolates were from France and Britain between the years 1985 (first isolation) and 1996. The maximum nucleotide and amino acid differences between the first isolate and the others were 4.1% and 7.6%, respectively, for the whole of S1 and 7.1% and 14.6%, respectively, in the HVR. Analysis within clearly recognisable subgroups suggested that even in the HVR the nucleotide mutation rate was only 0.3 to 0.6% per year. However, there was no evidence that mutations had become fixed in a progressive manner; this serotype did not appear to be evolving. Strains isolated several years apart could be more similar than those isolated in a given year. It is likely that the amino acid changes are largely at positions where amino acid differences are tolerated rather than as a consequence of immune pressure. Reasons for this conclusion are discussed.

Towards the routine application of nucleic acid technology for avian disease diagnosis.

The use of nucleic acid technology (polymerase chain reaction, probing, restriction fragment analysis and nucleotide sequencing) in the study of avian diseases has largely been confined to fundamental analysis and retrospective studies. More recently these approaches have been applied to diagnosis and what one might call real-time epidemiological studies on chickens and turkeys. At the heart of these approaches is the identification and characterisation of pathogens based on their genetic material, RNA or DNA. Among the objectives has been the detection of pathogens quickly combined with the simultaneous identification of serotype, subtype or genotype. Nucleic acid sequencing also gives a degree of characterisation unmatched by other approaches. In this paper we describe the use of nucleic acid technology for the diagnosis and epidemiology of infectious bronchitis virus, turkey rhinotracheitis virus (avian pneumovirus) and Newcastle disease virus.

Expression of bacteriophage T7 RNA polymerase in avian and mammalian cells by a recombinant fowlpox 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.

Investigation of the control of coronavirus subgenomic mRNA transcription by using T7-generated negative-sense RNA transcripts.

The subgenomic mRNAs of the coronavirus transmissible gastroenteritis virus (TGEV) are not produced in equimolar amounts. We have developed a reporter gene system to investigate the control of this differential subgenomic mRNA synthesis. Transcription of mRNAs by the TGEV polymerase was obtained from negative-sense RNA templates generated in situ from DNA containing a T7 promoter. A series of gene cassettes was produced; these cassettes comprised the reporter chloramphenicol acetyltransferase (CAT) gene downstream of transcription-associated sequences (TASs) (also referred to as intergenic sequences and promoters) believed to be involved in the synthesis of TGEV subgenomic mRNAs 6 and 7. The gene cassettes were designed so that negative-sense RNA copies of the CAT gene with sequences complementary to the TGEV TASs, or modified versions, at the 3' end would be synthesized in situ by T7 RNA polymerase. Using this system, we have demonstrated that CAT was expressed from mRNAs derived from the T7-generated negative-sense RNA transcripts only in TGEV-infected cells and only from transcripts possessing a TGEV negative-sense TAS. Analysis of the CAT mRNAs showed the presence of the TGEV leader RNA sequence at the 5' end, in keeping with observations that all coronavirus mRNAs have a 5' leader sequence corresponding to the 5' end of the genomic RNA. Our results indicated that the CAT mRNAs were transcribed from the in situ-synthesized negative-sense RNA templates without the requirement of TGEV genomic 5' or 3' sequences on the T7-generated negative-sense transcripts (3'-TAS-CAT-5'). Modification of the TGEV TASs indicated (i) that the degree of potential base pairing between the 3' end of the leader RNA and the TGEV negative-sense TAS was not the sole determinant of the amount of subgenomic mRNA transcribed and (ii) that other factors, including nucleotides flanking the TAS, are involved in the regulation of transcription of TGEV subgenomic mRNAs.

Characterization of the transmissible gastroenteritis virus (TGEV) transcription initiation sequence. Characterization of TGEV TIS.

The ability of the TGEV transcription initiation sequence (TIS) to produce subgenomic RNAs was investigated by placing a reporter gene, chloramphenicol acetyltransferase (CAT) under the control of either the mRNA 6 or the mRNA 7 TISs. Both constructs only produced CAT in TGEV infected cells and the amount of CAT produced from the mRNA 7 TIS was less than from the mRNA 6 TIS. Mutations were made within and around the TISs and the effect on CAT production assayed. THe results showed that the TGEV TIS acted as a initiator of transcription for CAT, though the degree of base pairing between the TIS and leader RNA was not the only factor implicated in the control transcription.

The cloning and sequencing of the virion protein genes from a British isolate of porcine respiratory coronavirus: comparison with transmissible gastroenteritis virus genes.

Previous analysis of porcine respiratory coronavirus (PRCV) mRNA species showed that mRNAs 2 and 3 were smaller than the corresponding transmissible gastroenteritis virus (TGEV) mRNA species (Page et al. (1991) J. Gen. Virol. 72, 579-587). Sequence analysis showed that mRNA 3 was smaller due to the presence of a new putative RNA-leader binding site upstream of the PRCV ORF-3 gene. However, this observation did not explain the deletion observed in PRCV mRNA 2. Polymerase chain reaction (PCR) was used to generate cDNA from the 3' coding region of the putative polymerase gene to the poly (A) tail of PRCV for comparison to the equivalent region from TGEV. The PRCV S protein was found to consist of 1225 amino acids, which had 98% similarity to the TGEV S protein. However, the PRCV S gene contained a 672 nucleotide deletion, corresponding to 224 amino acids (residues 21 to 245 in TGEV S protein), 59 nucleotides downstream of the S gene initiation codon. The PRCV genome from the ORF-3 gene to the poly (A) tail was sequenced for comparison to TGEV in order to identify other potential differences between the two viruses. Four ORFs were identified that showed 98% similarity to the TGEV ORF-4, M, N and ORF-7 genes. No other deletions or any PRCV specific sequences were identified.

Sequence comparison of the 5' end of mRNA 3 from transmissible gastroenteritis virus and porcine respiratory coronavirus.

Analysis of porcine transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) mRNA species indicated a deletion in mRNA 3 of PRCV. Polymerase chain reaction (PCR) was used to clone the 5' end of mRNA 3 from PRCV for comparison with the equivalent region in TGEV. Small deletions were observed within and around the PRCV sequence equivalent to the putative open reading frame (ORF) ORF-3a identified in TGEV. The potential RNA polymerase-leader complex binding site (leader RNA binding site), ACTAAAC, found upstream of ORF-3a in TGEV, was absent from the PRCV genome but a potential site was found in the PRCV genome upstream of a gene equivalent to TGEV ORF-3b. PCR analysis, using primers corresponding to sequences within the ORF-3b gene and the leader RNA sequence, confirmed that the leader RNA binding site was upstream of a gene equivalent to TGEV ORF-3b on PRCV mRNA 3 but upstream of ORF-3a on TGEV mRNA 3. The presence of the new leader RNA binding site would be responsible for generating the smaller mRNA 3 species found in PRCV-infected cells.