Paul Britton

Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism.

ABSTRACT A recombinant infectious bronchitis virus (IBV), BeauR-M41(S), was generated using our reverse genetics system (R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton, J. Virol. 75:12359-12369, 2001), in which the ectodomain region of the spike gene from IBV M41-CK replaced the corresponding region of the IBV Beaudette genome. BeauR-M41(S) acquired the same cell tropism phenotype as IBV M41-CK in four different cell types, demonstrating that the IBV spike glycoprotein is a determinant of cell tropism.

Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus

ABSTRACT Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for 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

Localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division.

ABSTRACT The subcellular localization of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) (group I and group II coronaviruses, respectively) nucleoproteins (N proteins) were examined by confocal microscopy. The proteins were shown to localize either to the cytoplasm alone or to the cytoplasm and a structure in the nucleus. This feature was confirmed to be the nucleolus by using specific antibodies to nucleolin, a major component of the nucleolus, and by confocal microscopy to image sections through a cell expressing N protein. These findings are consistent with our previous report for infectious bronchitis virus (group III coronavirus) (J. A. Hiscox et al., J. Virol. 75:506–512, 2001), indicating that nucleolar localization of the N protein is a common feature of the coronavirus family and is possibly of functional significance. Nucleolar localization signals were identified in the domain III region of the N protein from all three coronavirus groups, and this suggested that transport of N protein to the nucleus might be an active process. In addition, our results suggest that the N protein might function to disrupt cell division. Thus, we observed that approximately 30% of cells transfected with the N protein appeared to be undergoing cell division. The most likely explanation for this is that the N protein induced a cell cycle delay or arrest, most likely in the G2/M phase. In a fraction of transfected cells expressing coronavirus N proteins, we observed multinucleate cells and dividing cells with nucleoli (which are only present during interphase). These findings are consistent with the possible inhibition of cytokinesis in these cells.

The Coronavirus Infectious Bronchitis Virus Nucleoprotein Localizes to the Nucleolus

ABSTRACT The coronavirus nucleoprotein (N) has been reported to be involved in various aspects of virus replication. We examined by confocal microscopy the subcellular localization of the avian infectious bronchitis virus N protein both in the absence and in the context of an infected cell and found that N protein localizes both to the cytoplasmic and nucleolar compartments.

Universal oligonucleotides for the detection of infectious bronchitis virus by the polymerase chain reaction

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.

Experimental Evidence of Recombination in Coronavirus Infectious Bronchitis Virus

Abstract 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.

Interaction of the Coronavirus Nucleoprotein with Nucleolar Antigens and the Host Cell

ABSTRACT Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.

Molecular analysis of the 793/B serotype of infectious bronchitis virus in Great Britain.

Since the winter of 1990/91 respiratory disease of poultry in Great Britain has commonly been associated with the 793/B (or 4/91) serotype of infectious bronchitis virus (IBV). We have sequenced a variable part of the S1 region of the spike protein (5) gene. Comparison of up to 270 nucleotides of 12 British 793/B isolates, obtained in 1991 and 1993, revealed 94 to 100% nucleotide identity with each other. Eleven of them fell into one of two subgroups, A and B, one isolate forming subgroup C. Identity within subgroups A and B was > 98%. The whole S1 gene sequence (1617 nucleotides) was determined for five 793/B isolates, two from each of subgroups A and B and one from subgroup C; nucleotide identity between any two isolates was > 97%. A large proportion of the nucleotide differences corresponded to amino acid changes. The whole S1 amino acid sequence differed by 21 to 25% or more from that of all other published IBV sequences. This extensive difference has probably contributed to the persistence of the 793/B serotype in Britain even though het-erologous vaccines have been used. The finding that the 793/B isolates could be placed into three subgroups suggests that either (a) they had diverged from a common progenitor present, but undetected, in Britain prior to 1990/91 or (b) at least three different strains of the 793/B serotype had entered Britain in or prior to 1990/91.

Variation in the spike protein of the 793/B type of infectious bronchitis virus, in the field and during alternate passage in chickens and embryonated eggs

The degree of variation exhibited within the 793/B serotype (also known as 4/91 and CR88 serotypes) was investigated with nine French and 10 British isolates, collected between 1985 and 1994. The S1 part (1644 nucleotides) of the spike protein gene of the first known isolate of this serotype, FR/CR85131/85, had 95.9% to 97% nucleotide identity with the other isolates. Partial sequencing of isolates from Iran and Saudi Arabia, isolated in 2000, revealed approximately 95% nucleotide identity with European isolates, including the two live 793/B vaccinal strains, showing that they were not re-isolations of vaccinal virus. The data indicates that strains within the 793/B serotype have ≥96% nucleotide identity within the whole S1 gene and ≥93% nucleotide identity within the first 560 nucleotides, and ≥92% and ≥86% amino acid identities in the corresponding protein regions. This is similar to the identities exhibited within the Massachusetts serotype. Sequence analysis of a 793/B field isolate after passage in embryonated eggs, then in chickens and then again in eggs revealed selection for a serine and alanine at S1 amino acid position 95 in chicken-passaged and egg-passaged virus, respectively. There was no change in pathogenicity. This is the first demonstration at gene sequence level of host-driven selection for infectious bronchitis virus.