Humanitarian Resource Institute:  A U.S. & International Resource on the Scope of Humanitarian Assistance
.
.
.
.
March 30, 2003

Stephen M. Apatow
Director of Research and Development 
Humanitarian Resource Institute Biodefense Reference Library
Eastern USA: (203) 668-0282   Western USA: (775) 884-4680
Internet: http://www.humanitarian.net/biodefense 
Email: s.m.apatow@humanitarian.net

SARS - CORONAVIRUS SEQUENCES

In the context pf the findings by the Virology department of the
University of Frankfurt (ProMED: SARS - worldwide (16): etiology): 

"The random PCR products were sequenced, and 2 of the obtained sequences matched at the protein level to known coronavirus sequences deposited in GenBank. Both sequences (90 and 300 nucleotides in length) are located
in the polymerase gene but do not overlap with the sequence reported by CDC on 24 Mon 2003. Phylogenetic analysis of the larger fragment suggests that the novel virus is not related closely to any of the known phylogenetic lineages of coronaviruses (groups 1, 2, and 3). The sequence diverges by 50-60 percent from known coronavirus sequences and segregates phylogenetically between bovine coronavirus (group 2) and avian infectious bronchitis virus (group 3)."

The following reports demonstrate the range of challenges that can exist regarding research to study pathogenicity, attenuation and antigenicity for vaccine development, the creation of new pathogenic strains and potential use of such agents for bioterrorism. 

This work also illustrates the importance of veterinary research both in the isolation/definition of a potential infectious agent as well as vaccine development.  As outlined in the AVMA article: Biological terrorism against animals and humans: a brief review and primer for action: "Veterinarians have an important role in bioterrorism response preparation, surveillance for potential bioterrorism events, treatment of the ill, and in the control of disease."

The following serves as interesting reading (all reports now offline), but scratches the surface regarding the amount of research conducted on Avian infectious bronchitis virus (IBV).

1997: INSTITUTE FOR ANIMAL HEALTH
http://www.iah.bbsrc.ac.uk/reports/1996/aibv.html
[Edited]

Epidemiology (1 report)
Pathogenicity (1 report)
Virus replication (2 reports)
Publications

EPIDEMIOLOGY

Survey of Britain

Two species of virus are believed to be currently involved in respiratory disease in broilers and broiler parents in Britain, the coronavirus AIBV and the pneumovirus ARTV (see section Avian rhinotracheitis). The relative involvement of these viruses is not known. Furthermore, AIBV exists as many serotypes, ARTV as two (A and B). It is important to establish the sero/subtype of virus involved in outbreaks of disease in order that the most appropriate vaccines are used.

Supported by the British Chicken Association, we are using sero/subtype-specific RT-PCR to investigate flocks. Confirmation of identity and changes within each virus type are ascertained by nucleotide sequencing.

In the first four months of the survey, AIBV serotype Massachusetts was detected in a third of the samples received. Whether this was residual vaccine strain virus or field strain virus is not known. In another third of cases only AIBV 793/B serotype was detected. It is most likely that this was the cause of disease. In the remaining third of cases ARTV, subtype B, was
detected but usually in the presence of AIBV. In two cases AIBV of the D274 serotype were detected, revealing that this serotype, predominant during the 1980s, is still present.

PATHOGENICITY

Use of recombinants

In order to study pathogenicity, attenuation and antigenicity of the coronavirus AIBV, we wish to replace one gene of a given strain of AIBV (helper virus) with the corresponding (target) gene from a strain with different properties. The natural propensity for AIBV to undergo homologous recombination is the basis of our approach.

We have investigated rescue using six potential helper AIBV strains to see which may be suitable for recombination; a prerequisite for our approach is that an heterologous AIBV strain should be able to replicate CD-61. To-date CD-61 D-RNA has been rescued by five heterologous AIBV strains, as evidenced by Northern blotting.

It is likely that the 5' leader sequence and parts of the 5' and 3' UTRs of the genome are involved in replication in addition to playing a crucial role in transcription. Sequence analysis has revealed that the 5' and 3' UTRs of all six strains, including Beaudette, have only a few nucleotide differences, consistent with successful rescue.

