PCR Detection and Phylogenetic Analysis of Megalocytivirus Isolates in Farmed Giant Sea Perch Lates calcarifer in Southern Taiwan
Abstract
:1. Introduction
2. Materials and Methods
2.1. Fish and Virus
2.2. Viral DNA Extraction
2.3. PCR Assays for the Identification and Differentiation of GSPIV Isolates from Giant Sea Perch
2.4. Viral MCP Gene Cloning and Sequencing
2.5. Phylogenetic Analysis
3. Results
3.1. PCR Identification of GSPIV Isolates from Giant Sea Perch
3.1.1. The OIE-Registered Primer Sets
3.1.2. The Megalocytivirus Universal Primer Set
3.1.3. Primer Sets Respectively Specific to RSIV, ISKNV and TRBIV
3.1.4. The Hemi-Nested (hn) Primer Set
3.2. Phylogenetic Analysis of GSPIV Isolates from Giant Sea Perch
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Isolate a | Date (yr/mo) of Isolation | Fish Farm District b |
---|---|---|
GSPIV/TW1 | 2017/04 | PT |
GSPIV/TW2 | 2017/04 | PT |
GSPIV/TW3 | 2017/04 | PT |
GSPIV/TW4 | 2017/05 | K |
GSPIV/TW5 | 2017/05 | PT |
GSPIV/TW6 | 2017/05 | PT |
GSPIV/TW7 | 2017/06 | PT |
GSPIV/TW8 | 2017/06 | K |
GSPIV/TW9 | 2017/06 | K |
GSPIV/TW10 | 2017/06 | K |
GSPIV/TW11 | 2017/06 | K |
GSPIV/TW12 | 2017/06 | PT |
GSPIV/TW13 | 2017/06 | K |
GSPIV/TW14 | 2017/07 | PT |
GSPIV/TW15 | 2017/07 | PT |
GSPIV/TW16 | 2017/07 | PT |
GSPIV/TW17 | 2017/07 | PT |
GSPIV/TW18 | 2017/08 | PT |
GSPIV/TW19 | 2017/08 | PT |
GSPIV/TW20 | 2017/08 | PT |
GSPIV/TW21 | 2017/08 | PT |
GSPIV/TW22 | 2017/08 | PT |
GSPIV/TW23 | 2017/08 | PT |
GSPIV/TW24 | 2017/08 | PT |
GSPIV/TW25 | 2017/09 | PT |
GSPIV/TW26 | 2017/09 | PT |
GSPIV/TW27 | 2017/09 | PT |
GSPIV/TW28 | 2017/09 | PT |
GSPIV/TW29 | 2017/09 | PT |
GSPIV/TW30 | 2017/09 | PT |
GSPIV/TW31 | 2017/09 | PT |
GSPIV/TW32 | 2017/09 | PT |
GSPIV/TW33 | 2017/09 | PT |
GSPIV/TW34 | 2017/09 | PT |
GSPIV/TW35 | 2017/09 | PT |
GSPIV/TW36 | 2017/09 | PT |
GSPIV/TW37 | 2017/09 | PT |
GSPIV/TW38 | 2017/09 | PT |
GSPIV/TW39 | 2017/10 | PT |
Purpose | Primer | Primer Sequence (5′-3′) | PCR Conditions | Product | Target Gene | Reference |
---|---|---|---|---|---|---|
Detection | OIE RSIV/ISKNV | |||||
1-F | CTCAAACACTCTGGCTCATC | 94 °C, 5 min; 30 cycles: 95 °C, 30 s; 58 °C, 1 min; 72 °C, 1 min; 72 °C, 5 min | 570 bp | Pst I fragment | [28] | |
1-R | GCACCAACACATCTCCTATC | |||||
OIE RSIV | ||||||
4-F | CGGGGGCAATGACGACTACA | 94 °C, 5 min; 30 cycles: 95 °C, 30 s; 58 °C, 1 min; 72°C, 1 min; 72 °C, 5 min | 568 bp | DNA Polymerase | [29] | |
4-R | CCGCCTGTGCCTTTTCTGGA | |||||
RSIV/ISKNV/TRBIV | ||||||
MCP- uni332- F3 | AGGTGTCGGTGTCATTAACGACCT G | 94 °C, 5 min; 30 cycles: 95 °C, 30 s; 58 °C, 1 min; 72 °C, 1 min; 72 °C, 5 min | 777 bp | MCP | [6,31] | |
MCP- uni1108- R8 | TCTCAGGCATGCTGGGCGCAAAG | |||||
RSIV | ||||||
MCP- specR674- F4 | CCCGCACTGACCAACGTGTCC | 94 °C, 5 min; 30 cycles: 95 °C, 30 s; 58 °C, 1 min; 72 °C, 1 min; 72 °C, 5 min | 191 bp | MCP | [6] | |
MCP- specR888- R6 | CACAGGGTGACTGAACTCAGG TCG | |||||
ISKNV | ||||||
MCP- specI465- F3 | GGTGGCCGGCATCACCAACGG C | 94 °C, 5 min; 30 cycles: 95 °C, 30 s; 58 °C, 1 min; 72 °C, 1 min; 72 °C, 5 min | 413 bp | MCP | [6] | |
MCP- specI879- R3 | CACGGGGTGACTGAACCTG | |||||
TRBIV | ||||||
MCP- specT37- F1 | TTC ATC GAC ATC TCC GCT TTC | 94 °C, 5 min; 30 cycles: 95 °C, 30 s; 58 °C, 1 min; 72 °C, 1 min; 72 °C, 5 min | 453 bp | MCP | [6] | |
MCP-specT490- R1 | TST GAC CGT TGG TGA TAC CGG AG | |||||
hn-TRBIV | ||||||
MCP- specT37- F1 | TTC ATC GAC ATC TCC GCT TTC | 94 °C, 5 min; 30 cycles: 95 °C, 30 s; 58 °C, 1 min; 72 °C, 1 min; 72 °C, 5 min | 306 bp | MCP | Newly designed | |
hnMCP-specT342-R2 | CAC CGA CAC CTC CTC AAC C | |||||
β−actin | ||||||
β−actin−F | TAC CAC CGG TAT CGT CAT GGA | 94 °C, 5 min; 30 cycles: 95 °C, 30 s; 60 °C, 1 min; 72 °C, 1 min; 72 °C, 5 min | 150 bp | β−actin | [27] | |
