Abstract
By a broad-range PCR, we detected a novel herpesvirus (HV) in the specimen of a wels catfish (Silurus glanis) presenting disseminated, carp pox-like dermal lesions all over its body. The sequence analysis of the 463-bp PCR product from the viral DNA polymerase gene indicated the presence of a hitherto unknown virus, a putative member of the family Alloherpesviridae in the sample. Another PCR, targeting the terminase gene of fish HVs, provided an additional genomic fragment of over 1,000 bp. Surprisingly, the sequence of a co-amplified, off-target PCR product revealed its origin from a putative gene homologous to ORF87 and ORF45 of cyprinid HVs and anguillid herpesvirus 1 (AngHV-1), respectively. With specific primers, designed according to the genomic maps of the cyprinid and anguillid HVs, a genomic fragment of 15 kb was also amplified and sequenced by primer walking. In phylogeny inferences, based on several genes, the putative wels catfish HV clustered closest to various cyprinid HVs or to AngHV-1. The novel virus, named as silurid herpesvirus 2, represents a distinct species in the genus Cyprinivirus. However, its association with the skin disease remains unclear.
Introduction
Wels catfish (Silurus glanis), also called sheatfish, is one of the largest freshwater fishes native to parts of Europe and Asia in the wild. As an apex predator and bottom feeder in natural waters, it plays a role in maintaining healthy aquatic ecosystems (Vejrík et al., 2017; Cucherousset et al., 2018). Young catfish flesh is valued as human food hence the species is becoming increasingly important, besides fishing, also in aquaculture worldwide. With the intensification of fish breeding, the significance of knowledge concerning different viral diseases of fish has also increased markedly in recent years (Kibenge, 2019).
Herpesvirus-like agents, sometimes causing devastating diseases, have emerged as important pathogens of aquatic animals (Hanson et al., 2011). These viruses, occurring in fishes and amphibians, are only distantly related to the herpesviruses of mammals, birds and reptiles, i.e. members of the conventional family Herpesviridae, and are now classified in a separate family, Alloherpesviridae within the order Herpesvirales (Davison et al., 2009). Presently, the family Alloherpesviridae is divided into four genera that are named after the major host animal groups, from which the viruses therein had been described. Thus, the genus Batrachovirus has been established for the alloherpesviruses originating from frogs. Another genus, named Salmonivirus, has been created for the classification of alloherpesviruses of different salmonid fishes including the rainbow trout (Oncorhynchus mykiss), masou salmon (Oncorhynchus masou), coho salmon (Oncorhynchus kisutch), sockeye salmon (Oncorhynchus nerka) and the lake trout (Salvelinus namaycush) (Waltzek et al., 2009). The genus Ictalurivirus contains the very first piscine herpesvirus with fully sequenced genome, i.e. the channel-catfish herpesvirus, officially named as ictalurid herpesvirus 1 (IcHV-1) (Davison, 1992). Similar alloherpesviruses have been reported from fishes belonging to the Ictaluridae and Siluridae families of the order Siluriformes. The genome of a herpes-like virus from the black bullhead (Ameiurus melas), farmed in Italy (Borzák et al., 2018), as well as from the glass catfish (Kryptopterus bicirrhis) from Thailand (Henriquez et al., 2018) have also been sequenced entirely. The official species name of these virus types are Ictalurid herpesvirus 2 and Silurid herpesvirus 1, respectively. Both viruses have been isolated during mass mortality events among the respective farmed fish. It is worth mentioning that an additional alloherpesvirus, acipenserid herpesvirus 2 (AciHV-2) from an evolutionarily rather distant fish species, namely the white sturgeon (Acipenser transmontanus) has also been found to belong to the virus lineage that constitutes the genus Ictalurivirus (Doszpoly et al., 2011). This is an interesting fact, especially considering that another virus from the same host (AciHV-1) occupies a long, solitary branch in the phylogeny tree reconstructions, so that its genus affiliation has not yet been determined (Kurobe et al., 2008). The genus Cyprinivirus encompasses a number of herpes-like viruses of the common carp (Cyprinus carpio) along with other HVs, discovered in closely, or in a couple of cases in more distantly, related fishes (Davison et al., 2013; Borzák et al., 2020; Sellyei et al., 2020). Cyprinid HV-1 (CyHV-1) is the aetiological agent of the so-called carp pox disease, frequently seen among farmed carp. Besides skin lesions, this virus can cause acute septicaemia in young fish but chronic, latent or ‘dormant’ infections occur as well. Two additional viruses of cyprinid fish, CyHV-2 and CyHV-3, are associated with devastating diseases of the goldfish (Carassius auratus) and the ornamental koi carp, respectively (Hanson et al., 2011). The anguillid herpesvirus (AngHV-1) isolated from farmed European eels (Anguilla anguilla) in the Netherlands (Davidse et al., 1999) is also classified into the genus Cyprinivirus (van Beurden et al., 2010).
