WO2010006296A2 - New avian bornavirus - Google Patents

Abstract

The present invention relates to the discovery of a new avian bornavirus as the causative agent for psittacine proventricular dilation disease, methods of detecting the bornavirus and diagnosing bornavirus infection, methods of treating or preventing bornavirus infection, and methods for identifying anti-bornaviral compounds.

Description

NEWAVIAN BORNAVIRUS

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application Serial No. 61/134,630 filed on July 11, 2008 and U.S. Provisional Application Serial No. 61/224,407 filed on July 9, 2009 the disclosures of which are incorporated by reference in their entireties.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] Not applicable.

FIELD OF THE INVENTION

[0003] The present invention relates to the discovery of a new avian bornavirus as the causative agent for psittacine pro ventricular dilation disease, methods of detecting the bornavirus and diagnosing bornavirus infection, methods of treating or preventing bornavirus infection, and methods for identifying anti-bornaviral compounds.

BACKGROUND OF THE INVENTION

[0004] Proventricular dilation disease (PDD) is an inflammatory disease of birds, first described in the 1970s as Macaw Wasting Disease during an outbreak among macaws in North America and Germany (Gerlach S., Proc. Eur. Chap. Assoc. Avian. Vet., pp. 273- 281 (1991)). It has since been documented in 4 different continents in over 50 different species of Psittacines as well as captive and free-ranging species in at least 5 other orders of birds (Daoust PY et al., J Wildl Dis, 27(3):513-517 (1991); Gregory C et al., J Assoc Avian Vet, 8:69-75 (1994); Sullivan ND et al., Aust Vet J, 75(9):674 (1997); Gregory C et al., Progress in understanding provetricular dilation disease: a viral enormity, pp. 269- 275 (2000); Shivaprasad H., Proventricular dilation disease in peregrine falcon (Falco peregrinus), pp. 107-108 (2005)). PDD primarily affects the autonomous nerves of the upper and middle digestive tract, including the esophagus, crop, proventriculus, ventriculus, and duodenum. Microscopically, the disease is recognized by the presence of lymphoplasmacytic infiltrates within myenteric ganglia and nerves. Similar infiltrates may also be present in the brain, spinal cord, peripheral nerves, conductive tissue of the heart, smooth and cardiac muscle, and adrenal glands. Non-suppurative leiomyositis and/or myocarditis may accompany the neural lesions (Mannl A et al., Avian Dis, 31(1):214-221 (1987); Gerlach S., Proc. Eur. Chap. Assoc. Avian. Vet, pp. 273-281 (1991); Lutz ME and Wilson RB, J Am Vet Med Assoc, 198(11):1962-1964 (1991); Vice CA, Avian Dis, 36(4):1117-1119 (1992); Gregory C et al., J Assoc Avian Vet, 8:69-75 (1994); Berhane Y SD et al., Avian Pathol, 30(5):563-570 (2001)). Clinically, PDD cases present with GI tract dysfunction (dysphagia, regurgitation, and passage of undigested food in feces), neurologic symptoms (e.g. ataxia, abnormal gait, proprioceptive defects), or both (Gregory C et al., J Assoc Avian Vet, 8:69-75 (1994)). There is no proven therapy for PDD and although the clinical course of the disease can vary, most birds die within a year of developing clinical signs (Gregory C et al., J Assoc Avian Vet, 8:69-75 (1994); Gregory C et al., Progress in understanding provetricular dilation disease: a viral enormity, pp. 269-275 (2000)).

[0005] The cause of PDD is unknown, but several studies have raised the possibility that PDD may be caused by a viral pathogen. Early on, evidence of an infectious cause based on the appearance of clusters of cases of PDD was described among macaws (Gerlach S., Proc. Eur. Chap. Assoc. Avian. Vet, pp. 273-281 (1991)). Additionally, outbreaks of PDD have been reported (Phalen D., An outbreak ofpsittacine proventricular dilation syndrome (PPDS) in a private collection of birds and an atypical form of PDS in a nanday conure, pp. 27-34 (1986); Lublin A MS et al., IsraelJournal of Veterinary Medicine, 61(1):16-19 (2006)). Standard virological methods have revealed tantalizing leads, but none of these have been confirmed. Pleomorphic virus-like particles of variable size (30-250nm) have been described in tissues of affected birds (Mannl A et al., Avian Dis, 31(1):214-221 (1987)). These were suspected to be of the genus paramyxovirus (PMV); however, serological data has shown that PDD affected birds lack detectable antibodies against PMV of serotypes 1-4, 6, and 7, as well as against avian herpes viruses, polyomavirus, and avian encephalitis virus (Gerlach S., Proc. Eur. Chap. Assoc. Avian. Vet, pp. 273-281 (1991); Gregory C et al., J Assoc Avian Vet, 8:69-75 (1994)). More recently, PMV serotype-1 was isolated from the spinal cord of 6 of 32 affected birds. Based on partial sequence analysis these isolates were found to be closely related to the Hitchner Bl vaccine strain. However, subclinical infection with paramyxovirus serotype- 1 has subsequently been found to be common and PDD was not reproduced in two African gray parrots (a species highly susceptible to PDD) that were inoculated with the PMV isolates (Grand CH WO et al., J Vet Med B Infect Dis Vet Public Health, 49(9): 45-451 (2002); Grand CH MU and Korbel R., Pmc Assoc Avian Vet, 283-286 (2005)). An adeno-like virus was demonstrated within inclusion bodies in renal epithelium of one affected bird, while virus particles with morphological characteristics of enterovirus, coronavirus and reo virus have been sporadically documented in tissues or excretions of affected birds (Gregory C et al., J Assoc Avian Vet, 8:69-75 (1994); Gregory C et al., Proc Assoc Avian Vet, 43-52 (1997); Gregory C et al., Progress in understanding provetricular dilation disease: a viral enormity, pp. 269-275 (2000)). More consistently, enveloped virus-like particles of about 80nm in diameter have been demonstrated in the feces of affected birds and were absent from those of unaffected birds (Gough RE et al., Vet Rec, 139(1):24 (1996); Gregory C et al., Progress in understanding provetricular dilation disease: a viral enormity, pp. 269-275 (2000)). These virus-like particles are very similar to those isolated from tissues of affected birds using a macaw embryonic cell culture (Gough RE et al., Vet Rec, 139(1):24 (1996); Hartcourt-Brown et al., 1997). Tissue homogenates from an affected bird that contained such particles were used to inoculate susceptible birds, and have successfully reproduced the disease (Gregory C et al., Proc Assoc Avian Vet, 43-52 (1997)). This virus was initially suspected to be the eastern equine encephalitis virus (EEEV; an Alphavirus); however, further investigation has ruled this possibility out (Gregory CR LK et al., J Avian Medicine and Surgery, 11(3): 187-193 (1997)). Thus, whether PDD is indeed caused by an exogenous pathogen remains an open question.

[0006] To address this question, we have turned to a comprehensive, high throughput strategy to test for the presence of known or novel viruses in PDD affected birds. We have employed the Virochip, a DNA microarray containing representation of all viral taxonomy to interrogate 2 independently collected PDD case/control series for the presence of viral pathogens. We report here the detection of a novel bornavirus signature in 62.5% of the PDD cases and none of the controls. These bornavirus-positive samples were confirmed by virus-specific PCR testing, and the complete genome sequence has been recovered by ultra-high throughput sequencing combined with conventional PCR- based cloning. [0007] Bornaviruses are a family of negative strand RNA viruses whose prototype member is Borna Disease Virus (BDV), an agent of encephalitis whose natural reservoir is primarily horses and sheep (Durrwald R et al., Microbes Infect, 8(3):917-929 (2006)). Although experimental transmission of BDV to many species (including chicks (Rott R and Becht H, Curr Top Microbiol Immunol, 190:17-30 (1995))) has been described, there is little information on natural avian infection, and existing BDV isolates are remarkable for their relative sequence homogeneity. The agent reported here, which we designate ABV (avian bornavirus) is highly diverged from all previously identified members of the Bornaviridae family and represents the first full-length bornavirus genome cloned directly from avian tissue. Subsequent PCR screening for similar ABVs among an independently collected set of PDD cases and controls again yielded 57% ABV detection rate among PDD cases and none among the controls. Sequence analysis of a single complete genome and all of the additional partial sequences that we have recovered from these ABV isolates suggests that the viruses detected in cases of PDD form a new, genetically diverse arm of the Bornaviridae.

BRIEF SUMMARY OF THE INVENTION

[0008] The present method relates to the Applicants' discovery of avian bornaviruses that are etiologic agents for PDD. Accordingly, the invention provides compositions and methods useful in the detection, treatment, and diagnosis of PDD in birds. In a first aspect, the invention provides an isolated avian bornavirus comprising a genomic nucleic acid substantially identical to that of Accession No. EU781967 (see, Figure 15).

[0009] In addition, the invention provides isolated nucleic acid molecules substantially identical to a nucleic acid sequence of Accession No. EU781967; or its complement, hi some embodiments, the isolated nucleic acid comprises a nucleotide sequence at least 12 nucleotides in length that has at least 90% or95% sequence identity over its length to a sequence of Accession No. EU781967;or its complement. In some embodiments, the nucleotide sequence identity is with respect to the full length of the nucleic acid of Accession No. EU781967. In other embodiments, the isolated nucleotide sequence is a primer which has at least 90%, 95%, or 100% sequence identity its length to a sequence of Accession No. EU781967. In some embodiments, the primer is not identical in sequence to a non-avian Borna Virus sequence (e.g., No/98, H1766, He/80, V/Ref). In some embodiments, the nucleic acid is substantially identical to a sequence of any one of the avian bornavirus sequences of Figs. 14 to 18.

[0010] In some embodiments, the nucleic acid sequence encodes an open reading frame. In some embodiments, the invention provides an expression vector comprising a nucleic acid substantially identical to the nucleic acid sequence of Accession No. EU781967 or any one of the avian bornavirus sequences of Figs. 14 to 18. In additional, the invention provides a host cell which comprises the expression vector.

[0011] In another aspect, the invention provides a protein (e.g., N, X, P, M, G, or L protein ) encoded by the nucleotide sequence of Accession No. EU781967 or a nucleic acid substantially identical thereto. In other embodiments, the invention provides an isolated antibody (e.g., a polyclonal antibody, a monoclonal antibody) that specifically binds to protein encoded by a nucleotide sequence of Accession No. EU781967. In yet other embodiments, the invention provides a purified serum comprising polyclonal antibodies that specifically bind to a protein encoded by a nucleotide sequence of Accession No. EU781967. Such antibodies can be employed in the diagnosis or screening of a bird for PDD or ABV infection by obtaining a sample from the bird and using the antibody as a probe to detect any bornaviral protein in the sample.

[0012] In another aspect, the invention provides methods of diagnosing or screening for PDD in a subject by detecting the presence of ABV (nucleic acid (e.g., one substantially identical to that of Accession No. EU781967) or protein(e.g., N, X, P, M, G, or L protein)) in a sample from the subject, wherein the presence of ABV can be used in diagnosing PDD in the subject, identifying the subject as a carrier of PDD, or to identify the subject as being at risk of developing PDD. In a related aspect, the invention provides methods of determining that a subject does not have PDD, is not a carrier of PDD, and is not likely to develop PDD by testing for the presence of ABV in the subject and not detecting ABV.

[0013] In still another aspect, the invention provides a method of detecting a bornaviral nucleic acid by a) contacting a sample suspected of comprising a bornaviral nucleic acid with a nucleic acid molecule having a nucleotide sequence at least 12 nucleotides in length that is substantially identical or has at least 90% identity over its length to a corresponding segment of Accession No. EU781967 or is substantially identical to a sequence of any one of the avian bornavirus sequences of Figs. 14 to 18; and b) detecting the presence or absence of specific binding of the nucleic acid molecule having the nucleotide sequence to the bornaviral nucleic acid. Such methods can be employed in the diagnosis or screening of a subject for PDD or ABV infection by obtaining a sample from the subject and detecting the bornaviral nucleic acid as described above.

[0014] In another embodiment, the invention provides a method of detecting an avian bornaviral nucleic acid by a) contacting a sample suspected of comprising the bornaviral nucleic acid with at least one primer that hybridizes to a nucleotide sequence of Accession No. EU781967 is substantially identical to a sequence of any one of the avian bornavirus sequences of Figs. 14 to 18; b) performing a PCR reaction; and c) detecting the presence or absence of the bornaviral nucleic acid. Such methods can be employed in the diagnosis or screening of subject for PDD or ABV infection by obtaining a sample from the subject and detecting the bornaviral nucleic acid as described above. In some embodiments, the subject is a bird. The subject can be, for instance, a parrot, lorie, parakeet, cockatoo, or macaw or another species susceptible to infection by ABV. In other embodiments, the bird is an African grey parrot, pionu, eclectus parrot, conure, or cockatiel. The method may also be used on birds more distantly related to parrots, including the spoonbills, toucans, peregrine falcon, Canadian goose, weavers, and ostriches.

[0015] In still another embodiment, the invention provides a method of detecting a bornavirus infection in a sample by a) contacting a sample suspected of comprising a bornavirus protein with an antibody that specifically binds a polypeptide encoded by a nucleic acid of Accession No. EU781967 (e.g., AVB proteins N, X, P, G, M, L) or encoded by a nucleic acid of any one of the avian bornavirus sequences of Figs. 14 to 18; and b) detecting the presence or absence of the bornavirus protein. Such methods can be employed in screening subjects for PDD or ABV infection. In some embodiments, the subject is a bird (e.g., a parrot, a cockatoo, or a macaw). In other embodiments, the bird is an African grey parrot, pionu, eclectus parrot, conure, or cockatiel. The method may also be used on birds more distantly related to parrots, including the spoonbills, toucans, peregrine falcon, Canadian goose, weavers, and ostriches.

[0016] In some embodiments of the above, a bird found to be infected according to the detection of a ABV protein or nucleic acid in a sample from the bird is administered a therapy for the ABV infection or is euthanized. [0017] The invention also provides kits for detecting a bornaviral nucleic acid. In one embodiment, the kit can comprise a nucleic acid molecule having a nucleotide sequence at least 12 nucleotides in length that has substantial sequence identity to the corresponding segment of Accession No. EU781967. In another embodiment, the kit comprises at least one primer hybridizes to a nucleotide sequence of Accession No. EU781967; under highly stringent PCR conditions comprising a denaturation phase of 90⁰C - 95°C for 30 sec - 2 min., an annealing phase of 5O⁰C to about 65⁰C lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min., and an extension phase of about 72°C for 1 - 2 min for 20-40 cycles. In another embodiment, the kit comprises an antibody that detects a polypeptide encoded by an ORF of Accession No. EU781967. In further embodiments, the antibody can be monoclonal antibody or a polyclonal antibody. The kits can be used in the diagnosis of PDD or in screening birds for ABV infection.

[0018] In yet a further aspect the invention provides a method of assaying for an anti- bornaviral compound by a) contacting a sample comprising a bornavirus, the bornavirus comprising a genome that is substantially identical or has at least 90% identity over its length to the corresponding segment of Accession No. EU781967; and b) determining whether the compound inhibits the bornavirus.

[0019] In another aspect, the invention provides a method of treating or preventing a bornaviral infection or clinical PDD in a subject, by administering to the subject an antigen encoded by a bornavirus, the bornavirus comprising a genome that is substantially identical or has at least 90% identity over its length to the corresponding segment of Accession No. EU781967; thereby treating or preventing infection in the subject. In some embodiments, the antigen is provided in the form of a killed virus or as a protein encoded by the virus, or fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1. Clinical presentation of pro ventricular dilation disease cases and controls. Representative gross pathology, fluoroscopy, and histopathology from case/control specimens utilized in this study. A. Gross pathology from necropsy of control (left panel) African gray parrot (Psittacus erithacus) that died due to an air-sac infection. The normal-sized proventriculus is not visible in this view as it lies under the left liver lobe (L). Note the normal proportions of the organs in control (left panel) compared with case (right panel) Necropsy view of a great green macaw (Ara ambiguus) with proventricular dilatation disease. The proventriculus (PV) is markedly distended and extends laterally well beyond the left lobe of the liver (L). The ventriculus (V) was only slightly distended in this case. The location of the heart (H) is marked for orientation. B. Contrast fluoroscopy view of representative case and control. Left panel, African gray parrot (Psittacus erithacus) control 1.5 hours after administration of barium sulfate. The kidney (K) is marked for orientation. The outline of both the proventriculus (PV) and ventriculus (V) is clearly visible, showing normal size and shape. Within the intestinal loops (IL), wider and thinner sections represent active peristalsis. Right panel, representative PDD case, Eclectus parrot (Eclectus roratus) 18 hours after administration of barium sulfate. The proventriculus (PV) is markedly distended and contains most of the contrast material (dark color). A lesser amount is present in the ventriculus (V) and within intestinal loops (IL). A large filling defect (*) representing impacted food material is visible in the proventriculus. The location of the kidney (K) is shown for orientation. C. Proventriculus histology. Hematoxylin and eosin staining of proventriculus histological sections from a blue and yellow macaw (Ara ararauna) with proventricular dilatation disease, with a proventricular gland (G) shown for orientation. Left panel shows a normal appearing myenteric ganglion detected within the proventriculus of this case (arrow); right panel shows marked lympoplasmacytic infiltration present within a myenteric ganglion (arrows). Right panel inset shows a higher magnification of pathology where the typical wavy appearance of a small section of this myenteric nerve is preserved (arrows), but the rest of it has been almost completely destroyed by the infiltrating lymphocytes and plasma cells. D. CNS histology. Hematoxylin and eosin staining of a cerebral section from a control (left panel) African gray parrot (Psittacus erithacus) which died of other causes. Right panel, ceran African gray parrot (Psittacus erithacus) with proventricular dilatation disease. Perivascular cuffing is evident around blood vessels (arrows). Inset, higher magnification of perivascular cuffing (arrows).

[0021] Figure 2. Genome- wide comparison of avian bornavirus (ABV) genome sequences recovered by ultra-high throughput sequencing and PCR to available Borna disease virus (BDV) genome sequences. A. Bornaviridae genome schematic. Grey bar at base represents non-segmented negative sense viral RNA (vRNA) of Bornaviridae genome, with coordinates of major sequence landmarks highlighted below. Green bars and dashed lines, transcription initiation sites (TISs); red bars, transcription termination sites. Distinct ORF-encoding transcription products and the gene products they encode are diagrammed above: TISl transcripts encoding nucleocapsid (N) gene, pink; TIS2 transcripts encoding phosphoprotein (P) and X genes, green; TIS3 transcripts encoding the matrix (M), glycoprotein (G) and polymerase (large or 'L') gene, blue. Exon sequences, thick solid black lines; introns, thin solid black lines; dashed black lines, 3 'ends of transcripts generated transcription termination read-through; solid colored boxes indicate location of coding regions of transcripts, with reading frames other than +1 indicated at right. Array probes track shows location of Bornaviridae oligonucleotide 70mer probes from the Virochip array. PCR primers track shows location of primers generated for PCR follow up and screening of specimens in this study for detection of Bornaviridae species with expected product diagrammed below. vRNA RT-PCR track shows overlapping vRNA clones and RACE products recovered directly from RNA extracted from crop tissue of a histologically confirmed case of PDD. Solexa reads track shows distribution of 33mer reads with at least 15bp sequence identity to recovered ABV genome sequence. Sequence identity with BDV genomes track shows scanning average pairwise nucleotide sequence identity (window size of 100 nucleotides, advanced in single nucleotide steps) shared between ABV and all BDV genome sequences in NCBI. A dashed line on the graph indicates 50% identity threshold marker. B. Phylogenetic analysis of ABV genome and the 4 representative BDV genome isolates. Neighbor- joining phylogenetic trees based on nucleotide sequences of the ABV genome sequence (Accession number EU781967) and the following representative BDV genome sequences: H1766 (AJ311523), V/Ref (NC_001607), He/80 (L27077), and No/98 (AJ311524). Scale bar indicates genetic distance.

[0022] Figure 3. Comparison of ABV sequences recovered from PCR screening of PDD cases and controls to 4 representative genetic isolates of BDV. Neighbor joining Phylogenetic tree of ABV nucleotide sequences recovered by PCR screening with ABV consensus primers that map just internal to those shown in Figure 2, PCR probes track, that map to the L gene coding region (positions 3724-4257 of BDV reference sequence NCJ)01607) (A).

[0023] Figure 4. Alignment of bornavirus genomes 5' and 3 'ends: Bornavirus genome organization overview diagrammed above as in Figure 1. Sequences in alignments shown are complementary to vRNA sequence, genome isolate names shown at left. 3' end sequence recovered for ABV genome and other BDV genomes is shown in left panel, 5' end sequence recovered for ABV genome and other BDV genomes is shown in right panel. NCBI accession numbers for genomes aligned: hu2Pbr (AB258389), Bo/04w (AB246670), H1766 (AJ311523), Ref (NC_001607), V (U04608), V/FR (AJ311521), CRNP5 (AYl 14163), CRP3B (AYl 14162), CRP3A (AYl 14161), He/80/FR (AJ311522), He/80 (L27077), pBRT7-HrBDVc (AY705791), No/98 (AJ311524), ABV (EU781967).

