Comparative analysis of both genomic segments of betanodaviruses isolated from epizootic outbreaks in farmed fish species provides evidence for genetic reassortment J. G. Olveira, 1 S. Souto, 1 C. P. Dopazo, 1 R. Thie ´ ry, 2 J. L. Barja 1 and I. Bandı ´n 1 Correspondence Isabel Bandı ´n isabel.bandin@usc.es 1 Unidad de Ictiopatologı ´a-Patologı ´a Viral, Departamento de Microbiologı ´a y Parasitologı ´a, Instituto de Acuicultura, Universidad de Santiago de Compostela, Spain 2 French Food Safety Agency, BP111, F-06902 Sophia Antipolis, France Received 12 June 2009 Accepted 24 August 2009 Sequencing of the full coding region of both genomic segments of seven betanodavirus strains isolated from different farmed species in Spain and Portugal revealed that six were reassortants, exhibiting a red-spotted grouper nervous necrosis virus (RGNNV)-type RNA1 and a striped jack nervous necrosis virus (SJNNV)-type RNA2. Analysis of sequences of reassortant strains at both the genomic and protein levels revealed the existence of differences compared with type strains of both genotypes. These differences were greater in the polymerase sequence, which is remarkable because viral structural proteins generally diverge more rapidly than non-structural proteins. Changes in two amino acids observed in the SJNNV capsid protein might be involved in the colonization of new host species by these reassortant strains. In addition, a more extensive phylogenetic analysis, including partial sequences of both RNA segments of 16 other Iberian nodaviruses, confirmed the existence of reassortment between RGNNV and SJNNV. INTRODUCTION The family Nodaviridae contains small (25–30 nm), non- enveloped, icosahedral RNA viruses, grouped into two genera, Alphanodavirus and Betanodavirus, which infect a wide range of insects and fish, respectively (Schneemann et al., 2005). Betanodaviruses are the aetiological agents of the disease known as viral nervous necrosis or viral encephalopathy and retinopathy, a devastating neuro- pathological condition that affects marine fish worldwide (Munday et al., 2002). The genome of betanodaviruses consists of two single- stranded, positive-sense RNA molecules. The larger genomic segment, RNA1 (3.1 kb), encodes the RNA- dependent RNA polymerase (RdRp) of approximately 100 kDa, also named protein A (Nagai & Nishizawa, 1999; Tan et al., 2001). The smaller segment, RNA2 (1.4 kb), encodes the capsid protein of about 42 kDa (Delsert et al., 1997; Nagai & Nishizawa, 1999). In addition, a subgenomic RNA3 is synthesized during RNA replication from the 39 terminus of RNA1. RNA3 encodes a protein called B2 (Sommerset & Nerland, 2004; Iwamoto et al., 2005), which suppresses cellular RNA interference (Iwamoto et al., 2005; Fenner et al., 2006a, b). To date, a limited number of full-length betanodavirus RNA1 sequences have been published (Nagai & Nishizawa, 1999; Iwamoto et al., 2001, 2004; Tan et al., 2001; Sommerset & Nerland, 2004; Okinaka & Nakai, 2008), whereas complete nucleotide sequences of RNA2 are available for a larger number of isolates. RNA2 segment contains a highly variable region (the so- called T4 region) (Nishizawa et al., 1995), which encodes the C-terminal half of the capsid protein. Comparison of nucleotide sequences of the T4 region was used by Nishizawa et al. (1997) to classify betanodaviruses into four genotypes: striped jack nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus (TPNNV), red-spotted grouper nervous necrosis virus (RGNNV) and barfin flounder nervous necrosis virus (BFNNV). This classification is currently used to genotype new isolates in different countries (Aspehaug et al., 1999; Starkey et al., 2000; Dalla Valle et al., 2001; Skliris et al., 2001; Johnson et al., 2002; Chi et al., 2003; Gagne ´ et al., 2004; Johansen et al., 2004). A different nomenclature for the genotypes was proposed by Thie ´ry et al. (2004), with the groups named I, II, III and IV corresponding to RGNNV, BFNNV, TPNNV The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are FJ803911–FJ803923, FJ829452 and GQ131547–GQ131571. A table of primer sequences used for sequencing is available with the online version of this paper. Journal of General Virology (2009), 90, 2940–2951 DOI 10.1099/vir.0.013912-0 2940 013912 G 2009 SGM Printed in Great Britain