More than four decades ago, an outbreak, accompanied by high mortality and skin erosions resembling the signs of the so-called carp pox disease, was observed among 2-year-old wels catfish cultured in net cages in Hungary. Based on light and electron microscopic studies, the causative agent was suspected to be a herpesvirus (Békési et al., 1981), but molecular confirmation of the virus has not been described since then. Here, we present the detection, partial sequencing and phylogenetic analysis of a hitherto unknown herpesvirus, obtained from a wels catfish showing striking skin lesions.
Materials and methods
Origin of the fish and the test material
During an interim sampling process of a farm-scale feeding trial, conducted in collaboration with a Hungarian fish farm, disseminated skin rashes were noticed on the body surface of two of the six experimental fish, which were sent to our laboratory for health monitoring and evaluation. In the fish farm, no other obvious signs of disease or mortality were observed among the 2-year-old wels catfish. One fish with the most prominent rash was used for PCR testing for a possible viral origin of the skin disease. A pea-sized piece from the damaged skin region was placed in 1 mL 1 × TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and homogenised in a TissueLyser LT disruption instrument (Qiagen, Germany) at 50 Hz for 10 min. Purification of the DNA from the sample was made as described previously (Dán et al., 2003).
PCR, molecular cloning and sequencing
Since the skin rash resembling the so-called carp pox disease suggested the involvement of a herpes-like virus, first the amplification of a fragment from the herpesviral DNA-dependent DNA polymerase gene (pol) was attempted by using a broad-range PCR with consensus primers (Hanson et al., 2006). As the sample tested positive, further PCRs were performed to acquire fragments from other conserved genes, such as the terminase and helicase of alloherpesviruses. We used consensus primers and PCR protocols that had been designed and published previously (Doszpoly et al., 2015). Additionally, a new consensus forward primer (5′-TTYGTNTWYAAYTAYGAYTTYGA-3′), designed on the basis of a stretch of conserved amino acids (aa) with the possible fewest codons in the DNA polymerase alignments (unpublished results) was used for the amplification of a longer part from the pol gene. The PCR was performed with an initial denaturation step at 98 °C for 180 s, followed by 45 cycles consisting of denaturation at 98 °C for 10 s, annealing at 46 °C for 30 s and elongation at 72 °C for 60 s. The final elongation step was at 72 °C for 300 s. We used the Phusion Green High-Fidelity DNA Polymerase enzyme (Thermo Scientific™) with reaction mixtures as described before (Doszpoly et al., 2015). Coincidentally, an unexpected PCR product was also amplified from a genomic region close enough to the pol fragment so it seemed worthwhile to try a bridging PCR between the two. Based on the genome maps of CyHVs and AngHV-1, two pairs of specific, nested primers were designed: outfo: 5′-AACATCGTCGATTCGAGCCTGC-3′; outre: 5′-GACGTTCGCCATCAACTACCTG-3′ and info: 5′-TCGAGTTCGTGATGCGTCTGAG-3’; inre: 5′-TCGATCAAGGGCACGTCTACAC-3’. The successful amplification was performed with the PrimeSTAR® GXL DNA Polymerase (Takara Bio Inc.) enzyme according to the manufacturer's recommendations, in 50 µL final volume consisting of 31 µL MilliQ water, 10 µL 5 × PrimeSTAR GXL Buffer, 4 µL dNTP mixture (10 mM), 1 µL (15 pM) of each (forward and reverse) primer, 2 µL PrimeSTAR GXL DNA Polymerase and 1 µL target DNA. The PCR program consisted of an initial denaturation at 98 °C for 180 s, followed by 45 cycles with denaturation at 98 °C for 10 s, annealing at 60 °C for 15 s and elongation at 68 °C for 200 s. The final elongation was performed at 68 °C for 600 s.
