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동아시아 지역에 있어 전염성조혈기괴사증 바이러스 (IHNV)의 분자역학 및 병독성의 변화
니시자와토요히코 ( Toyohiko Nishizawa ) 한국어병학회 2018 한국어병학회지 Vol.31 No.1
Causative agent of infectious hematopoietic necrosis (IHN) belonging to genus Novirhabdovirus (Rhabdoviridae). Economic losses caused by IHNV are serious in mainly Oncorhynchus spp. including rainbow trout O. mykiss and Atrantic salmon Salmo salar. IHNV was initially found by endemic presence in U.S. West Coast for sockeye salmon fry O. nerka and chinook salmon fry O. tshawytscha in the 1950s, and it has spread to Japan, Korea and Taiwan in the 1970s, and also to Italy and France in the 1990s. Currently, IHNV is detectable in many parts of the world, including Russia and South America. Mortality due to IHNV infection in fish with ≤0.5 g of body weight reaches 60% to 100%, while the mortality reduces by fish growing. In recent years, onset of IHNV infection has increased also in fish with large sizes. Here, we introduce molecular epidemiology and virulence changes of IHNV in East Asia, furthermore, we discuss on future prospects in IHNV researches.
Shift of Phylogenic Position in Megalocytiviruses Based on Three Different Genes
권세련,Toyohiko Nishizawa,Jong-Won Park,오명주 한국미생물학회 2011 The journal of microbiology Vol.49 No.6
Major capsid protein (MCP), the adenosine triphosphatase (ATPase), and the PstI fragment genes from five Japanese and three Korean megalocytivirus isolates were sequenced and phylogenetically analyzed with known megalocytiviruses. Phylogenetic trees formed three major clusters (M1, M2, and M3 or P1, P2, and P3), and genogroup I was divided into two minor clusters (M1a/M1b and P1a/P1b) using three target genes. Sequence identity was >97% within each cluster, except cluster II of the PstI fragment (>94% of sequence identity). Interestingly, different genotyping patterns were observed for the same isolates depending on the gene analyzed. The JPN-YelTail and JPN-BfTuna isolates located in the minor M1a cluster, based on MCP and ATPase nucleotide sequences, appeared in the minor P1b cluster based on the PstI fragment, suggesting a shift of phylogenic position in megalocytiviruses. Further study will be conducted to compare the viral antigenicity and pathogenicity between the two isolates showing the shift of phylogenic position and the other isolates clustered within genogroup I.
계현정,이한솔,Toyohiko Nishizawa 한국어병학회 2023 한국어병학회지 Vol.36 No.1
Surface protrusions on nervous necrosis virus (NNV) particles play crucial roles in NNV antigenicity and infectivity. Pocket structures at the apex of surface protrusions are most likely responsible for initiating NNV infection by functioning as cell receptor binding sites. To create a scientifically robust model, we developed mouse monoclonal antibodies (MAbs) that recognize common epitopes on pocket structures shared among serologically distinct NNVs. We obtained 108 hybridoma clones that produced NNV-specific MAbs by immunizing eight mice using highly purified red-spotted grouper NNV (RGNNV) with and without physicochemical treatments. Finally, 13 hybridoma clones that produced MAbs which recognized different epitopes on RGNNV and/or striped jack NNV (SJNNV) were obtained. NNV-neutralizing activity was not observed in any of the MAbs. We believe that insufficient amounts of NNV used for immunization might be one of the factors responsible for preferential suppression of the generation of NNV-neutralizing antibodies.
