2.2 Primers design
Eighteen genome nucleotide sequences of PBoVs were downloaded from the
GenBank and subjected to perform homology analysis using the MegAlign
program of DNAStar software (version 7.1, DNASTAR Inc., Madison, USA).
The highly conserved sequences within the NS1 gene were
determined from an alignment of nine PBoV1/2 genomic sequences (GenBank
accession number HM053693, HM053694, HQ291309, KF025392, KF025393,
KF206155, KF206157, KF206161 and KM402139), and a pair of primers
PBoV1/2-F/R was designed using the Primer Premier (version 6.0, USA)
software based on the sequence of GenBank accession number HM053693,
which gave an amplification product of 327 bp (Table 1). The specific
primers for PBoV3/4/5 were found from an alignment of nine sequences of
PBoV3/4/5 (GenBank accession number JF429834, JF713714, KC473563,
JF713715, JN681175, JF429836, KF206158, JN621325 and JN831651), and a
pair of primers PBoV3/4/5-F/R targeting the VP 1 gene of PBoV3/4/5
was designed based on the sequence of GenBank accession number JF429834,
amplifying a fragment of 209 bp (Table 1). In addition, three pairs of
specific PCR primers (Q1F/Q1R, Q2F/Q2R and Q3F/Q3R) were designed
according to the reference sequence (GenBank accession number HM053693)
to amplify three overlapping fragments covering the entire viral genome
(Table.1).