First report of natural Cyprinid herpesvirus-2 infection associated with fatal outbreaks of goldfish (Carassius auratus) farms in Thailand

Abstract

Two separate fatal outbreaks occurred in ornamental goldfish (Carassius auratus) farms, marked by clinical signs including depression, scale protrusion, massive mucus production, and mortality rates exceeding 50% were identified in Thailand. Detailed histological examination revealed extensive necrosis of hematopoietic tissues in kidneys and spleen, together with marked necrotizing branchitis and brachium hyperplasia. Furthermore, characteristic intranuclear inclusion bodies with margination of chromatin were observed in hematopoietic cells in the spleen and kidneys, and in gill epithelial cells. Conventional polymerase chain reaction (PCR) detected Cyprinid herpesvirus-2 (CyHV-2) DNA in the affected populations, with no presence of CyHV-1 or CyHV-3 DNA. Using quantitative PCR (qPCR), we found that viral DNA largely localized within the spleen and kidneys, followed by gills, heart, brain, and liver. In situ hybridization demonstrated CyHV-2 tropism, with localization identified in hematopoietic cells in the kidney and spleen, and in epithelial cells in the gill lamellae and skin. Apart from hematopoietic cells in kidneys and spleen, transmission electron microscopy confirmed herpesviral particles within the epithelial cells in the gills and skin of infected fish. Genomic analysis was conducted on partial genome sequences of CyHV-2 DNA polymerase and helicase genes, and the tandem repeats region were obtained and subsequently characterized using next generation sequencing-based approach. This data suggested that these two CyHV-2 Thai strains were nearly identical and closely related to other CyHV-2 strains found in Asia. Genomic analysis based on the helicase gene revealed that the CyHV-2 Thai strains were unique and distinct by creating monophyletic clade. Our findings report the first identification of CyHV-2 in Thailand for first time, and highlight its potential pathological implications. While these Thai sequences of CyHV-2 are indeed unique, further investigations are in progress to confirm the definitive role of this genetic variability in relation to its virulence.