Grass carp reovirus (GCRV) is responsible for viral hemorrhagic disease in cultured grass carp (Ctenopharyngon idellus). GCRV is a non-enveloped, double-stranded RNA virus in the genus Aquareovirus, of the family Reoviridae, which encodes seven structural proteins (VP1-VP7) and five nonstructural proteins (NS80, NS38, NS31, NS26, and NS16). To date, the mechanism of GCRV entry into CIK Ctenopharyngon idellus kidney (CIK) cells remains poorly understood. Here, we present a study of the GCRV internalization mechanism in CIK cells. Our results indicated that GCRV infection was inhibited by chlorpromazine, the specific inhibitor for clathrin-mediated endocytosis. Colocalization of GCRV virions with endogenous clathrin was observed during early infection by confocal microscopy. Moreover, GCRV infection of CIK cells depended on acidification of the endosome. This was indicated by significant inhibition of viral infection following prophylactic treatment with the lysosomotropic drugs chloroquine or ammonium chloride. In addition, the disturbance of dynamin activity blocked GCRV entry, which confirmed the dynamin-dependent nature of clathrin-mediated endocytosis. Our findings suggest that GCRV might enter CIK cells via clathrin-mediated endocytosis in a pH-dependent manner. Additionally, dynamin is critical for efficient viral entry.

The channel catfish virus (CCV, Ictalurid herpesvirus 1) has caused sustained economic losses in the fish industry because of its strong infectivity and pathogenicity. Thus, it is necessary to determine the function of viral proteins in the CCV infection process. The present study aimed to characterize CCV glycoprotein ORF59 and explore its impact on virus infection in host cells. Firstly, its exclusive presence in the membrane fraction of the cell lysate and subcellular localization verified that CCV ORF59 is a viral membrane protein expressed at late-stage infection. A protein blocking assay using purified His6 tagged ORF59, expressed in sf9 insect cells using a baculovirus expression system, indicated a dose-dependent inhibitory effect of recombinant ORF59 protein on virus invasion. Knockdown of the ORF59 using a short hairpin (shRNA) showed that ORF59 silencing decreased the production of infectious virus particles in channel catfish ovary cells. The results of this study suggest that recombinant ORF59 protein might inhibit CCV entry into the host cells. These findings will promote future studies of the key functions of glycoprotein ORF59 during CCV infection.

Cyprinid herpesvirus 2 (CyHV-2) is a causative factor of herpesviral hematopoietic necrosis (HVHN) in farmed crucian carp (Carassius carassius) and goldfish (Carassius auratus). In this study, we analyzed the genomic characteristics of a new strain, CyHV-2 SH-01, isolated during outbreaks in crucian carp at a local fish farm near Shanghai, China. CyHV-2 SH-01 exhibited a high sensitivity to goldfish and crucian carp in our previous research. The complete genome of SH-01 is 290,428 bp with 154 potential open reading frames (ORFs) and terminal repeat (TR) regions at both ends. Compared to the sequenced genomes of other CyHVs, Carassius auratus herpesvirus (CaHV) and Anguillid herpesvirus 1 (AngHV-1), several variations were found in SH-01, including nucleotide mutations, deletions, and insertions, as well as gene duplications, rearrangements, and horizontal transfers. Overall, the genome of SH-01 shares 99.60% of its identity with that of ST-J1. Genomic collinearity analysis showed that SH-01 has a high degree of collinearity with another three CyHV-2 isolates, and it is generally closely related to CaHV, CyHV-1, and CyHV-3, although it contains many differences in locally collinear blocks (LCBs). The lowest degree of collinearity was found with AngHV-1, despite some homologous LCBs, indicating that they are evolutionarily the most distantly related. The results provide new clues to better understand the CyHV-2 genome through sequencing and sequence mining.