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Research


A better understanding of the mode of action of certain avian viruses, most notably the virus causing Psittacine Beak and Feather disease.

A major part of our current research involves the understanding how a number of different diseases affect birds, mainly parrots. A practical aspect of these investigations is the development of diagnostic, DNA-based as well as serological tests for these diseases.

Identification of disease causing organisms, such as bacteria, viruses, fungi, protozoans etc. has been done classically by selective growth, microscopic and serological procedures. Currently most procedures are based on the specific analysis of the genomes of these organisms by the polymerase chain reaction (PCR). This latter procedure has the advantage of being highly specific, sensitive, requiring only very limited amounts of test material and fast, because time consuming culture of the often fastidious organisms or viruses is not needed. Although the PCR technique has these advantages, the transient nature of many infections which is often accompanied by the disappearance of the detectable infectious genetic material (DNA or RNA) causes that the history of an infection often cannot be revealed. The defense of the host towards a disease however generates often long lasting immune responses, which can be seen in the sometimes rather high levels of specific antibodies, which are maintained in the circulatory system of the affected animal long after the animal has recovered from the disease. Hence antibodies can give an indication of a previous infection. This can be important because the possibility that an animal remains a carrier of a disease causing organism without this organism being detectable is real, and exemplified by the dormant continuance of some herpesviruses such as the virus causing shingles in humans.

Research at Avian Biotech is mostly focused on the understanding of the interaction between the virus causing Beak and Feather Disease (BFD) and its psittacine hosts. BFD is caused by a circovirus. Circoviruses are small viruses (diameter approx. 20 nm) with the relatively unique characteristic that their genome consists of a 2000 nt long single-stranded circular DNA molecule, instead of the far more common double-stranded DNA. The short length of the genome makes that it carries only two genes, one for a coat protein and one for a replication factor. Circoviruses show some relationship to plant geminiviruses, which has led some to suggest that circoviruses arose early in vertebrate history as a result of recombination between part of a geminivirus and a animal calicivirus.

The circoviridae can be divided into two genera. Members of the genus circovirus are found to infect many different avian families. In addition to beak and feather disease virus, which is specific for psittacines other circoviruses are found in canaries (Canary circovirus, CaCV), pigeons (Pigeon circovirus, CoCV), domestic geese (goose circovirus, GoCV), mallards (duck circovirus DuCV) gulls, ostriches etc. One of the circoviruses is found in pigs (Sus scrofa) and causes porcine wasting disease (PCV). The other genus within the Circoviridae, Gyrovirus, consists of a single species, chicken anaemia virus (CAV) which is specific for chickens. This virus bears no further structural relationship with the true circoviruses else than that it also has a single stranded circular DNA genome. Finally the human TT virus also has a single stranded circular DNA genome, which is approximately twice as long as that of the circoviridae. This virus belongs again to another family the Circinoviridae, unrelated to the circoviridae.

The BFD viral genome is recognized by a considerable amount of sequence variation between the different strains, which can lead to a twenty per cent difference in amino acid sequence of the coat protein of different strains. The sequence of the replicase gene is considerably less variable. Different BFD strains show a complex psittacine host specificity (6). A difficulty in the study of the BFD virus is that it has thusfar not been possible to culture the virus in tissue culture. Therefore biochemical analysis of the virus has thusfar only been possible using virus isolated from infected birds. This has rendered much fundamental as well as applied work on BFD virus very difficult. In order to study the virus further, but also to generate a serological test for BFD specific antibodies, we have begun to use cloning in E. coli and yeast to obtain useful amounts of the coat and the replicase protein. Although the latter protein can readily be made in a bacterial host such as E. coli, the coat protein proved more difficult. In part the reason may be the somewhat different nature of the protein translation machinery e.g. codon usage in bacteria and eukaryotes, such as the psittaciform host of the virus, in part it may also be the nature of the coat protein itself which is recognized by the high arginine content of its N-terminal segment, which may bind to bacterial DNA, a binding which may interfere with its biosynthesis. Nevertheless by using specially engineered E. coli hosts as well as by using truncated proteins which lack the arginine rich N-terminal fragment we have succeeded in obtaining usable amounts of replicase and coat proteins. Since recombinant proteins made in E.coli almost always contain contaminating E. coli proteins, which may interfere with downstream applications, we have also synthesized these proteins in the yeast Kluyveromyces lactis, which has the advantage that the manufacturing process can be relatively easily scaled up. The replicase as well as the coat proteins have been used to generate specific antibodies in chickens, which allows us to use egg yolk to generate large amounts of antibodies in an animal friendly way. The BFD specific chicken antibodies have been used to establish a highly specific and sensitive Elisa test for PBFD. This test is specific for BFD and although the antibodies have been prepared by immunization with coat protein of one strain of BFD virus, other strains with about twenty percent difference in the coat protein amino acid sequence are also readily recognized. The antibodies do not interact with recombinant coat protein of pigeon circovirus or polyoma virus. Of considerable interest is that sera of birds which test positive in the coat protein Elisa assay, also test positive in a replicase Elisa, although the amount of replicase specific antibody in these sera was much lower that the levels of antibodies specific for the coat protein. This observation is in agreement with findings with other viruses, where structural proteins, such as the coat protein, generate much stronger antibody responses than internal, nonstructural proteins, like the replicase. Future research into BFD virus will be directed towards a study of the function of the replicase protein in DNA replication and the generation of viral like BFD particles in vitro with isolated, purified recombinant viral coat protein made in E. coli or yeast and the possible use of these particles as well as the replicase protein as a BFD vaccine.


References

1) De Kloet, E., and S. R. de Kloet. 2004. Analysis of the beak and feather disease viral genome indicates the existence of several genotypes which have a complex psittacine host specificity. Arch. Virol. 149:2392-2412.


Avian Biotech International
1336 Timberlane Road    Tallahassee, FL 32312-1766
850-386-1145 or 800-514-9672 (Office)  850-386-1146 (Fax)

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