[0024] Figure 5. Alignment of transcription initiation and termination sites in bornavirus genomes: Panel A, alignment of the 3 bornavirus transcription initiation sites (TIS) and 6 nucleotides of flanking sequences. Panel B, alignment of the 4 bornavirus transcription termination sites. Source genomes for alignments are shown at left; ABV sequence is highlighted with black triangle.

[0025] Figure 6. Alignment of splice donor and splice acceptor sequences in bornavirus genomes: Panel A, alignment of splice donor 1 and splice acceptor 1 sequences; Panel B, alignment of splice donor 2 and splice acceptor 2 sequences; Panel C, alignment of splice acceptor 3 sequences. Source genomes for alignments are shown at left.

[0026] Figure 7. Phylogenetic relationships of sub-genomic loci in ABV and representative BDV genomes: Neighbor joining trees generated for the indicated nucleotide sequences of ABV and a representative set of BDV genomes are shown for each ORF in the bornavirus genome. NCBI accession numbers of representative BDV genomes are: Ref/V(NC_001607), H1766 (AJ311523), He/80 (AY705791), No/98 (AJ311524).

[0027] Figure 8. - Body weights of the ABV4-inoculated and sham-inoculated cockatiels during the study period. The ABV4-inculated birds (cockatiels 1-3), are shown in bald lines with solid symbols. Cockatiels 4,5 are the control birds; "*" marks the first observation of undigested seeds in the faeces; "#" marks the first detection of ABV4 RNA in a choanal or cloacal swab by RT-PCR. Note the continuous decrease in BW of cockatiels 1 and 3 starting on day 21 PI and 31 PI, respectively.

[0028] Figure 9. - Macroscopic pathological findings in an ABV4-inoculated cockatiel. Bar size = lcm; F= peritoneal fat; H= heart; L= right liver lobe; V= ventriculus; A) Markedly reduced pectoral muscle mass and subcutaneous fat stores are clearly seen in the ABV4-inoculated cockatiel (cockatiel 3). B) Normal pectoral muscle mass and subcutaneous fat stores in a control bird (cockatiel 5). C) The peritoneal cavity of cockatiel 3, showing a severely distended and thin- walled pro ventriculus (arrows) that is visible well beyond the left liver lobe. The ventriculus is mildly distended and the peritoneal fat is dramatically reduced. D) In cockatiel 5, the proventriculus is of normal size, and; therefore, completely hidden behind the left liver lobe. Note the abundant peritoneal fat stores. E) The proventriculus and intestine of cockatiel 3. The thin wall has been cut, exposing a large amount of undigested seeds. Whole seeds are also visible through the intestinal wall of cockatiels 1 and 3 starting on day 21 PI and 31 PI, respectively.

[0029] Figure 10. - Histological and immunohistochemical findings in AB V4- inoculated cockatiels. A) Myenteric ganglioneuritis (arrow) in the crop of cockatiel 2 (hematoxyline and eosin [H&E] staining); B) Myenteric ganglioneuritis (arrow) in the ventriculus of cockatiel 3 (H&E staining). C) Positive IHC staining for ABVN associated with focal gliosis (arrow) in the cerebrum of cockatiel 1. Here, the staining appears to be mainly extracellular. D) For negative control, a section parallel to that in C was stained, using pre-immune rabbit serum instead of the anti-ABVN antibody. E) Widespread positive IHC staining for ABVN of neurons and glial cells of the cerebrum of cockatiel 2. At greater magnification, staining of a large neuron and its dendrites is shown (inset). F) For negative control, a section parallel to that in E was stained, using pre-immune rabbit serum instead of the anti-ABVN antibody.

[0030] Figure 11. - Transmission electron microscopy images. A) Brain homogenate from an ABV4(+) PDD(+) African grey parrot (the inoculum used in this study). Three spherical virus-like particles approximately 60nm in diameter are shown [arrows] (negative staining with uranyl acetate). B) A virus-like particle from the same specimen in "A" shown at greater magnification. This particle is 98nm in diameter (negative staining with uranyl acetate). C) A virus-like particle from the brain of cockatiel 1. This particle is 99nm in diameter and is showing bold projections on its circumference (negative staining with uranyl acetate).

[0031] Figure 12. - Recovery of sequences matching ABV from the inoculum by high throughput pyrosequencing and RT-PCR. The location of seven unique RNA sequences, recovered from the inoculum by high throughput pyrosequencing, is shown. These sequences share 73-100% sequence identity with existing ABV sequences (Additional file 2). In addition, the location of the RT-PCR primers for the N, M, and genes is shown, all of which yielded products that were consistent with AB V4 genome. [0032] Figure 13. ABV RNA and protein detection in tissues harvested from a fatal case of PDD. RT-PCR for ABVN RNA (A, top panel) and GAPDH mRNA control (A, bottom panel) on a panel of RNA extracted from tissues harvested from bird 3 at necropsy. Tissue types examined are labeled above top gel lanes. Western blot showing tissues testing positive with ABVN, ABVP, and GAPDH control proteins. Tissue types examined are labeled above each lane shown.

[0033] Figure 14: Nucleotide sequence alignment of partial ABVN gene sequences recovered by RT-PCR during cloacal swab screening or necropsy.

[0034] Figure 15. ABV N.X, P, M, G, L protein and genomic nucleic acid sequences.

[0035] Figure 16. AB V4 sequence data recovered from deep sequencing of the brain inoculum used in the ABV challenge experiments described herein.

[0036] Figure 17. ABV N Sequences for Outbreak study [0037] Figure 18. Bird 1 Necropsy ABV M Sequence data

DETAILED DESCRIPTION OF THE INVENTION

[0038] It has been almost 40 years since the first description of PDD. Although a viral etiology has long been suspected, a convincing lead for a responsible viral pathogen has been lacking. By combining veterinary clinical investigation with genomics and molecular biology, we have identified a genetically diverse set of novel avian bornaviruses (ABVs) that plays a significant role in this disease. Through microarray analysis and follow-up PCR, we detected ABV sequences in 62.5% of the PDD cases in a set of specimens derived from 2 carefully collected PDD case/control series derived from two different continents. We confirmed that these assays faithfully reflect the presence of full-length bornavirus in ABV PCR positive specimens through cloning of the complete ABV vRNA sequence directly from RNA extracted from one of these ABV PCR positive PDD case specimens. We next found evidence for a significant association between the presence of ABV and clinically confirmed PDD in follow-up blinded PCR screening of a set of additional PDD cases and controls, with ABV was detected in 57% of PDD cases and none of the controls (P=0.006, Fisher's Exact Test; O.R. lower limit=17.3, 95% CL=I.4-216.6). [0039] Almost all prior sightings of bornaviruses in nature have been among mammals, and the mammalian isolates have been remarkably homogeneous at the sequence level (Table 2 and (reviewed in Durrwald R et al, Microbes Infect, 8(3):917-929 (2006))). The latter is a surprising feature for RNA viruses, whose RNA-dependent RNA polymerases typically have high error rates. By contrast, the ABV isolates reported here are quite diverged from their mammalian counterparts, and show substantial heterogeneity among themselves. We note with interest that a single earlier report suggesting a potential avian reservoir for bornaviruses exists (Berg M JM et al., Epidemiology and Infection, 127(1):173-178 (2001)). In this study, RT-PCR based on mammalian BDV sequences was used to recover partial sequences from stool collected in the wild near ponds, putatively from asymptomatic mallards and jackdaws in a setting where BDVs had been isolated from affected domesticated cats. The resulting sequences share a high level of sequence homology to the mammalian BDVs (average % predicted amino acid for best match is 98% with equine isolates). In contrast, the ABVs isolated here directly from tissues of affected birds are actually more distant from these putative avian BDV isolates (average predicted amino acid identity for best matches with those isolates, 72%) than they are from the closest matching mammalian isolates (average predicted amino acid identity for best mammalian matches, 82%). Given the lack of endemic BDV in Sweden, the high similarity to mammalian BDVs and uncontrolled sampling of specimens for this prior study, the possibility that the wild avian isolates might result from possible laboratory contamination has been raised (Durrwald R et al., Microbes Infect, 8(3):917- 929 (2006)), but no additional experiments to verify or refute this possibility have been performed. Thus, whether avian species can serve as asymptomatic carriers of bornaviruses that are similar to mammalian BDVs, or clinically susceptible hosts for the highly diverged ABVs we detect here, or hosts for both of these types of bornaviruses, remains an open question.

[0040] The known neurotropism of bornaviruses makes them attractive and biologically plausible etiologic agents in PDD, since (i) PDD cases have well-described neurologic symptoms such as ataxia, proproceptive defects and motor abnormalities; and (ii) the central GI tract pathology in the disorder results from inflammation and destruction of the myenteric ganglia that control peristaltic activity. However, despite our success in ABV detection in PDD, we did not observe ABV in every PDD case analyzed. There are several possible explanations for this result. First, although our PCR testing is sensitive in vitro, we do not know the tissue distribution (tropism) of ABV infection, or how viral copy number may vary at different sites as a function of the stage of the disease. By weighting our sample collection towards clinically overt PDD, we may have biased specimen accrual towards advanced disease. At this stage, where destruction of myenteric ganglial elements is often extensive, loss of infected cells may have contributed to detection difficulties (We note with interest in this context that in one of our case collections from Israel, virus detection occurred preferentially in CNS rather than in GI specimens). There are many precedents for such temporal variation in clinical virology - for example, in chronic hepatitis B viral loads typically decline by several orders of magnitude over the long natural history of the infection (Ganem and Prince 2004). It is also possible that our 57% detection rate may merely reflect suboptimal selection of PCR primers employed for screening; after all, our consensus primer selection was based on the first ABV sequences we recovered. We now recognize that there is substantial sequence variation within the ABVs (see Fig. 3); as more sequence diversity is recognized, better choices for more highly conserved primers will become apparent and could impact upon these prevalence estimates.

[0041] In accordance with the case-control studies, we have also have demonstrated an association between ABV infection and clinical PDD, indicating that ABV may be the causative agent of this devastating disease. Here, we provide further evidence consistent with this possibility. Through analysis of an outbreak of PDD in a mixed psittacine nursery, we provide real-time evidence of transmission of PDD signs and symptoms correlated with ABV infection. Through molecular analysis of both ABV RNA and protein in available necropsied tissue of infected birds, we also have gained a detailed view of ABV tropism in end-stage PDD. This real-time analysis has also allowed us some of the first insights into the development of PDD signs and symptoms in naturally acquired ABV infections. In combination with our recent work demonstrating that ABV (+) brain homogenates derived from PDD positive birds can confer ABV infection and signs and symptoms of PDD, this outbreak analysis provides further evidence that naturally occurring ABV infection and transmission correlates closely in time with the development of PDD. DEFINITIONS

[0042] Avian Bornaviras or ABV refers to both the genetic components of the virus, e.g., the genome and RNA transcripts thereof, proteins encoded by the genome (including structural and nonstructural proteins), and viral particles. The term "avian bornavirus " or a nucleic acid encoding " avian bornavirus " refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have a nucleotide sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater nucleotide sequence identity, preferably over a region of over a region of at least about 25_r 50, 100, 200, 500, 1000, or more nucleic acids, up to the full length sequence, to the nucleotide sequence of Accession No. EU781967; (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against an immunogen comprising an amino acid sequence of a protein encoded by an open reading frame of Accession No. EU781967, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence of Accession No. EU781967, and conservatively modified variants thereof; (4) encoding a protein having an amino acid sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater nucleotide sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a protein (e.g., a N, X, P, M, G, L protein) encoded by an open reading frame of Accession No. EU781967. A polynucleotide or polypeptide sequence is typically from a bird including, but not limited to, a bird of the psittacine group, e.g., parrots, lories, cockatoos, parakeets. The nucleic acids and proteins of the invention include both naturally occurring or recombinant molecules. Partial sequence include ABVl_6b (EU781953); ABV2_BIL (EU781954); ABV3JCD (EU781955); ABV4_ALV (EU781956); ABV4_7 (EU781957); ABV4_9 (EU781958); ABV4_14 (EU781959); ABV4_17 (EU781960); ABV4_18 (EU781961); ABV2_3 (EU781962); ABV2_5 (EU781963); ABV2_12 (EU781964); ABV2_30 (EU781965); ABV2_31 (EU781966) and also those of Figures 14 and 15 and Table 5.

[0043] "Protein encoded by avian bornavirus" or "protein encoded by avian bornavirus open reading frame (ORF)" refers to structural and non-structural bornaviral proteins encoded by nucleic acids that: (1) have a nucleotide sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater nucleotide sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleic acids, up to the full length sequence, to the nucleotide sequence of Accession No. EU781967; (2) bind to antibodies, e.g., polyclonal or monoclonal antibodies, raised against an immunogen comprising an amino acid sequence of a protein encoded by an open reading frame of Accession No. EU781967, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence of Accession No. EU781967, and conservatively modified variants thereof, including those of Figs 14 to 18; (4) encoding a protein having an amino acid sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater nucleotide sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a protein encoded by an open reading frame of Accession No. EU781967. The amino acid sequence of the structural and non-structural viral proteins encoded by ABV can be easily identified by one of skill in the art, using the algorithms disclosed herein, by aligning the ABV sequence with other bornavirus sequences, including B 19. Such proteins include the ABV proteins M, G, L, P, X, and N.

[0044] . A "biological sample" or "sample" includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, cloacal swabs, mucosa, tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, biological fluids, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a bird, including, but not limited to, a bird of the psittacine group, e.g., parrots, lories, cockatoos, parakeets. The tissue sampled can be, for instance, skin, brain (e.g., cerebrum, cerebellum, optic lobe), spinal cord, adrenals, pectoral muscle, lung, heart, liver, crop, proventriculus, ventriculus, duodenum, small intestine, large intestine, cloaca, kidney, bursa of fabricus, spleen, pancreas, adrenal gland, bone marrow, lumbosacral spinal cord, or blood.

[0045] In some embodiments, the subject from which the sample is taken is a bird, including but not limited to psittacines (e.g., African grey parrot, pionu, eclectus parrot, conure, cockatiel parrot, lorie, parakeet, cockatoo, or macaw). The method may also be used on birds more distantly related to parrots, including the spoonbills, toucans, peregrine falcon, Canadian goose, weavers, and ostriches. The subject may have been selected based upon a potential exposure to ABV or PDD. The subject may also be a human or immunocompromised human or another immunocompromised non-avian host suspected of exposure to ABV.

[0046] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be "substantially identical" and are embraced by the term "substantially identical.' This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists for a specified entire sequence or a specified portion thereof or over a region of the sequence that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0047] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. [0048] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat 'I. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0049] A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al, J. MoI. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0050] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0051] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et ah, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et ah, J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., MoI. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0052] A particular nucleic acid sequence also implicitly encompasses "splice variants." Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid. "Splice variants," as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. An example of potassium channel splice variants is discussed in Leicher, et ah, J. Biol. Chem. 273(52):35095-35101 (1998).

[0053] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0054] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0055] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0056] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0057] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0058] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) {see, e.g., Creighton, Proteins (1984)).

[0059] Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et ah, Molecular Biology of the Cell (3^rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. "Tertiary structure" refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.

[0060] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.

[0061] The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0062] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein). [0063] The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-1O⁰C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_n, is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42⁰C, or, 5x SSC, 1% SDS, incubating at 65⁰C, with wash in 0.2x SSC, and 0.1% SDS at 65⁰C.

[0064] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37⁰C, and a wash in IX SSC at 45⁰C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al.

[0065] For PCR, a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length. For high stringency PCR amplification, a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50°C to about 65°C, depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 9O⁰C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N. Y.).

[0066] "Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.

[0067] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively.

[0068] Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'_2; a dimer of Fab which itself is a light chain joined to V_H-C_HI by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'₂ dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries {see, e.g., McCafferty et al, Nature 348:552-554 (1990))

[0069] For preparation of antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies, many technique known in the art can be used {see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity {see, e.g., Kuby, Immunology (3^rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Patent 4,946,778, U.S. Patent No. 4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al, Bio/Technology 10:779-783 (1992); Lonberg et al, Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al, Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93/08829, Traunecker et al, EMBOJ. 10:3655-3659 (1991); and Suresh et al, Methods in Enzymology 121 :210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Patent No. 4,676,980 , WO 91/00360; WO 92/200373; and EP 03089). [0070] Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers {see, e.g., Jones et ah, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0071] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0072] In one embodiment, the antibody is conjugated to an "effector" moiety. The effector moiety can be any number of molecules, including labeling moieties such as radioactive labels or fluorescent labels, or can be a therapeutic moiety. In one aspect the antibody modulates the activity of the protein.

[0073] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to a avian bornavirus, polymorphic variants, alleles, orthologs, and conservatively modified variants, or splice variants, or portions thereof, can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with avian bornavirus and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein {see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Preferred antibodies are those which can distinguish an AVB protein from a non-avian bornavirus protein(e.g., any one or all of He/80, No/98, V, and Hl 766 with respect to proteins encoded by the N,X, P, M, G, and L genes thereof).

[0074] By "therapeutically effective dose" herein is meant a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques {see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0075] The phrase "functional effects" in the context of assays for testing compounds that modulate activity of a avian bornavirus includes the determination of a parameter that is indirectly or directly under the influence of a avian bornavirus, e.g., a phenotypic or chemical effect, such as the ability to increase or decrease viral genome replication, viral RNA and protein production, virus packaging, viral particle production (particularly replication competent viral particle production), cell receptor binding, viral transduction, cellular infection, antibody binding, inducing a cellular or humoral immune response, viral protein enzymatic activity, etc. "Functional effects" include in vitro, in vivo, and ex vivo activities. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index); hydrodynamic (e.g. , shape); chromatographic; or solubility properties for a protein; measuring inducible markers or transcriptional activation of a protein; measuring binding activity or binding assays, e.g. binding to antibodies; measuring changes in ligand or substrate binding activity; measuring viral replication; measuring cell surface marker expression; measurement of changes in protein levels; measurement of RNA stability; identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP and the like), e.g. , via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, and inducible markers.

[0076] "Inhibitors", "activators", and "modulators" of avian bornavirus nucleic acid and polypeptide sequences are used to refer to activating, inhibitory, or modulating molecules identified using in vitro and in vivo assays of the bornavirus nucleic acid and polypeptide sequences. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of avian bornavirus, e.g., antagonists. "Activators" are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate avian bornavirus activity, e.g., agonists. Inhibitors, activators, or modulators also include genetically modified versions of avian bornavirus, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, substrates, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g., expressing avian bornavirus in vitro, in cells, or cell membranes, applying putative modulator compounds, and then determining the functional effects on activity, as described above.

[0077] Samples or assays comprising avian bornavirus that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%. Inhibition of avian bornavirus is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%. Activation of avian bornavirus is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200-500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.

[0078] The term "test compound" or "drug candidate" or "modulator" or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleotide, oligonucleotide, etc., to be tested for the capacity to directly or indirectly modulation tumor cell proliferation. The test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.

[0079] A "small organic molecule" refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons.

[0080] An "siRNA" molecule or an "RNAi molecule refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA expressed in the same cell as the gene or target gene. "siRNA" thus refers to the double stranded RNA formed by the complementary strands. The complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA. The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferable about preferably about 20-30 base nucleotides, preferably about 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. See also PCT/US03/07237, herein incorporated by reference in its entirety. [0081] An siRNA molecule or RNAi molecule is "specific" for a target nucleic acid if it reduces expression of the nucleic acid by at least about 10% when the siRNA or RNAi is expressed in a cell that expresses the target nucleic acid.

ISOLATION OF AVIAN BORNAVIRUS GENOME AND GENES [0082] This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994)).

[0083] Avian bornavirus, polymorphic variants, orthologs, and alleles that are substantially identical to an amino acid sequence encoded by nucleic acids of Accession No. EU781967 can be isolated using nucleic acid probes and oligonucleotides under stringent hybridization conditions, by screening DNA libraries or by using PCR. Genes encoding bornaviral proteins can be isolated using cDNA libraries. Alternatively, expression libraries can be used to clone the avian bornavirus, polymorphic variants, orthologs, and alleles by detecting expressed homologs immunologically with antisera or purified antibodies made against human avian bornavirus or portions thereof.

[0084] To make a cDNA library to clone bornavirus genes expressed by the genome, one should choose a source that is rich in the RNA of choice. The mRNA is then made into cDNA using reverse transcriptase, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning. Methods for making and screening cDNA libraries are well known {see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al, supra; Ausubel et al, supra).

