Cyprinid herpesvirus 2 (CyHV-2), which infects goldfish and crucian carp causing high mortality, is an emerging viral pathogen worldwide. The genome of CyHV-2 is large and comprises double-stranded DNA, including several genes similar to cyprinid herpesvirus 1, ictalurid herpesvirus-1, cyprinid herpesvirus 3, and ranid herpesvirus-1. Genes of DNA viruses are expressed in three temporal phases: immediate-early (IE), early (E), and late (L) genes. Viral IE genes initiate transcription as soon as the virus enters the host, without viral DNA replication. IE gene products enable the efficient expression of E and L genes or regulate the host to initiate virus replication. In the present study, five IE genes of CyHV-2 were identified, including open reading frame (ORF)54, ORF121, ORF141, ORF147, and ORF155. Time course analysis and reverse transcription polymerase chain reaction confirmed five IE genes, thirty-four E genes, and thirty-nine L genes. In addition, all 150 ORFs identified in the CyHV-2 genome are transcribed, and are expressed in chronological order, similar to other herpesviruses. This study is the first to identify the IE genes of CyHV-2, which will provide more information for viral molecular characterization.

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Cyprinid herpesvirus 2 (CyHV-2) has caused great economic loss to the crucian carp breeding industry. Upon viral stimulation, eukaryotic cells generally activate the expression of anti-oxidative genes to maintain the intracellular oxidative balance and resist viral infection. Here, intracellular reactive oxygen species (ROS) levels in CyHV-2-infected cells were monitored to show that CyHV-2 induced the increase of intracellular ROS during early infection, and intracellular excessive accumulation of ROS was ameliorated during late infection, which was accompanied by activated expression of genes related to Nrf2 signaling pathway. In order to explore the interaction between CyHV-2 infection and ROS production, RyuF-2 cells were treated with either antioxidant epigallocatechin-3-gallate (EGCG) or berberine hydrochloride (BBH) and then infected with CyHV-2. Both BBH and EGCG could effectively inhibit the amplification of CyHV-2 while inhibiting the accumulation of intracellular ROS. Consistent with this, the oxidant stress-related genes were up-regulated by CyHV-2 infection and down-regulated in cells treated with either BBH or EGCG, through which the production of intracellular ROS was modulated. These results collectively demonstrated that early ROS accumulation favored the replication of CyHV-2, while antioxidants (BBH and EGCG) could inhibit the amplification of CyHV-2 by inhibiting ROS induction.

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