Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
2000-09-12
2003-03-11
Salimi, Ali R. (Department: 1640)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S204100, C424S229100, C435S320100, C435S325000, C435S235100, C435S091100, C435S091330, C435S069100
Reexamination Certificate
active
06531136
ABSTRACT:
INTRODUCTION TO INVENTION
This invention relates to the equine rhinovirus 1 (ERhV1) which has been sequenced and characterized. In particular, the invention relates to nucleotide and protein sequences of ERhV1 and a range of clinical and diagnostic products derived from ERhV1.
BACKGROUND OF INVENTION
Equine rhinovuris 1 (ERhV1) was first isolated from horses in the United Kingdom and subsequently from horses in mainland Europe, the USA and Australia. Most isolates were from the nasopharynx of horses with an acute, febrile respiratory disease. Virions had the characteristic size and morphology of picornaviruses and were acid-labile. Two other serologically distinct, acid-labile picornaviruses, ERhV2 and ERhV3, have also been isolated from horses.
Considerable uncertainity has surrounded the classification of ERhV1. Physicochemical studies have shown that the nucleic acid density and base composition of ERhV1 differ from those of rhinoviruses. In contrast to rhinoviruses, ERhV1 has a broad host-cell range in vitro and in vivo and there is no evidence of extensive antigenic variation. Infection of horses with ERhV1 causes a disease characterized by an acute febrile respiratory disease accompanied by anemia, fecal and urine shredding and viral persistance. The signs of systemic infection and persistance are not characteristic of rhinovirus infections in other species. The known host range of ERhV1 is broad and includes rabbits, guinea pigs, monkeys and humans, although in these species the virus does not appear to spread horizontally. There is both experimental and epidemiological evidence of ERhV1 infection of humans. A human volunteer inoculated intranasally with ERhV1 developed severe pharyngitis, lymphadenities, fever and viremia, and high ERhV1 antibody titers were found in the sera of 3 of 12 stable workers whereas no ERhV1 antibody was found in the sera of 159 non-stable workers.
In order to clarify the taxonomic status of ERhV1, a detailed study was undertaken to determine the nucleotide and amino acid sequence of ERhV1. The resultant studies provided the complete nucleotide sequence of the gene encoding the ERhV1 polyprotein and the 3′-nontranslated region (NTR) as well as part of the nucleotide sequence of the 5′NTR. The amino acid sequence of the various ERhV1 proteins was deduced from the nucleotide sequence.
The analysis of the nucleotide sequence of ERhV1 confirmed previous studies which indicated that many properties of ERhV1 are not consistent with those of other members of the genus Rhinovirus. Indeed many of the physiochemical and biological properties of ERhV1 have suggested ERhV1 is more closely related to foot-and-mouth disease virus (FMDV) the sole member of the Alpthovirus genus. In addition to the overall sequence similarity, several features of the ERhV1 genome are similar to those of FMDV. The ERhV1 L protein is most similar to its counterpart in aphthoviruses in both length, 207 amino acids in ERhV1 and 201 and FMDV, and in amino acid sequence identity. In aphthoviruses, the L protein catalyses its own cleavage from the polyprotein, and mediates cleavage of the p220 component of the cap-binding complex leading to inhibition of translation of capped mRNAs. Cardiovirus L proteins are only 67-76 amino acids long and are not auto catalytic. In contrast to the cardioviruses, aphthoviruses utilize two distinct initiation codons, which results in different forms of the L protein, Lab and Lb, differing from each other by 28 amino acids at their N-termini.
The second initiation codon occurs in a more favourable contexts, which is presumably the reason why Lb, the smaller of the two proteins, is the predominant species. Thus far, differences in the function of the two FMDV L proteins have not been detected. ERhV1 also possesses a second ATG, 63 bases downstream from the first optimal ATG, which is also present in a context optimal for initiation of translation. Translation from this ATG would result in an L protein with 21 fewer amino acids at its N-terminus. Therefore, it is probable that ERhV1 possesses a second species of L protein similar to the FMDV Lb protein. If so, the reason for the existence and conservation of two forms of the L protein in ERhV1 and FMDV is an intriguing question. Curiously, ERhV1 has tandemly repeated ATG codons at each of the possible initiation sites, where the first ATG in each case does not occur in a context optimal for translation. The role of these ATGs may be to ensure that translation is initiated from both possible initiation sites.
The 2A protease is only 16 amino acids in length in both FMDV and ERhV1, compared to 142-149 amino acids in other picornaviruses. In FMDV 2A protease cleaves at its C-terminus but, unlike the 2A protease of other picornaviruses, appears not to have a role in shut down of host cell macromolecular synthesis. The high degree of conservation of the FMDV and ERhV1 2A proteins is intriguing and suggests an important role for this protein in the diseases produced by these viruses.
It may be expected that the tree derived from the complete polyprotein coding sequence would provide the most representative view of the taxonomic status of ERhV1 by reducing any bias imparted by using restricted parts of the genome with highly variable evolutionary rates. However, such analysis is restricted because there are only a few complete polyprotein sequences available. The polymerase genes are the most conserved genes in positive strand RNA viruses and they have been used to construct a taxonomy, and to predict the ancient roots, of these viruses. In contrast to the polymerase gene, the VPI gene encodes the major antigenic determinants of the virus and evolves more rapidly than other regions in the genome. The diversity of VP1 regions make them useful for the study of closely related picornaviruses. Thus, trees based on the polymerase and VP1 genes presumably reflect the extremes of evolutionary rates from which the taxonomic status and evolutionary origin of ERhV1 could be identified. The ERhV1 VP1 amino acid sequence was more similar to FMDV that to any other sequence in the data base; this was true even when representative segments across the entire sequence were separately analysed.
Therefore, we consider that the difference in the topology of the VP1, compared to the other two trees, is most unlikely to be a consequence of genetic recombination. The topographic differences between the three ERhV1 trees compared to those of aphthoviruses, particularly the VP1 derived trees, as well as the presence of only one VPg gene in ERhV1 genome, leads us to conclude that ERhV1 is probably a member of a distinct genus proposed to be called Equirhinovirus.
The reassessment of the taxonomic status of ERhV1 focuses on a requirement to reassess the biology of the virus particularly with respect to the nature of clinical disease as well as means for control by vaccination and improved methods of diagnosis. For example, cardioviruses and aphthoviruses cause viremic infections accompanied by myocarditis. Clinical disease caused by ERhV1 is generally considered to be confirmed to the respiratory tract even though there is a viremia and the virus is shed in faces and urine. Whether ERhV1 infection produces systemic disease similar to that observed in apthovirus or cardiovirus infections, including the production of myocarditis, needs to be investigated. There is serological evidence that the incidence of ERhV1 infection is as high as 50% in some horse populations however, the number of reported isolations of ERhV1 is very small. We have clear evidence that primary isolation of the virus from clinical specimens is known to be difficult, suggesting that the true incidence of ERhV1 disease is much greater than reported.
The determination of the complete nucleotide sequence of ERhV1 polyprotein has important practical applications in developing novel methods for the diagnosis and control of ERhV1 disease in horses and other species.
OBJECT AND STATEMENT OF INVENTION
In one aspect, the invention provides a substantially pure n
Crabb Brendan S
Feng Li
Studdert Michael J
Foley & Lardner
Salimi Ali R.
The University of Melbourne
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