[0085] For a genomic library, the DNA is extracted from the tissue and optionally mechanically sheared or enzymatically digested. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in suitable vectors. These vectors are packaged in vitro. Recombinant vectors can be analyzed, e.g., by plaque hybridization as described in Benton & Davis, Science 196:180-182 (1977). Colony hybridization is carried out as generally described in Grunstein et al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975). [0086] A preferred method of isolating avian bornavirus and orthologs, alleles, mutants, polymorphic variants, splice variants, and conservatively modified variants combines the use of synthetic oligonucleotide primers and amplification of an RNA or DNA template (see Example 1, below, see also U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Methods such as polymerase chain reaction (PCR and RT-PCR) and ligase chain reaction (LCR) can be used to amplify nucleic acid sequences directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. Degenerate oligonucleotides can be designed to amplify homologs using the sequences provided herein. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of avian bornavirus encoding mRNA in physiological samples, for nucleic acid sequencing, or for other purposes. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.

[0087] Gene expression of avian bornavirus can also be analyzed by techniques known in the art, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or poly A⁺ RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, high density polynucleotide array technology, e.g., and the like.

[0088] Nucleic acids encoding a avian bornavirus genome or protein can be used with high density oligonucleotide array technology (e.g., GeneChip™) to identify avian bornavirus, orthologs, alleles, conservatively modified variants, and polymorphic variants in this invention. In the case where the homologs being identified are linked to modulation of the cell cycle, they can be used with GeneChip™ as a diagnostic tool in detecting the disease in a biological sample, see, e.g., Gunthand et ah, AIDS Res. Hum. Retroviruses 14: 869-876 (1998); Kozal et al, Nat. Med. 2:753-759 (1996); Matson et al, Anal. Biochem. 224:110-106 (1995); Lockhart et al, Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al, Genome Res. 8:435-448 (1998); Hacia et al, Nucleic Acids Res. 26:3865-3866 (1998).

[0089] The gene of choice is typically cloned into intermediate vectors before transformation into prokaryotic or eukaryotic cells for replication and/or expression. These intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors.

EXPRESSION IN PROKARYOTES AND EUKARYOTES [0090] To obtain high level expression of a cloned gene or genome, one typically subclones the nucleic acid into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et ah, and Ausubel et al, supra. Bacterial expression systems for expressing the protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al, Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. In one preferred embodiment, retroviral expression systems are used in the present invention.

[0091] Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

[0092] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells. A typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding the nucleic acid of choice and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

[0093] In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. [0094] The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc. Sequence tags may be included in an expression cassette for nucleic acid rescue. Markers such as fluorescent proteins, green or red fluorescent protein, β-gal, CAT, and the like can be included in the vectors as markers for vector transduction.

[0095] Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, retroviral vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculo virus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, S V40 early promoter, S V40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0096] Expression of proteins from eukaryotic vectors can be also be regulated using inducible promoters. With inducible promoters, expression levels are tied to the concentration of inducing agents, such as tetracycline or ecdysone, by the incorporation of response elements for these agents into the promoter. Generally, high level expression is obtained from inducible promoters only in the presence of the inducing agent; basal expression levels are minimal.

[0097] In one embodiment, the vectors of the invention have a regulatable promoter, e.g., tet-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al, Gene Ther. 5:491-496 (1998); Wang et al, Gene Ther. 4:432-441 (1997); Neering et al, Blood 88:1147-1155 (1996); and Rendahl et al, Nat. Biotechnol. 16:757-761 (1998)). These impart small molecule control on the expression of the candidate target nucleic acids. This beneficial feature can be used to determine that a desired phenotype is caused by a transfected cDNA rather than a somatic mutation.

[0098] Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a sequence of choice under the direction of the polyhedrin promoter or other strong baculovirus promoters.

[0099] The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable. The prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.

[0100] Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101 :347-362 (Wu et al., eds, 1983).

[0101] Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing avian bornavirus proteins and nucleic acids.

[0102] After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of the protein of choice, which is recovered from the culture using standard techniques identified below.

PURIFICATION OF POLYPEPTIDES

[0103] Either naturally occurring or recombinant avian bornavirus proteins can be purified for use in diagnostic assays, for making antibodies (for diagnosis and therapy) and vaccines, and for assaying for anti-viral compounds. Naturally occurring protein can be purified, e.g., from bird tissue samples. Recombinant protein can be purified from any suitable expression system. **

[0104] The protein may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others {see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al, supra; and Sambrook et al, supra).

[0105] A number of procedures can be employed when recombinant protein is being purified. For example, proteins having established molecular adhesion properties can be reversible fused to the protein. With the appropriate ligand or substrate, a specific protein can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally, protein could be purified using immunoaffinity columns. Recombinant protein can be purified from any suitable source, include yeast, insect, bacterial, and mammalian cells.

A. Purification of protein from recombinant bacteria

[0106] Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Promoter induction with IPTG is one example of an inducible promoter system. Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein.

[0107] Proteins expressed in bacteria may form insoluble aggregates ("inclusion bodies"). Several protocols are suitable for purification of protein inclusion bodies. For example, purification of inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells, e.g., by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3 passages through a French Press, homogenized using a Polytron (Brinkman Instruments) or sonicated on ice. Alternate methods of lysing bacteria are apparent to those of skill in the art (see, e.g., Sambrook et ah, supra; Ausubel et al, supra). [0108] If necessary, the inclusion bodies are solubilized, and the lysed cell suspension is typically centrifuged to remove unwanted insoluble matter. Proteins that formed the inclusion bodies may be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M). Some solvents which are capable of solubilizing aggregate-forming proteins, for example SDS (sodium dodecyl sulfate), 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of immunologically and/or biologically active protein. Other suitable buffers are known to those skilled in the art. Human proteins are separated from other bacterial proteins by standard separation techniques, e.g., with Ni-NTA agarose resin.

[0109] Alternatively, it is possible to purify recombinant protein from bacteria periplasm. After lysis of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art. To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To Iy se the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 niM MgSO₄ and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.

B. Standard protein separation techniques for purifying proteins

Solubility fractionation

[0110] Often as an initial step, particularly if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration will precipitate the most hydrophobic of proteins. The precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.

Size differential filtration

[0111] The molecular weight of the protein can be used to isolate it from proteins of greater and lesser size using ultrafiltration through membranes of different pore size (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cutoff than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.

Column chromatography

[0112] The protein can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands or substrates. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).

IMMUNOLOGICAL DETECTION OF POLYPEPTIDES AND NUCLEIC ACIDS

[0113] In addition to the detection of a avian bornavirus gene and gene expression using nucleic acid hybridization technology, one can also use immunoassays to detect avian bornavirus proteins, virus, and nucleic acids of the invention. Such assays are useful for, e.g., therapeutic and diagnostic applications. Immunoassays can be used to qualitatively or quantitatively analyze protein, virus, and nucleic acids. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).

A. Production of antibodies

[0114] Methods of producing polyclonal and monoclonal antibodies that react specifically with avian bornavirus protein, virus and nucleic acids are known to those of skill in the art {see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al, Science 246:1275-1281 (1989); Ward et al., Nature 341 :544-546 (1989)).

[0115] A number of immunogens comprising portions of a avian bornavirus protein, virus or nucleic acid may be used to produce antibodies specifically reactive with the avian bornavirus. For example, a recombinant avian bornavirus protein or an antigenic fragment thereof, can be isolated as described herein. Recombinant protein can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above. Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies. Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen. Naturally occurring protein may also be used either in pure or impure form. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.

[0116] Methods of production of polyclonal antibodies are known to those of skill in the art. An inbred strain of mice (e.g., BALB/C mice) or rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the beta subunits. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see, Harlow & Lane, supra).

[0117] Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse, et al, Science 246:1275-1281 (1989).

[0118] Monoclonal antibodies and polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Typically, polyclonal antisera with a titer of 10⁴ or greater are selected and tested for their cross reactivity against non- avian bornavirus proteins and nucleic acids, using a competitive binding immunoassay. Specific polyclonal antisera and monoclonal antibodies will usually bind with a Kd of at least about 0.1 mM, more usually at least about 1 μM, preferably at least about 0.1 μM or better, and most preferably, 0.01 μM or better. Antibodies specific only for a particular avian bornavirus protein can also be made by subtracting out other cross-reacting proteins, e.g., from other human bornaviruses or other non-human bornaviruses. In this manner, antibodies that bind only to the protein of choice may be obtained.

[0119] Once the specific antibodies against a avian bornavirus protein, virus or nucleic acid in are available, the antigen can be detected by a variety of immunoassay methods. In addition, the antibody can be used therapeutically. For a review of immunological and immunoassay procedures, see Basic and Clinical Immunology (Stites & Terr eds., 7⁴ ed. 1991). Moreover, the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, supra.

B. Immunological binding assays

[0120] Protein which is either associated with or separate from an ABV viral particle, can be detected and/or quantified using any of a number of well recognized immunological binding assays {see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168). ABV viral particles may be detected based on an epitope defined by the viral proteins as presented in a viral particle and/or an epitope defined by a viral protein that is separate from a viral particle (e.g., such as may be present in an infected cell). As used in this context, then, "antigen" is meant to refer to an ABV polypeptide as well as ABV viral particles. For a review of the general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991). Immunological binding assays (or immunoassays) typically use an antibody that specifically binds to a protein or antigen of choice. The antibody may be produced by any of a number of means well known to those of skill in the art and as described above.

[0121] Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen. The labeling agent may itself be one of the moieties comprising the antibody/antigen complex. Thus, the labeling agent may be a labeled avian bornavirus protein nucleic acid or a labeled anti- avian bornavirus antibody. Alternatively, the labeling agent may be a third moiety, such a secondary antibody, that specifically binds to the antibody/ antigen complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived). Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent. These proteins exhibit a strong non- immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, e.g., Kronval et al., J. Immunol. 111 :1401-1406 (1973); Akerstrom et ah, J. Immunol. 135:2589-2542 (1985)). The labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art. [0122] Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, optionally from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.

Non-competitive assay formats

[0123] Immunoassays for detecting avian bornavirus protein, virus and nucleic acid in samples may be either competitive or noncompetitive, and may be either quantitative or non-quantitative. Noncompetitive immunoassays are assays in which antigen is directly detected and, in some instances the amount of antigen directly measured. In a "sandwich" assay, for example, the anti- avian bornavirus antibodies can be bound directly to a solid substrate on which they are immobilized. These immobilized antibodies then capture the avian bornavirus antigen present in the test sample. Proteins thus immobilized are then bound by a labeling agent, such as a second anti- avian bornavirus antigen antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second or third antibody is typically modified with a detectable moiety, such as biotin, to which another molecule specifically binds, e.g., streptavidin, to provide a detectable moiety.

Competitive assay formats

[0124] In competitive assays avian bornavirus antigen present in a sample is detected indirectly by detecting a decrease in a detectable signal associated with a known, added (exogenous) avian bornavirus antigen displaced (competed away) from an anti- avian bornavirus antigen antibody by the unknown avian bornavirus antigen present in a sample. In this manner, such assays can also be adapted to provide for an indirect measurement of the amount of ABV antigen present in the sample. In one competitive assay, a known amount of avian bornavirus antigen is added to a sample and the sample is then contacted with an antibody that specifically binds to the avian bornavirus antigen. The amount of exogenous avian bornavirus antigen bound to the antibody is inversely proportional to the concentration of avian bornavirus antigen present in the sample. In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. The amount of avian bornavirus antigen bound to the antibody may be determined either by measuring the amount of avian bornavirus antigen present in avian bornavirus antigen /antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein. The amount of avian bornavirus antigen may be detected by providing a labeled avian bornavirus antigen.

[0125] A hapten inhibition assay is another competitive assay. In this assay the known avian bornavirus antigen is immobilized on a solid substrate. A known amount of anti- avian bornavirus antigen antibody is added to the sample, and the sample is then contacted with the immobilized avian bornavirus antigen. The amount of anti- avian bornavirus antigen bound to the known immobilized avian bornavirus antigen is inversely proportional to the amount of avian bornavirus antigen present in the sample. Again, the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.

Cross-reactivity determinations

[0126] Immunoassays in the competitive binding format can also be used for crossreactivity determinations. For example, a avian bornavirus antigen can be immobilized to a solid support. Proteins are added to the assay that compete for binding of the antisera to the immobilized antigen. The ability of the added proteins to compete for binding of the antisera to the immobilized protein is compared to the ability of the avian bornavirus antigen to compete with itself. The percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the added proteins listed above are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the added considered proteins, e.g., distantly related homologs.

[0127] The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps an allele or polymorphic variant of a avian bornavirus antigen, to the immunogen protein. In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required to inhibit 50% of binding is less than 10 times the amount of the avian bornaviras antigen that is required to inhibit 50% of binding, then the second protein is said to specifically bind to the polyclonal antibodies generated to avian bornavirus antigen.

Other assay formats

[0128] Western blot (immunoblot) analysis is used to detect and quantify the presence of avian bornavirus antigen in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind the avian bornavirus antigen. The anti- avian bornavirus antigen antibodies specifically bind to the avian bornavirus antigen on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti- avian bornavirus antigen antibodies.

[0129] Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al., Amer. Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of non-specific binding

[0130] One of skill in the art will appreciate that it is often desirable to minimize nonspecific binding in immunoassays. Particularly, where the assay involves an antigen or antibody immobilized on a solid substrate it is desirable to minimize the amount of nonspecific binding to the substrate. Means of reducing such non-specific binding are well known to those of skill in the art. Typically, this technique involves coating the substrate with a proteinaceous composition. In particular, protein compositions such as bovine serum albumin (BSA), nonfat powdered milk, and gelatin are widely used with powdered milk being most preferred.

Labels

[0131] The particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well- developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).

[0132] The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

[0133] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to another molecules (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. The ligands and their targets can be used in any suitable combination with antibodies that recognize avian bornavirus antigen, or secondary antibodies that recognize anti- avian bornavirus antigen.

[0134] The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems that may be used, see U.S. Patent No. 4,391,904. [0135] Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Colorimetric or chemiluminescent labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

[0136] Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies, hi this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.

DIAGNOSTIC ASSAYS AND KITS FOR ABV PROTEINS AND NUCLEIC ACIDS

[0137] The present invention provides diagnostic assays to detect ABV, ABV nucleic acids (genome and genes), ABV antibodies in an infected subject, and ABV proteins. In one embodiment, ABV nucleic acid is detected using a nucleic acid amplification-based assay, such as a PCR assay, e.g., in a quantitative assay to determine viral load. In another embodiment, ABV antigens are detected using a serological assay with antibodie. ABV antibodies in a sample can be detected using ABV antigens. These methods can also be used for removing the bornaviras from a blood sample. Donated blood contaminated with avian bornavirus can be dangerous for immunocompromised recipients or other susceptible individuals such as pregnant women.

A. Assays for ABV proteins and antibodies to ABV antigens [0138] In one embodiment of the present invention, the presence of bornavirus, bornavirus nucleic acid, or bornavirus protein in a sample is determined by an immunoassay. Enzyme mediated immunoassays such as immunofluorescence assays (IFA), enzyme linked immunosorbent assays (ELISA) and immunoblotting (western) assays can be readily adapted to accomplish the detection of the bornavirus or bornaviral proteins. An ELISA method effective for the detection of the virus can, for example, be as follows: (1) bind an anti-paroviral antibody or antigen to a substrate; (2) contact the bound receptor with a fluid or tissue sample containing the virus, a viral antigen, or antibodies to the virus; (3) contact the above with an antibody bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the above with the substrate for the enzyme; (5) contact the above with a color reagent; (6) observe color change. The above method can be readily modified to detect presence of an antibornaviral antibody in the sample or a specific bornaviral protein as well as the virus.

[0139] Another immunologic technique that can be useful in the detection of bornaviruses is the competitive inhibition assay, utilizing monoclonal antibodies (MABs) specifically reactive with the virus. Briefly, serum or other body fluids from the subject is reacted with an antibody bound to a substrate (e.g. an ELISA 96- well plate). Excess serum is thoroughly washed away. A labeled (enzyme-linked, fluorescent, radioactive, etc.) monoclonal antibody is then reacted with the previously reacted bornavirus virus- antibody complex. The amount of inhibition of monoclonal antibody binding is measured relative to a control. MABs can also be used for detection directly in samples by IFA for MABs specifically reactive for the antibody-virus complex.

[0140] Alternatively, a bornavirus antigen and/or a patient's antibodies to the virus can be detected utilizing a capture assay. Briefly, to detect antibodies to bornavirus in a patient sample, antibodies to the patient's immunoglobulin, e.g., anti-IgG (or IgM) are bound to a solid phase substrate and used to capture the patient's immunoglobulin from serum. A bornavirus, or reactive fragments of a bornavirus, are then contacted with the solid phase followed by addition of a labeled antibody. The amount of patient bornavirus specific antibody can then be quantitated by the amount of labeled antibody binding.

[0141] Additionally, a micro-agglutination test can also be used to detect the presence of bornavirus in test samples. Briefly, latex beads are coated with an antibody and mixed with a test sample, such that bornavirus in the tissue or body fluids that are specifically reactive with the antibody crosslink with the receptor, causing agglutination. The agglutinated antibody- virus complexes form a precipitate, visible with the naked eye or by spectrophotometer. Other assays include serologic assays, in which the relative concentrations of IgG and IgM are measured.

[0142] In the diagnostic methods described above, the sample can be taken directly from the patient or in a partially purified form. The antibody specific for a particular bornavirus (the primary reaction) reacts by binding to the virus. Thereafter, a secondary reaction with an antibody bound to, or labeled with, a detectable moiety can be added to enhance the detection of the primary reaction. Generally, in the secondary reaction, an antibody or other ligand which is reactive, either specifically or nonspecifically with a different binding site (epitope) of the virus will be selected for its ability to react with multiple sites on the complex of antibody and virus. Thus, for example, several molecules of the antibody in the secondary reaction can react with each complex formed by the primary reaction, making the primary reaction more detectable.

[0143] The detectable moiety can allow visual detection of a precipitate or a color change, visual detection by microscopy, or automated detection by spectrometry, radiometric measurement or the like. Examples of detectable moieties include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for either light or electron microscopy and biochemical detection), biotin-streptavidin (for light or electron microscopy) and alkaline phosphatase (for biochemical detection by color change). The detection methods and moieties used can be selected, for example, from the list above or other suitable examples by the standard criteria applied to such selections (Harlow and Lane, (1988)).

B. Assays for ABV nucleic acids

[0144] As described herein, a ABV infection may also, or alternatively, be detected based on the level of an ABV RNA or DNA in a biological sample. Primers from ABV can be used for detection of ABV, diagnosis, and determination of ABV viral load. Any suitable primer can be used to detect the genome, nucleic acid sub sequence, ORF, or protein of choice, using, e.g., methods described in US 20030104009. For example, the subject nucleic acid compositions can be used as single- or double-stranded probes or primers for the detection of ABV mRNA or cDNA generated from such mRNA, as obtained may be present in a biological sample (e.g., extracts of human cells). The ABV polynucleotides of the invention can also be used to generate additional copies of the polynucleotides, to generate antisense oligonucleotides, and as triple-strand forming oligonucleotides. For example, two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of ABV cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) the ABV polynucleotide. The primers are preferably at least or about 12, 15, 16, 18, 20, 22, 24, 25, 30, 35, 40, 45, or 50 nt or are, for instance, from about 12 to 50 nt in length, 15 to 30 nt in length, 15 to 25 nt in length, or 20 to 30nt in length) fragments of a contiguous sequence of SEQ ID NO: 1 or other polynucleotide sequence encoding an ABV nucleic acid or polypeptide.The amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to a ABV polynucleotide may be used in a hybridization assay to detect the presence of the ABV polynucleotide in a biological sample. These and other uses are described in more detail below.

[0145] Nucleic acid probes or primers specific to ABV can be generated using the polynucleotide sequences disclosed herein. The probes are preferably at least about 12, 15, 16, 18, 20, 22, 24, or 25 nt fragments of a contiguous sequence of SEQ ID NO: 1 or other polynucleotide sequence encoding an ABV nucleic acid or polypeptide. Nucleic acid probes can be less than about 200 bp, 150 bp, 100 bp, 75 bp, 50 bp, 60 bp, 40 bp, 30 bp, 25 bp 2 kb, 1.5 kb, 1 kb, 0.5 kb, 0.25 kb, 0.1 kb, or 0.05 kb in length. The probes can be produced by, for example, chemical synthesis, PCR amplification, generation from longer polynucleotides using restriction enzymes, or other methods well known in the art.

