Diverse Head-to-Tail Sequences in the Circular Genome of Human Bocavirus Genotype 1 Among Children with Acute Respiratory Infections Implied the Switch of Template Chain in the Rolling-Circle Replication Model

Head-to-tail sequences have been reported in human bocavirus (HBoV) 1-4. To reveal their features and functions, HBoV DNA was screened among respiratory specimens from pediatric patients with an acute respiratory infection (ARI) between April 2020 and December 2022, followed by HBoV genotyping. Head-to-tail sequences were detected using nested PCR, TA cloning, and Sanger sequencing, and these findings were confirmed by mNGS and amplicon sequencing. The secondary structure was predicted using the Mfold web server. The results indicated that head-to-tail sequences were detected in 42 specimens through TA cloning from 351 specimens positive for HBoV1 DNA, yielding 92 sequences into 32 types and 2 categories. Additionally, head-to-tail sequences were detected in 16 specimens by amplicon sequencing, yielding 60 sequences categorized into 23 types. The 374nt type, detected in 13 specimens, contains variants 374a and 374b, which differ in the unpaired loop regions of the palindrome or complementary reverse sequences, implying a switch of template chains during the replication process. The mNGS results in three specimens confirmed the presence of circular genome in copies below 1%. In conclusion, head-to-tail sequences of HBoV1 were common in children with ARI and were highly diverse in length and sequences. The variants may be generated by the switch of the template chain in the rolling-circle replication model.