Vol. 83: 97–103, 2009 DISEASES OF AQUATIC ORGANISMS Published February 12 doi: 10.3354/dao02015 Dis Aquat Org B2 protein from betanodavirus is expressed in recently infected but not in chronically infected fish Kjersti B. Mézeth1,*, Sonal Patel1, 2, Håvard Henriksen1, Anne Marie Szilvay1, Audun H. Nerland1, 2, 3 1Department of Molecular Biology, University of Bergen, Postbox 7803, 5020 Bergen, Norway 2Institute for Marine Research, Postbox 1870 Nordnes, 5817 Bergen, Norway 3Present address: The Gade Institute, University of Bergen, 5021 Bergen, Norway ABSTRACT: Betanodavirus infects both larvae and juvenile fish and can cause the disease viral encephalopathy and retinopathy (VER). During an acute outbreak of VER, infected individuals display several clinical signs of infection, i.e. abnormal swimming pattern and loss of appetite. Beta- nodaviruses can also cause chronic or persistent infection where the infected individuals show no clinical signs of infection. During infection the viral sub-genomic RNA3 and the RNA3-encoded B2 protein are expressed. Antibodies against the B2 protein from Atlantic halibut nodavirus were raised and used together with antibodies against the capsid protein to detect the presence of these 2 viral proteins in infected cells in culture and at different stages of infection in Atlantic halibut Hippoglos- sus hippoglossus and Atlantic cod Gadus morhua. The B2 protein was detected in recently infected, but not in chronically infected fish. Results suggest that the detection of B2 may be used to discrimi- nate a recent and presumably active infection from a chronic and presumably persistent infection. KEY WORDS: Fish nodavirus · Atlantic halibut · Atlantic cod · B2 protein · Viral nervous necrosis Resale or republication not permitted without written consent of the publisher INTRODUCTION the environment, indicating the possibility of horizon- tal transmission (Grotmol et al. 1999). Survivors of an The Nodaviridae family includes viruses of the outbreak can become subclinical carriers when the genus Alphanodavirus, which infect insects, and of virus persists in the nervous system for a long period the genus Betanodavirus, which infect fish (Munday of time (Johansen et al. 2004). This may lead to verti- et al. 2002, Venter & Schneemann 2008). Betanoda- cal transmission, as the virus may be reactivated dur- viruses are neurotrophic and affect the retina, the ing sexual maturation. Betanodaviruses are small, central nervous system and the ganglia of the periph- naked icosahedral viruses with a capsid composed of eral nervous system. Infection results in lesions char- multiple units of a single protein, the capsid protein acterized by cellular vacuolation and neuronal degen- (Delsert et al. 1997). The genome consists of 2 posi- eration, the disease called viral nervous necrosis (VNN) tive RNA strands, RNA1 and RNA2, which are or viral encephalopathy and retinopathy (VER). The capped but not polyadenylated (Sommerset & Ner- disease particularly affects larvae and juvenile fish, land 2004). RNA1 encodes the viral RNA dependent causing mortality rates up to 100%, and has been a RNA polymerase (RdRp) whereas RNA2 encodes the major obstacle for successful farming of several fish capsid protein (Tan et al. 2001, Sommerset & Nerland species (Grotmol et al. 1997, Lin et al. 2001, Munday 2004). During RNA replication, a sub-genomic RNA et al. 2002). During acute outbreaks, high numbers of segment called RNA3 is synthesized from the 3’ viruses are released into the environment (Nerland et region of RNA1 (Nagai & Nishizawa 1999). RNA3 al. 2007). Fish larvae are susceptible to the virus in encodes a polypeptide called B2, which is essential *Email: [email protected] © Inter-Research 2009 · www.int-res.com 98 Dis Aquat Org 83: 97–103, 2009 for both alpha- and betanodavirus replication (John- Plasmids. Total RNA was isolated from brain tissue of son et al. 2004, Fenner et al. 2006b). Atlantic halibut infected by AHNV and transcribed to RNA interference (RNAi) is a conserved evolution- cDNA for use as a template in a PCR reaction with the ary mechanism widely found in nature and is consid- primers 5’-CAC CAT GGA ACA AAT CCA ACA GGC- ered to be a cellular defense mechanism against for- 3’ and 5’-TAC ACG GGA GTG CGA CGT TGT CTG-3’. eign genes and viral infection (Cullen 2002, Plasterk The 276 bp PCR product was cloned into the pET200/ 2002). The B2 proteins from the alphanodaviruses D-Topo vector (Invitrogen) creating a plasmid encoding Flock House virus (FHV) and Nodamura virus (NoV) a B2 protein with a 6xHis tag; the resulting plasmid was and the betanodavirus greasy grouper nervous necro- named pETB2-His. Using pCMV-RNA1-myc (Mézeth et sis virus (GGNNV) have been shown to suppress RNAi al. 2007) as the template and the primers 5’-GCG GCC by binding to double stranded RNA, thus preventing GCA TAC TCT GTC TCC ATC GGC T-3’ and 5’-AAA cleavage, leading to enhanced accumulation of viral CTG ACA ACC ATG GAA CA-3’, a plasmid expressing RNAs in plant, insect and mammalian cells (Johnson et Myc-tagged B2 protein from a eukaryotic promoter was al. 2004, Lingel