Mass mortality caused by Cyprinid Herpesvirus 2 ( CyHV-2 ) in Prussian carp ( Carassius gibelio ) in China

From 2011 to 2012, hematopoietic necrosis associated with Cyprinid herpesvirus 2 (CyHV-2) caused serious mortality of farmed Prussian carp (Carassius gibelio included lethargy and inappetence, gill hemorrhages, haemorrhagic spots on body surface. Internal gross pathology included hyperaemia, hepatic hypertrophy and splenomegaly. The spleen showed extensive necrosis of the white pulp and arteries in both the kidney and spleen. Large numbers of viral particles, approximately 90-170nm in diameter with a spherical shape, were observed by liver, spleen and kidney. Sequencing of the PCR product showed more than 98% nucleotide identity with the published sequence of CyHV-2. Experimental infection with healthy Prussian carp using cause of the high mortalities. * Corresponding author’s email: liuhong@szciq.gov.cn Introduction Cyprinid herpesvirus 2 (CyHV-2), also known as herpesviral hematopoietic necrosis virus sis virus (GFHNV), is a very important viral Carassius auratus auratus L.) haematopoietic necrosis and high mortality (100%) in the spring of 1992 and 1993 (Waltzek et al., 2005). Subsequently, CyHV-2 was reported occurring in private ponds and aquaria or in et al., 1998), Taiwan (Chang et al., 1999), Australia (Stephens et al., 2004), southern England Prussian carp (Carassius gibelio) are closely a large scale in the past decade in China. Despite Bull. Eur. Ass. Fish Pathol., 32(5) 2012, 165 the wide culture and distribution of Prussian carp, few viral diseases have been reported 2012, an acute haematopoietic necrosis disease outbreak in farmed Prussian carp in central China caused serious loss, with mortality levels of 100% in some cases. This paper reports the report of CyHV-2 causing large-scale mortalities in cultured Prussian carp. Material and methods Fish samples Moribund Prussian carp, ranging from 240 to 260g, were collected between September and October, 2011, from farms undergoing a disease outbreak. During this period the pond water For experimental infection, virus-free Prussian Table 1) were obtained from Nanquan station, freshwater research center, Wuxi, China and then reared in a circulating water system with Bacteriology For bacteriological analysis, specimens of gill, liver, spleen and kidney were inoculated onto Brain Heart Infusion (BHI; Shanghai Haoran Bio Technologies Co.,Ltd, China) agar using Parasitology Smears of skin mucous, and pieces of gill tissue were examined directly for presence of parasites by light microscopy. Histological and electron microscopic examination Tissues (gill, brain, spleen, kidney and liver) processed routinely for histopathological examitions (5-7μm) were stained with haematoxylin and eosin (Sangon Biotech, China). For electron microscopy examination, tissue sections were fered to 1% aqueous osmium tetroxide (OSO4) (Sangon Biotech, China), dehydrated though a graded ethanol series and embedded in epoxy resin. Ultrathin sections were double stained with 1% uranyl acetate and 1% lead citrate. Virology – tissue culture Cell lines, fathead minnow (FHM; Gravell and Malsberger, 1965), epithelioma papilloin E-MEM (MEM prepared with Earle’s salt) (GIBCO, USA) with 10% foetal bovine serum and antibiotics (100IU/ml penicillin and 100μg/ ml streptomycin). Virus isolation Spleen and kidney were pooled individually mortar, pestle and sterile sand, suspended in E-MEM medium (containing 10% foetal bovine gation at 2057g for 15min. Supernatants were diluted to 1:100 and 1:1000 (w/v) with culture medium, and incubated on monolayer cultures of the cell lines in 25 cm2 days incubation. Cultures were examined daily 166, Bull. Eur. Ass. Fish Pathol., 32(5) 2012 using light microscopy for the appearance of a Virus transmission experiments groups were injected