Emergence of Reassortment between a New and Reported Types of Betanodavirus in Shellfish
Abstract
:1. Introduction
2. Results
2.1. Identification of Reassortment between the Types of Betanodavirus in Shellfish
2.2. Isolation of Reassortant Viruses in Shellfish
2.3. Comparision of the Full-Sequence Homology with Other Betanodaviruses
2.4. Pathogenicity of Reassortant Viruses against Sevenband Grouper Larvae
3. Discussion
4. Materials and Methods
4.1. Betanodaviruses in Shellfish and Finfishes
4.2. Primer Design for Genotyping of Betanodaviruses Based on RNA1 Segment
4.3. RT-PCR
4.4. Quantitative RT-PCR
4.5. Determination of Full-Genomic Sequences of Reassortant Virus
4.6. Comparison of Full-Genomic Sequences and Construction of Phylogenetic Tree
4.7. Isolation of Reassortant Virus in Shellfish
4.8. Transmission Electron Microscopy (TEM)
4.9. Pathogenicity of Two Reassortant Viruses to Fish in the Larval Stage
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Samples | No. of Single Betanodavirus Positive 1 | No. of Homologous Type 2 | No. of Reassortant Virus 3 | ||
---|---|---|---|---|---|
RG/KSNNV | KS/RGNNV | SJ/RGNNV | |||
Domestic shellfish | 306 | 302 | 1 | 1 | 2 |
Pacific oyster Crassostrea gigas | 179 | 178 | 1 | 0 | 0 |
Mussel Mytilus edulis | 49 | 49 | 0 | 0 | 0 |
Manila clam Ruditapes philippinarum | 29 | 29 | 0 | 0 | 0 |
Common orient clam Meretrix meretrix | 28 | 26 | 0 | 0 | 2 |
Granular ark Tegillarca granosa | 21 | 20 | 0 | 1 | 0 |
Domestic finfish | 35 | 35 | 0 | 0 | 0 |
Olive flounder Paralichthys olivaceus | 23 | 23 | 0 | 0 | 0 |
Rock bream Oplegnathus fasciatus | 3 | 3 | 0 | 0 | 0 |
Red sea bream Pagrus major | 2 | 2 | 0 | 0 | 0 |
Rock fish Sebastes schlegeli | 2 | 2 | 0 | 0 | 0 |
Stone flounder Platichthys stellatus | 5 | 5 | 0 | 0 | 0 |
Imported shellfish from China | 48 | 47 | 1 | 0 | 0 |
Manila clam Ruditapes philippinarum | 14 | 13 | 1 | 0 | 0 |
Common orient clam Meretrix meretrix | 13 | 13 | 0 | 0 | 0 |
Adams venus clam Mercenaria mercenaria | 13 | 13 | 0 | 0 | 0 |
Venus clam Mercenaria stimpsoni | 3 | 3 | 0 | 0 | 0 |
Wrinkled venus clam Callista brevisiphonata | 3 | 3 | 0 | 0 | 0 |
Scallop Saxidomus purpurata | 1 | 1 | 0 | 0 | 0 |
Chinese cyclina Cyclina sinensis | 1 | 1 | 0 | 0 | 0 |
Imported shellfish from Japan | 31 | 31 | 0 | 0 | 0 |
Manila clam Ruditapes philippinarum | 5 | 5 | 0 | 0 | 0 |
Venus clam Mercenaria stimpsoni | 5 | 5 | 0 | 0 | 0 |
Granular ark Tegillarca granosa | 9 | 9 | 0 | 0 | 0 |
Scallop Patinopecten yessoensis | 12 | 12 | 0 | 0 | 0 |
Total | 420 | 415 | 2 | 1 | 2 |
