Molecular detection and characterisation of herpesviruses in asymptomatic Russian sturgeon (Acipenser gueldenstaedtii) from European aquaculture

A total of 123 Russian sturgeon (A. gueldenstaedtii) without any clinical signs were cohabited twice for at least 65 days in total with tench (Tinca tinca), Nile tilapia (Oreochromis niloticus), crucian carp (Carassius carassius) and common carp (Cyprinus carpio) for investigation of propensity to infection with tilapia lake virus. This TiLV experiment (authorised by veterinary authorities and animal welfare committee under number 7221.3-2-025/19, FLI 23/19) was considered to have no influence on the disease of sturgeon subsequently taken under investigation. Individual specimens of the fish species cohabited with the sturgeon were also tested for acipenserid herpesvirus in random samples. Sturgeon started to die after 30 days of the experiment in both infected groups and contact-free uninfected controls.

At the start it was not known if the agent infecting sturgeon lethally contained DNA or RNA. Therefore, both nucleic acids were extracted. The primer pairs used are presented in Table 1.

DNA was extracted from samples with a QIAamp DNA-Mini Kit and RNA with a QIAamp Viral RNA Mini Kit (both Qiagen, Hilden, Germany) according to the manufacturer’s instructions. All samples were processed twice with both kits. This supported the assumption that the infectious agent was one containing DNA, and therefore only DNA was subsequently measured. This was carried out with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

For PCRs and nested PCR, a Go Taq Flexi kit (Promega, Madison, WI, USA) was used according to the recommendations of the manufacturer. The composition of the master mix for all first-round PCRs is shown in Table 2. The thermal profile for the reaction with the Stör-HV-F and Stör-HV-R primers as well as for the nested PCR using Stör-HV-Fn and Stör-HV-Rn was an initial step at 95°C for 2 min and 35 cycles at 94°C for 15 sec, 60°C for 30 sec and 72°C for 1 min. The PCR was finalised with 72°C for 10 min and adjusted to 4°C for overnight runs.

Five μL of extracted DNA was used as template for the PCR, and 1 μL was allocated for nested PCRs and for re-amplification if necessary. In the latter case, the water content was increased to 25 μL. All other PCRs with the primer pairs in Table 1 proceeded with an initial step at 95°C for 10 min, followed by 35 cycles at 95°C, 55°C and 72°C for 1 min each and were finalised with a step at 72°C for 7 min. All PCR results were visualised with 1.8% agarose gels containing 1% ethidium bromide and running for 60 min at 60 V according to Reinhard (18), and the size of the amplified fragments was assessed with a DNA ladder (Invitrogen, Carlsbad, CA, USA).

Signals from PCRs which were positive after nested PCR or re-amplification were cut from the agarose gels under UV light and purified using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions.

The purified DNA isolates were analysed using an ABI 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) at the Friedrich-Loeffler-Institut (Insel Riems, Germany). All sequences were identified with the National Center of Biotechnology Information (NCBI) database using the basic local alignment search tool NucleotideBLAST Blastn program.

The ISH procedure was as described by Bergmann et al. (3) with minor changes. Probe production was carried out using the PCR conditions for Stör-HV-F/R or Stör-HV-Fn/Rn as appropriate. As an unspecific hybridisation control, a koi herpesvirus (KHV) probe with the primer pair KHV 1Fn/1Rn was applied.

When the TiLV experiment had been running for 30 days, an approximately 10 to 15% mortality suddenly occurred in TiLV-infected sturgeon and in the negative control uninfected sturgeon, the latter being housed in another room and maintained by different people. Only some weak petechiae around the fins and the ventral part were visible in fish, both infected and control group individuals. No other clinical signs were observed. In all cases dead fish were lying at the bottom of the tanks with bent bodies (Fig. 1). All dead fish were sampled and frozen at -20°C until further use. Video was recorded of the TiLV experiment over 75 days; however, it brought no additional information regarding the mor tality event in sturgeon. No other fish were affected.

Fig. 1.

Dead sturgeon lying on the bottom of the tank with bent bodies

Both RNA and DNA extraction kits were used with the same samples twice. The extraction kits for RNA operate only on this nucleic acid, but may extract the DNA which is inside the RNA. Measured with the NanoDrop spectrophotometer, it was visible that the DNA extraction kit had up 10 times more DNA inside the eluate than the RNA kit had in its eluate (Table 3), which is guaranteed by the manufacturer.

Positive PCR signals were observed with the Stör-HV-1F, Stör-HV-1R, Stör-HV-Fn and Stör-HV-Rn primers. Re-amplification with 2 μL of product from the first-round PCR using Stör-HV-1F and Stör-HV-1R was also successful. An attempt was made to identify the target organ for the suspected herpesvirus in five sturgeons. The organs in which the infectious agent was determined are shown in Table 4. Almost no positive signals were observed in other sturgeon organs.

Positive signals were most frequently found in heart and liver tissue and less frequently in pancreata and swim bladders. Only in two kidneys were positive signals found.

In total 120 sturgeon were sampled and additionally 5 tench, 6 crucian carp, 4 Nile tilapia and 4 common carp. The positive results in PCRs are summarised in Table 5.

The PCR products of eight sturgeon, two crucian carp, one common carp and one tench were chosen for sequence analysis. The results were compared with the NCBI GenBank database. All resulted sequence data of the four species had a similarity of 99.7%. According to NCBI database matches with accessions up to 2023, all the obtained sequences were identified as Acipenser ruthenus genome assembly, chromosome 48, accession No. OV754656.1. The detected sequences were, however, very similar to that of AciHV-3 published by Clouthier et al. (6) in 2023 (Table 6). Using the results from sequence analysis, all positive fish samples contained parts of the AciHV-3-like genome; these parts were not of the sturgeon genome, and specifically were not chromosome 48 of the sterlet.

To confirm that the amplicon was not a genomic part of the animal, ISH was carried out. While all controls including the irregular KHV DNA probe showed no specific labelling of cells, an often single-cell positive reaction was found in different organ tissues. Positive signals were blackish-blue or blackish-purple with yellow negative cells (Fig. 2A) in the surrounding tissue and had to be strongly bound to a cell structure. As examples, sturgeon herpesvirus-positive cells were found in the spleen (Fig. 2B) and kidney (Fig. 3). In Fig. 3 the brownish cells are melanomacrophages after BBY counterstaining and the blackish-blue spots are herpesviral DNA bearing cells. The AciHV-3-like virus was also detected in the tissues of common carp and crucian carp (data not shown).

Fig. 2.

Detection of an AciHV-like virus in spleen tissue. A – irregular KHV probe; B – AciHV-HV probe

Fig. 3.

AciHV-like signals in kidney tissues. Red arrow – positive signals; black arrows – melanomacrophages