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Isolation, Identification, and Comprehensive Genomic Characterization of a Bovine Rotavirus G10P[11] Strain in China

JIAN LIU

JIAN LIU

Shanghai Animal Disease Prevention and Control CenterShanghai, China
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LIU, JIAN
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XIANCHAO YANG

XIANCHAO YANG

Shanghai Animal Disease Prevention and Control CenterShanghai, China
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YANG, XIANCHAO
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YAPING GUI

YAPING GUI

Shanghai Animal Disease Prevention and Control CenterShanghai, China
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GUI, YAPING
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QIQI XIA

QIQI XIA

Shanghai Animal Disease Prevention and Control CenterShanghai, China
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XIA, QIQI
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GUIDAN FENG

GUIDAN FENG

Shanghai Animal Disease Prevention and Control CenterShanghai, China
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FENG, GUIDAN
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DEQUAN YANG

DEQUAN YANG

Shanghai Animal Disease Prevention and Control CenterShanghai, China
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YANG, DEQUAN
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PING XU

PING XU

Shanghai Animal Disease Prevention and Control CenterShanghai, China
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XU, PING
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JUN TAO

JUN TAO

Shanghai Jiading District Agricultural Technology Extension Service CenterShanghai, China
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TAO, JUN
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YULING MA

YULING MA

Shanghai Jiading District Agricultural Technology Extension Service CenterShanghai, China
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MA, YULING
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JUN MA

JUN MA

School of Biological and Pharmaceutical Sciences, Shaanxi University of Science and TechnologyXi’an, China
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MA, JUN
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WENWEI SHENG

WENWEI SHENG

Shanghai Animal Disease Prevention and Control CenterShanghai, China
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SHENG, WENWEI
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JIAN WANG

JIAN WANG

Shanghai Animal Disease Prevention and Control CenterShanghai, China
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WANG, JIAN
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WEIDONG QIAN

WEIDONG QIAN

School of Biological and Pharmaceutical Sciences, Shaanxi University of Science and TechnologyXi’an, China
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QIAN, WEIDONG
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SHIXIN HUANG

SHIXIN HUANG

Shanghai Animal Disease Prevention and Control CenterShanghai, China
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HUANG, SHIXIN
  
16 set 2025
Isolation, Identification, and Comprehensive Genomic Characterization of a Bovine Rotavirus G10P[11] Strain in China's Cover Image
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Categoria dell'articolo: Original Paper
Pubblicato online: 16 set 2025
Pagine: 318 - 328
Ricevuto: 27 mag 2025
Accettato: 25 lug 2025
DOI: https://doi.org/10.33073/pjm-2025-027
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© 2025 JIAN LIU et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1.

Inverted phase-contrast microscopy of BRVA-infected MA104 cells (100× magnification). A)Infected, 24 h; B) infected, 48 h; C) infected, 72 h; D) uninfected, 24 h; E) uninfected, 48 h; F) uninfected, 72 h.Cells were cultured in 24-well plates and incubated with or without BRVA for different durations. Cytopathic effects began at 48 h and intensified by 72 h compared to the negative controls. Scale bars represent 100 μm.
Inverted phase-contrast microscopy of BRVA-infected MA104 cells (100× magnification). A)Infected, 24 h; B) infected, 48 h; C) infected, 72 h; D) uninfected, 24 h; E) uninfected, 48 h; F) uninfected, 72 h.Cells were cultured in 24-well plates and incubated with or without BRVA for different durations. Cytopathic effects began at 48 h and intensified by 72 h compared to the negative controls. Scale bars represent 100 μm.

Fig. 2.

Results of agarose gel electrophoresis for RT-PCR amplification of BRVA.M – DL 2000 DNA marker; 1 – MA104 cells infected with SHH2023001; 2 – negative control (uninfected MA104 cells). The presence of a specific band at approximately 1,060 bp confirms the successful amplification of the BRVA VP7 gene in the infected sample (lane 1). The negative control (lane 2) shows no amplification, indicating the absence of viral RNA.
Results of agarose gel electrophoresis for RT-PCR amplification of BRVA.M – DL 2000 DNA marker; 1 – MA104 cells infected with SHH2023001; 2 – negative control (uninfected MA104 cells). The presence of a specific band at approximately 1,060 bp confirms the successful amplification of the BRVA VP7 gene in the infected sample (lane 1). The negative control (lane 2) shows no amplification, indicating the absence of viral RNA.

Fig. 3.

