Isolation and partial characterization of a novel ... - Wiley Online Library

doi:10.1111/jfd.12150

Journal of Fish Diseases 2014, 37, 559–569

Isolation and partial characterization of a novel virus from different carp species suffering gill necrosis – ultrastructure and morphogenesis € tze1, M Lenk2, B Dresenkamp3, H Nieper4 and H Granzow1, D Fichtner1, H Schu T C Mettenleiter5 1 Institute of Infectology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Insel Riems, Germany 2 Department of Experimental Animal Husbandry and Biorisk Management, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Insel Riems, Germany 3 Department of Veterinary Medicine, State Institute for Consumer Protection of Saxony-Anhalt, Stendal, Germany 4 Landesuntersuchungsanstalt für das Gesundheits- und Veterin€arwesen Sachsen, Leipzig, Germany 5 Institute of Molecular Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Insel Riems, Germany

Abstract

Two isolates of a novel enveloped RNA virus were obtained from carp and koi carp with gill necrosis. Both isolates behaved identically and could be propagated in different cyprinid cell lines forming large syncytia. The virus was sensitive to lipid solvents and neither exhibited haemadsorption/ haemagglutination nor reverse transcriptase activity. Mature virus particles displayed a spherical shape with diameter of 100–350 nm after negative staining and 100–300 nm in ultrathin sections, covered by short projections of 8–10 nm in length. Maturation of virus progeny was shown to occur by budding and envelopment of the filamentous helical nucleocapsids at the cell surface. A detailed comparison of ultrastructure and morphogenesis of the novel virus isolates with selected arena-, ortho- and paramyxoviruses as possible candidates for evaluation of taxonomic classification yielded no consistency in all phenotypic features. Thus, on the basis of ultrastructure the novel virus isolates could not be assigned unequivocally to any established virus family. Correspondence H Schu¨tze, Institute of Infectology, FriedrichLoeffler-Institut, Federal Research Institute for Animal Health, Su€ dufer 10, D-17493 Insel Riems, Germany (e-mail: heike. [email protected]) Ó 2013 John Wiley & Sons Ltd

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Keywords: carp, electron microscopy, gill necrosis, morphogenesis, novel virus, ultrastructure.

Introduction

Over the past decades many viral pathogens of finfish have been identified and characterized. The reason for appearance and recognition of ever more fish diseases and pathogens is partly due to the increase in aquaculture with high concentrations of individuals favouring infectious processes and partly to concomitant development of sensitive diagnostic assays. The biological properties, molecular biological data and ultrastructural features of newly detected fish virus species frequently allowed a direct assignment to established taxa (Pilcher & Fryer 1980; Wolf 1988; Ahne & Kurstak 1989; Hetrick & Hedrick 1993; Scharschmidt 1993) as all members of a virus family exhibit identical structure and morphogenesis. Pathogenic viruses of fish have been classified by the International Committee on Taxonomy of Viruses (ICTV) as members or tentative members of 10 virus families (Wolf 1981, 1988; Hetrick & Hedrick 1993; King et al. 2012). The Iridoviridae and Herpesviridae, comprise viruses with a DNA genome, whereas the Picornaviridae, Birnaviridae, Reoviridae, Rhabdoviridae, Orthomyxoviridae,


H Granzow et al. Isolation and ultrastructure of a novel virus from carp

Paramyxoviridae, Retroviridae and Coronaviridae encompass viruses with an RNA genome. In 2000, a novel virus was isolated from white bream, Blicca bjoerkna (L.), and other cyprinids, for example, goldfish, Carassius auratus auratus (L.), and tench, Tinca tinca (L.) (Granzow et al. 2001). Subsequently, this cyprinid virus was named white bream virus (WBV) and classified by the ICTV into the new genus Bafinivirus within the subfamily Torovirinae of the Coronaviridae, (Sch€ utze et al. 2006; King et al. 2012). Five syncytia-inducing isolates from koi carp suffering from gill necrosis were analysed by Body et al. (2000) and based on their ultrastructural analyses, proposed to represent paramyxoviruses. During our attempts to isolate viruses from diseased fish, in 2000 and 2002, cultivation of organ homogenates on different cyprinid cell lines from common carp, Cyprinus carpio (L.), and koi carp Cyprinus carpio carpio, suffering gill necrosis led to the isolation of an enveloped, syncytia-inducing infectious agent with an RNA genome. In a first ultrastructural analysis of cells infected with these new isolates, virus morphogenesis steps were reminiscent of arenaviruses. Members of the Arenaviridae are lipid-enveloped, pleomorphic viruses with a diameter of mature virions in ultrathin sections between 50 and larger than 300 nm. Arenaviruses possess an envelope derived from the plasma membrane with short external surface projections of about 6–10 nm in length. In the interior, electron-dense granules (∅ 20 nm) were observed in most particles that have subsequently been identified as host-cell-derived ribosomes that, however, are apparently not important for virus replication. Aggregations of electron-dense filamentous structures in the cytoplasm, regularly covered by ribosome-like particles, possibly represent viral nucleocapsids (Murphy et al. 1970, 1976; Murphy & Whitfield 1975). In ultrathin sections of virus particles or virus infected cells, internal structural details concerning the arrangement of the nucleocapsid have been difficult to resolve. Only after negative staining, threadlike circular nucleocapsid strands could be observed after solubilization of the virion envelope by detergents (Vezza et al. 1977; Compans 1993). Assembly, maturation and egress of arenaviruses occur by budding at the plasma membrane where the clubshaped surface projections of 6–10 nm in length are visible on the virus envelope (for review see Dubois-Dalcq, Holmes & Rentier 1984; Ó 2013 John Wiley & Sons Ltd

