Molecular epidemiology of betanodavirus-Sequence analysis strategies and quasispecies influence outbreak source attribution

The quasispecies structure of nervous necrosis virus (NNV) was determined to investigate an outbreak of viral nervous necrosis disease at a barramundi (Lates calcarifer) hatchery. A traditional epidemiological investigation indicated horizontal transmission of infection between two cohorts of fish. However, variation in the viral capsid protein gene sequence from cell culture-derived viral populations and from individual fish suggested that each cohort was infected with a different virus. Molecular support for the correct epidemiological conclusion was provided by determining the consensus NNV sequence directly from multiple fish, to show that each cohort was infected with the same quasispecies. Variation in the capsid gene of isolates obtained from this quasispecies was up to 3.3% compared with sequences determined directly from fish tissue, and ≤1.7% between individual fish within each cohort. Determination of the NNV quasispecies structure supported implementation of biosecurity measures to protect fish in the hatchery from environmental sources of infection.