Isolation, Identification, and Genomic Analysis of a Novel Reovirus from Healthy Grass Carp and Its Dynamic Proliferation In Vitro and In Vivo.

Ke Zhang,Wenzhi Liu,Lingbing Zeng,Yan Meng,Yong Zhou,Yiqun Li,Yuding Fan,Vikram N Vakharia

doi:10.3390/v13040690

Abstract

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5' and 3' termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57–96.71% protein sequence identity) but shared only 22.65–45.85% and 23.37–43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus-C (AqRV-C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 104 copies/μL.

Highlights

Grass carp (Ctenopharyngodon idella) is an important freshwater aquaculture fish that is cultured widely in Asian countries
Given that the grass carp were healthy, we hypothesized that the carp might be carriers, the virus is nonpathogenic, or it has low virulence in grass carp. We identified this new reovirus strain as healthy grass carp reovirus (HGCRV), and its biological characteristics were studied
HGCRV was cultured in a grass carp ovary (GCO) cell line from homogenates of grass carp tissues at

Summary

Introduction

Grass carp (Ctenopharyngodon idella) is an important freshwater aquaculture fish that is cultured widely in Asian countries. Serious outbreaks of grass carp hemorrhagic disease (GCHD) occur frequently in many freshwater farms in southern China, which is associated with high mortality (up to 85% of fingerlings and yearlings), causing great economic losses [1,2]. The pathogen that causes GCHD was named grass carp reovirus (GCRV) and was identified as a new member of the family Reoviridae in 1983 [3]. Aquareovirus was initially classified in the family Reoviridae in 1991 [4]. To the best of our knowledge, there is no standard criterion for the species classification of the Aquareovirus; seven species (AqRV-A to AqRV-G) and some other unassigned species have been recognized, mainly based on the RNA-RNA hybridization analyses, sequence analysis, antigenic properties, and other characteristics. Recent studies have found that the identification of species belonging to the Aquareovirus genus has been mainly based on the RNA-dependent

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