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This project is designed to initiate research on the avian circoviruses by (1) developing assays for detection of viruses of the genus Circovirus, (2) obtaining and characterizing US avian circoviruses, with particular emphasis on ducks and geese, (3) attempting to grow avian circoviruses in vitro, and (4) if suitable material is found, attempted development of an infection study model of circovirus infection in ducks or geese, or alternatively (i.e. if no suitable virus inoculum obtained), continue efforts to grow virus in a larger range of cell culture systems. <P>
The first 0.5 year will be spent on Objective 1. Work to fulfill Objective 2 will occur throughout the entire 3 year program. It is anticipated that work on Objective 3 will commence towards the end of Year 1, and will continue throughout year 2. Fulfillment of Objective 4 will start in Year 3. <P>
Expected outputs include determining if circovirus infections are common in US birds (other than psittacines, which will not be included as part of this study) and the provision of training and mentoring to a graduate student leading to a post graduate qualification. Findings will be published in peer reviewed journals and at at least one conference.
NON-TECHNICAL SUMMARY: New viruses of humans and animals continue to be discovered every year. The circoviruses are a recently discovered such group of viruses. They have been found in pigs and birds, but none have been detected as yet in humans, or mammals other than pigs. During the last 15 years, several new circovirus species have been found in birds, and they tend to be very host specific. None of the avian circoviruses have been grown yet in vitro, and their effect on the host, if any, is poorly understood. They are however suspected to be immunosuppressive based on their presence in lymphoid tissues of infected birds. With the exception of psittacine beak and feather disease virus, and occasional case reports, there appears to be no research on circoviruses of birds in US; with one exception the new viruses of the last 15 years are being discovered by researchers in other countries. This project is designed to initiate research on the avian circoviruses with a view to understanding their occurrence, and their effects in US birds, in particular of the farmed and game species. <P>
APPROACH: Objective 1. Develop detection assays for viruses of genus Circovirus. Methods developed will be PCR, real-time PCR, dot blot and in situ hybridization. For PCR, species specific consensus primer PCR technology will be used for duck, pigeon and goose. Degenerate broad spectrum primers will be developed to detect potentially novel circovirus species. <P>
Objective 2. Sick and dead birds from local duck and geese (species of commercial interest), and avians submitted to the Diagnostic Laboratory (housed in the PI's building) will be examined for evidence of circovirus infection using the above methods. Any viruses identified, will be partially or fully characterized, and sequences compared to that in Genbank. <P>
Objective 3. Attempt to grow avian circoviruses in vitro. Using infected materials identified at 2, concerted attempts will be made to grow avian circoviruses. Firstly, 2 chicken lymphocyte cell lines will be used as well a PK-15(porcine kidney), (the latter support the growth of porcine circoviruses). Techniques such as electroporation will be used in an attempt to make these cell lines more permissive to circoviruses of other animal species. Uninoculated and inoculated cells will be maintained, examined for cytopathic effect, and tested for circoviral DNA by PCR. For goose and duck circovirus infected samples, cells from the same host species in which the virus was identified will be examined as above, since the circoviruses are very host specific. <P>
Objective 4. Attempt experimental circovirus infection in ducks or geese. Virus grown in vitro, is the preferred inoculum. If virus cannot be grown, homogenates of known circovirus positive tissue would be used as inoculum. Such material will be treated with detergent to destroy any enveloped viruses such as Newcastle Disease virus and Avian Influenza, and clarified. Flock history and necropsy findings of birds/flock of origin will be assessed, to exclude the possibility of highly virulent undesirable agents being present. The building proposed for use is a secure BSL2 containment facility with filtration of extracted air. Birds, bedding materials etc would be disposed of through the university biohazardous waste stream (Stericycle). If no duck or geese circoviruses are found, other readily available species such as pheasant or quail will be used, should circovirus be found in one of these species. Animal license cannot be applied for at this time because it is not known if (a) material will be found with which to attempt animal modeling, or (b) if it is found, in which animal species it will be found, and therefore, in which species modeling would be attempted (c) animal work would could not commence until October 2010. A license will be obtained for when these variables are known. No circovirus infected material from a readily available species for experimental reproduction, means that attempts to grow circoviruses as outlined in Objective failed, so additional cell lines will then be substituted as Objective 4 eg the B-cell line LSCC-1104B1, since it is known that there is variation in ability of CAV to grow in different cell lineages and the same may be true for the avian circoviruses.