Molecular Characterization of Budgerigar Fledgling Disease Virus SC-YB19, with an 18-Nucleotides Deletion, in China

Background: Budgerigar edgling disease virus (BFDV) poses a serious threat to the Chinese psittacine industry, causing enormous economic losses. This study aims to reveal the etiological role of BFDV and evaluate the molecular characterization. Results: We report on BFDV, designated SC-YB19, which had an 18-nucleotide (nt) deletion in the enhancer region, corresponding to the sequence position 164–181 nt, when compared with other BFDV strains. Sequence analyses suggested that 19 nucleotide substitutions were identied with the domestic strains, APV7 and AF118150. Phylogenetic analysis indicated that SC-YB19, along with three domestic strains, formed a unique cluster, and were closely related to Polish isolates. Conclusion: Taken together, these results demonstrate that a BFDV genotype variation was co-circulating in China, and provide important insights on evolution of BFDV.


Background
Budgerigar fledgling disease virus (BFDV) is a polyomavirus, and is the causative agent of budgerigar fledgling disease, which is an important viral disease of budgerigars (Melopsittacus undulatus). The disease was rst reported in 1981, with typical symptoms including abdominal distention, lack of down feathers on the back and abdomen, subcutaneous hemorrhage of nestling budgerigars, and acute death [1][2][3]. Polyomavirus has a wide host range, with novel polyomaviruses identified in vertebrate hosts including humans [4,5], bats [6,7], non-human primates [8], and horses [9]. In 1994, the rst BFDV report was made in Hubei province, China, however the virus still causes sporadic infections across China, and in icts signi cant losses to the budgerigar breeding industry [10].
BFDV, also called avian polyomavirus (APV), was identi ed as the rst non-mammalian member of the genus polyomavirus [11,12]. BFDV is a circular, double-stranded molecule with a genome of 4981 nt, which can be divided into an early and a late region. The early region codes for two nonstructural proteins, the large T and the small t antigen. The late region contains four structural proteins, VP1, VP2, VP3, and VP4 [13]. Similarly, the genome also contains four regulatory elements, a promoter, polyadenylation signals, origin of DNA replication and enhancer [14].
In the present study, we report a BFD infection in a budgerigar from Sichuan province for the rst time. To better understand the molecular characteristics of this strain, sequencing analysis and the phylogenetic tree was constructed based on its complete genome, which may assist in elucidating the genetic evolution of BFDV in China.

Ethics statement
No animals were sacri ced speci cally for this study.

Clinical case
During the spring of 2019, there was a budgerigar dead in the farm. Samples of hearts, livers, lungs and faeces were collected from the dead budgerigars. Each sample was subjected to grind and centrifuged at 8,000 rpm for 10 min. The DNA was extracted from supernatant, then identi ed with the primers [15].

Sequence alignment and phylogenetic analysis
Multiple sequence alignments were conducted using ClustalW in MEGA 6.0. A phylogenetic tree, based on complete sequences, was constructed using the neighbor-joining (NJ) method using MEGA software (version 4.0). Bootstrap values were estimated for 1,000 replicates. The sequences obtained in this study were assembled and submitted to GenBank under the accession number MT119153.

Phylogenetic analysis
Phylogenetic analysis of the complete sequence showed that BFDV strains could be divided into three groups ( Figure 1D). The SC-YB19 had a close relationship with domestic WF-GM01, SD18, APV-P strains and is more distant evolutionarily from the AY672646 strains group and strain QDJM-01.

Discussion
In 2019, a privately owned two-week-old male budgerigar was submitted for laboratory investigation, after its untimely death. A necropsy revealed subcutaneous hemorrhage, ascites and hydropericardium. Multiple areas of hemorrhage were observed in the liver. Tissue samples from the liver and lungs were positive for BFDV. No other viruses were identi ed in samples (data not shown).
To analyze genomic characteristics, PCR assay was employed to amplify and sequence the complete viral genome (termed SC-YB19), which was 4963 nt. The BFDV genome incorporated an early region, a late region, promoter, polyadenylation signals, origin of DNA replication and enhancers. The enhancers consisted of two parts at 80-126 nt and 131-178 nt, when compared with APV7.
The genetic diversity of SC-YB19 and APV isolates was investigated using multiple alignment analyses of whole genome sequences from APV1-7 isolates, which were isolated from Japan between 2003 and 2006. There were common and unique features between SC-YB19 and BFDV strains. Firstly, an 18-nt deletion was detected in the latter part of the enhancer section in SC-YB19, but not in the other BFDV isolates (Fig. 1A and B). Deletions in this region have not been previously described for BFDV, and may be associated with transcription activities [14]. Further experiments will be required to con rm the biological effects of this sequence deletion. In addition, nucleotide substitutions were found in the SC-YB19, AF118150, APV-7, AF241168-70, M20775 and NC004764 (Table 2). Notably, no amino acid substitutions were observed (Table 1). These data indicate that SC-YB19 sequences were consistent with at least two out of ve domestic strains in terms of mutation positions, but distinct to foreign strains (Fig. 1C). These observations further suggest that ve domestic strains showed genetic diversity and underwent evolution at some point in the past.
Phylogenetic analyses based on complete sequences, suggested that SC-YB19, along with the domestic strains, WF-GM01, SD18 and APV-P formed a unique cluster and were closely related to Polish virus isolates. However, QD-JM01 was located in a cluster of APV1, APV2, APV4 and APV5 strains, which were isolated from the Japanese black-headed caique (Pionites melanocephalus). AY672646 was distinct with ve domestic strains and did not belong to any cluster (Fig. 1D). Our data showed that different BFDV genotypes were co-circulating in China.

Conclusion
We report for the first time a novel mutation in BFDV, in mainland China. The strain, along with ve domestic strains forms a unique cluster. We theorize these viruses were co-circulating in China, and underwent a similar evolution.