Isolation and characterization of new betanodaviruses from shellfish as pathogenic agents of viral nervous necrosis (VNN) in fish

Abstract

Shellfish could act a vector or a reservoir in propagating and spreading of viral diseases in marine organisms by accumulating agents into the body from neighboring water. The purpose of this study was to analyze the frequency and genotypic distribution of betanodavirus belonging to major aquatic virus in marine environment by using domestic and imported live shellfish. Using a newly developed consecutive methods that combines detection and discrimination PCR of betanodavirus, the first emergence of BFNNV genotype was identified in 446 NNV positive samples out of 969 domestic and imported shellfishes. Although BFNNV was not detected in cultured fish, it showed a high rate of prevalence in shellfish (18%) and a constant rate throughout the year unlike the RGNNV genotype showing variation by season. It was also confirmed for the first time that betanodavirus accumulates in the shellfish digestive tract, preserving its infectivity, as virus were isolated from shellfish by providing optimized virus proliferation conditions. The presence of 32 NNV in shellfish that were clearly detected in DSN-2 RT-PCR but not in DMT-2 RT-PCR for discrimination of 4 major genotype was noted, and we identified a completely new Betanodavirus (KSNNV) by analyzing the 26 partial sequences of viruses that were successfully isolated from these samples. The complete sequence of KSNNV, which has a lower level of identity (RNA1 81-83%, RNA2 76-77%) than the major four genotypes (RNA1 82-92%, RNA2 79-83%), was the longest of all known betanodaviruses and constituted the formation of a new, seperated cluster from the other genotypes in phylogenetic analysis. Thus, we proposed KSNNV as the fifth new genotype Betanodavirus. Using the mDMT-2 RT-PCR that enables discrimination of KSNNV, we confirmed the prevalence of KSNNV not only in Korea but also in imported shellfish, which strongly suggested the possibility of occurrence of KSNNV worldwide. KSNNV showed susceptibility to broad host species by inducing asymptomatic infection with vacuolation in nervous tissues of 7 out of 14 species of juvenile including freshwater and marine fishes. Surprisingly, in a pathogenicity test of KSNNV to a just-hatched larval fish, main target of Betanodavirus, it was shown that KSNNV is a highly pathogenic agent by rapidly inducing 100% mortality to sevenband grouper and mandarin fish. The emergence of diverse genotypes in the waters of Korea and neighboring countries could elevate the chance of producing evolved forms by reassortment in aquatic envrionment. Because the generation of new forms of recombinant viruses can lead to unpredictable propagation to new host species and confer a high risk of acquiring new characteristics such as strong pathogenicity, there is a need to develop a detection method for all segments of betanodaviruses. The double segment detection system of Betanodavirus using shellfish developed in this study reported the first appearance of not only reassortant SJ/RGNNV (RNA1: SJNNV, RNA2: RGNNV), which is known only in European Mediterranean coastal waters, in Korea, but also two new reassortant strains of KS/RGNNV and RG/KSNNV, both of which have not been globally known for the first time. We have confirmed the existence of two new reassortants (KS/RGNNV, RG/KSNNV) that have been successfully isolated and confirmed their genetic background by phylogenetic analysis, so we reported two new types of Betanodavirus reassortants for the first time. Interestingly, the combinatorial forms of the segments possessed by these new reassortants enabled the analysis of the characteristics of Betanodavirus in various angles. In vitro characterization of 2 heterogeneous reassortants and 2 homologous genotypes (RGNNV and KSNNV) in E-11 confirmed that betanodavirus did not completely dominate the segment, but comprehensively influenced the optimal virus growth temperature and proliferation peak concentration. And serological analysis using sera from mouse and fish injected with these viral antigens showed a common result that the neutralizing epitope of betanodavirus was dependent on RNA2. KSNNV was shown to contain a neutralizing epitope that is different from the serotype of RGNNV and BFNNV, suggesting the possibility of a new classification of a new serotype group of Betanodavirus. In addition, both reassortants not only showed high infectivity to four common susceptible hosts (sevenband grouper, rock bream, olive flounder and mandarin fish) of KSNNV and RGNNV, but also induced 100% mortality in both fish species (sevenband grouper and mandarin fish) in larvae stage, the main attack target of betanodavirus. These results showed that reassortants have high pathogenicity and similar infection characteristics againt common fish species of each homologous genotype virus. In conclusion, using shellfish in marine environment, we are the first to report the prevalence of KSNNVs belonging a new taxon of Betanodavirus and new evolutionary forms generated by the recombination of these viruses and existing viruses, and to analyze the risks to the fisheries industry worldwide.

패류는 수산생물의 바이러스 질병의 전파 및 확산에 있어 질병 원인체를 체내에 축적하여 vector 또는 reservoir로서의 역할을 할 수 있다. 본 연구는 국내 양식장 주변 수역 내 활패류와 수입 활패류를 검출시료로 활용하여 국내외 해양 생태계 내 존재하는 주요 위험 어류 바이러스군에 속하는 betanodavirus의 출현빈도 및 유전형 분포를 분석하고자 하였다. 수집된 국내 및 수입 패류 1,000여 마리의 조직 내 betanodavirus의 검출 시도를 통해 국내 미보고 exotic genotype인 BFNNV 유전형 바이러스의 국내 및 주변국에서의 최초 출현을 알리고 이들의 높은 출현율을 보고하였다. 뿐만 아니라 패류 내 유전형 분류가 되지 않는 다수의 betanodaviruses의 존재를 밝히고 이들이 세계적으로 미보고된 새로운 바이러스임을 검증하여, 본 연구는 새로운 5번째 betanodavirus 유전형 그룹으로서 KSNNV 유전형의 신규 분류를 제안하였다. 이로써 국내 및 주변국가의 수역 내 다양한 유전형의 동시 출현으로 인해 segment의 재조합을 통하여 진화된 reassorted 바이러스의 발생 가능성이 강력히 시사되었으며, 본 연구는 다양한 형태로 재조합된 reassortant betanodavirus의 생성 가능성에 초점을 두고 이를 탐색하고자, RNA2뿐만 아니라 RNA1 segment까지도 검출 및 유전형 판별이 동시에 가능한 종합적인 betanodavirus 검출시스템을 확립하였다. 본 연구의 확립된 검출시스템을 패류에 적용함으로써 어류에서는 검출되지 않았던 exotic reassortant인 SJ/RGNNV의 출현, 그리고 본 연구에서 확인한 새로운 KSNNV 유전형과의 유전적 조합을 이룬 새로운 진화 형태인 KS/RGNNV, RG/KSNNV reassortant의 등장을 처음으로 알렸다. 본 연구를 통해 확인된 국내 및 수입 패류 내 보존되어 있는 이들 바이러스의 높은 감염활성, 그리고 해담수어를 포함한 광범위한 감수성 숙주범위 및 부화자어에 대한 높은 병원성 특성은 이들 바이러스의 수생태계 내 높은 유행 가능성과 이로 인한 세계 양식 산업에서의 산업적 위험성을 강력하게 시사하였다. 이로써 본 연구는 새로운 병원성 바이러스 탐색이라는 획기적인 개념의 검출 시스템을 구축하는 데 있어서 패류 활용의 강점을 강조하고, 또한 reassortants가 보유한 각 segment의 유전형의 조합을 활용한 본 논문의 연구는 Betanodavirus의 근원적인 생리 및 병원성 특성의 종합적 분석을 가능케하여 Betanodavirus 특성 연구의 접근에 있어 새로운 frontier를 제시한다.