The few nucleotide differences revealed that the leader sequence of the rescued D-RNA had been derived from the helper virus genome rather than from the input D-RNA. This indicated that a recombination event had occurred between the replicating D-RNA and the genomic RNA of the helper virus, analogous to the discontinuous mechanism involved in the transcription of subgenomic coronavirus mRNAs. This suggests that the alternative model for the replication of D-RNAs, involving the direct copying and subsequent amplification of the D-RNA, does not take place.

Our results also indicated that there was indeed an intimate interaction between the replicating D-RNA and genomic RNA, an essential requirement for recombination involving a target gene inserted into CD-61.

We have identified a unique restriction site within a region of CD-61 predicted not to be involved in replication or packaging of the D-RNA. We are investigating if this is a suitable site for insertion of target genes, without impairment of the replication of CD-61. We have inserted the reporter gene luciferase (circa 2 kb), to produce CD-61LUC, under the control of an AIBV transcription associated sequence (TAS) for the expression of a mRNA by the IBV polymerase. The TAS was derived from the AIBV Beaudette gene 5.

All six AIBV helper strains and Beaudette expressed luciferase from CD-61LUC and packaged the modified D-RNA, as evidenced by rescue of CD-61LUC. Rescue of CD-61LUC was confirmed by a sensitive luciferase assay although not by
Northern blotting. This indicated that either replication or packaging of CD-61 had been diminished by inclusion of the luciferase gene.

This work has shown that heterologous strains of AIBV can replicate CD-61 containing an inserted gene, a requirement for the introduction of AIBV target genes into helper virus to produce recombinants. Studies are continuing to improve the efficiency of the system.

One such approach is to insert the CD-61 vector, with target gene, into the genome of fowlpox virus (FPV) such that AIBV-infected cells infected with the recombinant FPV-CD-61 will express CD-61 RNA. It is anticipated that this will result in more cells producing CD-61 and in larger quantities than in our current approach in which CD-61 is electroporated into IBV-infected cells.

We have constructed a cDNA version of CD-61, containing a T7 RNA polymerase promoter at the 5' end and a hepatitis d virus antigenome ribozyme (HdVR) followed by a T7 RNA polymerase termination sequence at the 3' end. CD-61 RNA will be expressed from this recombinant FPV using T7 polymerase provided by another recombinant FPV that we have constructed. To-date we have shown that the ribozyme-containing CD-61, transcribed in vitro, was replicated and packaged after electroporation into AIBV helper virus-infected cells, demonstrating that the additional T7 and HdVR sequences had not adversely affected CD-61, presumably because they had been successfully removed by self-cleavage by the ribozyme. This CD-61 construct, with and without the luciferase gene, is currently being recombined into FPV.

VIRUS REPLICATION

Control of replication and transcription

Coronaviruses have the largest genomes (27.6 kb for AIBV) of any known RNA virus. No full length clones, from which to generate infectious RNA, have been produced, for technical reasons. Consequently we are using smaller, naturally-occurring, defective RNAs to identify sequences likely to be essential for replication of the genomic RNA and its incorporation into virus particles (packaging).

A 9.1 kb defective RNA (CD-91) has 1.1 and 1.6 kb from the 5' and 3' ends of the genome, respectively, including the approximately 500 base long 5' and 3' UTRs, and a large central region comprising part of the polymerase gene.
Electroporation of CD-91 RNA into AIBV strain Beaudette-infected CK cells results in its replication and packaging (rescue), revealed by a series of passages in fresh CK cells.

We have previously shown that removal of 3.0 kb from the central region of CD-91, to produce CD-61, did not deleteriously affect rescue although removal of a further 1.4 kb region did do so. Translation of CD-61-specific ORFs is not required for its replication, suggesting that the 1.4 kb region might be essential for packaging. Recent removal of bases 803 to 2955 (including
0.4 kb of the 1.4 kb region), but not nucleotides 803 to 1683, inhibited rescue, indicating the essential nature of at least part the 1.4 kb region and possibly of nucleotides between 803 and the start of the 1.4 kb region. The latter is not merely a 'stuffer' sequence; reinsertion in the opposite orientation was not sufficient to restore rescue.