et al. 2005, Sullivan & Ganem 2005, constructed. The B2 stop codon TAG was changed to Fenner et al. 2006a). TAT, followed by cloning of the 248 bp fragment with Atlantic halibut nodavirus (AHNV) was first iso- NcoI and NotI overhangs into the NcoI and NotI sites lated from hatchery-reared larvae of Atlantic halibut in the pCMV/myc/cyto vector (Clontech). The result- in 1995 (Grotmol et al. 1995). The clinical signs of ing plasmid was named pcB2-Myc. Constructions were AHNV infection are loss of appetite, abnormal swim- confirmed by DNA sequencing. ming pattern, and changes in pigmentation (Munday Expression and purification of His-tagged B2. Re- et al. 2002). Mass mortality in farmed Atlantic cod combinant B2-His was expressed from the pETB2-His and diseased juveniles with similar clinical signs to vector in Escherichia coli. The protein was purified on a AHNV-infected Atlantic halibut led to the identifica- fast protein liquid chromatography (FPLC) system tion of Atlantic cod nodavirus (ACNV) (Patel et al. (Amersham Pharmacia) using a modified, scaled up, 2007, Nylund et al. 2008). The RdRp and capsid pro- Ni-NTA procedure (Qiagen) on a HiTrap chelating col- tein of AHNV and ACNV are more than 97% identi- umn (Amersham) loaded with copper. Recombinant B2- cal (GenBank accession nos. AJ401165, AJ245641, His protein was dialyzed against phosphate-buffered EF617332, EF617326), and both strains belong to the saline (PBS) and concentrated using PEG6000. barfin flounder genotype of nodavirus (Nishizawa et Cell lines, infection and transfection. The fibroblast al. 1997). Diagnosis of VER is performed by histo- cell line (SSN-1) from whole fry tissue of striped snake- pathology with identification of necrosis and vacuo- head Channa striatus was infected with AHNV lation in the brain and retina, often combined with (Frerichs et al. 1996). The cells were grown on poly-L- immunohistochemistry (IHC) using antibodies against lysine coated coverslips in a 24-well plate and infected the capsid protein (Grotmol et al. 1997). Other diag- as described previously (Mézeth et al. 2007). COS-7 nostic tests, including enzyme-linked immunosorbant cells were transfected with Lipofectamine 2000 (Gibco) assays (ELISA) and reverse transcriptase polymerase using 0.25 µg plasmid DNA and 1.25 µl Lipofectamine chain reactions (RT-PCR) for detection of the capsid 2000 per coverslip. protein and viral RNAs, respectively, have been Antibodies. Purified recombinant His-tagged B2 developed (World Organisation for Animal Health protein was used for immunization of rabbits. Bio- 2006). In the present study we found that the capsid Genes (Berlin) performed immunization and testing of protein was detected in both recently infected fish the rabbit serum (rabbit anti-B2 6073) by ELISA. and in chronically infected fish, whilst the B2 protein Before use the serum was diluted 1/10 in PBS and was only detected in recently infected fish. This sug- absorbed against COS-7 cells. The monoclonal anti- gests that detection of B2 expression can be used to body 9E10 (IgG1) recognizes the c-Myc epitope (Evan discriminate between different stages of nodavirus et al. 1985). A mouse anti-capsid serum was raised by infection. immunizing mice with purified His-tagged recombi- nant capsid protein, and testing of the serum was done by ELISA (Jordal 2004). The rabbit anti-capsid MATERIALS AND METHODS serum was described previously (Sommerset et al. 2005). For immunofluorescence (IF) analysis of sec- Sequence analysis. The deduced amino acid tions and transfected and infected cells, all primary sequences (GenBank, NCBI, Accession numbers and secondary (Texas Red and fluorescein isothio- AF319555 and NOD401165) were compared using the cyanate [FITC] conjugated species-specific from ClustalX1.83 software, applying standard default Southern Biotechnology) antibodies were diluted in parameters. 0.5% BSA in PBS. Mézeth et al.: B2 protein from betanodavirus 99 Sampling of virus-infected fish tissue. AHNV- mary antibodies anti-B2 6073 and the mouse anti-cap- infected Atlantic halibut were sampled from 2 groups. sid serum were used. Labeled cells were examined for One group consisted of larvae hatched and cultivated fluorescence and images were captured using the in 6-well Nunc plates. The larvae were infected with Leica DM RXA confocal scanning microscope with a AHNV using homogenates prepared from infected 63x oil immersion objective and Leica PowerScan soft- halibut brains (diluted 1/100) as described previously ware. Sections of brain and eye from non-infected, (Grotmol et al. 1999). The larvae were sampled 10 to chronically or recently infected fish were subjected to 15 d post-infection while mortality was observed. This IF analysis. After rehydrating, sections were blocked group is further referred to as ‘recently infected’. The with 0.5% BSA in PBS for 15 min at room temperature. other group of Atlantic halibut consisted of survivors of Primary antibody (anti-B2 6073 or anti-capsid K233) a natural VER outbreak (during larval stage) at a com- was added to the sections and incubated overnight at mercial sea farm.