The amplicons were purified with the Nucleospin® Gel and PCR Clean-up Kit (Macherey-Nagel) and Zymoclean™ Large Fragment DNA Recovery Kit (Zymo Research), then sequenced directly with the PCR primers on both strands. In case of mixed DNA sequence results, molecular cloning was performed using the CloneJET PCR Cloning Kit (Thermo Scientific™) according to the manufacturer's recommendation. The nucleotide (nt) sequence of the large product from the bridging PCR was determined by primer walking. The sequencing reactions were carried out with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems™). Subsequently, the reaction mixtures were sent to a commercial service for electrophoresis.
Sequence analyses, annotation and phylogeny inference
The identity of the newly obtained sequences was confirmed with the BLAST algorithms run at the NCBI GenBank database. The sequence reads were quality controlled and assembled using the Staden package (Staden, 1996). Deduced aa sequences were generated with the JavaScript DNA Translator 1.1 program (Perry, 2002). From the DNA polymerase and terminase proteins of alloherpesviruses, multiple aa sequence alignments were made online using the MAFFT v7 (Katoh et al., 2019) with default parameters and were then edited manually as described recently (Doszpoly et al., 2020). For phylogeny inference, the maximum-likelihood (PhyML) method of the TOPALi v2.5 program package (Milne et al., 2004) was used with the WAG aa substitution model and 1,000 samplings. Bayesian inference was also performed using MrBayes 3.1.1 program (Ronquist and Huelsenbeck, 2003) within the TOPALi v2.5 package with 1,000,000 generations, sampling frequency of 10 and 25% burn in. Phylogenetic trees were edited and visualised using the MEGA X (Kumar et al., 2018).
Results
Clinical signs and pathological findings
Small nodules, of up to 1 cm in diameter, were seen all over the body including the head and the mouth of the affected fish. In certain body regions, the spots were covered with intact skin whereas the majority of them possessed a crater-like, wounded surface (Fig. 1). Other visible clinical signs or mortality were not recorded in the fish stock. Gross pathological examination of the six, humanely exterminated specimens revealed that the internal organs were normal, except for the fish with the most prominent skin rash, in which the liver was pale and friable, giving the appearance of fatty liver. On the gills of the same fish, a remarkable amount of the monogenean ectoparasite, Thaparocleidus vistulensis was noted.
