Isolation and identification of a new strain of hirame rhabdovirus (HIRRV) from Japanese flounder Paralichthys olivaceus in China

In this study, diseased flounder juveniles (average weight of 30 g, average body length of 15 cm) come from a fish farm in Shandong province, China. The fish suffering from a hemorrhage disease were kept at a temperature ranged from 8 to 10 °C, and the cumulative mortality rate was approximately 20% in a month. Five diseased fish and five healthy fish were taken for parasitological, bacteriological and virological examinations. The gills and mucus scrapings from the skin were inspected for the presence of parasites. The kidneys and spleens were homogenized and subjected to bacteria culture on brain–heart infusion agar plates at 15 °C for 7 days. Histopathological examinations were carried out using the tissue sections.

The kidney, spleen, brain and gill tissues from five diseased fish were individually homogenized at a ratio of 1:10 (w/v) in M199 medium supplemented with 10% FBS, and 1% Penicillin-Streptomycin (Gibco). The tissue homogenates were filtered through a 0.22 μm filter membrane and were inoculated on four fish cell lines including EPC, FHM, FG and CAR cell lines, cultured in M199 medium supplemented with 4% FBS and 1% Penicillin-Streptomycin at 15 °C. The inoculated cell cultures were incubated at 15 °C for 7 days and examined daily for the presence of CPE. The cultures were centrifuged at l,500 × g at 4 °C for 20 min and the supernatants were immediately stored at −80 °C. The obtained virus was named CNPo2015.

Three fish cell lines (EPC, FHM and FG) were used for virus titering. Prior to infection, the cells were transferred into 96-well plates and grew to monolayer cells. Serial 10-fold dilutions of the tissue homogenate were inoculated on three fish cell lines. The cultures were maintained in M199 medium supplemented with 4% FBS and 1% Penicillin-Streptomycin at 15 °C. The virus titers on the cell lines were determined by 50% endpoint dilution assays (TCID₅₀) according to the method described by Reed and Muench [13].

EPC cell monolayers were incubated with the virus culture at a MOI of 0.1 for 2 days. Then the cultures were collected and fixed in 2.5% glutaraldehyde in phosphate buffer overnight. Ultrathin sections were prepared as previously described [14]. The sections were placed on grids and examined under a Jeol JEM-1200 EX electron microscope.

500 μL of culture supernatants was used for RNA extraction using TRIzol (Takara). The quality and quantity of RNA were checked using a NanoDrop ND-8000 spectrophotometer (Thermo Scientific). cDNA was synthesized from 1 μg of RNA using M-MLV kit (Takara), according to the manufacturer’s instructions. The resulting cDNA was used for PCR analyses of HIRRV, VHSV, IHNV, VNNV. The PCR was performed in a total volume of 25 μL containing 1 μL of cDNA, 2.5 U of Taq DNA polymerase (Takara), 5 μL of PCR reaction buffer, 0.5 μL of each primer (10 pmol) and RNase-free water. Normal EPC cell sample was used as the negative control. All the PCR amplifications were performed according to previous studies [9, 15‐18], and the primer sets and PCR conditions were shown in Table 1. The products were examined by electrophoresis using a 1.0% agarose gel. Then the products were sequenced using the BigDye® v3.1 dye terminator and were analyzed on an ABI PRISM 3730 XL DNA Analyzer (Applied Biosystems, USA), following the manufacturer instruction. The obtained sequences were submitted in the NCBI website and blasted for similar sequences in the GenBank database.

The ORF of the P gene was amplified for phylogenetic analysis. A pair of specific PCR primers (P-F: 5′-ATGTCTGATAACGAAGGAG-3′ and P-R: 5′-CTACCTCATGGTCTTCTTG-3′) were designed using Primer premier 5.0. The amplification was performed as follows: 94 °C for 5 min, followed by 30 cycles of denaturing at 94 °C for 1 min, annealing at 53 °C for 1 min, extension at 72 °C for 1 min and finally incubation at 72 °C for 10 min. The obtained PCR products were sequenced as described above, and the sequence was submitted to the GenBank database. The P gene sequence of CNPo2015 was compared with five other HIRRV isolates in GenBank. The sequences were aligned using Clustal software (version 1.81). Phylogenetic tree based on the P gene was constructed by MCMC method using the BEAST software (version 2.4.5) as previously described [19], and the phylogenetic tree was visualized using FigTree (version 1.4.3).