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Compans 1993; Southern 1996; King et al. 2012). Interestingly, our two novel isolates exhibited morphogenetic features similar to arenaviruses prompting the hypothesis that they may indeed represent (the first?) fish viruses of this family. For more detailed characterization and possible assignment of the taxonomic status, parallel comparative ultrastructural analyses by electron microscopy were performed on cell cultures infected by the novel isolates as well as representative members of the Paramyxoviridae, Orthomyxoviridae and Arenaviridae. Besides molecular biological and biochemical similarities, members of an established virus family exhibit common ultrastructural features and steps of morphogenesis (King et al. 2012). Thus, electron microscopic data on morphology and/or morphogenesis of a virus isolate continue to yield important details concerning its taxonomic position.

Materials and methods

Cell cultures For virus isolation and propagation, the following cell lines were used: EPC (CCLV-RIE 173), FHM (fathead minnow; CCLV RIE 57; Fryer & Lannan 1994), CCB (Common carp brain, CCLV RIE 816; Neukirch, Boettcher & Bunnajirakul 1999), CaPi (pituitary gland of 3 years old carp, CCLV RIE 112) and To/F (Black-head minnow, CCLV RIE 116). All cell lines were obtained from the Collection of Cell Lines in Veterinary Medicine (CCLV) of the FriedrichLoeffler-Institut, Greifswald-Insel Riems. Fish cells were grown to confluency in 25 cm2 cell culture flasks at 26 °C in Eagle‘s minimal essential medium (MEM) supplemented with 10% foetal bovine serum. Viruses Isolate DF 20/00 was obtained from a diseased common carp originating from fish farms in the province of Saxony (Germany) with high morbidity and mortality. Clinical signs were reddening of the skin with a rough texture, focal loss of epidermis, overproduction of mucus on skin and gills, as well as necrosis and inflammation in the gills. Swelling of the kidney and spleen and reddening of the intestine was also observed. Isolate DF 26/ 02 was obtained from a diseased koi carp in the



province of Saxony-Anhalt, which had been imported from Belgium displaying distinct petechiae in the gills and skin and yellow foci in liver, kidney and spleen. Both isolates were obtained by incubation of suitable cell cultures with different organ homogenates in accordance with the requirements of the EU (Anonymous 1992). All infection experiments were performed with supernatants of cell cultures exhibiting cytopathogenic alterations, diluted in medium 1:100 and incubated at 26 °C. Cells were harvested for further analysis when cellular alterations, syncytia and plaques became visible. For comparative studies with representative members of selected RNA virus families, Vero cells were cultivated in T 25 flasks in a 1:1 mixture of minimal essential medium (MEM, Hanks’ salts) and MEM (Earle’s salts) supplemented with non-essential amino acids (Roth), sodium pyruvate (Sigma) and 10% foetal bovine serum (Invitrogen) and infected with the arenavirus lymphocytic choriomeningitis virus (LCMV) strain ‘Armstrong E 350’. Between 48 and 96 h p.i., cells were harvested and processed for electron microscopy. MDBK cells (CCLV RIE 261) were infected with the paramyxovirus. Newcastle disease virus strain ‘La Sota’ and salmon head kidney cells (SHK-1) were inoculated with the orthomyxovirus infectious salmon anaemia virus strain ‘ISAV 11’. After onset of cytopathogenic alterations, the monolayers were fixed and processed for electron microscopy as described by Granzow et al. (1997, 1999). Inactivation studies To further characterize the infectious agent, the lipid solvent sensitivity was determined as viruses with a lipid envelope are inactivated by chloroform. To this end, 10% chloroform (Roth, Germany) was added for 4 h at 4 °C to suspensions of DF 20/00 and DF 26/02 as well as birnavirus (infectious pancreatic necrosis virus, IPNV, European reference strain Sp, obtained from Dr Olesen, Denmark; Jørgensen & Grauballe 1971) and rhabdovirus (viral haemorrhagic septicaemia virus, VHSV strain F1; Deuter & Enzmann 1986) as positive controls. After centrifugation at 2000 g, the supernatant was titrated and compared to untreated samples. Ó 2013 John Wiley & Sons Ltd

