Characterization of a virulent ranavirus isolated from marine ornamental fish in India

Abstract

A viral agent implicated in the mortality of marine ornamental "Similar Damselfish" (Pomacentrus similis Allen, 1991) was isolated and characterized. The virus grew well in marine and freshwater fish cell lines from seabass and snakehead. The virus was sensitive to chloroform, acidic pH (3.0) and heat treatment at 56 °C. Biochemical characterisation indicated that the virus had double stranded DNA genome. Transmission electron microscopic analysis of ultrathin sections of infected cell pellets showed iridovirus-like icosahedral virus particles of 120-130 nm. Purified virus had six structural protein bands that ranged from of 44 to 132 kDa. PCR analysis confirmed the presence of viral DNA in infected cell cultures and sequence analysis of the major capsid protein gene showed an identity of 99.82% to that of largemouth bass virus. Serum neutralization studies involving the viral agent and koi ranavirus (KIRV) indicated partial homogeneity between the two isolates. Experimental infection of seabass (Lates calcarifer) and similar damselfish (P. similis) fingerlings with the similar damselfish virus showed cumulative mortalities of 68.75 and 93.33%. The biophysical and biochemical properties of the viral agent isolated, serological characteristics, size of major capsid proteins and the sequence similarity of the MCP gene proved that the virus belongs to the genus Ranavirus of the family Iridoviridae. Ability of the virus to grow in marine and freshwater fish cell lines and its pathogenicity to one of the cultivable marine fish shows the wide host range of the virus. This is the first report of ranavirus induced mortality in marine fish in India.

Keywords: Aquaculture; India; Iridoviridae; Ornamental fish; Ranavirus; Seabass.

Fig. 1

Transmission electron micrographs of SRDV. a Electron micrograph of SRDV-infected SNKD2a cells showing virus particles in the assembly site in the cytoplasm (scale bar 500 nm) and b higher magnification of the virus particles in the cytoplasm with sizes ranging from 120 to 130 nm (scale bar 100 nm). c Virus particles budding from the plasma membrane of an infected cell acquiring the envelope (scale bar 200 nm)

Fig. 2

Acridine orange staining of SRDV infection in SNKD2a cells. a Uninfected control SNKD2a cells. b SRDV infected SNKD2a cells. Yellow coloured extrachromosomal intracytoplasmic inclusion bodies indicated by arrow

Fig. 3

SDS-PAGE analysis of structural proteins of SRDV in 12% acrylamide gel stained with 0.1% Coomassie brilliant blue. Lane 1: purified SRDV; Lane 2: medium range molecular mass markers (GeNei). Two major structural proteins of 63 and 51 kDa (thick arrow) and 4 minor proteins of 132, 101, 46 and 44 kDa (thin arrow) are indicated

Fig. 4

Agarose gel electrophoretic analysis of the PCR products obtained from SRDV infected cell culture pellet preparation with primers targeting major capsid protein gene of EHNV. a with M151 (EHNMCP1FW) and M152 (EHNMCP1RE) primers. Lane 1: SRDV infected SBCP2 cell culture pellet; Lane 2: uninfected SBCP2 cell culture pellet; Lane 3: PCR negative control; Lane 4: 100 bp molecular marker (GeneRuler, Fermentas, Thermo Scientific). b with M151 (EHNMCP1FW) and M154 (EHNMCP2RE) primers. Lane 1: SRDV infected SBCP2 cell culture pellet; Lane 2: uninfected SBCP2 cell culture pellet; Lane 3: PCR negative control; Lane 4: 100 bp molecular marker

Fig. 5

Phylogenetic tree constructed using partial MCP gene sequences of five similar ranaviruses LMBV (FR682503), KIRV (KJ939444), EHNV (FJ433873) and FV3 (AY548484) by neighbour-joining method using CLC Main Workbench indicating the relationship of SRDV with other similar ranaviruses including the type virus of the genus, FV3

Fig. 6

a Cumulative mortality of seabass fingerlings experimentally challenged with SRDV. b Cumulative mortality of similar damselfish fingerlings experimentally challenged with SRDV