[0146] The polynucleotides of the invention, particularly where used as a probe in a diagnostic assay, can be detectably labeled. Exemplary detectable labels include, but are not limited to, radiolabels, fluorochromes,(e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4',5^l-dichloro-6-carboxyfluorescein, 6-carboxy-X-rhodamine (ROX), 6- carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5 -carboxy fluorescein (5-FAM) or N,N,N',N'-tetramethyl-6-carboxyrho- damine (TAMRA)), radioactive labels, (e.g. .sup.32p, .sup.35S, and .sup.3H), and the like. The detectable label can involve two stage systems (e.g., biotin-avidin, hapten-anti-hapten antibody, and the like). [0147] Preferred primers and probes are identical to an AVB nucleic acid sequence and different from a non-avian bornavirus sequence (e.g., any one or all of bornaviruses No. 98/, He/80, H1766, V).

[0148] The invention also includes solid substrates, such as arrays, comprising any of the polynucleotides described herein. The polynucleotides are immobilized on the arrays using methods known in the art. An array may have one or more different polynucleotides.

[0149] Any suitable qualitative or quantitative methods known in the art for detecting specific ABV nucleic acid (e.g., RNA or DNA) can be used. ABV nucleic acid can be detected by, for example, in situ hybridization in tissue sections, using methods that detect single base pair differences between hybridizing nucleic acid (e.g., using the Invader.RTM. technology described in, for example, U.S. Pat. No. 5,846,717), by reverse transcriptase-PCR, or in Northern blots containing poly A+mRNA, and other methods well known in the art. For detection of ABV polynucleotides in blood or blood-derived samples, the use of methods that allow for detection of single base pair mismatches is preferred.

[0150] Using the ABV nucleic acid as a basis, nucleic acid probes (e.g., including oligomers of at least about 8 nucleotides or more) can be prepared, either by excision from recombinant polynucleotides or synthetically, which probes hybridize with the ABV nucleic acid, and thus are useful in detection of ABV virus in a sample, and identification of infected individuals, as well as further characterization of the viral genome(s). The probes for ABV polynucleotides (natural or derived) are of a length or have a sequence which allows the detection of unique viral sequences by hybridization. While about 6-8 nucleotides may be useful, longer sequences may be preferred, e.g., sequences of about 10-12 nucleotides, or about 20 nucleotides or more. Preferably, these sequences will derive from regions which lack heterogeneity among ABV viral isolates.

[0151] Nucleic acid probes can be prepared using routine methods, including automated oligonucleotide synthetic methods. A complement to any unique portion of the ABV genome will be satisfactory, e.g., a portion of the ABV genome that allows for distinguishing ABV from other viruses that may be present in the sample, e.g., other bornavirus such as B 19. For use as probes, complete complementarity is desirable, though it may be unnecessary as the length of the fragment is increased. [0152] For use of such probes as diagnostics, the biological sample to be analyzed, such as blood or serum, may be treated, if desired, to extract the nucleic acids contained therein. The resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques; alternatively, the nucleic acid sample may be dot blotted without size separation. The probes are usually labeled with a detectable label. Suitable labels, and methods for labeling probes are known in the art, and include, for example, radioactive labels incorporated by nick translation or kinasing, biotin, fluorescent probes, and chemiluminescent probes. The nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions of suitable stringencies.

[0153] The probes can be made completely complementary to the ABV genome or portion thereof (e.g., to all or a portion of a sequence encoding an ABV GAG polypeptide). Therefore, usually high stringency conditions are desirable in order to prevent or at least minimize false positives. However, conditions of high stringency should only be used if the probes are complementary to regions of the viral genome which lack heterogeneity among ABV viral isolates. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, length of time, and concentration of formamide. These factors are outlined in, for example, Sambrook et al. (1989), "Molecular Cloning; A Laboratory Manual", Second Edition (Cold Spring Harbor Press, Cold Spring Harbor, N. Y.).

[0154] Generally, it is expected that the ABV sequences will be present in a biological sample (e.g., blood, cells, and the liked) obtained from an infected individual at relatively low levels, e.g., at approximately 10²-10⁴ ABV sequences per 10⁶ cells. This level may require that amplification techniques be used in hybridization assays. Such techniques are known in the art.

[0155] For example, the Enzo Biochemical Corporation "Bio-Bridge" system uses terminal deoxynucleotide transferase to add unmodified 3'-poly-dT-tails to a DNA probe. The poly dT-tailed probe is hybridized to the target nucleotide sequence, and then to a biotin-modified poly-A. PCT Publication No. WO84/03520 and European application no. EPA 124221 describe a DNA hybridization assay in which: (1) analyte is annealed to a single-stranded DNA probe that is complementary to an enzyme-labeled oligonucleotide; and (2) the resulting tailed duplex is hybridized to an enzyme-labeled oligonucleotide. EPA 204510 describes a DNA hybridization assay in which analyte DNA is contacted with a probe that has a tail, such as a poly-dT tail, an amplifier strand that has a sequence that hybridizes to the tail of the probe, such as a poly-A sequence, and which is capable of binding a plurality of labeled strands.

[0156] Non-PCR-based, sequence specific DNA amplification techniques can also be used in the invention to detect ABV sequences. An example of such techniques include, but are not necessarily limited to the Invader assay, see, e.g., Kwiatkowski et al. MoI Diagn. December 1999;4(4):353-64. See also U.S. Pat. No. 5,846,717.

[0157] A particularly desirable technique may first involve amplification of the target ABV sequences in sera approximately 10,000 fold, e.g., to approximately 10 sequences/mL. This may be accomplished, for example, by the polymerase chain reactions (PCR) technique described which is by Saiki et al. (1986), by Mullis, U.S. Pat. No. 4,683,195, and by Mullis et al. U.S. Pat. No. 4,683,202. Other amplification methods are well known in the art.

[0158] The probes, or alternatively nucleic acid from the samples, may be provided in solution for such assays, or may be affixed to a support (e.g., solid or semi-solid support). Examples of supports that can be used are nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidine fluoride, diazotized paper, nylon membranes, activated beads, and Protein A beads.

[0159] In one embodiment, the probe (or sample nucleic acid) is provided on an array for detection. Arrays can be created by, for example, spotting polynucleotide probes onto a substrate (e.g., glass, nitrocellulose, and the like) in a two-dimensional matrix or array. The probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions. Samples of polynucleotides can be detectably labeled (e.g., using radioactive or fluorescent labels) and then hybridized to the probes. Double stranded polynucleotides, comprising the labeled sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is washed away. Techniques for constructing arrays and methods of using these arrays are described in EP 799 897; WO 97/29212; WO 97/27317; EP 785 280; WO 97/02357; U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP 728 520; U.S. Pat. No. 5,599,695; EP 721 016; U.S. Pat. No. 5,556,752; WO 95/22058; and U.S. Pat. No. 5,631,734. Arrays are particularly useful where, for example a single sample is to be analyzed for the presence of two or more nucleic acid target regions, as the probes for each of the target regions, as well as controls (both positive and negative) can be provided on a single array. Arrays thus facilitate rapid and convenience analysis.

C. Kits

[0160] The invention further provides diagnostic reagents and kits comprising one or more such reagents for use in a variety of diagnostic assays, including for example, immunoassays such as ELISA and "sandwich"-type immunoassays, as well as nucleic acid assay, e.g., PCR assays. In a related embodiment, the assay is performed in a flow- through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. Such kits may preferably include at least a first peptide, or a first antibody or antigen binding fragment of the invention, a functional fragment thereof, or a cocktail thereof, or a first oligo pair, and means for signal generation. The kit's components may be pre-attached to a solid support, or may be applied to the surface of a solid support when the kit is used. The signal generating means may come pre-associated with an antibody or nucleic acid of the invention or may require combination with one or more components, e.g., buffers, nucleic acids, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use.

[0161] Kits may also include additional reagents, e.g., blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, enzymes, and the like. The solid phase surface may be in the form of microtiter plates, microspheres, or other materials suitable for immobilizing nucleic acids, proteins, peptides, or polypeptides. An enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is one such component of the signal generating means. Such enzymes are well known in the art. Where a radiolabel, chromogenic, fluorigenic, or other type of detectable label or detecting means is included within the kit, the labeling agent may be provided either in the same container as the diagnostic or therapeutic composition itself, or may alternatively be placed in a second distinct container means into which this second composition may be placed and suitably aliquoted. Alternatively, the detection reagent and the label may be prepared in a single container means, and in most cases, the kit will also typically include a means for containing the vial(s) in close confinement for commercial sale and/or convenient packaging and delivery.

ASSAYS FOR MODULATORS OF AVIAN BORNAVIRUS

A. Assays

[0162] Modulation of a avian bornavirus, and corresponding modulation of the cell cycle, e.g., tumor cell, proliferation, can be assessed using a variety of in vitro and in vivo assays, including cell-based models. Such assays can be used to test for inhibitors and activators of avian bornavirus. Modulators of avian bornavirus are tested using either recombinant or naturally occurring protein of choice, preferably human avian bornavirus.

[0163] Preferably, the avian bornavirus will have the sequence as encoded by a sequence as shown in Accession No. EU781967 or a conservatively modified variant thereof. Alternatively, the avian bornavirus of the assay will include an amino acid subsequence having substantial amino acid sequence identity to a sequence as shown in Accession No. EU781967. Generally, the amino acid sequence identity will be at least 60%, preferably at least 65%, 70%, 75%, 80%, 85%, or 90%, most preferably at least 95%.

[0164] Measurement of modulation of a avian bornavirus or a cell expressing avian bornavirus, either recombinant or naturally occurring, can be performed using a variety of assays, in vitro, in vivo, and ex vivo, as described herein. A suitable physical, chemical or phenotypic change that affects activity, e.g., enzymatic activity, cell surface marker expression, viral replication and proliferation can be used to assess the influence of a test compound on the polypeptide of this invention. When the functional effects are determined using intact cells or animals, one can also measure a variety of effects.

In vitro assays

[0165] Assays to identify compounds with avian bornavirus modulating activity can be performed in vitro. Such assays can used full length avian bornavirus or a variant thereof, or a mutant thereof, or a fragment thereof. Purified recombinant or naturally occurring protein can be used in the in vitro methods of the invention. In addition to purified avian bornavirus, the recombinant or naturally occurring protein can be part of a cellular lysate or a cell membrane. As described below, the binding assay can be either solid state or soluble. Preferably, the protein or membrane is bound to a solid support, either covalently or non-covalently. Often, the in vitro assays of the invention are substrate or ligand binding or affinity assays, either non-competitive or competitive. Other in vitro assays include measuring changes in spectroscopic (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein.

[0166] In one embodiment, a high throughput binding assay is performed in which the protein or a fragment thereof is contacted with a potential modulator and incubated for a suitable amount of time. In one embodiment, the potential modulator is bound to a solid support, and the protein is added. In another embodiment, the protein is bound to a solid support. A wide variety of modulators can be used, as described below, including small organic molecules, peptides, antibodies, etc. A wide variety of assays can be used to identify avian bornavirus-modulator binding, including labeled protein-protein binding assays, electrophoretic mobility shifts, immunoassays, enzymatic assays, and the like. In some cases, the binding of the candidate modulator is determined through the use of competitive binding assays, where interference with binding of a known ligand or substrate is measured in the presence of a potential modulator. Either the modulator or the known ligand or substrate is bound first, and then the competitor is added. After the protein is washed, interference with binding, either of the potential modulator or of the known ligand or substrate, is determined. Often, either the potential modulator or the known ligand or substrate is labeled.

Cell-based in vivo assays

[0167] In another embodiment, the avian bornavirus is expressed in a cell, and functional, e.g., physical and chemical or phenotypic, changes are assayed to identify modulators of the cell cycle. Any suitable functional effect can be measured, as described herein. The avian bornavirus can be naturally occurring or recombinant. Also, fragments of the avian bornavirus or chimeric proteins can be used in cell based assays. In addition, point mutants in essential residues required by the catalytic site can be used in these assays. B. Modulators

[0168] The compounds tested as modulators of avian bornavirus can be any small organic molecule, or a biological entity, such as a protein, e.g., an antibody or peptide, a sugar, a nucleic acid, e.g., an antisense oligonucleotide or a ribozyme or RNAi, or a lipid. Alternatively, modulators can be genetically altered versions of a avian bornavirus. Typically, test compounds will be small organic molecules, peptides, circular peptides, RNAi, antisense molecules, ribozymes, and lipids.

[0169] Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.

[0170] In one preferred embodiment, high throughput screening methods involve providing a combinatorial small organic molecule or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

[0171] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. [0172] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al, Nature 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al. , Science 261 : 1303 (1993)), and/or peptidyl phosphonates (Campbell et al. , J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).

[0173] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

C. Solid state and soluble high throughput assays

[0174] In one embodiment the invention provides soluble assays using a avian bornavirus, or a cell or tissue expressing an avian bornavirus, either naturally occurring or recombinant. In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the avian bornavirus is attached to a solid phase. Any one of the assays described herein can be adapted for high throughput screening.

[0175] In the high throughput assays of the invention, either soluble or solid state, it is possible to screen up to several thousand different modulators or ligands in a single day. This methodology can be used for avian bornavirus in vitro, or for cell-based or membrane-based assays comprising a avian bornavirus. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay many plates per day; assay screens for up to about 6,000, 20,000, 50,000, or more than 100,000 different compounds are possible using the integrated systems of the invention.

[0176] For a solid state reaction, the protein of interest or a fragment thereof, e.g., an extracellular domain, or a cell or membrane comprising the protein of interest or a fragment thereof as part of a fusion protein can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage. A tag for covalent or non- covalent binding can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.

[0177] A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.) Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).

[0178] Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs. For example, agonists and antagonists of cell membrane receptors (e.g., cell receptor- ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherein family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g. which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.

[0179] Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.

[0180] Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to persons of skill in the art. For example, poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.

[0181] Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of, e.g., peptides); Geysen et al, J. Immun. Meth. 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank & Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor et ah, Science, 251:767-777 (1991); Sheldon et al, Clinical Chemistry 39(4):718-719 (1993); and Kozal et al, Nature Medicine 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.

VACCINES

[0182] Within certain aspects, ABV virus, proteins or peptides and immunogenic fragments thereof, and/or polynucleotides, as well as anti-ABV antibodies and/or T cells, may be incorporated into pharmaceutical compositions or immunogenic compositions (e.g., vaccines). Whole virus vaccine (live and attenuated, or replication incompetent, or killed) or subunit vaccines, such as structural or non-structural ABV proteins or immunogenic fragments thereof, can be used to treat or prevent ABV infections by eliciting an immune response in a subject. Alternatively, a pharmaceutical composition may comprise an antigen-presenting cell (e.g., a dendritic cell) transfected with a ABV polynucleotide such that the antigen-presenting cell expresses an ABV peptide.

[0183] Pharmaceutical compositions comprise one or more such vaccine compounds and a physiologically acceptable carrier. Vaccines may comprise one or more such compounds and a non-specific immune response enhancer. A non-specific immune response enhancer may be any substance that enhances an immune response to an exogenous antigen. Examples of non-specific immune response enhancers include adjuvants, biodegradable microspheres {e.g., polylactic galactide) and liposomes (into which the compound is incorporated; see, e.g., U.S. Patent No. 4,235,877). Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

[0184] Vaccine preparation is generally described in, for example, Powell and Newman, eds., Vaccine Design (the subunit and adjuvant approach), Plenum Press (NY, 1995). Vaccines may be designed to generate antibody immunity and/or cellular immunity such as that arising from CTL or CD4+ T cells.

[0185] Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other antigens may be present, either incorporated into a fusion polypeptide or as a separate compound, within the composition or vaccine. Polypeptides may, but need not, be conjugated to other macromolecules as described, for example, within US Patent Nos. 4,372,945 and 4,474,757. Pharmaceutical compositions and vaccines may generally be used for prophylactic and therapeutic purposes.

[0186] Nucleic acid vaccines encoding a genome, structural protein or non-structural protein or a fragment thereof of ABV can also be used to elicit an immune response to treat or prevent ABV infection. Numerous gene delivery techniques are well known in the art, such as those described by Rolland (1998) Crit. Rev. Therap. Drug Carrier Systems 75:143-198, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). In a preferred embodiment, the DNA may be introduced using a viral expression system {e.g., vaccinia, pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et al. (1989) Proc. Natl. Acad. Sci. USA 55:317-321; Flexner et al. {\9%9) Ann. NY. Acad. Sd. 569:86-103; Flexner et al. (1990) Vaccine 5:17-21 ; U.S. Patent Nos. 4,603,112, 4,769,330, 4,777,127 and 5,017,487; WO 89/01973; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner (1988) Biotechniques 6:616-621; Rosenfeld et al. (1991) Science 252:431-434; Kolls et al. (1994) Proc. Natl. Acad. Sci. USA 97:215-219; Kass-Eisler et al. (1993) Proc. Natl. Acad. Set USA 90:11498-11502; Guzman etal. (1993) Circulation 55:2838-2848; and Guzman et al. (1993) Cir. Res. 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al. (1993) Science 259:1745-1749 and reviewed by Cohen (1993) Science 259:1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. It will be apparent that a vaccine may comprise both a polynucleotide and a polypeptide component. Such vaccines may provide for an enhanced immune response.

[0187] Vaccines and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a vaccine or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.

PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION

[0188] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered (e.g., nucleic acid, protein, modulatory compounds or transduced cell), as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington 's Pharmaceutical Sciences, 17 ed., 1989). Administration can be in any convenient manner, e.g., by injection, oral administration, inhalation, transdermal application, or rectal administration.

[0189] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.

[0190] The compound of choice, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0191] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. Parenteral administration and intravenous administration are the preferred methods of administration. The formulations of commends can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.

[0192] Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using well known technology.

[0193] Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.

[0194] The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. The dose will be determined by the efficacy of the particular vector employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient.

[0195] In determining the effective amount of the vector to be administered in the treatment or prophylaxis of conditions owing to diminished or aberrant expression of the protein, the physician evaluates circulating plasma levels of the vector, vector toxicities, progression of the disease, and the production of anti-vector antibodies. In general, the dose equivalent of a naked nucleic acid from a vector is from about 1 μg to 100 μg for a typical 70 kilogram patient, and doses of vectors are calculated to yield an equivalent amount of therapeutic nucleic acid.

[0196] For administration, compounds and transduced cells of the present invention can be administered at a rate determined by the LD-50 of the inhibitor, vector, or transduced cell type, and the side-effects of the inhibitor, vector or cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses.

EXAMPLES

[0197] The following examples are offered to illustrate, but not to limit the claimed invention. See, now published, Kistler et al., Virology Journal 5:88 (2008), incorporated herein by reference in its entirety with respect to its report of the work of Example 1.

Example 1 : Identification of a new avian bornavirus Materials and Methods

PDD case and control definitions, specimen collection, and RNA extraction for pan-viral microarray screening.

[0198] Two independent sets of PDD case and control specimens collected from two distinct geographic locations were independently prepared for pan- viral microarray screening and subsequent PCR screening. Sampling collection and inclusion criteria for each set are described below.

United States PDD case/control series.

[0199] Specimen collection: All specimens provided for initial screening were crop tissue biopsies obtained from live psittacine birds to be used as normal controls or multiple tissue samples collected from clinically diseased birds at the time of euthanasia. Specimens were collected from client-owned birds. All of these samples originated from the southeast region of Florida. Crop biopsy tissue was collected from live birds under isoflurane anesthesia. Following routine surgical preparation and sterile technique, the skin was incised over the center of the crop. The crop tissue was exposed and a section of tissue removed taking care to include large visible blood vessels. Fresh crop biopsy tissue was trimmed into tissue slices < 5mm thick and submersed in RNAlater (Qiagen, Inc., USA, Valencia, CA) solution immediately upon harvest and frozen within 2 minutes of collection at -20C to -80C according to manufacturer's protocol, and held in this manner until shipped. A duplicate sample was fixed in 10% buffered formalin for routein histological examination with H & E stain. Time of frozen storage varied (2 weeks to 12 months) as samples were accumulated prior to shipping frozen. Clinically affected birds submitted as positives were euthanized under isoflurane anesthesia and mixed tissues (proventriculus, ventriculus, heart, liver, spleen, kidneys, brain) were placed into RNAlater within 1 minute of death and frozen within 2 minutes of death. Duplicate samples were collected for histopathology diagnosis of PDD.

[0200] Inclusion criteria: PDD-positive cases were required to meet the following criteria 1) Clinical history of characteristic wasting/malabsorption syndrome with dilation of the proventriculus and/or ventriculus and presence of undigested food in the stool and in most cases, a clinical history of ataxia or other CNS signs consistent with clinical PDD, and 2) histopathology confirming the presence of moderate to extensive lymphoplasmacytic neurogangliitis affecting at least one of the following areas: crop, proventriculus, ventriculus, brain, adrenal gland, or myocardium. PDD-negative controls were required to be from birds with no evidence of lymphoplasmacytic neurogangliitis on histopathology derived either from 1) normal birds with no clinical history of PDD or no known exposure to PDD or 2) birds which died of other causes. Crop biopsies from samples from living birds classified as suspicious cases were also submitted. Suspicious cases were defined histologically as having lymphocytes and plasma cells surrounding neurons but not infiltrating into the neurons. An additional specimen derived from a live bird raised with two necropsy-confirmed PDD birds in Virginia was also collected for analysis. Here, only cloacal swab and blood specimens were available. Due to lack of histopathological confirmation and crop tissue, thus, this specimen was excluded from statistical analyses. However, ABV PCR was performed on these specimens and the resulting viral sequences isolated were included in the subsequent comparative sequence analysis.

