Molecular detection of a putatively novel cyprinid herpesvirus in sichel (Pelecus cultratus) during a mass mortality event in Hungary

Abstract

Summary. In the early summer of 2014, mass mortality of Sichel (Pelecus cultratus) was 34 observed in Lake Balaton, Hungary. Histological examination revealed degenerative changes 35 within the tubular epithelium, mainly in the distal tubules and collecting ducts in the kidneys 36 and multifocal vacuolisation in the brain stem and cerebellum. The routine molecular 37 investigations showed the presence of the DNA of an unknown alloherpesvirus in some 38 specimens. Subsequently, three genes were amplified and sequenced partially from the 39 putative herpesviral genome (DNA polymerase, terminase, and helicase). Phylogenetic tree 40 reconstruction, based on the concatenated sequence of these three conserved genes, implied 41 that the virus undoubtedly belongs to the genus Cyprinivirus within the family 42Alloherpesviridae. The sequences of the Sichel herpesvirus differ markedly from those of the 43 three known cypriniviruses (CyHV-1, CyHV-2 and CyHV-3); putatively representing the 44 fourth virus species in the genus. (CyHV-2), originally described as goldfish haematopoietic necrosis virus, was isolated also in 76Japan from goldfish (Carassius auratus) [15]. Cyprinid herpesvirus 3 (CyHV-3), is also 77 known as koi herpesvirus, was described from common and koi carp [13]. The complete 78 genome sequence of the above mentioned cypriniviruses and that of the Anguillid herpesvirus 79 1 (AngHV-1) was determined [1, 5, 24]. 80During the early summer of 2014, mass mortality occurred among Sichel in Lake 81Balaton (Hungary), approximately 20.000 cadavers were collected by fishermen, other species 82were not affected. The present study was aimed at genetically characterizing a novel 83 alloherpesvirus (AHV) detected in Sichel. 84Cadavers of six Sichel were sent to our laboratory (64.5-149.7 g) for histopathological 85 and molecular investigations. All cadavers were necropsied immediately after arrival with 86 routine tissue collection from the main organ systems (gills, brain, liver, kidney, spleen, 87 intestine and eyes) for histopathological examination. Tissues were fixed in 10% neutral 88 buffered formalin, routinely processed, embedded in paraffin, sectioned at approximately 5 89 micrometer, mounted on glass slides, and stained with hematoxilin and eosin. The exterior 90 mucus, gills and eyes were sampled for parasite examination through an optical microscope. 91For bacteria isolation, kidney tissue was inoculated onto blood agar plates. 92For the molecular investigations organ samples were homogenized using 93 the TissueLyser high-throughput disruption instrument (Qiagen, Hilden, Germany) according 94 to the manufacturer's recommendations. 200 µL of supernatants from organ homogenates 95 were extracted using the Roche MagNA Pure LC automated system with a Total Nucleic Acid 96Isolation Kit (Roche Diagnostics, Indianapolis, IN) and eluted in 100 µL of elution buffer. 97Subsequently, the samples were tested for the presence of CyHV-3 with an expansively used 98 PCR for the detection of cyprinid HVs [10]. 99 4 After real...

Cyprinid Herpesvirus 3

“…Abbreviated virus names are shown at the branch tips (see Table 1), and the names of genera are marked on the right. For (C), partial sequences (134-158 residues in length; some truncated from longer sequences) located between the highly conserved DF(A/T/S)(S/A)(L/M)YP and GDTDS(V/T/I)M motifs were derived from EF685904.1 for AciHV-1, HQ857783.1 (Marcos-Lopez et al, 2012) for GadHV-1, KM357278.1 (Doszpoly et al, 2015) for CyHV-4, FJ641907.1 (Doszpoly et al, 2008;Waltzek et al, 2009) for IcHV-2, FJ641908.1 (Waltzek et al, 2009) for SalHV-2, and EU349277.1 (Waltzek et al, 2009) for SalHV-3. Abbreviated virus names are shown at the branch tips (see Table 1), and the names of genera are marked on the right.…”

“…However, the positions of these viruses, and others not included in Fig. 1C, have been examined with greater discrimination using sequences from other genes (Doszpoly, Benko, Bovo, Lapatra, & Harrach, 2011;Doszpoly et al, 2008Doszpoly et al, , 2015Doszpoly, Somogyi, et al, 2011;Kelley et al, 2005;Kurobe, Kelley, Waltzek, & Hedrick, 2008;Marcos-Lopez et al, 2012). There has been some consideration of establishing subfamilies in the family Alloherpesviridae, as has taken place in the family Herpesviridae.…”

Identification of an essential virulence gene of cyprinid herpesvirus 3

“…ORF57 is conserved among the cypriniviruses, which currently comprise CyHV-1, CyHV-2, CyHV-3 and AngHV-1, with the likelihood of an expanded list in future ( Doszpoly et al., 2015 ). Amino acid sequence similarity in ORF57 ranges from 67.5 to 72.4% among the three cyprinid herpesviruses, and from 57.9% to 60.4% between these viruses and AngHV-1 ( Table S2 ).…”

“…It currently contains 12 species distributed into four genera. One of these genera, Cyprinivirus , comprises a growing list ( Doszpoly et al., 2015 ) of phylogenetically related viruses, some of which cause severe economic losses to the aquaculture industry. These include cyprinid herpesvirus 2 (CyHV-2; also known as goldfish hematopoietic necrosis virus), which causes a lethal disease in goldfish ( Carassius auratus ) and gibel carp ( C. gibelio ) ( Xu et al., 2013 , Xu et al., 2014 , Ito and Maeno, 2014 ), anguillid herpesvirus 1 (AngHV-1), which is responsible for mortalities of up to 30% in cultured and wild European and Japanese eel populations ( Anguilla anguilla and A. japonica ) ( van Beurden et al., 2012 ), and cyprinid herpesvirus 3 (CyHV-3; also known as koi herpesvirus), which is the etiological agent of a lethal disease in common carp ( Cyprinus carpio ) and koi carp ( Adamek et al., 2014 , Rakus et al., 2013 ).…”

Molecular detection of a novel cyprinid herpesvirus in roach (Rutilus rutilus) and asp (Leuciscus aspius) showing typical signs of carp pox disease

“…Few years ago, a novel cyprinid herpesvirus was reported from sichel (Pelecus cultratus) and was tentatively named "cyprinid herpesvirus 4" (CyHV-4). In the studied fish, microscopic lesions were observed in the epithelium of renal tubules, as well as congestion and multifocal vacuolisation in the brain stem and cerebellum [10].…”