Identification and characterization of a novel reovirus strain isolated fromgrass carp (Ctenopharyngodon idella)

Abstract

Background: Grass carp (Ctenopharyngodon idella) hemorrhagic disease (GCHD) is a devastating disease that leads to substantial economic losses in the freshwater aquaculture industry.

Results: In this study, we investigated an outbreak of GCHD in large-scale grass carp and identified GCRV-II infection. Notably, hematoxylin and eosin (H&E) staining showed severe histopathological changes in the spleen, head kidney, gill, and gut. Furthermore, we sequenced the entire genome of the viral isolate, and multiple sequence alignment and phylogenetic tree analysis indicated that it represents a novel strain of GCRV-II, provisionally named GCRV-YX246. Finally, artificial infection experiments confirmed the strong virulence, high mortality, and severe pathological damage caused by GCRV-YX246, as demonstrated through artificial infection.

Conclusions: A novel reovirus from large-scale grass carp cultured in China was identified. The discovery of this novel GCRV-II strain enhances our understanding of GCRV-II biology and provides valuable insights for developing more effective prevention strategies for GCHD.

Keywords: Genome; Grass carp reovirus; Identification; Pathogenicity; Phylogenetic analysis.

Fig. 1

Pathogenic characteristics and identification of GCRV-YX246. A Clinical symptoms observed in diseased grass carp (Ctenopharyngodon idellus). B Nested PCR products showing bands at 408 bp and 363 bp from GCRV cDNA. C Viral loads detected in different tissues of diseased grass carp (n = 6). Muscle and brain exhibited the highest viral loads, followed by skin and gut. D Immunofluorescence analysis of the SP, HK, gut, and gills under natural infection conditions. The white arrows indicate the presence of GCRV-II virus. Scale bar, 50 μm. E TEM images of the SP, HK, gut, and gills under natural infection conditions. The red arrows point to GCRV-II virions. Scale bar, 500 nm

Fig. 2

Histopathological changes in the SP, HK, gill, and gut of fish naturally infected with GCRV-II. Histological examination of the SP (A), HK (B), gill (C), and gut (D) from naturally infected fish. Red arrows indicate erythrocyte infiltration, while black arrows mark areas of cellular necrosis. Scale bar, 50 μm

Fig. 3

Circular map of the GCRV-YX246 genome. Each circular map represents a replicon from the complete genome. GCRV-YX246 genome consists of 11 segments RNA strand (scale variable). Labeling from outside to the inside: Contigs; COGs on the forward strand; CDS, tRNAs, and rRNAs on the forward strand; CDS, tRNAs, and rRNAs on the reverse strand; COGs on the reverse strand; GC content; GC skew

Fig. 4

Sequence analysis of GCRV-YX246. Relationships between predicted proteins in the three GCRV genotypes. Double-headed arrows indicate equivalent proteins across the genotypes

Fig. 5

Phylogenetic relationship of GCRV-YX246 to the Reoviridae family. Phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstrap replicates based on RNA-dependent RNA polymerase (RdRp) amino acid sequences

Fig. 6

Phylogenetic analysis of GCRV-YX246 and other GCRV strains was constructed based on the core protein VP6. Phylogenetic tree was performed using the neighbor-joining method with 1,000 bootstrap replicates

Fig. 7

Phylogenetic analysis of GCRV-YX246 and other GCRV-II strains was conducted using the full genome sequences, and a maximum likelihood (ML) phylogenetic tree was constructed to infer evolutionary relationships

Fig. 8

Pathogenicity analysis of highly pathogenic GCRV-YX246 in grass carp. A Hemorrhagic symptoms observed in grass carp 7 days post-infection with GCRV-YX246. B Survival curves of grass carp after GCRV-YX246 infection. n = 60. C Viral loads in various tissues of grass carp post GCRV-YX246 challenge (n = 6). The gill and gut exhibited the highest viral loads, followed by the SP and HK. D Immunofluorescence assay showing GCRV-II presence in the SP, HK, gut, and gill of infected grass carp (white arrow indicates GCRV-II virus). Scale bar, 50 μm. E Histopathological lesions in the SP, HK, gut, and gill of grass carp infected with GCRV-YX246. In the SP, the blue arrows indicate the MMCs, the red arrow shows erythrocyte infiltration, and the black arrows mark cellular necrosis. Scale bar, 50 μm

Fig. 9

Pathogenicity analysis of highly pathogenic GCRV-YX246 in rare minnows. A Hemorrhagic symptoms observed in rare minnows at 7 days post-infection with GCRV-YX246. B Survival curves of rare minnows after GCRV-YX246 infection; n = 30. C Viral loads in various tissues of rare minnows post GCRV-YX246 challenge (n = 6), with the gill and gut exhibiting the highest viral loads, followed by the SP and HK. D Immunofluorescence assay demonstrating GCRV-II presence in the SP, HK, gut, and gill of infected rare minnows (white arrow indicates GCRV-II virus). Scale bar, 50 μm. E Histopathological lesions in the SP, HK, gut, and gill of rare minnows infected with GCRV-YX246. In the SP, the blue arrow indicate the MMCs, the red arrow shows erythrocyte infiltration, and the black arrows mark cellular necrosis. Scale bar, 50 μm