[0201] RNA extractions: For RNA extractions, specimens were thawed in RNALater, sliced into 0.5mm x 0.5mm pieces, transferred to 2ml of RNABee solution (Tel-Test, Inc., Friendswood, TX), homogenized with freeze thawing and scapel mincing, then extracted in the presence of chloroform according to manufacterer's instructions. Resulting RNA was next incubated with DNase (DNA-free, Applied Biosystems/Ambion, Austin, TX) to remove any potential contaminating DNA present in the specimen.

Israeli case/control series.

[0202] Specimen collection: Tissue samples were obtained from psittacine birds submitted to the Division of Avian and Fish Diseases, Kimron Veterinary Institute (KVI) Bet Dagan, Israel, for diagnostic necropsy. A few additional specimens were obtained through private veterinarians. Some tissues were kept for nearly 4 years frozen either at - 2O⁰C or -80°C prior to testing, while others were fresh tissues from recent cases. The types of banked frozen tissue varied from case to case, while for some of the older cases only gastrointestinal content had been banked. Clinical histories for these birds were available from the submission forms or through communication with the submitting veterinarians. The results of ancillary tests performed at the KVI were available through the KVI computerized records.

[0203] Inclusion criteria: Only cases for which appropriate histological sections were available for inspection were considered for this study. These had to include brain and at least two of the following tissues: crop, proventriculus, ventriculus. The tissue-types examined for each bird for which specimens were provided are listed in Supplemental Data File 1. PDD-positive cases were required to have evidence of lymphoplasmacytic infiltration of myenteric nerves and/or ganglia within one or more of the upper GI tract tissues mentioned above. PDD-negative controls had no detectable lesions and no evidence of non-suppurative encephalitis. For most birds in the PDD-negative group, a cause of death (other than PDD) has been determined. Two birds that came from a known PDD outbreak, but showed only cerebral lymphoplasmacytic perivascular cuffing, were classified as 'suspicious'. These were excluded from the statistical analysis, as were all other birds for which a PDD status could not be clearly determined and classified as 'inconclusive' (e.g. due to poor tissue preservation, poor section quality, or scarcity of myenteric nerves within the tissues examined).

[0204] RNA extraction: When possible, a sample of brain as well as a combined pro ventricular/ventricular sample was prepared for RNA extraction for each bird. If not available, other tissues and/or gastrointestinal content were used. Frozen samples were allowed to thaw for 1-2 hours at room temperature prior to handling. Then, under a laminar flow biohazard hood and using aseptic technique, approximately Icm3 of each tissue was macerated by two passages through a 2.5ml sterile syringe and transferred into sterile test tubes containing 4ml nuclease-free PBS. The content of the tubes was mixed by vortex for 30sec, and the tubes were placed overnight at 4°C. RNA extraction was performed on the following day, using either the QIAamp® viral RNA kit (Qiagen, Valencia, CA; batch 1&2, specimens 1-8) or the TRI Reagent® kit (Molecular Research Center, Cincinnati, OH; all other specimens), following the manufacturers' instructions. The end product was either provided lyophilized (batches 1 and 2, samples 1-9) as a dry pellet, or re-suspended in 40ul nuclease-free DDW.

Virus chip hybridization experiments.

[0205] Microarray analysis of specimens was carried out as previously described (Chiu CY et al., Clin Infect Dis, 43(8):e71-76 (2006)). Briefly, 50-200ng of DNAse-treated total RNA from each sample was amplified and labeled using a random-primed amplification protocol and hybridized to the Virochip. Microarrays (NCBI GEO platform GPL3429) were scanned with an Axon 4000B scanner (Axon Instruments). Virochip results were analyzed using E-Predict (Urisman A et al., Genome Biol, 6(9):R78 (2005)) and vTaxi (K. Fisher et al., in preparation).

PCR primers for detection of avian bornaviruses.

[0206] Microarray-based Bornaviridae PCR primers. Initial PCR primers were generated based on two of the 70mer microarray probes with hybridization signal in the Bornaviridae positive arrays that localize to positions 3701-3770 and 4201-4270 of the Bornaviridae reference sequence [NC OO 1607]. Subsequences within each of these probes (BDV LconsensusF: 5'-CCTCGCGAGGAGGAGACGCCTC-3' and BDVJLconsensusR: 5' CTGCTCTTGGCTGTGTCTGCTGC-3'; positions 3710-3729 and 4252-4230, respectively of the NCBI Bornaviridae reference sequence) that are 100% conserved across the 12 other fully sequenced bornavirus genome isolates in NCBI (huP2br [AB258389], Bo/04w [AB246670], No/98 [AJ311524], H1766 [AJ311523], He/80/FR [AJ311522], V/FR [AJ311522], virus rescue plasmid pBRT7-HrBDVc [AY05791], CRNP5 [AYl 14163], CRP3B [AYl 14162], CRP3A [AYl 14161], He/80 [L27077], and V [U040608]) were utilized for initial follow-up PCR and sequence confirmation of microarray screening results. Briefly, IuI of the randomly amplified nucleic acid prepared for microarray hybridization from all specimens was utilized as template for 35 cycles of PCR, under the following conditions: 94°C, 30 seconds; 50°C, 30 seconds; 72°C, 30 seconds. Resulting PCR products were gel purified, subcloned into the TOPO TA cloning vector pCR2.1 (Invitrogen, USA, Carlsbad CA) and sequenced with M13F and M13R primers.

[0207] Generation of ABV consensus PCR primers. Sequences recovered from BDV LconsensusF and BDVJLconsensusR PCR products were aligned, and an additional set of ABV consensus primers biased towards the aBV sequences were identified: ABVJLconsensusF, 5'-CGCCTCGGAAGGTGGTCGG-S' (maps to positions aligning with residues 3724-3742 of BDV reference genome) and ABVJLconsensusR, 5'-GGCAYCAYCKACTCTTRAYYGTRTCAGC-S' (maps to positions aligning with residues 4233-4257 of BDV reference genome). Using identical PCR cycling conditions as described above for the microarray-based Bornaviridae PCR assay, these ABV consensus primers were found to be>100X more sensitive for ABV detection compared to BDV_LconsensusF and BDV_LconsensusR primers, and were thus utilized to re-screen the initial set of PDD case and control samples provided for microarray analysis (no additional positives identified) and all subsequently provided samples.

Ultra high-throughput sequencing.

[0208] Sample preparation and sequencing. 500ng of total RNA derived from one of the PDD case specimens was linearly amplified via modification of the MesssageAmp aRNA kit (Applied Biosystems/Ambion, Austin, TX). To ensure the amplification of both mRNA and vRNA present in the specimen, T7-tailed random nonamer was mixed in an equimolar ratio with the manufacturer-provided T7-oligo(dT) primer during the 1st strand synthesis step. The resulting aRNA was next used as input for a modified version of Genomic DNA sample preparation protocol for ultra high-throughput Solexa sequencing (Illumina, Hayward, CA). 400ng of the input aRNA was reverse-transcribed with reverse transcriptase (Clontech Laboratories, Inc., Mountain View, CA) using a random nonamer tailed with 19bp of the Solexa Long (5'-

CACGACGCTCTTCCGATCTNNNNNNNNN-3') primer sequence (Illumina, Hayward CA). Following termination of reaction, first strand cDNA products were purified from the reaction with Qiagen MinElute spin column (Qiagen USA, Valencia CA). To ensure stringent separation from primers, the MinElute eluate was then filtered through a Microcon YM30 centrifugal filter (Millipore Corp., Billerica, MA). The resulting eluate served as template for 2nd strand synthesis in a standard Sequenase 2.0 (USB, Cleveland, OH) reaction primed with a random nonamer tailed with 22bp (5'-GGCATACGA GCTCTTCCGATCTNNNNNNNNN-3') of the Solexa Short primer sequence (Illumina, Hayward CA). Double-stranded DNA products were separated from primers and very short products through a second Qiagen MinElute spin column run followed by a Microcon YM50 centrifugal filter. This eluate was used as template for 10 cycles of PCR amplification with the full length Solexa L and S primers using KlenTaq LA DNA polymerase mix (Sigma-Aldrich, St. Louis, MO). PCR product was purified from the reaction with a MinElute spin column. Following cluster generation, Solexa sequencing primer was annealed to the flow cell, and 36 cycles of single base pair extensions were performed with image capture using a IG Genome Analyzer (Illumina, Hayward, CA). The Solexa Pipeline software suite version 0.2.2.6 (Illumina, Hayward, CA) was utilized for base-calling from these images. Using software default quality filters, cycles 4-36 were deemed high quality, resulting in a total of 1.4 million 33mer reads for downstream sequence analyses.

[0209] Identification of Bornaviridae reads. Reads sharing 100% identity to each other or the Solexa amplification primers were filtered, reducing our initial set of 1.4 million reads to a working set of 600,000 unique reads. In order to quickly assess the homology of this set of reads to different sequence databases, we employed an iterative strategy using ELAND (Efficient Local Alignment of Nucleotide Data) and BLAST analyses. To filter reads from our analysis potentially derived from psittacine host tissue, the working set of reads were aligned to a database of all Aves sequences from NCBI (n=918,511) using ELAND, which tolerates no more than 2 base mismatches, and discards both low quality reads and reads with low sequence complexity. Reads that did not align to the Aves database by ELAND analysis were next re-aligned to the Aves database for high stringency blastn analysis (e=10-7, word size=l 1), followed by progressively lower stringencies (down to e=10-2, word size=8), corresponding to reads containing only 22 nucleotide identities to sequences in the Aves database. To identify reads with some homology to Bornaviridae sequences in the resulting set of 322,790 host-filtered reads, we re-implemented the ELAND/iterative blastn analysis strategy (down to > 15 nucleotides identity) using a database of all NCBI BDV sequences (n=207) augmented by our previously recovered aBV sequences (n=5). An additional iterative tblastx analysis was incorporated to capture distantly related reads that shared similarity to the known BDV sequences only at the level of predicted amino acid sequence (down to > 6 amino acid identity).

Complete ABVvRNA genome sequence recovery by RT-PCR.

[0210] Initial genome sequence recovery. Sequences from 33mer reads from the deep sequencing with a minimum of 91% sequence identity with known BDV sequences present in the NCBI database were utilized to generate a set of primers for additional cloning and sequence recovery by RT-PCR of both mRNA and vRNA present in the clinical specimen. In this manner, we generated a hybrid assembly derived from multiple overlapping clones and 5' RACE products encompassing the ABV genome sequence.

[0211] vRNA genome sequence recovery. To ensure recovery of accurate sequence across the ABV genome, especially at splice junctions and transcription initiation and termination sites, we utilized the sequence from ABV hybrid assembly to design primers for recovery of 3 overlapping products by RT-PCR directed against the vRNA present in the specimen. Aliquots of 500ng of DNAse-treated total RNA extracted from the clinical specimen were annealed with 3 primers complementary to the predicted vRNA sequences: ABVIr, 5'-ATGACCAGGACGAGGAGATG-S' (maps to residues 8831- 8812 of vRNA), ABV2r, 5'-CCTGTGAATGTCTCGTTTCTG-S' (maps to residues 5754-5733 of vRNA), and ABV3r 5-TTCTTTCAGCAACCACTGACG-3' (maps to residues 2563-2543 of vRNA). Reverse transcription was carried out at 5OC for lhr with SuperScriptIII (Invitrogen, Carlsbad CA) according to manufacturer's instructions. Following RNase H treatment, PCR was performed on the resulting cDNA with Phusion polymerase (NEB, Ipswich, MA) with the primers used for reverse transcription and the following primers: ABVIf: 5'-GGATCATTCCTTGATGATGTATTAGC-S', (maps to residues 5567-5589) ABV2f: 5'-CAAATGGAGAGCCTGATTGG-S' (maps to residues 2378-2397) ABVSf: 5'-AATCGGTAAGTCC AGAGTC AAGG-3' (maps to residues 155- 177). All products were amplified for 35 cycles under the following conditions: 98°C, 3 minutes; 98°C, 10 seconds, 50°C, 30 seconds, 72°C 3 minutes. Resulting products were gel purified, and subcloned into the TOPO T/A cloning vector pCR2.1 after incubation with Taq polymerase and dATP for 10 minutes at 72°C. For each product, 4 independent transformants were prepared for standard dideoxy sequencing on an ABI3730 sequencer (ElimBio, Hayward CA). Forward and reverse reads spanning each clone were generated using M13F and M13R and additional overlapping primers spaced at 600-800bp intervals across the each of the clones.

[0212] 5 ' and 3 ' RACE to sequence at vRNA termini. vRNA RT-PCR products containing uncapped vRNA termini were captured using the First Choice RLM RACE kit (Ambion, Austin TX) with the following modifications to the standard protocol: 1) tobacco acid phosphotase treatment was omitted, 2) a phosphorylated RNA, RNAligate, 5'-P-GUUAUCACUUUCACCC-S' was substituted for the 3' RNA ligation-mediated RACE primer provided in the kit and ligated to 3' ends as per manufacterer's 5' RACE protocol, and 3) in the 3' RACE reverse transcription reactions, two reverse transcription reactions were performed and carried forward in parallel: one with random decamers and one with a DNA oligo complementary to oJSmer utilized in the RNA ligation step (ligateRC, S'-p-GGGTGAAAGTGATAAC-S'). For 5' RACE, a single round of PCR was sufficient to generate a product using the vRNA specific primer ABV5RaceOuter, 5'- CAGTCGGTTCTTGGACTTGAAGTATCTAGG-S' (maps to residues 346-317 of vRNA) and manufacturer provided outer PCR primer. For 3' RACE, nested PCR was required to recover detectable PCR product of expected size using outer PCR primers oJSmerRC and the gene specific primer ABV3RaceOuter, 5'-

CCCGTCTACTGTTCTTTCGCCG-3' (maps to residues 8479-8497 of vRNA), followed by inner PCR using Tailed_RNAligateRC, 5'-

AAGCAGTGGTAACAACGCAGAGTACGGGTGAAAGTGATAAC-S' and the gene specific primer, ABV3RaceInner, 5'-GCAATCCAGGAATAAGCAAGCACAAA-S' (maps to residues 8595-8620 of vRNA). Both of the RACE PCR reactions were carried out with Platinum Taq polymerase (Invitrogen, Carlsbad, CA) in 35 cycles of gradient PCR (with varying annealing temperature): 94°C, 30 seconds; 55-58°C, 30 seconds; 72°C, 30 seconds. Resulting PCR products were gel purified and subcloned into TOPO T/A cloning vector pCR 4.0. For the 5' RACE products, 7 independent transformants from 3 independently generated PCR products were subcloned and sequenced with M13F and M13R primers. For the 3' RACE products, 6 independent transformants from 4 independently generated PCR products were subcloned and sequenced with M13F and M13R primers. Terminal sequences reported here reflect the majority consensus sequence obtained from these reads.

[0213] Genome sequence assembly. Genome sequence assemblies from both initial genome sequence recovery and vRNA genome sequence recovery were generated using Consed, version 16.0 software (Gordon D et al., Genome Res, 8(3): 195-202 (1998)). All bases from the resulting vRNA genome sequence assembly are covered at least 4X with a minimum Phred value of 20.

Blinded PCR screening of additional PDD cases and controls.

[0214] Beyond the initial set of 16 specimens provided for microarray analysis, specimens from a total of 38 additional PDD cases, PDD controls, and PDD suspicious birds with varied clinical histories were provided to us blinded by our 2 collaborators.

[0215] Sample processing: For specimens provided in tissue form from the US collaborators, total RNA was extracted as described above with RNABee, DNase treated, then reverse-transcribed and PCR-amplified according to our random amplification protocol for microarray sample preparation (Materials and Methods). Specimens provided from Israel in the form of extracted RNA were similarly DNAse-treated and amplified prior to PCR screening.

[0216] PCR screening: IuI of the randomly amplified material generated from these RNA samples was used as input template for aBV consensus PCR as described above. In parallel, as an independent control for input specimen RNA integrity, PCR for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was performed on all specimens using designed based on Friedman-Einat et al (Friedman-Einat M et al., Gen Comp Endocrinol, 115(3): 354-363(1999)) and Gallu gallus GAPDH sequence: Gg_GAPDHf: 5'-AGTCATCCCTGAGCTSAAYGG*GAAGC-3' (bp708-733 in Gallus gallus cDNA (NCBI accession NM 204305), * indicates the junction of GAPDH exon 8 and 9 spanned by this primer), Gg_GAPDHr 5'-ACCATCAAGTCCACAACACGG-S' (Spans bp 1037-1017 in Gallus gallus GAPDH cDNA (NCBI accession NM_204305), maps to GAPDH exon 12). After PCR results were tallied, clinical information on all specimens tested was unmasked.

[0217] Sample inclusion for association analysis: To reduce potential confounding due to differences in viral detection resulting from specimen tissue source, only specimens derived from upper GI tract tissue (crop, proventriculus/ventriculus) that tested positive by GAPDH mRNA PCR were included in association analysis presented in Table 3. This consisted of a total of 21 specimens. 7 of which were derived from histologically confirmed PDD cases and 14 derived from histologically negative control specimens.

[0218] Samples excluded from association analysis: The remaining 17 samples were excluded from the analysis because they were either 1) GAPDH-positive or GAPDH- negative samples derived from specimen other than upper GI tract tissue (GI content, brain, liver, or intestine) or 2) derived from cases that were histologically or clinically 'suspicious', but unconfirmed PDD cases. Five additional ABV PCR positives were identified among this set of samples excluded from the statistical analyses: 1 derived from GI content from a confirmed PDD case, and 4 derived from a variety of tissues from the PDD suspicious cases.

Phylogenetic and comparative sequence analysis.

[0219] Multiple sequence alignments of complete genome sequences or partial sequences derived from PCR screening studies were generated with ClustalW (Thompson JD et al, Nucleic Acids Res, 22(22):4673-4680 (1994)) version 1.83. Resulting alignments were used for scanning pairwise sequence analysis (window size, 100; step size 1 nucleotide steps). Additional ClustalW alignments and neighbor-joining phylogenetic trees were generated using Mega software, version 4.0.2 (Tamura K et al., MoI Biol Evol, 24(8): 1596-1599 (2007)).

Results

Microarray-based detection of a Bornaviridae signature in PDD

[0220] To identify a possible viral cause of PDD, we applied the Virus chip, a DNA microarray containing 70mer oligonucleotide probes representing all known viral sequences conserved at multiple nodes of the viral taxonomic tree (Chiu CY et al., Clin Infect Dis, 43(8):e71-76 (2006); Chiu CY et al., J Clin Microbiol, 45(7):2340-2343 (2007)) to identify viral signatures unique to histologically confirmed cases of PDD. At the outset of this study, specimens from two independently collected PDD case/control series were available for this investigation (Figure 1, Materials and Methods). The first series (n=8), from samples originating in the United States, consisted of crop biopsy specimens from 3 histologically confirmed PDD cases and 5 controls that were provided for nucleic acid extraction and follow-up Virus chip analysis. The samples from the second series (n=8) originated in Israel, where total RNA and DNA from proventriculus, ventriculus and brain specimens were extracted from 5 PDD cases and 3 controls. For each series, total RNA was reverse-transcribed with random primers, PCR-amplifϊed, and fluorescently labeled and hybridized to the Virus chip microarray as previously described (Chiu CY et al., CHn Infect Dis, 43(8):e71-76 (2006)).

[0221] In both of the PDD case/control series, a Bornaviridae signature was detectable in half the cases and none of the controls (Table 1). In the US cohort, which contained only GI tract specimens, we detected a bornavirus in 2 of 3 cases. Surprisingly, in samples from the Israeli PDD case/control series for which we had both GI tract and brain specimen RNA for each animal, we detected the Bornaviridae signature in 3 of the cases, but only in samples derived from brain tissue. These signatures were unambiguously confirmed by follow-up PCR and sequence recovery, using primers based on the sequences of the most strongly annealing Bornaviridae oligonucleotides on the microarray (Figure 2, Array probes and PCR probes tracks). These analyses revealed the presence of a set of surprisingly divergent avian bornaviruses (ABVs) in the PDD cases; these recovered sequences shared less than 70% sequence identity to any of the previously identified mammalian bornavirus isolates in the NCBI database.