Treatment with carbonate buffer decreases antigenicity of nervous necrosis virus (NNV)
Gye, Hyun Jung,Nishizawa, Toyohiko Elsevier 2019 Aquaculture Vol.500 No.-
<P><B>Abstract</B></P> <P>Antigens are commonly immobilized after suspending them in an alkaline buffer such as carbonate buffer (pH 9.6) for enzyme-linked immunosorbent assay (ELISA). The objective of this study was to determine the effect of 100 mM carbonate buffer (pH 9.6) on antigenicity of nervous necrosis virus (NNV) known to be pathogenic to fish. ELISA values of NNV antigens immobilized using carbonate buffer (pH 9.6) were ≤ 0.23, significantly lower than those by dry immobilization (ELISA values of approximately 1.0). NNV antigenicity was drastically decreased by treatment with carbonate buffer (pH 9.6), but not by treatment with Tris-HCl buffers at pH 8.0 or pH 9.6. Thus, the decrease of NNV antigenicity was due to carbonate/bicarbonate, not pH. NNV infectivity was also decreased after treatment with carbonate buffer (pH 9.6). The anti-NNV serum could recognize conformational structures of NNV surface protrusions related to NNV infection. Therefore, NNV surface protrusions might have been denatured by treatment with carbonate buffer (pH 9.6). Results of this study also demonstrate that coating NNV antigens by buffer immobilization might be far from practical. However, the sensitivity of ELISA detecting NNV antigens can be greatly improved by dry immobilization of antigens.</P> <P><B>Highlights</B></P> <P> <UL> <LI> NNV antigens were easily denatured by treatment with carbonate buffer (pH 9.6). </LI> <LI> Infectivity of NNV was also decreased by treatment with carbonate buffer (pH 9.6). </LI> <LI> The denaturation of NNV was due to carbonate/bicarbonate, not pH. </LI> <LI> Sensitivity of ELISA detecting NNV antigens can be improved by dry immobilization. </LI> </UL> </P>
김형준,조재권,황형규,오명주,Toyohiko Nishizawa 한국미생물학회 2012 The journal of microbiology Vol.50 No.5
Infectious pancreatic necrosis virus (IPNV), a type species of aquabirnaviruses in the family Birnaviridae, is an etiological agent of infectious pancreatic necrosis and has been isolated from epizootics of cultured salmonids. In the present study, an epithelioma papulosum cyprini (EPC) cell line persistently infected with IPNV (PI-EPC) was experimentally established by subculturing EPC cells surviving IPNV infection,and was characterized. PI-EPC cells were morphologically indistinguishable from EPC, but continued to grow and yield IPNV. PI-EPC cells showed no cytopathic effect due to IPNV inoculation, and susceptibility of PI-EPC cells against heterologous viruses was not different from that of EPC cells. Only one cell of 103.5 PI-EPC cells produced IPNV at approximately 100.5 50% tissue culture infectious dose (TCID50)/cell/day, which was approximately 1,000 times lower than that of normal EPC cells. PI-EPC cells that did not yield IPNV (N-PI-EPC) were screened. The IPNV genome was detected from both PI-EPC and N-PI-EPC cells,and the IPNV VP2 structural protein was detected from both cell lines, but no other IPNV proteins were observed by Western blot analysis with anti-IPNV serum. Thus, multiplication of IPNV in PI-EPC cells was regulated by some host cell factors, except interferon.
Gye, Hyun Jung,Nishizawa, Toyohiko Elsevier 2018 Aquaculture Vol.485 No.-
<P><B>Abstract</B></P> <P>Enzyme-linked immunosorbent assay (ELISA) for detecting fish antibodies is problematic due to its low reproducibility and high background optical density (OD). In this study, we investigated problematic sources using nervous necrosis virus (NNV, a fish pathogenic virus belonging to the genus <I>Betanodavirus</I>) and sevenband grouper (<I>Hyporthodus septemfasciatus</I>) as a model for detecting specific fish IgM through ELISA. It was revealed that both fish IgM and mammalian immunoglobulins were non-specifically adsorbed to purified NNV particles. This could be the problematic source of the high background OD and low reproducibility in ELISA. ELISA values of naïve fish IgM non-specifically adsorbed to NNV particles immobilized onto ELISA plate wells ranged from 0.09 to 0.15 (high background OD). However, ELISA values of NNV antigens non-specifically adsorbed to naïve fish IgM immobilized onto ELISA plate wells were all <0.03 (low background OD). Thus, we developed a sandwich ELISA by immobilizing fish sera. NNV-specific antibodies could be indirectly detected by detecting NNV antigens captured by fish IgM immobilized onto ELISA plate wells using anti-NNV serum. When anti-NNV and naïve fish sera were subjected to such sandwich ELISA, ELISA values of anti-NNV fish sera ranged from 0.24 to 2.48. Its reproducibility was sufficiently secured based on results obtained from five experiments performed on different days. Conversely, ELISA values of naïve fish sera were all <0.02, suggesting that background OD was completely regulated. The present sandwich ELISA does not require antiserum against fish IgM, meaning that NNV-specific antibodies are detectable from any fish species using only one antiserum against NNV.</P> <P><B>Highlights</B></P> <P> <UL> <LI> ELISA by immobilizing NNV antigens was problematic for detecting specific fish IgM. </LI> <LI> Fish IgM and mammalian immunoglobulins were non-specifically adsorbed to NNV particles. </LI> <LI> Sandwich ELISA by immobilizing fish sera was developed for detecting NNV-specific IgM. </LI> <LI> Reproducibility and background OD were greatly improved in such sandwich ELISA. </LI> </UL> </P>