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Haemagglutination assay Haemadsorption (red blood cell concentration 0.75%, incubation at 20 °C for 30 min) and haemagglutination (red blood cell concentration 0.5%, incubation at 4 °C for 30 min) were performed with erythrocytes from humans (group 0 and A), chicken, pig, rat and rabbit according to standard procedures (Mayr et al. 1977). Red blood cells from fish are not used for haemagglutination assays due to lack of standardized methods. Nucleic acid inhibition assay Cell cultures were infected with DF 20/00 and DF 26/02, an iridovirus [epizootic haematopoietic necrosis virus, EHNV Isolate 86/8774 (Langdon, Humphrey & Williams 1988), gratefully obtained from Dr Olesen, Denmark] and a birnavirus (IPNV, see above) at a multiplicity of infection (multiplicity of infection m.o.i. is the ratio of the number of infectious virus particles to the number of target cells) of 0.1 and grown under addition of 1000 lg mL 1 cell culture medium or 100 lg mL 1 cell culture medium 5-iodo-2’deoxyuridine (IDU, Serva). Infectivity was determined 4 days post-infection by titration. In contrast to RNA viruses, replication of DNA viruses is inhibited by IDU. Reverse transcriptase assay. The reverse transcriptase assay (Roche Diagnostics, Mannheim, Germany, # 11468120910) was conducted according to the manufacturer’s protocol. Isolation of virus particles from isolate DF 20/00 was done by ultracentrifugation of cell culture supernatant. The resulting pellet was lysed and added to the reaction mixture. Human immunodeficiency virus (HIV-1) revertase from the kit served as positive control and standard for the reaction, whereas lysate of bovine leukaemia virus (BLV)-infected cells served as positive control for sample preparation and reaction. Digoxigenin-labelled DNA synthesized by reverse transcriptase was detected by immunological and colorimetric means. Electron microscopy. For negative staining of virus particles in suspension, formvar-coated nickel grids were placed for 7 min on drops of cell culture supernatant or suspension of cell culture sediments. Negative staining was performed with 2% phosphotungstic acid (PTA, pH 6.0 or 7.4) for 7 min.



The appearance of syncytia and plaques in the infected cell cultures was observed by light microscopy 48–120 h p.i. Embedding of cell cultures at different times after infection in epoxy resin was performed as previously described (Granzow et al. 1997). Briefly, infected cell cultures were fixed after the appearance of CPE in 2.5% glutaraldehyde in 0.1M cacodylate buffer (300 mOsmol, pH 7.2) followed by 1% buffered osmium tetroxide. Cells were then dehydrated stepwise in a graded series of ethanol and embedded in glycid ether 100. Ultrathin sections were double stained with uranyl acetate and lead citrate (Reynolds 1963). Specimens were examined at 80 kV with a transmission electron microscope Tecnai 12 (Philips) or Tecnai Spirit (FEI).

Results

Biochemical analyses Chloroform treatment significantly reduced infectivity of the novel virus isolates indicating that a lipid envelope is required to maintain infectivity. In contrast, treatment with 5-iodo-2-deoxyuridin did not impair virus replication significantly in contrast to the included DNA-containing iridovirus EHNV (Table 1). Neither in cells infected with isolate DF 20/00 (Table 2) nor after infection with DF 26/02 (data not shown) was any reverse transcriptase activity detectable. Haemadsorption and haemagglutination assays performed with human erythrocytes (group 0 and A), chicken, pig, rat and rabbit were negative. As a positive haemagglutinating control, orthomyxovirus from koi carp was included. A comparison of all test results is shown in Table 3. Electron microscopical analyses Isolates DF 20/00 and DF 26/02. After negative staining of the cell culture supernatant, virus Table 1 Nucleic acid inhibition assay* Virus

1000 lg IDUmL 1

100 lg IDUmL

DF 20/00 DF 26/02 Iridovirus, EHNV Birnavirus (IPNV)

3.0/2.0 4.5/4.5 2.5/3.5 5.5/5.5