Macroscopic image of proliferative skin lesions on the head (A) and trunk (B) of a 2-year-old affected wels catfish (Silurus glanis). Arrows indicate nodules of different sizes, some of which have a crater-like, damaged surface. The scale bar corresponds to 1 cm
Citation: Acta Veterinaria Hungarica 70, 4; 10.1556/004.2022.00038
PCR, sequencing and annotation
PCR amplification of fragments from the catalytic subunit of the pol gene and from the DNA packaging terminase subunit were successful. After removing the primer sequences, 463 and 1,002 nt useful sequences were obtained, respectively. An additional 1,759-bp fragment from the pol was also amplified and sequenced. The PCR targeting the helicase gene was negative. The sequence of the pol gene showed homology to the corresponding genes of other alloherpesviruses. The results of the BLASTX application implicated AngHV-1 and several cyprinid HVs as the closest relatives. Interestingly, direct sequencing of the PCR product, obtained from the terminase gene, resulted in multiple and thus unreadable trace files, therefore molecular cloning of the amplicon was performed. After sequencing more than 10 clones, several sequences could be isolated. Besides the targeted and expected terminase gene fragment, certain bacterial and wels catfish chromosomal sequences were identified. Most surprisingly however, we found one clone that contained a DNA fragment of 711 bp, the sequence of which showed clear homology to a conserved alloherpesviral gene of yet unknown function. The homologous genes have been described as ORF87 and ORF45 in the CyHV-1 (Davison et al., 2013) and AngHV-1 (van Beurden et al., 2010), respectively. Although the genome organisation of these two, entirely sequenced viruses is not fully co-linear, the size and content of the conserved gene block between the pol gene and the homologues (ORF87 and ORF45) of the serendipitously amplified and cloned genomic fragment are comparable. Indeed, the nested PCR bridging the pol fragment with the putative ORF87/45 homologue was successful and yielded an approximately 15-kb product. After sequencing it by primer walking, a large contig encompassing 15,222 bp could be assembled. It was annotated by comparison with the corresponding genomic region of CyHV-1 and AngHV-1. As shown in Fig. 2, this genomic fragment contains seven full and two partial ORFs in the CyHV-1 and in the novel wels catfish virus, whereas in the AngHV-1, two additional minor ORFs are present. In this latter virus, the orientation of this genomic region is in the opposite direction, and it is situated closer to the left end of the genome, hence the lower designation number of the homologous ORFs. The role of the majority of the ORFs in the region is yet to be deciphered, nonetheless, the homology among the three viruses supports the assumption on their function as genes. The ORF79/55 codes for the catalytic subunit of the DNA polymerase, and ORF80/52 is the gene of a putative protein (Allo60) that also seems to be highly conserved as it is present in every alloherpesvirus genome sequenced to date (Davison et al., 2013). The genes ORF81/51, ORF82/50 and ORF83/49 encode homologous membrane proteins, however the role of the also well conserved product of ORF87/45, also present in our catfish virus, is unknown (Fig. 2). We submitted the new sequences to the GenBank with accession numbers MW732127 and MW732128.
Schematics of the genetic content of the PCR-amplified genome fragment of the novel silurid herpesvirus 2 (SiHV-2) compared with the corresponding genome region of the two most closely related viruses from the genus Cyprinivirus. Homologous open reading frames (ORFs) are marked with identical colours. The numbering is as described in the genome of cyprinid herpesvirus 1 (CyHV-1) and anguillid herpesvirus 1 (AngHV-1), respectively. Grey and black arrows mark ORFs without obvious homology to any known genes. The size of the arrows is proportional. The scaling is in base pairs. The GenBank accession number of each sequence is also shown
Citation: Acta Veterinaria Hungarica 70, 4; 10.1556/004.2022.00038
Phylogeny inference and classification
The topology of phylogenetic trees, calculated on several proteins was very similar, providing clear evidence that the novel wels catfish alloherpesvirus belongs to the genus Cyprinivirus. Figure 3A shows an ML tree, based on a 790-aa-long alignment from the DNA polymerase. The phylogenetic place of the newly found virus was further confirmed by the other tree, shown by Fig. 3B, which was calculated on an alignment made from the 273-aa sequences of the terminase subunit.
Phylogeny reconstruction of the family Alloherpesviridae based on amino acid (aa) sequence alignments. Maximum Likelihood and Bayesian analyses were performed. The trees were derived from a 790-aa alignment of the DNA polymerase (A) and from a 273-aa alignment of the terminase subunit (B). The tree topologies are supported by high bootstrap and posterior probability values as marked at the nods. The genus names are above the branches. The viruses are marked with their species name. Species names proposed but not yet officially approved are not in italics
Citation: Acta Veterinaria Hungarica 70, 4; 10.1556/004.2022.00038
We propose this virus as the prototype of a new alloherpesvirus species in the genus Cyprinivirus with a provisional name Silurid herpesvirus 2.
