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Natural transmission of the epizootic ulcerative syndrome (EUS) was conducted on naïve snakeheads Ophicephalus striatus (also known as Channa striata) kept (A) in aquifer water, (B) in lakewater, (C) cohabiting with EUS snakeheads in lakewater, and (D) cohabiting with apparently healthy snakeheads in lakewater during the 1994 to 1995 EUS season. The results showed that EUS-like lesions developed in 6 to 14 d among naïve snakeheads cohabiting with EUS snakeheads and with apparently healthy snakeheads in lakewater (Treatments C and D). Among naïve fish exposed to lakewater (Treatment B), similar lesions developed in 16 to 21 d, while naïve fish in aquifer water (Treatment A) did not develop EUS-like lesions. EUS signs began as Grade I (slight) lesions that gradually progressed to Grades III-IV (severe) 3 to 5 d from lesion onset, similar to the naturally affected EUS fish. The virus was recovered from some but not all naturally EUS-affected snakeheads, snakeheads with healing lesions and apparently healthy snakeheads, but not from naïve snakeheads. The results provide evidence of a waterborne horizontal transmission of the EUS-associated virus. This is the first report of a successful horizontal transmission of the EUS-associated virus from apparently healthy snakeheads to naïve fish under natural conditions and of virus recovery in tissue culture from naturally exposed experimental fish.

This is the first report of the isolation and characterization of a fish virus from the Philippines. The virus was isolated using snakehead spleen cells (SHS) from severely lesioned epizootic ulcerative syndrome (EUS)-affected snakehead Ophicephalus striatus from Laguna de Bay, in January 1991. The virus induced cytopathic effects (CPE) in SHS cells yielding a titer of 3.02 x 10(6) TCID50 ml(-1) at 25 degrees C within 2 to 3 d. Other susceptible cell lines included bluegill fry (BF-2), catfish spleen (CFS) and channel catfish ovary (CCO) cells. Replication in chinook salmon embryo cells (CHSE-214) was minimal while Epithelioma papulosum cyprini cells (EPC) and rainbow trout gonad cells (RTG-2) were refractory. Temperatures of 15 to 25 degrees C were optimum for virus replication but the virus did not replicate at 37 degrees C. The virus can be stored at -10 and 8 degrees C for 30 and 10 d, respectively, without significant loss of infectivity. Viral replication was logarithmic with a 2 h lag phase; viral assembly in the host cells occurred in 4 h and release of virus occurred 8 h after viral infection. A 1-log difference in TCID50 titer between the cell-free virus and the total virus was noted. Freezing and thawing the virus caused a half-log drop in titer. Viral exposure to chloroform or heating to 56 degrees C for 30 min inactivated the virus. Exposure to pH 3 medium for 30 min resulted in a more than 100-fold loss of viral infectivity. The 5-iododeoxyuridine (IUdR) did not affect virus replication, indicating a RNA genome. Neutralization tests using the Philippine virus, the ulcerative disease rhabdovirus (UDRV) and the infectious hematopoietic necrosis virus (IHNV) polyvalent antisera showed slight cross-reaction between the Philippine virus antiserum and UDRV but established no serological relationship with SHRV and IHN virus. Transmission electron microscopy (TEM) of SHS cells infected with the virus showed virus particles with typical bullet morphology and an estimated size of 65 x 175 nm. The Philippine virus was therefore a rhabdovirus, but the present study did not establish its role in the epizootiology of EUS.