Nodavirus infections in Israeli mariculture M Ucko, A Colorni and A Diamant Israel Oceanographic and Limnological Research Ltd, National Center for Mariculture, Eilat, Israel Abstract Viral encephalopathy and retinopathy (VER) infections were diagnosed in five fish species: Epi- nephelus aeneus, Dicentrarchus labrax, Sciaenops ocellatus, Lates calcarifer and Mugil cephalus cultured on both the Red Sea and Mediterranean coasts of Israel during 1998–2002. Spongiform vacuolation of nervous tissue was observed in histological sec- tions of all examined species. With transmission electron microscopy, paracrystalline arrays and pie- ces of membrane-associated non-enveloped virions measuring approximately 30 nm in diameter were observed in the brain and retina of all species. At the molecular level, the nodavirus was detected by using a primer set that amplified the T4 region of the coat protein gene. When the same set of primers was used to search for VER in an additional fish species, Sparus aurata, it was found to produce non-specific amplicons, giving rise to false-positive results. This problem was overcome by using a different primer set (F1/VR3), designed on a highly conserved region of the virus gene, which amplified a fragment of 254 bp, and confirmed that S. aurata was nodavi- rus-free. This set was validated on all five species of infected fish, as well as clinically healthy fish. Comparison of the coat protein genes from the Israeli isolated sequences indicated that more than one viral strain was involved. No strict host-specif- icity was evident. Red Sea and Mediterranean iso- lated sequences grouped in distinct clusters, together with several foreign isolates from the Mediterranean area and the Far East, as phylogenetically close to the Epinephelus akaara RGNNV type. Keywords: encephalopathy, nodavirus, Red Sea, retinopathy, RT-PCR, VER. Introduction Fish viral encephalopathy and retinopathy (VER) has emerged as a major constraint to the farming of many species of marine fish throughout the world (Munday, Kwang & Moody 2002). The first account of a similar viral-induced nervous necrosis was reported in larvae of the Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel), in Japan (Yoshikoshi & Inoue 1990), and the disease was named viral nervous necrosis (VNN). Severe outbreaks, affecting mainly Eur- opean sea bass, Dicentrarchus labrax (L.), have been reported since 1995 in Italy (Bovo, Maltese, Rosin, Borghesan, Mutinelli, Montesi & Comid 1999). VER may be the most important disease problem in halibut, Hippoglossus hippoglos- sus (L.), culture today (Bergh, Nilsen & Samuel- sen 2001). The aetiological agent was identified as a group of neuropathogenic viruses belonging to the family Nodaviridae, clearly distinguishable from insect nodaviruses (Tang, Lin, Krishna, Yeager, Schnee- mann & Johnson 2002), and lately classified in the genus Betanodavirus (Ball, Hendry, Johnson, Rue- chert & Scotti 2000). VER virus particles are spherical, non-enveloped and about 25 nm in diameter. Their genome consists of two single-stranded, positive-sense, non-polyadenylated RNA molecules with molecular weights of 1.01 · 10 6 Da (RNA1) and 0.49 · 10 6 Da (RNA2). RNA1 encodes a non-structural protein of about 100 kDa, while RNA2 is 1410 bases in length and contains a single open-reading frame (ORF), which encodes a coat protein of about 42 kDa (Mori, Nakai, Muroga, Arimoto, Journal of Fish Diseases 2004, 27, 459–469 Correspondence M Ucko, Israel Oceanographic and Limnological Research Ltd, National Center for Mariculture, PO Box 1212, Eilat 88112, Israel (e-mail: mucko@ocean.org.il) 459 Ó 2004 Blackwell Publishing Ltd