# Successful in vitro propagation of porcine bocavirus: Demonstrating dual respiratory-enteric tropism and pathogenicity

## TL;DR

Researchers successfully grew porcine bocavirus in a lab for the first time, showing it causes both gut and lung disease in pigs and may pose a zoonotic risk.

## Contribution

First successful in vitro propagation of porcine bocavirus and experimental confirmation of its pathogenicity and dual tissue tropism.

## Key findings

- PBoV-CNH was isolated and propagated in LLC-PK1 cells, the first such successful isolation in a continuous cell line.
- Experimental infection showed PBoV-CNH causes both respiratory and intestinal disease in piglets, depending on the infection route.
- Phylogenetic analysis showed PBoV-CNH clusters with human bocaviruses, suggesting potential for cross-species transmission.

## Abstract

The inability to propagate porcine bocavirus (PBoV) in vitro has severely impeded research into its fundamental biology and pathogenic potential since its discovery 15 years ago. This study reports the successful isolation and characterization of a novel PBoV strain, PBoV-CNH, from diarrheic piglets in China. Crucially, PBoV-CNH was isolated and propagated in LLC-PK1 cells, a kidney-derived cell line from 3-4-week-old pigs, matching the age of the susceptible host. This represents the first documented isolation of PBoV in a continuous cell line. The isolate exhibited typical bocavirus morphology (20–30 nm particles), shared 94.15% whole-genome nucleotide identity with the NCBI reference strain (NC_016031.1), and displayed hemagglutination activity (HA) characteristic of Parvoviridae. Phylogenetic analysis revealed that PBoV-CNH clusters within a clade containing human bocaviruses (HBoVs), highlighting close genetic relatedness. Experimental infection of piglets confirmed PBoV-CNH as a primary pathogen. Notably, the virus exhibited dual tissue tropism: orally infected piglets developed acute diarrhea with high intestinal viral loads, while intranasally infected piglets showed diarrhea, significant lung pathology, and the highest viral loads in respiratory tissues. This efficient replication and shedding in the respiratory tract, combined with phylogenetic proximity to HBoVs and a previously reported human-PBoV infection case, signals a tangible risk of cross-species transmission. To our knowledge, this work constitutes the first successful in vitro propagation of PBoV and provides definitive experimental evidence, fulfilling Koch’s postulates, of its in vivo pathogenicity and tissue tropism. These findings provide essential tools and foundational insights for future research into PBoV biology, transmission, and control strategies.

This study overcomes the 15-year barrier to PBoV research by achieving its first successful isolation and propagation in LLC-PK1 cells, providing an essential in vitro tool. Critically, experimental infection of naïve piglets establishes PBoV as a primary pathogen capable of causing both gastrointestinal and respiratory disease, with tropism dictated by infection route. The finding of high viral loads and pathology in respiratory tissues, combined with phylogenetic clustering of PBoV-CNH alongside HBoVs, signals a tangible risk for cross-species transmission. This work provides the fundamental platform and insights urgently needed to investigate PBoV biology, transmission dynamics, and zoonotic potential, enabling future mitigation strategies.