Isolates | Type | RNA1 | RNA2 | RNA3 | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Total | RdRp | 5′ UTR | 3′ UTR | Total | CP | 5′ UTR | 3′ UTR | Protein B2 | ||
KG1-reKS/RG | KS/RGNNV | 3123 | 2946 | 97 | 77 | 1434 | 1014 | 26 | 391 | 375 |
CM1-reRG/KS | RG/KSNNV | 3105 | 2946 | 78 | 78 | 1444 | 1020 | 30 | 391 | 375 |
KSNNV-KOR1 | KSNNV | 3123 | 2946 | 97 | 77 | 1444 | 1020 | 30 | 391 | 375 |
SGWak97 | RGNNV | 3105 | 2946 | 78 | 78 | 1434 | 1014 | 26 | 391 | 375 |
Segment | Part | Reassortant Strains | Homology to Corresponding Sequence (%) | ||||
---|---|---|---|---|---|---|---|
KSNNV-KOR1 (KSNNV) | SGWak97 (RGNNV) | JFIwa98 (BGNNV) | SJNag93 (SJNNV) | TPKag93 (TPNNV) | |||
RNA1 | Total | KG1-reKS/RG | 99.8 1 | 82.7 | 82.2 | 81.2 | 82.5 |
CM1-reRG/KS | 83.2 | 97.2 | 83.1 | 82.2 | 82.8 | ||
RdRp | KG1-reKS/RG | 99.6 (99.8) 2 | 87.3 (88.6) | 86.5 (87.3) | 86.2 (87.2) | 86.0 (87.1) | |
CM1-reRG/KS | 87.5 (89.6) | 98.0 (98.7) | 88.2 (89.6) | 87.6 (88.9) | 88.6 (89.5) | ||
RNA3 | KG1-reKS/RG | 99.5 | 81.0 | 80.6 | 80.9 | 81.5 | |
CM1-reRG/KS | 82.8 | 94.1 | 83.4 | 81.6 | 84.5 | ||
Protein B2 | KG1-reKS/RG | 99.6 (99.7) | 83.8 (84.5) | 83.3 (84.8) | 83.3 (84.7) | 82.5 (83.8) | |
CM1-reRG/KS | 85.5 (87.0) | 95.2 (96.4) | 86.8 (87.8) | 83.3 (85.1) | 87.3 (89.1) | ||
5′ UTR | KG1-reKS/RG | 100.0 | 70.1 | 72.4 | 71.1 | 82.7 | |
CM1-reRG/KS | 87.5 | 98.0 | 88.2 | 87.6 | 88.6 | ||
3′ UTR | KG1-reKS/RG | 98.7 | 84.6 | 79.0 | 84.6 | 84.6 | |
CM1-reRG/KS | 84.6 | 96.2 | 79.7 | 82.3 | 82.3 | ||
RNA2 | Total | KG1-reKS/RG | 76.8 | 97.4 | 83.0 | 79.6 | 80.3 |
CM1-reRG/KS | 98.5 | 76.6 | 78.2 | 77.4 | 78.0 | ||
CP | KG1-reKS/RG | 76.0 (77.4) | 99.7 (99.8) | 86.4 (87.6) | 81.5 (82.5) | 81.5 (82.8) | |
CM1-reRG/KS | 98.8 (99.1) | 76.3 (77.5) | 78.9 (80.2) | 77.5 (78.8) | 79.2 (80.4) | ||
5′ UTR | KG1-reKS/RG | 70.0 | 100.0 | 88.5 | 85.2 | 77.8 | |
CM1-reRG/KS | 100.0 | 70.0 | 63.3 | 70.0 | 70.0 | ||
3′ UTR | KG1-reKS/RG | 78.3 | 98.0 | 83.2 | 80.2 | 81.4 | |
CM1-reRG/KS | 98.7 | 77.7 | 82.2 | 80.2 | 79.8 |
Primer | Sequence (5′ to 3′) | Object | Reference |
---|---|---|---|
c1VNNR | CCGTCTAATGCGACAGACATC | 1st & 2nd round R1-DSN-2 RT-PCR | This study |
c1VNNF1 | GCGTTCCAAAAGAAAGAAGCATAC | 1st round R1-DSN-2 RT-PCR | This study |
c1VNNF2 | GTTCCGTGGTACATGCCAAC | 2nd round R1-DSN-2 RT-PCR R1-DMT RT-PCR | This study |
s1VNNKSR | AAGCTCGTCAGCCACGATG | R1-DMT RT-PCR (for KSNNV) | This study |
s1VNNRGR | TCTCATTAGCCAATAAAGTTGTTA | R1-DMT RT-PCR (for RGNNV) | This study |
s1VNNBFR | CAGTATCAGTGAGGAGGGTGTC | R1-DMT RT-PCR (for BFNNV) | This study |
