To understand ABV better, we began an epidemiological study of ABV in Japan. First, we collected blood samples from three birds: a Nymphicus hollandicus with clinically suspected PDD, an Eclectus roratus with FPD and a healthy duck (Anas platyrhynchos). We isolated total RNA from whole blood using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and reverse-transcribed with transcriptor reverse-transcriptase and random hexamer (Roche, Indianapolis, IN, USA). We designed degenerate primers based on the alignment of amino Doxorubicin chemical structure acid
sequences of the N of ABV genotypes 1 to 5 (ABV1–5) and L of ABV genotypes 1 to 4 (ABV1–4) for detection of ABV-specific nucleic acid (Table 1). In this study, we used six primer pairs (MH168–176, 169–176, 173–176, 174–176, 175–170 and 175–177) and two primer pairs (MH171–172 and 178–179) for ABV N and L, respectively. We carried out PCR with Ex-Taq Hot Start Version (TaKaRa, Shiga, Japan) and eight pairs of degenerate primers using the following program: denaturation for 2 min, 10 cycles of 94°C for 30 s, 60–55°C
(the GPCR Compound Library chemical structure annealing temperature was decreased by 0.5°C every other cycle) for 30 s, 72°C for 30 s and 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s followed by 72°C 3 min. Surprisingly, using degenerate primers for ABV N, we obtained bands of the expected sizes from the sample of the bird affected by FPD, but from the samples of the bird with PDD and the healthy duck. (Fig. 1a). On the other hand, using Nutlin-3 nmr primers for ABV L, we found no amplification of the expected bands in any of the samples (data not shown). The representative nucleic acid sequence of the amplicons (Acc. No. AB519142) showed 99% and 98% identity to two known ABV5 N sequences (Acc. No. FJ002318 and FJ002319) by BLAST and clustered into ABV5 according to phylogenetic analysis (data not shown). Furthermore, to confirm the above result, we carried out PCR with primers specific for the ABV5 M gene (MH180 and
181). An amplicon was generated as expected (Fig. 1b) and its nucleotide sequence (Acc. No. AB519143) showed 98% and 95% identity to two previously identified ABV5 M sequences (Acc. No. FJ002334 and FJ002335). These results indicated that the FPD-affected bird may have been infected with genotype 5 of ABV. We also tested the FPD bird’s blood for beak and feather disease virus (circovirus) and budgerigar fledgling disease virus (polyomavirus) by PCR, and found that it was negative for both viruses (data not shown). After 8 months of blood sampling, we collected feces from the ABV5 (+) bird and a healthy bird of the same species. At that time, the ABV5 (+) bird showed clinical signs of FPD but not of PDD. We suspended the fecal samples in PBS and centrifuged at 9,500 ×g for 10 min at 4°C. We isolated RNA from the supernatant using a viral RNA mini kit (Qiagen, Tokyo, Japan), and subjected it to RT-PCR with random hexamers and the MH175–170 primer pair.
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