Genome segments encoding capsid protein-like variants of Pyrus pyrifolia cryptic virus

According to previous studies, three double-stranded (ds) RNA molecules (dsRNA1, 2, and 3) detected in Japanese pear are transmitted to the next generation with high frequency through both ovules and pollen. Nucleotide sequence analysis of dsRNA1-encoding RNA-dependent RNA polymerase (RdRp) has suggested that these dsRNAs are related to a cryptovirus named Pyrus pyrifolia cryptic virus (PpCV). In this study, purified dsRNA prepared from a PpCV-infected Japanese pear cultivar was subjected to next-generation deep sequencing. This sequencing generated two de novo assembled contigs corresponding to dsRNA2 and 3, with BLAST analysis of the predicted amino acid sequences indicating homology to capsid proteins (CPs) of the cryptoviruses persimmon cryptic virus and Sinapis alba cryptic virus 1, respectively. Relationships between the two contigs and dsRNA2 and 3 were confirmed by northern blot hybridization with probes generated using primers designed from the assembled contigs. Rapid amplification of cDNA ends analyses of 5'- and 3'-terminal sequences of dsRNA2 and 3 revealed that these two dsRNAs consist of 1523 and 1481bp, respectively. The 5'-terminal sequences (AGAAUUUC) of dsRNA1, 2 and 3 were found to be conserved. Phylogenetic analysis of deduced amino acid sequences of the two CP-like variants indicated that PpCV belongs to Deltapartitivirus (Partitiviridae). Our results imply that PpCV is tri-segmented.