s1VNNSJR | CATCATCCATGCCAGCTTG | R1-DMT RT-PCR (for SJNNV) | This study |
s1VNNTPR | CAGCCAATATCCTCAATTTCG | R1-DMT RT-PCR (for TPNNV) | This study |
c2VNNR | TGGTCATCAACGATACGC | 1st & 2nd round R2-DSN-2 RT-PCR | [10] |
c2VNNF1 | GCTTCCTGCCTGATCCAAC | 1st round R2-DSN-2 RT-PCR | [10] |
c2VNNF2 | TGCCAAATGGTGGGAAAG | 2nd round R2-DSN-2 RT-PCR qRT-PCR | [10] |
q2VNNR | TTGTTGCCGACACACAGG | qRT-PCR | [10] |
s2VNNKSR | CGCTTCTGCGTTGTTTGG | R2-DMT RT-PCR (for KSNNV) | [11] |
s2VNNRGR | TTGAAGTTGTCCCAGATGC | R2-DMT RT-PCR (for RGNNV) | [10] |
s2VNNBFR | GGTAGAGCCAAGAAGTATTGATTTG | R2-DMT RT-PCR (for BFNNV) | [10] |
s2VNNSJR | GGCAACGGTTTGTCAGTGAC | R2-DMT RT-PCR (for SJNNV) | [10] |
s2VNNTPR | AAACCCAGAAGTGGCAGTTG | R2-DMT RT-PCR (for TPNNV) | [10] |
R1KS-5 GSPR1 | GGGTCACCAAATGGGGCCATCC | 5′ RACE for KSNNV RNA1 | [17] |
R1KS-5 GSPR2 | CACACAGAGCGGTGATGCCTGTGATGTC | [17] | |
R1KS-3 GSPF1 | GCGCAACAGCACTGCACGAACTGTCC | 3′ RACE for KSNNV RNA1 | [17] |
R1KS-3 GSPF2 | GAATGCCATCGTGGCTGACGAGCTTGG | [17] | |
R2KS-5 GSPR1 | GTTGCCAACGAGGATGGTCCGAAACG | 5′ RACE for KSNNV RNA2 | [17] |
R2KS-5 GSPR2 | CCGACCTGGAGACGTCAGTCGCTGCTC | [17] | |
R2KS-3 GSPF1 | CATGTGGAGAAGGCCGCAGGAGATGC | 3′ RACE for KSNNV RNA2 | [17] |
R2KS-3 GSPF2 | CAGCCGCGGCAGATACTGCTTCC | [17] | |
R1RG-5 GSPR1 | TCGCCGAAGGGCGCAATACCG | 5′ RACE for RGNNV RNA1 | This study |
R1RG-5 GSPR2 | CCACGAGGTCGGGAGCACACATACATGG | This study | |
R1RG-3 GSPF1 | TGGTGCCGGGCTTATCAGAGGAATTG | 3′ RACE for RGNNV RNA1 | This study |
R1RG-3 GSPF2 | GACTGGAATGACGTTGTAGCCAACGAG | This study | |
R2RG-5 GSPR1 | CGTGTTTGCGGGGCACATTGG | 5′ RACE for RGNNV RNA2 | This study |
R2RG-5 GSPR2 | GGCAGGAGGTCGGGGACGATGGTTG | This study | |
R2RG-3 GSPF1 | CAGCCCCGTCAAATCCTGCTGCCTGT | 3′ RACE for RGNNV RNA2 | This study |
R2RG-3 GSPF2 | CCGGTTCCCTAGTGCGTATCGTTGA | This study |
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Kim, Y.C.; Min, J.G.; Kim, K.I.; Jeong, H.D. Emergence of Reassortment between a New and Reported Types of Betanodavirus in Shellfish. Pathogens 2021, 10, 1232. https://doi.org/10.3390/pathogens10101232
Kim YC, Min JG, Kim KI, Jeong HD. Emergence of Reassortment between a New and Reported Types of Betanodavirus in Shellfish. Pathogens. 2021; 10(10):1232. https://doi.org/10.3390/pathogens10101232
Chicago/Turabian StyleKim, Young Chul, Joon Gyu Min, Kwang Il Kim, and Hyun Do Jeong. 2021. "Emergence of Reassortment between a New and Reported Types of Betanodavirus in Shellfish" Pathogens 10, no. 10: 1232. https://doi.org/10.3390/pathogens10101232