Healthy flounder juveniles (10 ~ 15 cm, about 30 g) were obtained from a farm in Rizhao, Shandong, China. Prior to the experiment, PCR assay was performed to confirm the fish free of HIRRV. All fish were held in seawater at a temperature of 10 °C. After acclimation for 7 days, a total of 150 fish were randomly divided into five groups (30 fish per group). Fish in each group were injected intraperitoneally with 100 μL of virus culture (10^7.5, 10^6.5, 10^5.5, 10^4.5TCID₅₀ per fish). In addition, fish in the control group were injected with equivalent amount of M199 media. All fish were checked daily for clinical signs and mortalities for 14 days.

The external clinical signs observed in the diseased fish were skin darkening, abdominal distension, fin reddening (Fig. 1a). After dissection, diseased fish showed visceral pallor, capillaries expansion and yellow ascitic fluid in the abdominal cavity (Fig. 1b). No bacteria or parasites were isolated from the fish samples. Histological examination showed that most of the kidney cells were characterized by marked cell degeneration and necrosis. The formation of broken nucleus and pathologic pigmentation were also present in kidneys (Fig. 1d).

The homogenate supernatants of kidney and spleen tissues induced positive CPE in the EPC, FHM, FG cells at 3 days post inoculation at 15 °C. The CPE was characterized by rounded and granular cells, grape-like clusters, cell detached and lysis. No CPE was observed in the CAR cell line (Fig. 2). In all cell lines, no CPE was induced by the brain and gill samples. At 7 days post inoculation, the EPC cells produced the highest titer of virus with a titer of 1 × 10^8.5 TCID₅₀/mL, while the titer was 1 × 10^8.1 TCID₅₀/mL for FHM cells, and 1 × 10^7.3 TCID₅₀/mL for FG cells, respectively. Virus titers in all cell lines stabilized for the remainder of the infection.

Transmission electron microscopy showed that abundant viral particles were aggregated on the surface of cells at 2 days post inoculation (Fig. 3a). Meanwhile, some particles were also found inside the cytoplasm with a vesicle encapsulated (Fig. 3b). The intact virion exhibited a bullet-shaped capsid enclosed with envelope. Moreover, the virion averaged approximately 160 nm in length and 80 nm in width.

As shown in Fig. 4, a specific band was amplified from virus culture supernatants using HIRRV specific primers, while no product was detected in the normal EPC cell samples (data not shown). No product was detected using primers of VHSV, IHNV, VNNV or LCDV. The G gene fragment of CNPo2015 revealed 100% sequence identity with the known HIRRV isolates.

The P gene of CNPo2015 strain was amplified and the complete ORF sequence was submitted to GenBank under accession number KY701726. The P gene sequence analysis showed CNPo2015 had the highest nucleotide sequence identity to 080113 strain and SSB13 strain (98.5%), followed by CA-9703 strain and 8401-H strain (98.1%) and Kor-TY15 strain (97.4%). A phylogenetic tree based on the P gene ORF sequences revealed that CNPo2015 strain was clustered in a clade with 8401-H strain and CA-9703 strain. It showed that CNPo2015 strain was farther to SSB13 strain, 080113 strain and Kor-TY15 strain, which were isolated from spotted sea bass, stone flounder and black seabream, respectively (Fig. 5).

Flounder juveniles were inoculated with different doses of the HIRRV CNPo2015 strain. Symptoms such as abdominal distension and fin reddening appeared on day 4 and began to die on day 5 post-infection. At 14 days post-infection, fish infected with 10^7.5, 10^6.5, 10^5.5, 10^4.5 TCID₅₀ of CNPo2015 strain had cumulative mortalities of 100, 60, 40 and 10%, respectively (Fig. 6). Mortality rates in each group were plotted and the LD₅₀ in juvenile flounder was determined to be 1.0 × 10^5.9 TCID₅₀/fish. No signs or mortalities were observed in the control fish during the experiment.