Sequencing of six strains, representing three serotypes, of AIBV revealed high sequence conservation of approx. 98% in the 5' UTR and in the final three-fifths of the 3' UTR. The first two-fifths of the 3' UTR were much more variable, including extensive deletions. The sequence immediately following the 5' UTR, ie the start of the polymerase gene, was less conserved than the
UTR. These data suggest that the first 500 and last 300 or so bases of the genome may be essential for replication of the genomic RNA.

In several experiments, new defective RNAs arose during passage. These varied in length but had one feature in common; all were large, at least 9 kb in length. Their size did not diminish on further passage, suggesting, counter to observations with some other viruses, that large defective RNAs of AIBV might have a selective advantage over smaller ones.

Analysis of replication

The polymerase gene of AIBV comprises approximately 20 kb. The encoded polyprotein is processed to a number of smaller polypeptides. The mechanisms by which this is achieved, and the nature of the products, is being investigated with Drs TDK Brown and K Tibbles, University of Cambridge.

An AIBV-encoded 3C-like protease (3CLpro) is responsible for several of the cleavages within the polyprotein. Trans processing studies have proved difficult, with the small amounts expressed in eukaryotic systems. In order to obtain greater expression, a cDNA of the 3CLpro gene has been amplified by PCR and cloned into the bacterial expression vector pMAL, downstream of, and in frame with, the inducible maltose-binding protein.

Earlier studies suggested the involvement of the cellular ubiquitin-mediated protein degradation system in the turnover of the AIBV polyprotein. A major impediment to the extension of these studies is the unavailability of sera with sufficient affinity and specificity to trace the fate of the viral polyprotein in vivo. Two approaches are being taken to overcome this problem. One is the introduction of epitope tags into the polyprotein to enable detection with anti-tag antibodies. A histidine tag has been introduced near the carboxy-terminus of the 3CLpro without impairment of processing of the polyprotein fragment bearing the mutated protease. Another tag being used, for which mAbs are available, corresponds to a peptide sequence (DA3) within the
nucleoprotein of transmissible gastroenteritis coronavirus

The second approach is the use of combinatorial library technology to produce synthetic antibodies with the desired reactivity. A clone has been made at the Laboratory for Molecular Biology, Cambridge, directed against ubiquitin. This has given promising results in terms of reactivity to ubiquitin in immune precipitations of multi-ubiquinated target proteins.

PUBLICATIONS (AVIAN INFECTIOUS BRONCHITIS VIRUS)

Cavanagh D & MacNaughton M (1995) Coronaviruses. In: Principles and Practice of Clinical Virology, 3rd ed, pp 325-336. Edit Zuckerman AJ, Banatvala JE & Pattison JR. Chichester, Wiley

Kottier SA (1995) Investigation into the use of recombination for the production of site-specific mutants of coronavirus infectious bronchitis virus. PhD thesis, University of Reading

Adzhar A, Shaw K, Britton P & Cavanagh D (1996) Universal oligonucleotides for the detection of infectious bronchitis virus by the polymerase chain reaction. Avian Pathology, 25: 817-836

Penzes Z, Wroe C, Brown TDK, Britton P & Cavanagh D (1996) Replication and packaging of coronavirus infectious bronchitis virus defective RNAs lacking a long open reading frame. Journal of Virology, 70: 8660-8668

Shaw K, Britton P & Cavanagh D (1996) Sequence of the spike protein of the Belgian B1648 isolate of nephropathogenic infectious bronchitis virus. Avian Pathology, 25: 607-611

Tibbles KW, Brierley I, Cavanagh D & Brown TDK (1996) Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus. Journal of Virology, 70: 1923-1930


Back to the Biodefense Reference Library Bioinformatics Pathological Diagnostics

Copyright © 1994-2003 Humanitarian Resource Institute.  All rights reserved