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.
|