intraperitoneally (i.p.) with the tissue homogenate supernatant from diseased above). The control group was challenged with tissue homogenate originating from non infected tissue. Fish in all groups were injected with 1ml ity and clinical signs were examined and recorded and processed for PCR detection. Molecular biology (PCR and sequence analysis) Total nucleic acid of the tissue homogenate supernatant and the infected cell monolayers were extracted using Dneasy Blood and Tissue kit (TaKaRa, China) using the manufacture’s performed according to published protocols (Hedrick et al., 2006). The primers used are electrophoresed on a 1.5% (w/v) agarose/TAE (40 mm Tris–acetate, pH 7.2, 1 mm EDTA) gel containing 1.0 mg mL ethidium bromide and visualized under UV light. Polymerase chain reaction products were Agarose Gel DNA Extraction Kit (TaKaRa, using ABI 3730xl DNA analyzer (Applied Biosystems) at Sangon Biotech (Shanghai) Co., Ltd. The obtained sequences were aligned with BLAST search at GenBank. Results Outbreaks and gross signs of disease Information supplied by the farm owners rewhich the water temperature had reduced Table 1. Primer sets used for the detection of the virus-genome. Virus Annealing temperature/ °C Fragment Length /bp Used for CyHV (1,2or 3) CyHVpol-FOR:5’CCCAGCAACATGTGCGACGG-3’ 55 362 PCR CyHVpol-REV:5’CCGTARTGAGAGTTGGCGCA-3’a GFHNV GFHNV-HelF:5’-GGACTTGCGAAGAGTTTGATTTCTAC-3’ 60 366 PCR GFHNV-HelR:5’-CCATAGTCACCATCGTCTCATC-3’b SVCV SVC-R4:5’-CTGGGGTTTCCNCCTCAAAGYTGY-3’ 55 606 RT-PCR SVC-F1:5’-TCTTGGAGCCAAATAGCTCARRTC-3’c KHV KHV-SPHF:5’-GACACCACATCTGCAAGGAG-3’ 63 292 PCR KHV-SPHR:5’-GACACATGTTACAATGGTCGC-3’d a – Bull. Eur. Ass. Fish Pathol., 32(5) 2012, 167 carp showed anorexia and lethargy. At the on a daily basis. Consequently, these losses increased and resulted in high mortalities, but grass carp (Ctenopharyngodon idellus) and silver carp (Hypophthalmichthys molitrix) mixed by PCR). Haemorrhagic spots on the external aemia present on the submaxilla and abdomen ment haemorrhages were observed in many splenomegaly, renal hypertrophy, empty intestine and frequently petechial hemorrhaging of the swimbladder (Figure 3). No pathogenic BHI agar plates and no evidence of parasitic Histological and electron microscopic examination The most pronounced histopathological changes were observed in the spleen and kidney. The haematopoietic cells of kidneys displayed both karyopyknosis and karyorrhexis and renal tubule epithelia occasionally exhibited cloudy degeneration (Figure 4). In the spleen, necrosis with intranuclear inclusions were seen in some cells. Necrotic splenocytes showing nuclear hypertrophy, margination of chromatin, or pyknosis was observed. Infected renal hematopoietic cells showed swelling of the cytoplasm and similar nuclear pathology (Figure 5). Ultra thin sections of the hematopoietic tissue merous sub-spherical virions in the cytoplasm with diameter between 90-170 nm (Figure 6), and many clusters of virions were observed in (Figure 7). Virus isolation Virus isolation was successful on KF-1 cells from apparent CPE was observed in FHM and EPC tive by PCR). The KF-1 cell cultures exhibited slight pyknosis and lysis on the 6th day postinfection, followed by aggregation of rounded, degenerate cells. Virus transmission experiments and group 2 began to die 3-4 days post-infection with most mortalities occurring at day 7 and the control group, no clinical signs or mortality was observed. Molecular biology (PCR and sequence analysis) PCR testing of tissue samples (brain, spleen, kidney) from diseased Prussian carp were found with the CyHV and GFHNV primers. More than 98% homology was shared with the published reference strain of GFHNV (GenBank Accesthe infected KF-1 cell harvests and the tissues 168, Bull. Eur. Ass. Fish Pathol., 32(5) 2012 Figure 1. Figure 2. Figure 3. 1