Evolutionary characterization and pathogenicity of a porcine G9P[23] rotavirus with gene segments linked to canine and giant panda strains

Abstract

Porcine rotavirus A (RVA) has emerged as an increasingly consequential zoonotic pathogen, causing severe intestinal disorders across diverse mammalian species, including humans. During of an outbreak that struck nursing piglets with diarrhea, a porcine G9P[23] rotavirus, named as RVA/Pig-wt/China/ZJ03/2022/G9P[23] (hereafter referred to as ZJ03), was identified. To further elucidate the evolutionary diversity of ZJ03, a comprehensive analysis of all genome segments was conducted. The genome constellation was identified as G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1. Nucleotide sequence identity and phylogenetic analyses indicated that the VP3 and NSP1 genes of ZJ03 are most closely related to the corresponding genes of the giant panda strain and the dog strain, respectively, showing the highest homology at 95.73 % identity and 94.64 %. The remaining genes demonstrated the most intimate relationship with porcine strains. Their highest homology levels ranged from 95.98 % to 99.49 % similarity. Therefore, evidence suggests interspecies transmission and genetic reassortment events between porcine, canine, and giant panda rotavirus strains. To evaluate the pathogenicity of ZJ03 strain, we experimentally infected 3-day-old piglets oral inoculation with the PoRV ZJ03 strain at a dose of 2 × 10^5.5 TCID₅₀/ml per piglet. The infection resulted in severe diarrhea in all piglets, which occurred at 48 h post-infection (hpi), accompanied by sustained viral shedding and characteristic small intestinal villous atrophy, indicating significant damage to the intestinal epithelium. In vitro, ZJ03 exhibited efficient replication kinetics in MA104 cells, reaching peak titers of 10^9.25 TCID₅₀/mL at 36 h post-infection. This study reports the first documented case of a novel porcine G9P[23] rotavirus with gene segments linked to canine and giant panda strains in mainland China, characterized by high viral titer and virulence. The findings highlight the emergence of a previously unrecorded RVA strain with significant virological and ecological implications.

Keywords: Genome analysis; Isolation; Pathogenicity; Porcine rotavirus; Whole-genome sequencing.

Fig. 1

Virus Isolation, identification, and growth curves of ZJ03. (A) CPE of MA104 cells infected with ZJ03. (B) Fluorescence were stained for RVA-VP6 (Green). (C) Electron microscopic images of purified PoRV particles. (D) Growth kinetics of ZJ03 in MA104 cells. Data was presented as mean ± SD by triplicates.

Fig. 2

Phylogenetic analysis based on the nucleotide sequences of the VP7,VP4,VP6 segments from ZJ03 and other strains by MEGA 7 using the neighbor-joining (NJ) method and 1,000 bootstrap replicates. (A) Phylogenetic analysis revealed that ZJ03 strain can be classified into the lineage III. (B) Phylogenic trees based on VP7 gene from ZJ03. (C) Phylogenic trees based on VP4 gene from ZJ03. (D) Phylogenic trees based on VP6 gene from ZJ03.

Fig. 3

Phylogenetic analysis based on the nucleotide sequences of the NSP1, NSP2, NSP3, NSP4, NSP5, VP1, VP2 and VP3 segments from ZJ03 and other strains by MEGA 7 using the neighbor-joining (NJ) method and 1,000 bootstrap replicates. Phylogenic trees based on the nucleotide of NSP1, NSP2, NSP3, NSP4, NSP5, VP1, VP2 and VP3 genes from ZJ03.

Fig. 4

Comparison of VP4 (VP5* and VP8*) and VP7 antigenic epitopes in different strains. (A) Neutralizing epitopes on the VP7 protein. (B) Neutralizing epitopes on the VP5 protein. (C) Neutralizing epitopes on the VP8 protein. The red letters represent the mutilation of amino acids. (D) The structure of the VP4 protein. (E) The structure of the VP8 protein. The antigenic domains 8-1, 8-2, 8-3, and 8-4 are represented by the colors green, purple, yellow, and blue, respectively. The conserved antigenic domain is shown in red. (F) The structure of the VP5 protein. The green represent the antigenic domains. The red represent the conserved antigenic domains. (G) The structure of the VP7 protein. The antigenic domains 7-1a, 7-1b, and 7-2 are represented by the colors green, blue and purple.

Fig. 5

Pathogenicity of PoRV strain ZJ03 in 3-day-old piglets. 3-day-old piglets were orally inoculated with 2 mL ZJ03 virus and 2 mL DMEM (Control group) respectively. (A) The Clinical signs and macroscopic lesions. (B) HE results for histopathological lesions. (C) IHC results for histopathological lesions. (D) Average weight gain from days 0 to 7 post-challenged. (E) Fecal score was recorded each 24 h after inoculation until necropsy. (F) Clinical score was recorded each 24 h after inoculation until necropsy. (G) Survival curves of the piglets in both group. (H) Viral load in intestinal tissues was detected using RT-qPCR after necropsy. (I) Viral shedding was detected using RT-PCR each 24 h after inoculation until necropsy.