Table 1: ABV detection in PDD cases^a controls totals

Virochip⁺ 5 0 5

Virochip^" 3 8 11 totals 8 8 16 a3 crop biopsies from US source and 5 brain and proventriculus/ventriculus biopsies from Israel source were examined, with ABV detected in 2 of crop specimens and 3 brain specimens. ^b5 crop biopsies from US source and 3 brain and proventriculus/ventriculus biopsies from Israel source were examined. Recovery of complete genome sequence of a divergent avian bornavirus (ABV) from a PDD case via ultra high-throughput sequencing and follow-up PCR

[0222] To determine if the sequence fragments we detected among specimens derived from PDD cases corresponded to the presence of a full-length bornavirus, we performed unbiased deep sequencing on a PCR-confirmed bornavirus positive PDD case that contained the highest concentration of RNA. To recover both mRNA and vRNA present in the sample, RNA from this specimen was linearly amplified with both oligo(dT) and random hexamer primers, and then PCR-amplified using a modified random amplification strategy compatible with the Solexa sequencing platform (Materials and Methods). An initial set of 1.4 million 33mer reads was obtained from this template material. Filtering on read quality, insert presence, and sequence complexity reduced this data set to 600,000 unique reads. Additional ELAND and iterative BLAST analyses ((Altschul SF et al, Nucleic Acids Res, 25(17):3389-3402 (1997)) Materials and Methods) of these reads against all avian sequences in NCBI (including ESTs, n= 918,511) identified reads in the dataset with at least 22 nucleotides of sequence identity likely derived from host transcripts randomly amplified during sequencing sample preparation. The 322,790 reads that passed this host filter were next screened for the presence of bornavirus sequence through similar ELAND and iterative BLAST analyses (Materials and Methods) using a database generated from all Borna Disease virus (BDV) sequences present in NCBI (n=207) and the sequences we had recovered from PCR follow-up of the PDD samples that tested positive for bornavirus by Virus chip microarray (n=5). These analyses provided us with 1400 reads with at least a match of 15 or more nucleotides (blastn) or 7 or more predicted amino acids (tblastx) to known BDV sequences.

[0223] Mapping these 1400 reads onto their corresponding positions on a consensus sequence for the 14 publicly available BDV genome sequences revealed spikes of high read coverage distributed discontinuously across the entire span of the BDV genome consensus. Reads containing blastn scores >90% identity to known BDV sequences were used a source sequences for primer design for PCR and sequence recovery of additional bornavirus sequence from both mRNA and vRNA templates present in the PDD specimen. Sequences recovered in this manner facilitated subsequent primer design for recovery of complete genome sequence via RT-PCR of 3 large overlapping fragments of the genome and 5'- and 3'-RACE (Figure 2A, vRNA RT-PCR track) directly from negative stranded vRNA present in the total RNA extracted from this clinical specimen.

[0224] As our initial PCR results suggested, the bornavirus genome sequence we recovered is quite diverged from all known BDV genomes, including the BDV isolate No/98, a divergent isolate sharing only 81% sequence identity with all other BDV genomes (Nowotny N et al., J Virol, 74(12): 5655-5658 (2000)). Overall, this newly recovered bornavirus genome sequence shares only 64% sequence identity at the nucleotide level to each of the complete BDV genomes. Scanning pairwise sequence identity analysis indicates this genetic divergence exists across the entire genome (Figure 2 A, Sequence identity shared with BDV genomes track). Given this divergence, we re- examined the depth and distribution of the 322,790 reads from this specimen that passed the host filter to determine if we had missed reads derived from the PDD-bornavirus in our initial screen against all BDV sequences. Not surprisingly, this retrospective BLAST analysis revealed an additional 2600 reads from across the recovered bornavirus genome that were missed in the initial BLAST analyses due to the lack of sequence conservation between the PDD-bornavirus and the available BDV sequences (Figure 2A, Solexa reads track). In total, approximately 1% of all the high throughput shotgun reads could be mapped to the recovered bornavirus genome.

[0225] Despite this sequence divergence, this avian bornavirus genomic sequence possesses all of the hallmarks of a Bornaviridae family member (Figure 2A): six distinct ORFs encoding homologs of the N, X, P, M, G, and L genes are detectable. Likewise, non-coding regulatory sequence elements (the inverted terminal repeat sequences ((Schneider U et al., Proc Natl Acad Sci USA, 102(9):3441-3446 (2005)) Figure 4), the transcription initiation and termination sites ((Schneemann A et al., J Virol, 68(10):6514- 6522 (1994)), Figure 5), and each of the signals for pre-mRNA splicing ((Schneider PA et al., J Virol, 68(8):5007-5012 (1994)), Figure 6) are all conserved in sequence and location, with the exception of the splice acceptor site 3 at position 4560 that has been previously found in a subset, but not all BDV genomes (Tomonaga K et al., Proc Natl Acad Sci USA, 97(23):12788-12793 (2000); Cubitt B et al., J Gen Virol, 82(Pt 3):641- 646 (2001)). Taken together, these data provide evidence that our analysis has uncovered a novel divergent avian bornavirus (ABV) present in cases of PDD. [0226] Phylogenetic and pairwise sequence analyses support this conclusion. Genomic and sub-genomic phylogenetic analyses of nucleotide sequences place the recovered ABV sequence on a branch distant from representative members of the 4 distinct genetic isolates of BDV for which complete genome sequences are available (Figure 2B, Figure 7). Strikingly, the ABV genome sequence segregates to a position virtually equidistant from both the set of 3 closely related BDV isolates (V/Ref, Hl 766, and He/80) and the divergent No/98 BDV isolate (Figure 2B). Broader phylogenetic analyses comparing sequence derived from the recovered ABV sequence to all publicly available BDV sequences confirms that the genetic divergence shown here extends to all known isolates of BDV. Moreover, in contrast to the previously identified divergent No/98 isolate, which retains a high level of conservation with other BDV isolates at the amino acid level, the ABV isolate also shows significant sequence divergence in the predicted amino acid sequence of every ORF in the genome (Table 2).

Table 2 - Predicted amino acid sequence similarity between ABV, the divergent BDV-No/98 and other BDV genomes

*V alues without parentheses have no deviation in % pairwise amino acid identity among compared isolates.

PCR screening of additional PDD cases and controls suggests an association between the presence of AB V and PDD [0227] Recovery of complete ABV genome sequence confirmed that the microarray hybridization signature we detected accurately reflected the presence of bornaviruses in our PDD specimens. With these results in hand, we next performed virus-specific PCR screening of an additional set of PDD case and control specimens with our ABV consensus primers to further examine whether the presence of ABV is associated with clinical signs and symptoms of PDD. An additional set of 21 samples derived from upper GI tract specimens (crop, proventriculus or ventriculus) from PDD cases and controls were screened for ABV sequences in a blinded fashion (Materials and Methods). For this analysis, we targeted the same region of the genome as was used for PCR confirmation of the initial microarray results, but here utilized a set of ABV consensus sequence primers (Materials and Methods) derived from more conserved ABV sequences we found upon sequence analysis of the original microarray-based primers (Figure 2, PCR probes track) used in our initial PCR follow-up work. PCR for glyceraldehydes 3 phosphatase (GAPDH) mRNA was performed in parallel with the ABV PCR on all specimens to control for integrity of RNA provided from each specimen. Of the 21 specimens analyzed, 4 were positive for ABV by PCR and confirmed by sequence recovery. Unmasking the clinical status of these samples revealed that 7 of the samples were derived from confirmed PDD cases and 14 samples were derived from PDD controls. Among the PDD cases, we found 57% (4/7) to be positive by ABV PCR (Table 3). All other specimens were negative by ABV PCR, and positive only for GAPDH mRNA. This PCR analysis provides an independent test of the statistical significance of the association between the presence of ABV and histologically confirmed PDD (P=0.006, Fisher's Exact Test; O.R.=17.3, 95% CL=I.4-216.6). Although we do not observe ABV in 100% of PDD cases in this series (see Discussion), our results nonetheless indicate a significant association of ABV with PDD.

Table 3 - Analysis of significance of ABV detection rate in PDD

P- 0.006, Fisher's Exact Test, O.R. = 17.3 (CI. = 1.4-216.6) Sequence analysis of additional ABV isolates indicates at least 4 divergent isolates in this branch of the Bornaviridae family

[0228] Recovery of partial sequence from additional isolates of ABV (from the above PDD case/control specimens as well as an additional samples derived from known or suspected PDD cases (Materials and Methods)) provided the opportunity to further investigate the genetic diversity within this new branch of the Bornaviridae. Here, our description of results is restricted to comparison with representative members of the 4 major isolates of BDV, but virtually identical results were obtained when all available BDV sequences from this region of the genome were compared (n=14).

[0229] As we observed for the complete ABV genome sequence, phylogenetic analysis of partial sequences recovered from the coding region of the L gene revealed that each of the 14 ABV isolates we have recovered resides on a branch distant from the BDV isolates. This analysis also indicated that there are 4 genetically distinct ABV branches among these isolates. Pairwise sequence analysis of the nucleotide and predicted amino acid sequence from this region of the genome provides additional evidence for surprising genetic diversity among the ABV branches compared to that seen among the BDV branches (Table 4). Although derived from coding sequences of one of the more divergent genes of the bornavirus genome (Table 2, L gene), the region we have used for PCR screening is relatively conserved among the BDV isolates, ranging from 81-98% at the nucleotide level, and 96-99% at the amino acid level (Table 4). In contrast, the sequence identity shared across this region of the genome among the ABV branches of the tree ranges from 77-83% at the nucleotide level and 86-94% at the amino acid level. Taken together with the phylogenetic analysis described above, these data provide evidence that these ABV isolates form a new, genetically diverse branch of the Bornaviridae phylogeny that is significantly diverged from the founder BDV isolates.

Table 4 - Average pairwise sequence identity shared between ABV and BDV isolates*

*PCR fragment examined corresponds to bp 3735-4263 of antigenomic strand of BDV V/Ref genome isolate [GenBank: NC_001607]. Bold text, average % nucleotide identity; plain text, average % predicted amino acid identity. ABVl isolate [GenBank:EU781953], AB V2 isolates [GenBank: EU781954 and GenBank:EU781962-66], ABV3 isolate [GenBank:EU781955], ABV4 isolates [GenBank:EU781956-61], Ref/V isolates [GeneBank:NC_001607, GenBank:AJ311521, GenBank:U04608], H1766 isolates GenBank:AJ311523, GenBank:AB258389, GenBank: AB246670], He/80 isolates [GenBank:L27077, GenBan:AJ311522, GenBank:AY05791, GenBank: AYl 14163, GenBank:AYl 14162, GenBank:AYl 14161], No/98 isolate [GenBank:AJ311524].

Example 2: Experimental induction of pro ventricular dilatation disease in cockatiels (Nvmphicus hollandicus) inoculated with brain homogenates containing avian bornavirus 4

[0230] In this study, the clinical and pathological manifestations of PDD were induced by inoculation of cockatiels with brain homogenates containing avian bornavirus 4. By using high throughput pyrosequencing an in-depth view of the viral content of the inoculum was achieved, revealing that of 3 candidate virus families detected, only the presence of ABV RNA correlated with the development of PDD. This study provides evidence of a causal association between AB V4 infection and PDD in cockatiels.

[0231] Five cockatiels were inoculated via multiple routes (intramuscular, intraocular, intranasal, and oral) with a brain homogenate derived from either a PDD(+) avian bornavirus-4 (AB V4) (+) case (n=3 inoculees) or from a PDD(-) ABV(-) control (n=2 inoculees). The control birds remained free of clinical or pathological signs of PDD, and tested ABV(-) by RT-PCR and immunohistochemistry (IHC). In contrast, all three cockatiels inoculated with ABV4(+) brain homogenate developed gross and microscopic PDD lesions, and two exhibited overt clinical signs. In numerous tissues, ABV RT-PCR and sequence analysis demonstrated the presence of ABV4 RNA nearly identical to that in the inoculum. ABV was detected in the central nervous system of the three ABV- inoculees by IHC. Pyrosequencing to investigate the viral flora in the ABV4(+) inoculum uncovered 7 unique reads sharing 73-100% nucleotide sequence identity with previously identified ABV sequences and 24 reads sharing 40-89% amino acid sequence identity with viruses in the Retro viridae and Astro viridae families. Of these candidate viral species, only ABV RNA was recovered from tissues of the inoculated birds.

METHODS Inoculation experiment

[0232] The inoculation experiment was approved by the animal care committee at Kimron Veterinary Institute (KVI), Bet Dagan, Israel. For the experiment, six male wild- type cockatiel parrots were purchased from a local breeder. The birds were determined to be in good health based on physical examination, complete blood-cell count and fecal cytology. To be included in this study, the cockatiels had to be ABV(-) by RT-PCR, and show no histological lesions suggestive of PDD in multiple crop biopsy sections. To test for pre-existing ABV-infection, whole blood as well as cloacal and choanal swabs were collected from each bird, submersed in an RNA preservative (RNAlater; Qiagen, Valencia, CA), and kept frozen at -8O⁰C. In addition, full thickness crop biopsies of about 10mm in diameter were surgically collected from all birds as previously described [Kistler AL et al., Virol. J, 5:88 (2008)]. Approximately one fourth of each biopsy was submersed in RNAlater and frozen at -8O⁰C until RT-PCR testing, while the rest was placed in 10% neutral buffered formalin, sectioned into 4-6 slices, and prepared for histopathological examination. The crop biopsy and several of choanal and cloacal swabs of the sixth bird tested positive for AB V2 by RT-PCR. This bird was therefore removed from the inoculation study; however, we continued to monitor it for ABV-shedding for the duration of the study period.

[0233] The cockatiels were housed in individual cages and placed in animal isolation units, where they were allowed to recover from surgery and acclimatize for 8 days prior to inoculation. Drinking water and a commercial seed-based diet were provided on an ad lib basis, and ambient temperature was kept at 28⁰C.

[0234] The inoculum was prepared from brain tissue of an African grey parrot (Psittacus erithacus) that had shown prior to death classical gastrointestinal signs of PDD (KVI# F45b). The bird was confirmed to be PDD(+) by histology and ABV4(+) by RT- PCR and subsequent sequencing. Approximately Ig tissue was macerated by two passages through a 2.5ml syringe and was then diluted 1 :4 in sterile saline. The preparation underwent two 24h freeze-thaw cycles at -8O⁰C, before centrifugation at 4⁰C at 4000xg for lOmin. The supernatant was collected and kept on ice until use (within 90min). This same methodology was used to prepare a sham inoculum from brain tissue of an African grey parrot that had died from causes other than PDD, and that was ABV(-) by RT-PCR (KVI# F27b).

[0235] To test for the presence of bacteria in the inoculum, routine microbial culture was attempted on blood-agar and McConkey's agar media, while high throughput pyrosequencing was employed to test the inoculum for the presence of viral RNA (see below). In addition, TEM was used to screen the inoculum for the presence of viral particles.

[0236] Birds included in the study were randomly assigned to one of two treatment groups. Three male cockatiels were inoculated with the ABV-containing homogenate, while the other two males received the sham inoculum. For inoculation/sham inoculation, a combination of the intramuscular (0.2ml injected by 28G needle into the left pectoral muscle), oral (0.2ml), intranasal (1 drop in each nostril), and conjunctival (one drop on each eye) routes was used. After inoculation, the cockatiels were monitored for 95 days by an observer, who was blinded to their treatment status. This included daily observation of the birds' general attitude, behavior, gait, feeding activity and uro-fecal output. BW was recorded weekly using an electronic scale. Whole blood, choanal and cloacal swabs were collected on days 1, 2, 4, 8, 11, 13, 21, 26, 35, 40, 57, 63, 70, 77, 85, 93 PL All samples were immersed in RNAlater and frozen at -8O⁰C. Birds that lost >25% BW and/or showed signs of advanced disease (e.g. marked lethargy, weakness, neurological signs) during the study were humanely euthanized by CO₂ inhalation All other birds were humanely euthanized at the end of the study. Diagnostic necropsies were performed for all birds under a biohazard hood, using aseptic technique. For each bird, a complete set of tissue samples was collected in RNAlater and frozen at -8O⁰C for RT-PCR testing. A second set of tissue samples was placed in 10% neutral buffered formalin for histopathology. In addition, tissue samples of brain and proventriculus were collected for TEM, and stored frozen at -8O⁰C with no additive.

ABV nucleocapsid gene cloning, expression and polyclonal antibody generation

[0237] The open reading frame (ORF) encoding the ABV nucleocapsid (ABVN) gene flanked with BamHI and Notl restriction sites was amplified from AB V2 total RNA [Kistler AL et al, Virol. J, 5:88 (2008)] by RT-PCR with the following primers: ABVN- BamHI, 5'GCGCGCCCCCGGATCCATGCCACCCAAAAGGCAAAG-S' and ABVN- Notl, 5'-GCGTGCTACGCCATGCGGCCGCCGTTTGCAAATCCAGTTACGCC-S' (restriction sites bolded, ABVN ORF overlap italicized). The resulting product was sequence-confirmed, digested with BamHI and Notl and subcloned into a BamHI/Notl- digested modified pMAL vector (gift from Matthew C. Good, UCSF), which contains a 6xHis tag on the C-terminus (His₆), and a maltose-binding protein (MBP) tag on the N- terminus. Ligation into this vector generated a TEV protease cleavage site (tev) between the N-terminal MBP tag and ABVN ORF. The sequence-confirmed, modified pMAL vector containing the ABVN ORF was transformed into pRIL÷ BL21(DE3) E. coli and recombinant MBP-tev- AB VN-HiS₆ protein expression was induced with 25OuM IPTG at 37°C for 4 hours. Cells were lysed in 5OmM Tris pH 8.0, 10OmM NaCl, and IX Roche Complete Protease Inhibitors (Roche Applied Science; Indianapolis, IN) using 3 cycles through a microfluidizer. MBP-tev-AB VN-HiS₆ protein was purified from cell lysates via Ni-NTA column chromatography followed by amylose column binding and elution with maltose. The resulting eluate was concentrated with a 5OkDa Amicon Ultra (Millipore; Billerica, MA) and incubated with 10 units of TEV protease for 1 day at 4°C. The cleavage reaction mixture was then diluted into 25mM Tris, pH 7.0, 10OmM NaCl buffer and loaded on a ImL RESOURCE S column (GE LifeSciences, Piscataway NJ) to separate the cleaved MBP tag and TEV protease from the ABVN- His₆ protein by ion exchange chromatography. Resulting fractions containing ABVN-HiS₆ protein were combined, concentrated using a 15kDa Amicon Ultra, and further purified based on size using a 24mL Superdex200 column (GE LifeSciences). Fractions containing purified ABVN-HiS₆ were combined and concentrated using a 15kDa Amicon Ultra. 2.5 mg of this purified ABVN-HiS₆ was used for polyclonal antibody generation in rabbits (Pacific Immunology; Ramona, CA).

Histopathology and immunohistochemical staining

[0238] Tissue specimens were processed for routine histopathology, sectioned at 6 μm, and stained with hematoxylin and eosin. To increase the sensitivity of PDD-specific lesion detection, multiple sections (3-5) were prepared for the crop, ventriculus and proventriculus of each bird. Crop biopsies were sectioned 5-6 times. For each bird, a second set of slides was prepared for immunohistochemical staining. Briefly, tissue sections underwent deparafinization and rehydration, followed by treatment with 3% H₂O₂ for lOmin. The sections were then washed twice with PBS, and incubated for 60min at room temperature with a rabbit anti-ABVN polyclonal antibody (see above) at 1 :500 or 1:1000 dilution. After rinsing in PBS for 5min, horse-radish peroxidase polymer- conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was added for 30min. The sections were again rinsed with PBS, and the substrate- chromogen solution (Zymed AEC; San Francisco, CA) was added for 3min at room temperature. Specimens were then rinsed, counterstained with hematoxylin, and allowed to dry at 60⁰C for 2h.

Transmission electron microscopy

[0239] Frozen tissue specimens (-8O⁰C; no additive) were allowed to thaw at room temperature and were then minced by a scalpel blade or macerated by 2 passages through a sterile 2.5ml syringe. Approximately Ig of each specimen was placed in a test tube containing 4ml PBS followed by vortexing. The preparations were centrifuged at 400Og at 4⁰C for lOmin, and the clarified supernatant was collected. Virus concentration was then attempted by ultra-centrifugation (Kubota 7800; Kubota, Japan) at 40,00Og for 5h, followed by discarding approximately 95% of the supernatant and re-suspending the sediment in the remaining fluid. End products were then stored at -8O⁰C until use.

[0240] For negative staining examination, carbon-stabilized and Formvar-coated 300- mesh copper grids were used. The grids were floated on a drop of suspect sample and allowed to adhere to the drop for 2min at room temperature. The grid was then removed and excess liquid was drained by blotting the edge of the grid with filter paper. Next, the grid was floated on a drop of 2% aqueous uranyl acetate solution for 30sec. The excess stain was removed as before and the specimens were examined by TEM, using a Tecnai G2 Spirit electron microscope (FEI company, Hillsboro, OR).