Discussion
Here we report the sequence analysis of a more than 15-kb-long genomic fragment from a novel alloherpesvirus, detected in the diseased skin sample of a farmed wels catfish. Similar skin lesions appeared in the same fish pond during the autumn of a couple of the following years. General clinical signs or mortality were not observed, but identical viral sequences were obtained by PCR from the newer samples, too. Attempts to isolate the virus in cell culture failed. Next-generation sequencing of a concentrated homogenate from skin samples prepared from diseased fish has resulted in only relatively short, low-coverage, scattered HV contigs (Surján et al., 2021). It revealed, however, the concomitant presence of a novel papillomavirus, the entire circular genome of which has been obtained (Surján et al., 2021). Retrospective PCR screening for the detection of papillomaviruses was negative in the sample examined in the present study. Thus, the association between the carp pox-like skin condition and either or both of the detected viruses requires further investigation. The possible identity of the novel HV with the virus reported by Békési et al. (1981) several decades ago is questionable especially because of the considerable difference between the severity of the clinical signs. The old outbreak was accompanied by high mortality. Therefore, further examinations, including histopathological ultrastructural studies with light and electron microscopy and possibly in situ DNA hybridisation, will be necessary to clarify the causative role of silurid HV-2 in the skin erosions.
The PCR primers and method, elaborated for the general detection of the helicase gene of cyprinid HVs (Doszpoly et al., 2015) proved to be unsuitable for the detection of every member of the genus Cyprinivirus. The novel wels catfish alloherpesvirus needs deeper characterisation, ideally determination of its complete genomic sequence. The data available currently provide sufficient evidence for its approval and taxonomic classification. We plan to submit a formal taxonomy proposal to the ICTV. This is the second HV found in a fish from the family Siluridae, therefore we propose to name it silurid herpesvirus 2. The specific new rules of virus taxonomy and nomenclature, presently undergoing radical transformation, are not elaborated yet, therefore the use of the strain name as a provisional species name (Silurid herpesvirus 2) seems to be a reasonable temporary solution.
Although the number of viruses known to occur in various fish is rapidly increasing, our knowledge of the viral pathogens of wels catfish is still limited. Among the group of DNA viruses, representatives of the iridoviruses (Ahne et al., 1989), circoviruses (Lőrincz et al., 2012) and papillomaviruses (Surján et al., 2021) have been reported so far. However, the potential pathogenic impact of these infections is far from being fully understood. As wels catfish culture intensifies, data on their viral pathogens will become increasingly important.
Furthermore, our results provide an interesting experience of the usefulness of a deeper investigation of PCR products that look false at first sight. The molecular cloning of an amplicon, producing hardly readable, if at all, sequences, led to an unexpected success. In fact, this was the second case when we encountered such a result. Previously, when working on the first genome sequences from another piscine virus, we also obtained, by chance, an off-target PCR product, which then proved to be a great help in the phylogenetic characterisation of the black bullhead herpesvirus (Doszpoly et al., 2008).
Our findings support the need to change, or at least fine-tune, the taxon names within the family Alloherpesviridae. The current genus names, derived simply from the host fishes, are clearly inappropriate. With the constant increase in the number of viruses and hosts, a deterioration of the situation is easily predictable. By now, almost all genera include viruses from various, often distantly related fishes, while the genus Batrachovirus, created for the classification of the amphibian lineage, seems to need further subdivision (Doszpoly et al., 2011). Changes in taxonomy and nomenclature usually cause some confusion, therefore they should be carefully considered and preferably limited to a necessary minimum.
Ethical statement
The third author, Edit Eszterbauer is an Editorial Board member and the last author, Mária Benkő is the Editor-in-Chief of Acta Veterinaria Hungarica.
Acknowledgements
The financial support, provided by grants NN140356 and K140348 from the National Research, Development and Innovation Office of Hungary, is greatly appreciated.
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