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<p>Since 2016, mass mortality events characterized primarily by severe abdominal swelling and ascites have emerged in Chinese soft-shelled turtle (<i>Pelodiscus sinensis</i>) farms in China, leading to substantial economic losses. However, the cause of the disease has remained elusive, as no bacteria or parasites were detected in the diseased turtles. Tissue homogenate filtrate was used to infect Vero, GCO, TSK, TSL, and C6/36 cells, all of which produced cytopathic effects (CPE). Spherical, suspected viral particles with diameters approximately 30 nanometers (nm) were observed in the kidney tissue of diseased turtles using transmission electron microscopy (TEM). The complete viral genome was obtained through a combination of sequence-independent single-primer amplification (SISPA) and high-throughput sequencing, with rapid amplification of cDNA ends (RACE) employed to determine the terminal sequences. The genome is a positive-sense single-stranded RNA comprising 10,956 nucleotides (nt), exhibiting classic Orthoflavivirus characteristics and encoding a single open reading frame (ORF). Phylogenetic analysis indicated that the virus was most closely related to <i>Trionyx sinensis</i> flavivirus isolate YH2018, member of the <i>Orthoflavivirus</i> genus. Consequently, the virus has been named <i>P. sinensis</i> flavivirus isolate HZ2016 (PSFV-HZ2016). In an artificial challenge experiment, injection of tissue filtrate resulted in 100% mortality among healthy turtles within 16 days post-infection. Infected turtles displayed symptoms similar to those of the primary cases. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed broad tissue tropism, with the highest viral load detected in the heart. Histopathological analysis confirmed multi-organ damage consistent with severe systemic infection. These findings identify PSFV-HZ2016 as a novel and highly virulent <i>Orthoflavivirus</i> responsible for ascites-associated mortality in <i>P. sinensis</i>, providing crucial insights for diagnosis, surveillance, and disease management in aquaculture.</p>

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Isolation and characterization of a highly pathogenic Orthoflavivirus associated with ascites syndrome in Chinese soft-shelled turtles (Pelodiscus sinensis)

  • 期刊论文

  • 发表日期：

    2026年5月20日

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  • Xuemei Yuan,
  • Jiayun Yao,
  • Jing Chen,
  • Aqin Zheng,
  • Jinbiao Jiao,
  • Haiqi Zhang

摘要

Since 2016, mass mortality events characterized primarily by severe abdominal swelling and ascites have emerged in Chinese soft-shelled turtle (Pelodiscus sinensis) farms in China, leading to substantial economic losses. However, the cause of the disease has remained elusive, as no bacteria or parasites were detected in the diseased turtles. Tissue homogenate filtrate was used to infect Vero, GCO, TSK, TSL, and C6/36 cells, all of which produced cytopathic effects (CPE). Spherical, suspected viral particles with diameters approximately 30 nanometers (nm) were observed in the kidney tissue of diseased turtles using transmission electron microscopy (TEM). The complete viral genome was obtained through a combination of sequence-independent single-primer amplification (SISPA) and high-throughput sequencing, with rapid amplification of cDNA ends (RACE) employed to determine the terminal sequences. The genome is a positive-sense single-stranded RNA comprising 10,956 nucleotides (nt), exhibiting classic Orthoflavivirus characteristics and encoding a single open reading frame (ORF). Phylogenetic analysis indicated that the virus was most closely related to Trionyx sinensis flavivirus isolate YH2018, member of the Orthoflavivirus genus. Consequently, the virus has been named P. sinensis flavivirus isolate HZ2016 (PSFV-HZ2016). In an artificial challenge experiment, injection of tissue filtrate resulted in 100% mortality among healthy turtles within 16 days post-infection. Infected turtles displayed symptoms similar to those of the primary cases. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed broad tissue tropism, with the highest viral load detected in the heart. Histopathological analysis confirmed multi-organ damage consistent with severe systemic infection. These findings identify PSFV-HZ2016 as a novel and highly virulent Orthoflavivirus responsible for ascites-associated mortality in P. sinensis, providing crucial insights for diagnosis, surveillance, and disease management in aquaculture.