RNA extraction

[0241] For RNA extractions from tissue and whole blood, specimens underwent two 24h freeze-thaw cycles at -8O⁰C followed by scalpel mincing (tissues only). Total RNA was then extracted using the TRI Reagent® kit (Molecular Research Center, Cincinnati, OH), following the manufacturer's instructions. RNA extractions from choanal and cloacal swabs were performed by the QIAamp viral RNA kit (Qiagen, Valencia, CA). The end product was lyophilized and stored at -8O⁰C until testing.

Pyrosequencing and analysis of RNA extracted from the challenge inoculum

[0242] Five independent random amplification reactions generated from approximately 50ng of total RNA derived from the ABV4-positive brain homogenate were pooled together for library generation for pyrosequencing using standard 454/Roche GS-FLX protocols [Margulies M et al, Nature, 437:376-380 (2005)]. After filtering primer sequences, exact duplicates, low complexity sequences and reads < 36bp long, a working set of 239,556 reads remained for analysis. To filter reads potentially derived from psittacine host tissue RNAs, the working set of reads was aligned to a database of all Aves sequences extracted from NCBI (n=918,511) using megablast (e = 10^"10; word size = 12), followed by progressively lower stringencies (down to e = 10^"4; word size = 12). The remaining 16,475 Aves-filtered reads were next aligned to all sequences in NCBI to identify viral and non- viral sequences via a high stringency megablast (e = 10^"10; word size = 12) followed by a lower stringency blastn (e = 10^"6; word size = 8), and blastx (e = 10^" ; word size = 4) were performed. A final low stringency tblastx alignment (e = 10^" ; word size = 3) to a database containing all viral sequences present in NCBI was performed to screen for potential divergent viral species missed in the prior screens. Candidate viral reads identified from this final screen were verified by re-blasting against all NCBI sequences. Reads that failed to yield viral sequence matches in this final re- blast were considered false positives and discarded as potential viral sequence. Reads that did yield viral sequences in the re-blast against NCBI were considered candidate viral sequences and were grouped according to viral species, aligned to identify regions of overlap useful for RT-PCR primer design.

RT-PCR for ABVRNA detection

[0243] Initial RT-PCR for ABV was performed in a blinded fashion on all RNA samples extracted for the challenge study. Each sample was used as input template for 1- step RT-PCR assay (Qiagen, USA, Valencia CA) using previously described primers [Kistler AL et al., Virol. J, 5:88 (2008)] for amplification of ABVN, ABVM and ABVL RNAs. Resulting RT-PCR products were gel purified, incubated with 0.25mM dATP and recombinant Taq polymerase (Invitrogen, Inc., Carlsbad CA, USA) at 72°C for 15 minutes, then subcloned into the pCR2.1 TOPO T/A cloning vector (Invitrogen, Inc., Carlsbad CA, USA). For each subcloned RT-PCR product, 3 independent transformants were amplified and sequenced using Ml 3 forward and Ml 3 reverse primers. Upon sequence confirmation of the identity of RT-PCR products, sample identities were unmasked and a follow-up control 1-step RT-PCR assay using primers directed against a highly conserved region of 18s rRNA sequences detected from the initial brain inoculum in 124 overlapping reads (rRNAF: 5'- CGGCGTCCAAC- TTCTTAGAG-3', rRNAR: 5'- AATGGGGTTCAACGGGTTAC-3') was performed on tissue-matched case and control RNAs. For all 1-step RT-PCR assays described above, 5uL of template RNA was used in a final reaction volume of 25uL, incubated at 50°C for 30 minutes, followed by a 95°C incubation for 15 minutes, and 35 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and elongation at 72°C for 30 seconds.

RT-PCR screening for non-ABV viral species detected in the inoculum

[0244] Primers for RT-PCR recovery of viruses other than ABV were designed based on candidate viral sequences recovered in the pyrosequencing analysis of the brain inoculum (Additional file 3). RT-PCR for each of these viral species was performed on RNA derived from matched tissues specimens from each bird to screen for the presence of these viruses in both inoculated and control birds.

List of abbreviations

[0245] ABV, avian bornavirus; ABV2, avian bornavirus 2; AB V4, avian bornavirus 4; PDD, proventricular dilation disease; GI, gastrointestinal; BW, body-weight; PI, post- inoculation; TEM, transmission electron microscopy; IHC, immunohistochemistry; ORF, open reading frame; ABVN, ABV nucleocapsid; His₆, 6xHis tag; MBP, maltose-binding protein.

RESULTS

Clinical observations and post-mortem macroscopic findings

[0246] Five cockatiels were each inoculated via multiple routes (intramuscular, intraocular, intranasal, and oral) with a brain homogenate derived from either a PDD(+) ABV(+) bird (n=3) or from a PDD(-) ABV(-) bird (n=2). The birds were then followed for 95 days for behavioural and clinical manifestations. Two out of three cockatiels in the ABV-inoculated group developed overt clinical manifestations typical of PDD during the study period. In the first bird to show clinical signs (cockatiel 1), a sharp decrease in body- weight (BW) and body condition was observed starting on day 21 post-inoculation (PI), and undigested seeds were present in the feces from day 50 PI. In the second bird (cockatiel 3) these same clinical signs were observed starting on days 31 and 85 PI, respectively (Figure 8). Interestingly, from day 9 PI onwards, cockatiel 1 had started plucking feathers over its entire trunk. The bird was also reluctant to move around in its cage, as evident by the accumulation of faeces on a single spot underneath its perch. These signs were not observed in any of the other study birds. After losing nearly 30% of its initial BW, and exhibiting signs of severe weakness, cockatiel 1 was humanely euthanized on day 64 PI. The other birds in the study were euthanized at the end of the study period (95 days PI). At this time, cockatiel 3 had lost >25% of its initial BW. The third ABV-inoculated bird (cockatiel 2) and the two control birds (cockatiels 4 and 5) did not show overt clinical signs of PDD during the study period. However, the BW of cockatiel 2 did appear to fluctuate while those of the control birds remained fairly stable (Figure 8).

[0247] In accordance with the clinical signs, on necropsy cockatiels 1 and 3 showed severe pectoral muscle atrophy and complete absence of peritoneal fat stores (Figure 9a). In contrast, cockatiels 2, 4, and 5 had normal pectoral muscle mass and extensive fat stores (Figure 9b). All three of the ABV-inoculated birds (cockatiels 1-3) showed dilatation of the proventriculus and to a lesser extent also of the ventriculus (compare Figure 9c and 9d). These findings were dramatic in cockatiels 1 and 3, where the giant thin- walled proventriculus was impacted with undigested seeds (Figure 9e). Whole seeds were also present throughout the intestine of these birds, and their livers appeared small and pale, consistent with chronic nutrient malabsorption (Figure 2c). Moderate but clear distension of the proventriculus and also the proventriculus/ventriculs transitional area (isthmus) was observed in cockatiel 2; however, no pro ventricular impaction was present, the liver appeared normal, and undigested seeds were not present along the bird's intestine.

Histopathology

[0248] Histopathologic lesions consistent with PDD were undetectable in the control inoculees. In contrast, all three of the ABV -inoculated cockatiels exhibited the PDD hallmarks of lymphoplasmacytic infiltrates within myenteric ganglia of the upper and middle GI tract (Figure 10). Interestingly, the most severely affected organ was the ventriculus, and in cockatiels 1 and 2 lymphoplasmacytic infiltrates were not limited only to ventricular nerves, but were also scattered throughout the thick tunica muscularis. This latter finding is notseen in all PDD cases, but was present in the bird from which the inoculum was prepared.

[0249] Beyond these general observations, a number of additional lesions were detectable in the ABV -treated birds. In cockatiel 1, lymphoplasmacytic infiltrates were present in the epicardium, epicardial ganglia, and one peri-adrenal ganglion, while in the cerebral grey matter of this bird multiple foci of gliosis, encircling small particles of amorphous eosinophilic material, were present along with mild lymphoplasmacytic perivascular cuffing. Similar histological findings were present in cockatiels 2 and 3, but with some differences. For example, in cockatiel-2 brain lesions were undetectable but marked lympho-plasmacytic perivascular cuffing was present in a section of the lumbosacral spinal cord. In cockatiel-3 mild lymphoplasmacytic perivascular cuffing was present in the cerebrum, but with no gliosis

Transmission electron microscopy screening of the inoculum and brain tissue of the study birds

[0250] The original homogenate as well as brain homogenates from the five study cockatiels were prepared for transmission electron microscopy (TEM) screening. Variable numbers of spherical virus-like particles 50-13 Onm in diameter were present in brain homogenates of all ABV-inoculated birds and in the original inoculum. Similar particles were not detected in the controls. Consistent with previously reported morphology of bornaviruses [Kohno T et al., J Virol, 73:760-766 (1999)], these particles appeared to be surrounded by a membrane, and in some cases filamentous structures akin to glycoproteins, appeared to protrude from the membrane (Figure 11).

Pyrosequencing of inoculum and analysis of ABV transmission

[0251] High throughput pyrosequencing was applied to the ABV4-positive brain homogenate to survey the diversity of viral species that might be present in the inoculum (Materials and Methods). From a total of 239,556 reads, we identified a set of 7 unique reads sharing 73-100% nucleotide sequence identity with existing ABV sequences (Figure 12, Table 5).

Table 5. Viral RNA sequences recovered from the inoculum by high throughput pyrosequencing

*amino acid identity of highest scoring tblastx match

_fraction of read covered by alignment to higiest scoring tblastx match

[0252] Twenty-four reads that shared 40-89% sequence identity at only the amino acid level with viruses in the Retroviridae and Astroviridae families were also detected (data not shown). An additional set of reads matched sequences derived from host (n=223,081) and non- viral sequences in NCBI (n=480). The remaining reads yielded no match to sequences present in NCBI (n=l 5,803).

[0253] To determine if the AB V4 present in the inoculum was transmitted and correlated with PDD signs and symptoms, we performed blinded RT-PCR of the ABV N gene (ABVN) on all RNA samples taken over the course of the study. After unmasking the identity of the samples, we found that we detected ABVN RNA only in the ABV- inoculated birds(data not shown). Independent RT-PCR for host RNAs on a set of matched tissue samples from ABV -inoculated and control birds confirmed the presence of intact RNA in the samples under study. Recovery of additional ABV RT-PCR products from the M and L gene provided further supporting evidence for ABV virus transmission only in the experimental birds (data not shown). Sequence analysis of these RT-PCR products revealed that the ABV recovered from the inoculees shared 99-100% sequence identity with the AB V4 sequences present in the original inoculum.

[0254] To assess the specificity of the correlation between the PDD symptoms and ABV transmission, we also performed independent RT-PCRs for the highly represented sequences for which we found detectable sequence similarity to Retroviridae and Astroviridae sequences (data not shown). Although RT-PCR products corresponding to these species were detectable in the original inoculum, none were recovered from the matched tissue specimens tested in the experimental or control inoculees.

Tissue distribution and localization of ABV detected in inoculees

[0255] Upon detection of transmission of ABV infection and PDD signs and symptoms in the ABV-inoculees, we next investigated the viremia, viral shedding, tissue distribution and subcellular localization of ABV4 by RT-PCR for ABVN RNA and immuno- histochemistry for ABVN protein. By RT-PCR, none of the blood samples collected during the study tested positive for the presence of ABV RNA. This was also the case for all the cloacal and choanal swab samples for cockatiel 1 (the first bird to develop clinical signs of PDD). In contrast, a single choanal swab collected from cockatiel 3 (the second bird to develop clinical PDD) tested ABV positive on day 85 PI, and both choanal and cloacal swabs collected from cockatiel 2 (ABV-inoculated, but sub-clinical bird) tested ABV-positive on days 85 PI and 91 PI. From the tissues collected at necropsy, the brain, lumbosacral spinal cord, kidney and small intestine of cockatiel 1, the heart, spleen, pancreas, proventriculus, small intestine, and brain of cockatiel 2, and the brain, pancreas, small intestine, and proventriculus of cockatiel 3, tested positive for ABV RNA by RT- PCR.

[0256] By immunohistochemistry (IHC) for the ABVN protein, the brains of all 3 ABV-inoculated cockatiels were positive (Figure 10). In birds 2 and 3, ABVN staining was widespread throuout the cerebrum, localizing to the nuclei of neurons and glial cells. Staining of the cytoplasm was also present, but to a lesser extent. In contrast, in bird 1 there appeared to be N protein staining associated with areas of gliosis; in those foci staining appeared to be extracellular (Figure 10). ABVN-IHC staining was also observed in a single neuron of one affected myenteric ganglion of cockatiel 1, and in the lumbrosacral spinal cord of cockatiel 2, but was not detected in blood cells (including lymphocytes within perivascular cuffs), endothelial cells, connective tissue and other mesenchymal cell types. Epithelial involvement was difficult to determine due to the presence of non-specific ABVN-IHC staining in many of the sections. Non of the tissues from the control birds stained positively for ABVN.

DISCUSSION

[0257] Our recent discovery of ABVs and their significant association with PDD [Kistler AL et al., Virol. J, 5:88 (2008)] as well as the results of two other independent studies [Honkavuori KS et al., Emerg Infect Dis., 14:1883-1886 (2008); Rinder M et al., J Virol. [Epub ahead of print] (2009)] offer for the first time in over 30 years a compelling etiological candidate for this disease. Here, we have successfully reproduced the clinical and pathological changes typical of PDD in cockatiels inoculated with brain homogenates containing AB V4. Our strategy for this study represents a modification of Koch's postulates, where high throughput pyrosequencing was used to obtain in-depth information on potential virus candidates in the inoculum, and this information was followed up by PCR testing of the study birds for all suspected viruses. This strategy may be of use in cases where virus isolation is difficult, or its methods are still under development, as was the case for ABV. Although we found viral sequences showing variable degree of homology to known genomes within the Retroviridae and Astroviridae families were detected in the ABV (+) brain homogenates, none of these sequences were detected in tissues of the inoculated birds, making their role in PDD pathogenesis unlikely.

Experimental ABV inoculation strategy

[0258] The inoculum used for this study was prepared from the brain of a PDD(+) ABV4(+) African gray parrot. We chose to use only brain tissue for an inoculum source, as it is easy to collect aseptically, should be free of the potentially contaminating flora of other body systems (e.g. GI flora), and is known to be a major target site of ABV [Honkavuori KS et al., Emerg Infect Dis., 14:1883-1886 (2008); Rinder M et al., J Virol. [Epub ahead of print] (2009); Kistler AL, unpublished data]. For inoculees, we utilized cockatiels because they were readily available and have been previously used for PDD research [Ritchie BW et al, Proc. Assoc. Avian Vet., pp. 41-45 (2004); Gregory CR et al, Pr oc. Assoc. Avian Veterinarians, pp. 43-52 (1997)]. A combined intramuscular-oral- conjunctival-intranasal inoculation route was employed since the natural infection route of ABV is not yet known and prior work with an uncharacterized mixed tissue homogenate from a PDD(+) was previously used to reproduce PDD [Gregory CR et al., Proc. Assoc. Avian Veterinarians, pp. 43-52 (1997)].

Disease conferred by experimental inoculation with ABV(+) brain homogenates

[0259] All 3 cockatiels in our study that were inoculated with the ABV4(+) brain homogenate developed pathological lesions typical of PDD. Two of the three also showed overt clinical signs of PDD. Based on their weight loss patterns (Figure 8), the symptom-free or incubation period was 21 days in one bird and 31 days in the other, although for one cockatiel abnormal behavior (reduced ambulation, feather picking) was seen as early as day 9 PI. These birds reached the advanced stages of PDD at 64 and 95 days PI, respectively, showing a relatively slow progression of the disease. The third bird was symptom-free on day 95 PI despite suffering from moderate distension of the proventriculus. It is likely that this bird would have also eventually developed clinical PDD, but may have taken longer to do so. This spectrum of clinical findings is consistent with what is seen in naturally infected psittacine birds [Gregory C et al., J Assoc Avian Vet, 8:69-75 (1994)], and underline the great difficulty of identifying sick birds and preventing introduction of PDD into naive collections.

[0260] The ABV-inoculated cockatiels in this study developed mainly lesions of the GI tract, with the most severe lesions being in the ventriculus. Brain lesions were mild or completely absent, and the clinical signs were those of the GI form of PDD. These findings are very similar to those seen in the original bird from which the inoculum was prepared, and may therefore reflect the role of the ABV strain type in determining lesion distribution patterns and the clinical manifestation of PDD. Differences in pathology and virulence of different ABV strains have not been studied to date, and warrant further experimental investigation. ABV tropism detected in inoculees

[0261] A variety of tissues of the ABV4-inoculated cockatiels were AB V4 RNA(+) by RT-PCR. Of these, the brain, spinal cord and GI tract, were most commonly represented. IHC staining showed the presence of ABVN in nuclei and to a lesser extent also in the cytoplasm of neurons and glial cells of the brain and lumbosacral spinal cord. In other tissues the IHC results were more difficult to interpret, as epithelial tissues often showed non-specific staining. However, a recent independent study of necropsied cases of PDD demonstrated broad tissue and cell tropism of ABV in PDD cases was demonstrated by RT-PCR and IHC with cross-reacting polyclonal antisera raised against the Borna disease virus P protein [Rinder M et al., J Virol. [Epub ahead of print] (2009)].

Antemortem ABV-RNA detection in inoculees

[0262] In mammals, the initial site of bornavirus infection is thought to be the upper respiratory tract [Morales JA et al., Med Microbiol Immunol, 177:51-68 (1998); Sauder C and Staeheli P, J Virol, 77:12886-12890 (2003)]. Moreover, PDD has been proposed to be transmitted via the oral-fecal route [Gregory C et al., J Assoc Avian Vet, 8:69-75 (1994)]. Thus, we obtained weekly oronasal and cloacal swab specimens from the experimentally inoculated birds to probe for the presence of ABV. In the experimentally inoculated birds, we detected ABV in choanal and cloacal specimens no earlier than day 85 PI, while viremia could not be detected at all during the study period. In contrast, the majority of choanal and cloacal specimens as well as 18% of blood samples collected during the same period from an asymptomatic cockatielnaturally infected with AB V2 (see Methods), tested positive for ABV-RNA. One potential explanation for this finding is that ABV shedding may be limited in symptomatic PDD cases by the host's immune response (seen as lymphoplasmacytic infiltrates). Alternatively, the route of experimental inoculation used and/or other conditions present in this study may have resulted in reduced or variable ABV-RNA shedding compared with the naturally infected bird. Finally, the difference in ABV strain between the inoculees (AB V4) and the naturally infected bird (AB V2) may have resulted in this difference. Antemortem diagnosis of ABV infection and any factors that may affect it are of great clinical and epidemiological importance, and should be at high priority for further investigation.

[0263] The detection of a naturally occurring clinically asymptomatic ABV2-infected cockatiel at the start of this study is of particular interest in light of the results mentioned. To date (9 months after its purchase), this bird remains symptom-free, and althoughit may still develop PDD in the future, it is also possible that this cockatiel is not susceptible to this particular virus strain, or that this ABV strain is of low pathogenicity in general, or in cockatiels. These observations raise the possibility that ABV infections may not always confer clinically overt signs of PDD, and long-term asymptomatic carriers may play a role in the epidemiology of ABV. Further investigation of outcomes associated with both naturally and experimentally ABV-infected psittacines are required to better understand these findings.

Example 3. Real-time evidence on the role that ABV plays in PDD transmission as well as insight into the natural history of ABV infection

[0264] An acute outbreak of PDD in a psittacine nursery provided an opportunity to garner real-time evidence on the role that ABV plays in PDD transmission as well as insight into the natural history of ABV infection. During the outbreak, we detected AB V2 RNA and proteins in numerous tissues from the central nervous system and gastrointestinal tract of a symptomatic chick exposed to a histologically confirmed case of PDD. Follow-up RT-PCR screening of 46 birds housed with the PDD case identified 12 additional ABV(+) birds. Sequence analysis of the recovered ABV RT-PCR products indicated that all 12 birds were infected with the same AB V2 isolate found in the symptomatic chick. Additional chicks boarded at the nursery during the outbreak also contracted signs of PDD. RT-PCR testing of 2 available chicks confirmed AB V2 infection, and a similar tissue distribution at necropsy. Within 4 weeks of exposure, 5/8 contacts of the exposed chicks were ABV2(+) by cloacal swab RT-PCR testing and follow-up sequence analysis.