Virus identification by indirect immunofluorescence assay.MA104 cells grown on coverslips in 6-well plates were infected with BRVA. The cells were fixed and incubated with a monoclonal antibody against the BRVA p42 antigen (1:200) and a goat anti-mouse fluorescent secondary antibody. Images were captured post-infection. BRVA infection (SHH2023001); uninfected (normal cells). Scale bars represent 50 μm. The signal indicates cytoplasmic sub-cellular localization, which is expected for BRVA infection. The negative control (uninfected) was used to validate the assay, and multiple fields of view were checked for consistency.
Virus identification by indirect immunofluorescence assay.MA104 cells grown on coverslips in 6-well plates were infected with BRVA. The cells were fixed and incubated with a monoclonal antibody against the BRVA p42 antigen (1:200) and a goat anti-mouse fluorescent secondary antibody. Images were captured post-infection. BRVA infection (SHH2023001); uninfected (normal cells). Scale bars represent 50 μm. The signal indicates cytoplasmic sub-cellular localization, which is expected for BRVA infection. The negative control (uninfected) was used to validate the assay, and multiple fields of view were checked for consistency.

Fig. 4.

Transmission electron microscopy (TEM) observation of BRVA-infected MA104 cell cultures. SHH2023001 virions were visualized under TEM (Scale bar = 200 nm).
Transmission electron microscopy (TEM) observation of BRVA-infected MA104 cell cultures. SHH2023001 virions were visualized under TEM (Scale bar = 200 nm).

Fig. 5.

Phylogenetic analysis of the SHH2023001 strain based on nucleotide sequences of VP1 (a), VP2 (b), VP3 (c), VP4 (d), VP6 (e), and VP7 (f) genes. Trees were constructed using the neighbor-joining method in MEGA 7.0, with bootstrap values (1,000 replicates) above 70 indicated. The scale bar represents nucleotide substitutions per site. Genotypes are shown on the right. Strains detected in this study are marked with a black circle.
Phylogenetic analysis of the SHH2023001 strain based on nucleotide sequences of VP1 (a), VP2 (b), VP3 (c), VP4 (d), VP6 (e), and VP7 (f) genes. Trees were constructed using the neighbor-joining method in MEGA 7.0, with bootstrap values (1,000 replicates) above 70 indicated. The scale bar represents nucleotide substitutions per site. Genotypes are shown on the right. Strains detected in this study are marked with a black circle.

Fig. 6.

Phylogenetic analysis of the SHH2023001 strain based on nucleotide sequences of NSP1 (a), NSP2 (b), NSP3 (c), NSP4 (d), and NSP5 (e) genes. Trees were constructed using the neighbor-joining method in MEGA 6.0, with bootstrap values (1,000 replicates) above 70 indicated. The scale bar represents nucleotide substitutions per site. Genotypes are shown on the right. Strains detected in this study are marked with a black circle.
Phylogenetic analysis of the SHH2023001 strain based on nucleotide sequences of NSP1 (a), NSP2 (b), NSP3 (c), NSP4 (d), and NSP5 (e) genes. Trees were constructed using the neighbor-joining method in MEGA 6.0, with bootstrap values (1,000 replicates) above 70 indicated. The scale bar represents nucleotide substitutions per site. Genotypes are shown on the right. Strains detected in this study are marked with a black circle.

Nucleotide sequence identity between strain SHH2023001 and other strains for each gene segment_

Gene Closest strain Accession No. Homology (%) Genotype
VP7 RVA/Human-wt/THA/DB2015-66/2015/G10P[14] LC367319 95.41 G10
VP4 RVA/Cow-tc/USA/B223/1983/G10P[11] LC133550 96.62 P[11]
VP6 RVA/Bovine-tc/KOR/KJ9-1/2010/G6P[7] HM988974 97.20 I2
VP1 RVA/Human-wt/JPN/HKD0825/2016/G1P[8] LC384330 95.12 R2
VP2 RVA/Sheep-wt/CHN/GS2023/2023/G6P[1] PP115427 97.77 C2
VP3 RVA/Bovine-tc/CHN/DQ-75/2008/G10P[11] GU384193 94.25 M2
NSP1 RVA/Sheep-wt/CHN/GS2023/2023/G6P[1] PP115432 96.47 A11
NSP2 RVA/Human-wt/VNM/NT0578/2008/G2P[4] LC060821 96.98 N2
NSP3 RVA/Human-wt/HUN/BP1062/2004/G8P[14] FN665695 96.14 T6
NSP4 RVA/Horse-wt/IND/ERV2/2015/G6P[1] OK651114 97.44 E2
NSP5 RVA/Human-wt/HUN/BP1062/2004/G8P[14] FN665698 97.20 H3
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