VNN is the biggest challenge in terms of the sustainability and development of aquaculture, and it poses a significant risk to aquaculture globally [3, 18]. VNN is endemic in the Mediterranean basin and numerous studies have demonstrated its occurrence in both farmed and wild fish [3, 41]. RGNNV is the most frequently detected genotype in the Mediterranean [18], however, the SJNNV genotype was also reported in the Iberian Peninsula in 2009 [27]. Reassortant RGNNV / SJNNV and SJNNV / RGNNV strains have only been reported in the Mediterranean [42].
Turkey has 42% of sea bass production in the world [43] and a strategic position in terms of sea bass production both in Europe and worldwide. The sea bass production in Turkey has high rate of 32.8% within all fish species [44]. The first detection of betanodavirus in Turkey was from sea bass in 2011 and were genotyped as RGNNV [45]. Subsequently, the RGNNV genotype betanodavirus was detected in sea bass and sea bream in the monitoring and screening studies conducted in 2012 and 2014 [46].
Since VNN is not a notifiable disease in animal diseases reporting systems such as OIE and ADNS, there are difficulties in evaluating the epidemiological situation of countries. However, it is seen that the VNN is endemic in the Mediterranean basin in the light of literatures. When the few studies conducted to determine epidemiological prevalence of betanodavirus are examined, it is seen that ranges from 0.23–88.5%. [25, 29–31, 47]. Here, we investigated betanodavirus prevalence based on epidemiological approach in all hatcheries and some farms in Turkey. Betanodavirus prevalence in hatcheries and farms were found to be 6.25% and 5%, respectively.
It has been reported that water temperature affects the activity of the RdRp which is necessary for the replication of the virus, and that different optimum temperatures are required for the epidemiology of different genotypes [31]. It is known that betanodaviruses are highly resistant to both freshwater and seawater environment and also to be highly resistant to external environment [48]. For this reason, larvae or fry-sized sea bass could be exposed to the virus through horizontal transmission in farm or hatchery used contaminated sea water [49]. It is seen that the water temperatures of the hatcheries in the study are lower than the optimum growth temperature of the RGNNV genotype. However, the main purpose of the study in hatcheries is to detect the vertical transmission of the virus that can be found persistently in the spawner and the virus that can be found in the environmental water, if any. The virus has not been detected in FE, preL, postL and fry sized fish, which supports the absence of persistent virus infection in spawners. When the decontamination methods of the water used in hatcheries are evaluated, it is thought that the facilities imlement biosecurity measures by using the filter and UV method and these help the water used to be free from virus. Viruses detected in hatcheries or farms were isolated from fish in units outside the closed system where sea water is used directly. It is thought that the detected viruses might be due to horizontal transmission from sea water where virus endemic region.
Betanodaviruses cause VNN disease in more than 120 fish species and with four generally accepted genotypes [3, 19, 20]. However, each genotype or isolates of the same genotype in different geographies show different pathogenicity on different fish species [50]. A certain amount of viral load should be present in the tissue to cause pathology [50–52]. In experimental infection in sea bass, it was shown that clinical signs did not occur up to 106.55 DKID50, 8.96 x 108 copies for RNA2 and 1.21 x 107 copies for RNA1 in brain samples. In addition, it was determined that those who remained alive and did not show clinical symptoms carried 104.55 DKID50, 1.29 x 107 copies for RNA2, and 6.25 x 105 copies for RNA1 [53]. The DKID50 used in experimental infections appears to be 104.55 [53] and 104 [50]. Considering that the viral loads obtained in this study are between 3.10 and 4.82 x 102 which is thought that the infectious loads are not sufficient. This hypothesis is supported by the absence of any disease in the fish from which the two isolates were obtained, and the absence of VNN-related mortality in the farm or hatcheries.
In this study, samples found positive by RT-qPCR were primarily inoculated onto the E-11 cell line, but could not be isolated. Subsequently, isolation was provided in the SSN-1 cell line. In the study conducted by Sakamoto et al. [25] for the detection of betanodavirus in wild marine species, samples found positive by Nested PCR could not be isolated in the E-11 cell line. The researchers attributed this to the fact that positive samples either did not have sufficient virus titer to generate CPE, or that only virion or infective capsid may not be present despite having viral RNA [25]. Panzarin et al. [36] compared virus isolation in cell culture and RT-qPCR test and reported that although the method of inoculation was unknown, samples with ≥ 31 Ct value could not be isolated in SSN-1 cell line. A similar situation has been experienced in this study. All of the samples were collected from healthy appearance of the sea bass and the low Ct values of the positives were attributed to the low virus titer. The compatibility of field and laboratory data supports the hypothesis that there is no active infection in farms or hatcheries where the virus is detected, and this may be due to low viral load. However, in this study, as different from Sakamoto et al [25] SSN-1 cell line was used in addition to E-11 cell line and virus isolation was achieved. In the light of this experiences, although it has been reported that the E-11 cell line is more suitable for quantitative tests such as virus titration, it is thought that choosing the E-11 cell line for virus isolation in infections with low viral load may have negative consequences such as failure to isolation of the virus from cell line.
In conclusion, the presence and prevalence of betanodavirus was investigated in all age groups in both sea bass hatcheries and sea bass farms in Turkey. It was evaluated that there was no active infection in the juvenile sea bass and that they could be subclinically infected due to low viral load. It has been evaluated that biosafety measures prevent water-borne transmission of the virus, and it is suggested that biosafety measures implememented in hatcheries and farms should continue and seawater should not be used without sanitation. In addition, it is thought that the SSN-1 cell line should be used beside of E-11 cell line in the isolation of betanodavirus in subclinical infections. However, more and more specific studies are needed to determine the different cell culture adaptations of betanodaviruses detected as persistent or subclinical.