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

Fig 1. Global distribution of PBoV-positive samples and four major events following its initial detection. The base map was created using public domain data from Natural Earth (https://www.naturalearthdata.com/downloads/110m-cultural-vectors/).

Fig 2. NGS analysis identified PBoV as the likely causative agent in an outbreak of diarrhea of unknown origin. (A) PCR results confirmed that PBoV existed in intestine samples. (B) NGS statistical results revealed bocavirus as the only identified virus in diarrhea samples.

Fig 3. Cell isolation and in vitro characterization of PBoV. (A and B) Virus-induced CPE in PBoV-infected LLC-PK1 cells that are visible on light microscopy (A) and in plaque assays (B). Scale bar represents 75 μm. (C) Negatively stained virions purified from PBoV-infected LLC-PK1 cells. Scale bar represents 200 nm. (D and E) Growth curves of PBoV in LLC-PK1 cells, with titers peaking mostly at 48 to 60 hpi determined by TCID50, viral DNA copies peaking at 84 hpi determined by qPCR. (F) PBoV-infected LLC-PK1 cells detected by IFA, the infected cells were stained with a mouse serum raised against the PBoV NP1 protein. Scale bar represents 125 μm. (G) Nuclear localization signal of NP1 protein was observed in PBoV-infected cells. Scale bar represents 5 μm. (H) The expression levels of NP1 protein increased in a time-dependent manner in PBoV-infected cells. (I) PBoV propagated in LLC-PK1 cells can agglutinate 0.6% fresh guinea pig red blood cells with a HA titer of 28.

Fig 4. Genomic characteristics and phylogeny of PBoV-CNH. (A) The genome organization of PBoV-CNH. The diagram is drawn to scale, and the scale bar is shown at the bottom. The abbreviation nt denotes nucleotides. (B) Phylogenetic analysis of PBoV isolates based on the VP1 gene. This analysis employed a neighbour-joining method using the MEGA 6.0 software. Bootstrap probabilities for each node were calculated using 1,000 replicates. The gray background highlights the PBoV isolated in this study. Scale bars indicate the number of nt substitutions per site. The virus strains detected in China, Europe, America, Africa are highlighted in red, green, blue and purple, respectively. (C) Summary table of amino acid coding differences between PBoV and NCBI reference strain NC_016031. (D) Phylogenetic trees of the VP1 gene sequence of PBoV-CNH in the present study and corresponding sequences of other parvoviruses. The green background highlights the PBoV strains. Scale bars indicate the number of nt substitutions per site. The tree was constructed by MEGA software, with neighbour-joining method, 1,000 bootstrap replications. (E) Phylogenetic analysis of complete genome sequences of bocavirus from…

Fig 5. Pathogenicity of PBoV-CNH in piglets. (A-C) Onset of diarrhea (A), diarrhea severity scores (B) and body temperature change (C) were documented once every six hours. (D and E) Viral DNA copies in fecal and nasal swabs were collected for the detection of virus shedding time by qPCR. (F) (a, b and c) 3-day old piglets were orally- and intranasally-inoculated with PBoV-CNH (1 × 105TCID50/pig) exhibited diarrhea symptom, watery diarrhea was observed in both orally and intranasally-infected piglets; (d, e and f) there are bleeding spots in the lungs of intranasally-infected piglets; (g, h and I) pathological changes were observed in intestines of challenged piglets. (G) Histopathological changes in the intestines and lungs of piglets challenged with PBoV. (H) Morphometric analysis for VH:CD ratio for the villous atrophy. P-value < 0.01 was considered as significant (**P < 0.01). (I) Piglets were euthanized, and different organs were collected for viral load determinations.

Fig 6. Susceptibility of various cell lines to PBoV-CNH. (A) Effect of trypsin on PBoV replication in LLC-PK1 cells. Virus titers were detected following trypsin treatment at doses of 0, 4, 8, 16, and 32 μg/mL at 12, 24, and 48 hpi. (B) PBoV-infected different cells were detected by IFA. The cells were infected with PBoV-CNH (MOI = 1) in the presence of 16 μg/mL trypsin, and then incubated with mouse polyclonal antibodies against the PBoV-NP1 protein. Scale bar represents 125 μm. (C) Growth kinetics of PBoV in different cell lines. Supernatants were collected at the indicated time points (0, 12, 24 and 48 hpi), and cycle threshold (CT) values for viral DNA copies were measured at each time point using qPCR. Mean ± SD (error bars) are shown (n = 3).