Genomic classification of new betanodavirus isolates by phylogenetic analysis of the coat protein gene suggests a low host-fish species specificity Richard Thie ´ ry, 1 Joe ¨ lle Cozien, 1 Claire de Boisse ´ son, 2 Soasig Kerbart-Boscher 1 and Laurent Ne ´ varez 1 Correspondence Richard Thie ´ry r.thiery@brest.afssa.fr 1 French Food Safety Agency, Fish Infectious Diseases and Parasitology Unit, BP 70, F-29 280 Plouzane ´ , France 2 French Food Safety Agency, Viral Genetics and Biosafety Unit, BP 53, F-22 440 Ploufragan, France Received 7 May 2004 Accepted 5 July 2004 Viral encephalopathy and retinopathy is a devastating disease that causes neurological disorders and high mortality in a large number of cultivated marine fish species around the world. It is now established that several viral strains classified in the genus Betanodavirus of the family Nodaviridae are the aetiological agents of this disease. Betanodaviruses can be classified into four genotypes based on the coat protein gene sequence. Here, the coat protein genes of the three major strains isolated from sea bass (Dicentrarchus labrax) in France were found to be different. In addition, 21 novel strains of betanodavirus from several fish species from France, Spain, Tunisia and Tahiti were classified by using phylogenetic analysis of a partial sequence (383 nt) of the coat protein gene. Most of the isolates were grouped in the red-spotted grouper nervous necrosis virus type, which was subdivided into two subtypes, one of them containing only French isolates. Furthermore, an isolate obtained from sea bass during an outbreak at low temperature (15 6C) was classified as the barfin flounder nervous necrosis virus type. This is the first reported isolation from sea bass of such a strain, which is known to infect several cold-water marine fish species. In addition, a betanodavirus belonging to the striped jack nervous necrosis virus type was detected in Senagalese sole (Solea senegalensis) farmed in Spain, which is the first indication of the presence of this genotype outside Japan. These findings suggest that the different genotypes can infect a variety of fish species and thus have a low host-fish species specificity. INTRODUCTION Betanodavirus is a recently recognized genus of the family Nodaviridae, which had previously been found only in insects (Ball et al., 2000). Viruses belonging to this genus are the causative agent of viral encephalopathy and retinopathy (VER), also known as viral nervous necrosis, a devastating disease of many species of marine fish cultured worldwide (Munday et al., 2002). Affected fish commonly display neurological disorders, which are often associated with strong vacuolization of the central nervous system and the retina. Betanodaviruses are small, spherical, non-enveloped viruses with a genome that is composed of two single-stranded, positive-sense RNA molecules. The larger genomic segment, RNA1 (3?1 kbp), encodes the RNA-dependent RNA poly- merase (Chi et al., 2001; Nagai & Nishizawa, 1999; Tan et al., 2001), whilst the coat protein is encoded by RNA 2 (1?4 kbp) (Delsert et al., 1997; Nishizawa et al., 1994). Comparison of the coat protein gene of five fish nodaviruses identified a highly conserved region (aa 83–216) and a variable region (aa 235–316) with amino acid sequence identities of 93 and 62 %, respectively (Nishizawa et al., 1995). A classification of betanodaviruses based on comparison of the variable region of the coat protein gene among 25 isolates from farmed fish from Japan, Thailand, Italy and Australia was proposed by the same group (Nishizawa et al., 1997). According to this, betanodaviruses could be classified into four types, designated striped jack nervous necrosis virus (SJNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV) and red-spotted grouper nervous necrosis virus (RGNNV). The same classification was subsequently used The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ698093–AJ698113. 0008-0264 G 2004 SGM Printed in Great Britain 3079 Journal of General Virology (2004), 85, 3079–3087 DOI 10.1099/vir.0.80264-0