Initial outbreak ofproventricular dilatation disease

[0265] A hobbyist breeder with a mixed species psittacine handfeeding nursery began seeing crop stasis and feed refusal in a 5 -week old umbrella cockatoo (Cacatua alba) chick. The breeder started the chick on fluconazole and saw no improvement after 3 days of treatment. At that time the bird was cultured and E. coli enteritis was diagnosed. After 10 days of treatment with fluconazole, subcutaneous fluids, metaclopramide, and lactulose and an antibiotic chosen based on sensitivity testing (enrofloxacin), the bird died. The breeder did not seek a necropsy. The same day this chick died 1 sibling and 2 unrelated brooder mates of this chick began regurgitating and shaking their heads. These chicks' crops were emptying normally. The breeder had again started them on fluconazole. The sibling to the dead chick regurgitated during a feeding, aspirated, and died suddenly. Again, no necropsy was sought. The two surviving umbrella chicks were examined after 4 to 5 days of treatment with fluconazole, showing no improvement. Very large numbers of Candida-like yeast were found on fecal and crop smears. E. coli was isolated from a cloacal swab. The breeder was advised that these infections were likely secondary infections and an underlying primary infectious disease remained undiagnosed. The chicks were started on itraconazole solution and amikacin injections (based on sensitivity testing). The chicks slowly began recovering.

[0266] Eight days later, a scarlet macaw (Ara macaό) chick refused to be fed. As the breeder tried to feed it, it aspirated and died. Necropsy showed gross acute aspiration as the cause of death, moderate degree of breast muscle atrophy, evidence of a possible preexisting aspiration pneumonia (small areas of the lung with dark congestion and edema), and an atrophic gizzard. The breeder declined histopathology. At the same time this macaw was seen 2 jenday conures (Aratinga jandaya) presented with regurgitation and head shaking. The birds appeared depressed and moderately underweight. High numbers of Candida-like yeast were found on crop and fecal smears and Klebsiella pneumonia enteritis was diagnosed from cloacal swab. Based on sensitivity testing, the birds were started on itraconazole and amikacin.

[0267] Five days after the scarlet macaw and jendays were examined, 2 scarlet macaw chicks died. The breeder submitted these birds for necropsy and histopathology testing. Histopathology confirmed a diagnosis of proventricular dilatation disease (PDD) based on characteristic lymphoplasmacytic ganglioneuritis lesions in crop, proventriculus, ventriculus, and heart. Non-suppurative perivascular cuffing, glial cell activation, demyelination, and Purkinje cell necrosis that were found in the brain and spinal cord. By the time the diagnosis was made, the 2 jenday conures (Aratinga jenday a) had died and several more birds had begun showing clinical signs of regurgitation, feed refusal, and crop stasis. The breeder had begun treating any sick bird with itraconazole and amikacin as soon as it started showing clinical signs.

[0268] An additional scarlet macaw chick that was not recovering with itraconazole and amikacin and began showing CNS signs was euthanized. Gross necropsy showed an extremely enlarged, thin-walled proventriculus and atrophic gizzard. Two more birds, a Moluccan cockatoo (Cacatua moluccensis) and a jenday conure, did not recover and began showing CNS signs. They were euthanized and the Moluccan cockatoo was necropsied for further study. This bird also showed a greatly enlarged, thin walled proventriculus and atrophic gizzard. No other birds at this premise died in the outbreak. As of late October, 2008 they had apparently recovered and were lost to follow-up.

Expansion of the PDD outbreak

[0269] Part of this outbreak involved a second breeder's nursery. A scarlet macaw and 2 umbrella cockatoo chicks were boarded at the first breeder's premise when they were 3 weeks old and still required hand feeding. This occurred the week of July 10-17, 2008, just before the first breeder's index case of PDD died. Two weeks after returning from the first breeder, the scarlet macaw began showing crop stasis and lethargy. The second breeder thought the chick had aspirated and began treating them with Nystatin and enrofloxacin. The bird began to improve, then worsened and died. Three days after the macaw died, the 2 umbrella cockatoo chicks began showing the same clinical signs. She treated the umbrellas for 10 days with Nystatin and enrofloxacin and they seemed to recover. Due to the exposure of the birds to the PDD outbreak in the first breeder's flock, the second breeder was warned to isolate these chicks and any other chicks that were in her nursery while these were being handfed. The breeder elected euthanasia and although both birds had seemed to recover clinically by the time they were euthanized, on necropsy they both showed an extremely enlarged, thin walled proventriculus, an atrophic gizzard, and a dilated duodenum.

Index case trace back

[0270] Upon diagnosis of PDD, we began to explore how the disease entered the nursery. The parents of the first umbrella chick to become ill and die had never had chicks before but all the other chicks in the nursery came from parents who had been producing chicks for years and had had no new exposure to other birds. The umbrella chicks had been artificially incubated and hatched. Although egg transmission has been suggested to occur with PDD, this seemed like an unlikely possibility.

[0271] Further discussions revealed the breeder had brought an African grey (Psittacus erithacus) hen into the house periodically for topical treatment of severe dermatitis sometime during the 3 -week period before the first chick became ill. This bird was treated on the same kitchen counter where chicks were routinely hand-fed. Routine cleaning and disinfection of the counter after treating the African grey was not performed after handling the African grey, or generally before handling the chicks.

[0272] The breeder had obtained the African grey hen in 1992. She had always been thin but in late 2005 and early 2006, the breeder noticed she looked thinner and was producing fewer chicks. The bird was moved to a cage on her own in an outbuilding and remained apparently normal but thin. In June of 2008, the breeder noticed undigested seeds in her feces. Complete blood count (CBC), blood chemistry panel, cultures, and gram stains, and direct smears uncovered only a high number of Candida-like yeast in the gastrointestinal tract. The bird was started on fluconazole and the droppings returned to normal. However, 10 days after fluconazole was initiated the bird began to self-mutilate her feet. The bird was started on amoxicillin/clavulanic acid and gabapentin, and meloxicam. Wounds were topically treated with 1% silver sulfadiazine cream. Sometime in late June 2008, the breeder began routinely bringing the hen indoors to apply the sufadiazine cream to treat her wounds.

[0273] The African grey hen died on July 15, 2008. Necropsy findings included an atrophic gizzard in addition to extensive and severe dermatitis. Histopathology confirmed characteristic lesions of PDD in the crop, proventriculus, ventriculus, upper small intestine, and brain. A Candida-like yeast was found throughout the skin lesions. The first chick began showing clinical signs compatible with PDD on July 16, 2008. A summary of the outbreak chronology is presented in Table 6.

Table 6. PDD Outbreak Chronology

Molecular testing for avian bornavirus in necropsied cases of PDD [0274] This outbreak of several confirmed cases of PDD provided us with the opportunity to obtain real-time information related to the association previously reported between avian bornavirus (ABV) infection and clinical signs and symptoms of PDD [Daoust PY et al., J Wildl Dis, 27:513-517 (1991); Doneley RJ et al, Aust Vet J, 85:119- 123 (2007); Gregory C et al., J Assoc Avian Vet, 8:69-75 (1994)]. We first sought to determine if ABV was present in the earliest available confirmed cases of PDD. Tissue specimens were obtained at necropsy for the histologically confirmed case of PDD in one of the first breeder's scarlet macaw chicks. RT-PCR analysis of the RNA derived from a full panel of tissues obtained from this case demonstrated the presence of ABV2 RNA in a wide variety of tissues (Figure 13 A, top panel). Similar to recently reported results from a distinct set of PDD cases in Germany [Gregory C et al., J Assoc Avian Vet, 8:69-75 (1994)], we detected AB V2 RNA across a wide variety of tissues sampled at necropsy, with the exception of liver tissue and blood cell pellet, which exhibited only weakly positive or negative AB V2 RNA signal, respectively. Parallel RT-PCR for GAPDH mRNA served as a control for input RNA quality (Figure 13 A, bottom panel) for both these and the entire set of specimens analyzed. Similar profiles of ABV2 RNA and protein were observed in a later necropsy tissue panel obtained from the second breeder's umbrella cockatoo chicks that were exposed to the PDD outbreak in first breeder's home (Table 7, birds 2 and 3).

All samples were assayed using 1 step RT-PCR for ABVN fragment as previously described [Doneley RJ et al., Aust Vet J, 85:119-123 (2007)]. Results shown are qualitative values based on RT-PCR product signal intensity on agarose gel (+++, strong positive; ++, clear positive; +, faint positive; -, negative).

Vt. = not tested; ^cn.d. = not determined, RT-PCR for both control (GAPDH mRNA) and ABVN RNA were negative. dAll 3 Necropsied chicks were exposed to histologically confirmed case of PDD while being hand fed at Breeder 1 's home: Necropsy 1, Breeder l's scarlet macaw chick (severe clinical signs of PDD at necropsy); Necropsy 2, Breeder 2's umbrella cockatoo chick (milder clinical signs of PDD at necropsy); Necropsy 3, Breeder 2's umbrella cockatoo chick (severe clinical signs of PDD at necropsy).

[0275] We also examined the same panel of necropsy tissue specimens with polyclonal antisera we have generated against purified recombinant ABVN and ABVP proteins (see Methods). In general, we observed that the highest detectable ABVN and ABVP protein expression levels were found in tissues of the central nervous system and gastrointestinal tract (Figure 13B and 13C); in other tissues, these two viral proteins were not detectable or undetermined due to lack of signal in our control GAPDH Western blot analyses performed in parallel (data not shown). Surprisingly, although we were able to detect ABV RNA in the RT-PCR assay for the two necropsied birds with severe signs of PDD, one of the necropsied birds from the second breeder, an umbrella cockatoo chick with less severe clinical signs (Table 7, bird 2)had no detectable ABVN and ABVP protein by Western blot analysis in all tissues examined, despite the presence of intact GADPH protein (data not shown). Molecular analysis of ABV transmission during the PDD outbreak [0276] Having detected AB V2 RNA in the available symptomatic cases of PDD, we next investigated if additional birds in both breeders' flocks that were exposed to the PDD cases were detectably infected with ABV. At the site of the initial outbreak, 46 other birds were housed indoors and potentially exposed to the index and subsequent transmitted cases of PDD. Cloacal swabs and blood samples were obtained from each of these birds. Two of the tested birds harbored detectable ABV RNA in both blood pellet and cloacal swab specimens. Ten additional birds tested positive for ABV infection, but only in their cloacal swab specimens. Follow-up sequence analysis of recovered ABV RT-PCR products indicated that all these birds harbored AB V2 that was virtually identical to that detected in the necropsied tissue from the first breeder's histologically confirmed case of PDD (Figure 14) RT-PCR analysis of blood and cloacal swabs from the 8 potentially exposed indoor birds and two unexposed birds housed outdoors at the second breeder's home showed that within 4 weeks 5/8 of the exposed indoor birds had detectable ABV RNA in their cloacal swab specimens. In contrast, ABV RNA was not detectable in any of the collected blood specimens or the cloacal specimens derived from the unexposed outdoor birds. Follow-up sequence analysis demonstrated that all the ABV RT-PCR products recovered from this second set of birds corresponded to virtually identical (>95% nucleotide sequence identity) isolates of ABV2 detected in the first breeder's birds (Figure 14).

[0277] This outbreak of PDD was also significant in the context of the clinical presentation of the affected birds. Here, PDD was detected first in unweaned psittacine chicks. The signs and symptoms as well as course of disease were particularly vigorous in these birds, with many developing CNS signs within 48 hours of initial feed refusal and rapidly deteriorating to death within 3 days. Although this rapid course of disease was observed for many of the affected chicks, a number of exposed chicks appeared to recover with supportive therapy for secondary infections. Despite this, our ABV RT-PCR testing indicated these exposed but apparently recovered asymptomatic birds harbored ABV infections. Moreover, necropsy analysis of ABV(+) chicks that seemed to have recover from their initial gastrointestinal signs and symptoms also harbored gross pathology (enlarged and thin-walled proventriculus and atrophied ventriculus - e.g., bird #2 from breeder 2)) consistent with subclinical PDD. These observations are especially intriguing in light of the fact that the index case for this outbreak, an adult African grey hen, suffered only mild symptoms for many years before the onset of overt clinical PDD.

[0278] Through our molecular analyses of ABV RNA and protein, we have also gained insight into the tissue tropism of ABV in end-stage cases of PDD. The RT-PCR analysis for the highly expressed ABVM and ABVN RNAs demonstrated the presence of ABV in virtually every tissue we examined (Figure 13). Only liver and blood cells seem to be particularly poor sources for ABV growth at this stage of PDD. These results corroborate recent analyses of case control specimens obtained at necropsy (see, Rinder M, et al., J Virol 2009, 83:5401-5407), and provide further evidence that unlike the founding member of the bornavirus family, the mammalian Borna disease virus (BDV), ABV tropism does not appear to be restricted to neural cells. Interestingly, Western blot analysis for ABV proteins indicates that the expression of ABVN and ABVP proteins is enriched in the CNS and GI tissues These results are more consistent with the clinical signs and symptoms associated with disease. Whether the difference in these results merely reflects an artifact of the different sensitivity of these two types of assays versus a bona fide difference in the levels of expression of viral proteins or actively replicating ABV in different tissues is unclear.

[0279] Given the reproducible detection of ABV in GI tissues and the lack of consistent detection of ABV in blood specimens in our analysis of available necropsy tissues, it is not completely surprising that we observed a higher sensitivity of ABV detection in cloacal swab specimens compared to blood specimens during the follow-up screening of exposed birds. However, it is notable that we were able to detect ABV infection in a small number of blood specimens in the ABV(+) birds. The significance of these results is also unclear, especially in light of the detection of ABV in cloacal swabs of acutely exposed but clinically asymptomatic birds.

Methods for ABV detection in outbreak specimens

[0280] ABV RNA detection. RNA was extracted from clinical specimens with RNABee (Tel-Test, Inc. Texas), followed by isopropanol precipitation. Resulting pellets were resuspended in RNAse and DNAse free water and subjected to ABVN or ABVM RT-PCR as previously described (see, Gancz, AY et al., 2009 Virology J., in press) using the Qiagen 1 step RT-PCR kit (Qiagen, USA). Control RT-PCRs for GAPDH niRNA were performed in parallel using the previously described primers (see, Kistler AL, et al., Virol J 2008, 5:88). All candidate RT-PCR positives were gel-purified, subcloned into the pCR2.1 TOPO T/A vector and sequenced confirmed using flanking M 13 forward and reverse primers.

[0281] ABV protein detection. 20-100mg of tissue was sonicated in 200-400 uL of RIPA buffer (5OmM Tris 8.0, 15OmM NaCl, 1% NP40, 0.5% Sodium deoxycholate, 0.1% SDS) and incubated on ice for 30 minutes. Extracted protein was centrifuged at 16,00Og for 30 minutes at 4 degrees and the supernatant was quantitated using BCA assay (BioRad). 30ug of total protein was run on a NuPage 4-12% Bis-Tris polyacrylamide gel (Invitrogen) at 130 V for 90 minutes, transferred using XCeIl II Blot Module (Invitrogen) at 30V for 80 minutes, and blocked in 0.5X Odyssey Buffer in 0.5X PBS. Western blot analysis was performed using polyclonal antisera raised against ABVN (Gancz AY et al., 2009 Virology Journal, in press) diluted 1:2000, ABVP (see below) diluted 1:2000 and commercially supplied monoclonal antibodies to GAPDH diluted 1 :5000 in 5% milk in TBST and imaged on the Odyssey Imager (LI-COR Biosciences).

Methods for ABV phosphoprotein gene cloning, expression and polyclonal antibody generation.

[0282] The open reading frame (ORF) encoding the ABV phosphoprotein (ABVP) gene flanked with Ndel and Xhol restriction sites was amplified from ABV2 total RNA [Kistler AL et al., Virol. J., 5:88 (2008)] by RT-PCR with the following primers: ABVP- Ndel, 5'-C4L4rGATGGCACGGCCCTCG-3' and ABVP-XhoI, 5'- CTCGA GTT ATGGT ATT ATGTCGAG-3' (restriction sites italicized). The resulting product was sequence-confirmed, digested with Ndel and Xhol and subcloned into an Ndel/XhoI-digested pET15b (Novagen), which contains a 6xHis tag on the N-terminus (His₆). The sequence-confirmed pET15b vector containing the ABVP ORF was transformed into pRIL+ BL21(DE3)LysS E. coli and recombinant HiS₆-ABVP protein expression was induced with 50OuM IPTG at 37⁰C for 5.5 hours. Cells were lysed in 5OmM Tris pH 8.0, 10OmM NaCl, and IX Roche Complete Protease Inhibitors (Roche Applied Science; Indianapolis, IN) with 0.5 mg/mL lysozyme for 30 min in ice and 3 cycles through a microfluidizer. HiS₆-ABVP protein was purified from cell lysates via Ni- NTA column chromatography. The resulting eluate was then loaded on a ImL RESOURCE Q column (GE LifeSciences, Piscataway NJ) to separate any remaining products from the HiS₆-ABVP protein by ion exchange chromatography. Fractions containing purified HiS₆-ABVP were combined and concentrated using a 1OkDa Amicon Ultra. 2 mg of this purified HiS₆-ABVP was used for polyclonal antibody generation in rabbits (Pacific Immunology; Ramona, CA).

[0283] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WE CLAIM:

1. An isolated nucleic acid comprising a nucleotide sequence at least 12 nucleotides in length that has at least 90% sequence identity over its length to Accession No. EU781967 or its complement.

2. The nucleic acid of claim 1, wherein the nucleotide sequence has at least 90% identity over the foil length of Accession No. EU781967.

3. The nucleic acid of claim 1 , wherein the nucleotide sequence is a primer.

4. The nucleic acid of claim 1 , wherein nucleotide sequence comprises at least 95% identity to Accession No. EU781967.

5. The nucleic acid of claim 1 , wherein nucleotide sequence comprises a sequence of Accession No. EU781967.

6. The nucleic acid of claim 1, wherein nucleotide sequence encodes an open reading frame.

7. A protein encoded by a nucleotide sequence of claim 1.

8. A composition comprising a protein encoded by a nucleotide sequence of claim 1.

9. A composition comprising a nucleotide sequence of claim 1.

10. An isolated antibody that specifically binds to a protein encoded by a nucleotide sequence of claim 1.

11. The antibody of claim 10, wherein the antibody is a polyclonal antibody.

12. The antibody of claim 10, wherein the antibody is a monoclonal antibody.

13. Purified serum comprising polyclonal antibodies that specifically binds to protein encoded by a nucleotide sequence of claim 1.

14. An isolated bornavirus comprising a genomic nucleic acid of claim 1.

15. An expression vector comprising the nucleic acid of claim 1.

16. A host cell comprising the expression vector of claim 15.

17. A method of detecting a bornaviral nucleic acid, the method comprising the steps of: a) contacting a sample suspected of comprising a bornaviral nucleic acid with a nucleotide sequence at least 12 nucleotides in length that has 90% identity over its length to the corresponding segment of Accession No. EU781967 ; and b) detecting the presence or absence of specific binding of the nucleotide sequence to a bornaviral nucleic acid.

18. A method of detecting a bornaviral nucleic acid, the method comprising the steps of: a) contacting a sample suspected of comprising the bornaviral nucleic acid with at least one primer that hybridizes to a nucleotide sequence of Accession No. EU781967 b) performing a PCR reaction; and c) detecting the presence or absence of the bornaviral nucleic acid.

19. A method of detecting a bornavirus infection in a sample, the method comprising the steps of: a) contacting a sample suspected of comprising a bornavirus protein with an antibody that specifically binds a polypeptide encoded by Accession No. EU781967 ; and b) detecting the presence or absence of the bornavirus protein.

20. A kit for detecting a bornaviral nucleic acid, the kit comprising a nucleotide sequence at least 12 nucleotides in length that has at least 90% identity over its length to the corresponding segment of Accession No. EU781967 .

21. A kit for detecting a bornaviral nucleic acid, the kit comprising at least one primer hybridizes to a nucleotide sequence of Accession No. EU781967 under highly stringent PCR conditions comprising a denaturation phase of 90°C - 95°C for 30 sec - 2 min., an annealing phase of 50°C to about 65°C lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min., and an extension phase of about 72°C for 1 - 2 min for 20-40 cycles.

22. A kit for detecting a bornavirus in a sample, the kit comprising an antibody that detects a polypeptide encoded by an ORF of Accession No. EU781967 .

23. The kit of claim 22, comprising a monoclonal antibody.

24. The kit of claim 22, comprising a polyclonal antibody.

25. A method of assaying for an anti-bornaviral compound, the method comprising the steps of: a) contacting a sample comprising a bornavirus, the bornavirus comprising a genome that has at least 90% identity over its length to the corresponding segment of Accession No. EU781967; and b) determining whether the compound inhibits the bornavirus.

26. A method of treating or preventing a bornaviral infection in a subject, the method comprising the step of: administering to the subject an antigen encoded by a bornavirus, the bornavirus comprising a genome that has at least 90% identity over its length to the corresponding segment of Accession No. EU781967; thereby treating or prevention infection in the subject.

27. A method of diagnosing or confirming a diagnosis of PDD in a subject, said method comprising detecting an ABV virus or protein in a sample from the subject.

28. A method of preventing PDD transmission by detecting ABV protein or nucleic acid in a sample from a bird and isolating any bird which tests positive for ABV protein or nucleic acid from other birds which are not known to be infected with ABV.