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A disease outbreak was reported in adult koi, Cyprinus carpio koi, from a fish farm in Kerala, India, during June 2015. The clinical signs were observed only in recently introduced adult koi, and an existing population of fish did not show any clinical signs or mortality. Microscopic examination of wet mounts from the gills of affected koi revealed minor infestation of Dactylogyrus sp. in a few koi. In bacteriological studies, only opportunistic bacteria were isolated from the gills of affected fish. The histopathological examination of the affected fish revealed necrotic changes in gills and, importantly, virus particles were demonstrated in cytoplasm of gill epithelial cells in transmission electron microscopy. The tissue samples from affected koi were negative for common viruses reported from koi viz. cyprinid herpesvirus 3, spring viraemia of carp virus, koi ranavirus and red sea bream iridovirus in PCR screening. However, gill tissue from affected koi carp was positive for carp edema virus (CEV) in the first step of nested PCR, and sequencing of PCR amplicons confirmed infection with CEV. No cytopathic effect was observed in six fish cell lines following inoculation of filtered tissue homogenate prepared from gills of affected fish. In bioassay, the symptoms could be reproduced by inoculation of naive koi with filtrate from gill tissue homogenate of CEV-positive fish. Subsequently, screening of koi showing clinical signs similar to koi sleepy disease from different locations revealed that CEV infection was widespread. To our knowledge, this is the first report of infection with CEV in koi from India.

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Cyprinid herpesvirus-2 (CyHV-2) is a linear double-stranded DNA virus in the genus Cyprinivirus of family Alloherpesviridae. The virus is known to be highly pathogenic to ornamental goldfish (Carassius auratus), crucian carp (C. carassius) and Gibel carp (C. auratus gibelio), and also to the hybrids of goldfish and other carps. Cyprinid herpesvirus-2, having the smallest genome (290.3 kb) among Cyprinivirus, causes herpesviral hematopoietic necrosis disease (HVHND) that results in huge economic losses in aquaculture industry as the disease can cause high mortality (50-100%) among the affected fish. The disease was initially reported as the cause of epizootics in juvenile goldfish of Japan during 1992 and 1993. To date, this disease has been reported around the world including Europe, North America, Oceania and Asia. Huge economic losses due to the CyHV-2 infection among cultured gibel carp in China, during 2011-2012, mass mortality in crucian carp during 2012 in Italy, 95% mortality in goldfish during 2014 in France, 85% mortality in goldfish during 2016 in Poland had been reported. Strategies for controlling the spread of CyHV-2 are thus urgently required to limit economic damage. Furthermore, the review will shed light on lacunae in current knowledge as well as on the perspectives that merits further investigations on CyHV-2 research. The paper forms the first comprehensive overview of CyHV-2 causing a serious economically significant fish disease and, will be helpful for the researchers to get all related information from a single manuscript.

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In this study, a new cell line derived from the caudal fin of the freshwater angelfish Pterophyllum scalare was developed and characterized. The cell line was designated angelfish fin (AFF) and subcultured 44 times since its development. These cells grew well in Leibovitz's -15 medium supplemented with 10% foetal bovine saline (FBS) at 28° C and the modal chromosome number (2n) was 48. The AFF cell-line is mainly comprised of epithelial cells as confirmed by immunocytological technique using anti-cytokeratin antibodies, an epithelial cell marker. This cell line was tested for growth in a temperatures range from 20 to 37° C and at various FBS concentrations of 5-20% at 28° C. The cell line was cryopreserved at different passage levels and revived successfully with 80% survival rate. Polymerase chain reaction amplification and sequencing of partial mitochondrial 16s rRNA and coI genes confirmed that the AFF cell-line originated from angelfish. Mycoplasma sp. contamination was not detected in AFF cells and checked by Hoechst 33258 fluorescence staining. At the 42nd passage the cells were transfected with 2 μg of pAcGFP1-N1 expression vector. The AFF cells exhibited cytotoxic effects when exposed to the bacterial extra cellular products from Serratia marcescens and Proteus hauseri. The AFF cells and cells from kidney and brain did not show cytopathic effect when exposed to cyprinid herpes virus2 and viral nervous necrosis virus. The newly developed AFF cell line will be useful for the isolation of viruses affecting angelfishes, such as iridoviruses, in the future.