Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: Identification of a candidate etiologic agent

Kistler, Amy L; Gancz, Ady; Clubb, Susan; Skewes-Cox, Peter; Fischer, Kael; Sorber, Katherine; Chiu, Charles Y; Lublin, Avishai; Mechani, Sara; Farnoushi, Yigal; Greninger, Alexander; Wen, Christopher C; Karlene, Scott B; Ganem, Don; DeRisi, Joseph L

Recovery of the 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 designed a set of PCR primers to perform ABV-specific PCR screening of an independent set of PDD case and control specimens to investigate the association between the presence of ABV and 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 three regions of the genome: 1) the L gene region of the genome that we used for PCR confirmation of the microarray results, (Figure 2 , PCR probes track), 2) a subregion within the N gene and 3) a subregion within the M gene (Materials and Methods). PCR for glyceraldehyde 3 phosphate dehydrogenase (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, 5 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 71% (5/ 7) to be positive by ABV PCR (Table 3 ). In contrast, all PDD controls 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 = 0.01, Fisher's Exact Test). 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.