β−actin−R | CCA CGC TCT GTC AGG ATC TTC | |||||
Cloning | JM-MCP | |||||
JM-MCP-F | AGG TGC GAA CGT AAC CAG T | 94 °C, 5 min; 30 cycles: 95 °C, 30 s; 58 °C, 1 min 30 s; 72 °C, 1 min; 72 °C, 8 min | 1348 bp | MCP | Newly designed | |
JM-MCP-R | TTA CAG GAT AGG GAA GCC TGC |
Virus Name | Genus | MCP Accession No. | Country (origin) | Year | Host |
---|---|---|---|---|---|
Giant sea perch iridovirus (GSPIV/TW2/PT) | Megalocytivirus | MH237932 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Giant sea perch iridovirus (GSPIV/TW4/K) | Megalocytivirus | MH237933 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Giant sea perch iridovirus (GSPIV/TW5/PT) | Megalocytivirus | MH237934 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Giant sea perch iridovirus (GSPIV/TW7/PT) | Megalocytivirus | MH237935 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Giant sea perch iridovirus (GSPIV/TW8/K) | Megalocytivirus | MH237936 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Giant sea perch iridovirus (GSPIV/TW11/K) | Megalocytivirus | MH237937 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Giant sea perch iridovirus (GSPIV/TW17/PT) | Megalocytivirus | MH237938 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Giant sea perch iridovirus (GSPIV/TW22/PT) | Megalocytivirus | MH237939 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Giant sea perch iridovirus (GSPIV/TW25/PT) | Megalocytivirus | MH237940 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Giant sea perch iridovirus (GSPIV/TW29/PT) | Megalocytivirus | MH237941 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Giant sea perch iridovirus (GSPIV/TW30/PT) | Megalocytivirus | MH237942 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Giant sea perch iridovirus (GSPIV/TW35/PT) | Megalocytivirus | MH237943 | Taiwan | 2017 | Giant sea perch (Lates calcarifer) |
Rock bream iridovirus (RBIV-HD) | Megalocytivirus | KT031401 | Korea | 2015 | Rock bream (Oplegnathus fasciatus) |
Grouper iridovirus | Megalocytivirus | KT989778 | Taiwan | 2015 | Grouper (Epinephelus sp.) |
Rock bream iridovirus (RBIV-C1) | Megalocytivirus | HQ105005 | China | 2009 | Rock bream (Oplegnathus fasciatus) |
Red sea bream iridovirus (RIE12-1) | Megalocytivirus | AP017456 | Japan | 2016 | Red sea bream (Chrysophrys major) |
Sea bass iridovirus | Megalocytivirus | AY310917 | China | 1993 | Sea bass (Lateolabrax sp) |
Red sea bream iridovirus (RSIV-2) | Megalocytivirus | AB666329 | Japan | 1994 | Greater amberjack (Seriola dumerili) |
Red sea bream iridovirus (3GG1) | Megalocytivirus | AB666326 | Hong Kong | 2004 | Orange-spotted grouper (Epinephelus coioides) |
Red sea bream iridovirus (7GG) | Megalocytivirus | AB666322 | Hong Kong | 2004 | Orange-spotted grouper (Epinephelus coioides) |
Red sea bream iridovirus (GIG42) | Megalocytivirus | AB666323 | Hong Kong | 2002 | Giant grouper (Epinephelus lanceolatus) |
Spotted knifejaw iridovirus (SKIV-ZJ07) | Megalocytivirus | GQ202216 | China | 2009 | Spotted knifejaw (Oplegnathus punctatus) |
King grouper iridovirus (KGIV-05) | Megalocytivirus | EU847414 | Taiwan | 2005 | Giant grouper (Epinephelus lanceolatus) |
Barramundi perch iridovirus (BPIV-08) | Megalocytivirus | EU847418 | Taiwan | 2008 | Barramundi perch (Lates calcarifer) |
Stone flounder iridovirus (724) | Megalocytivirus | HQ263620 | China | 2010 | Stone flounder (Kareius bicoloratus) |
Rock bream iridovirus (SBIV-KOR-TY) | Megalocytivirus | AY532613 | Korea | 2000-2001 | Sea bass (Lateolabrax japonicus) |
Infectious spleen and kidney necrosis virus (ISKNV-HT) | Megalocytivirus | HQ317464 | China | 2006 | Mandarin fish (Siniperca chuatsi) |
Rock bream iridovirus (CNU-2) | Megalocytivirus | AY849394 | South Korea | 2004 | Rock bream (Oplegnathus fasciatus) |
Olive flounder iridovirus | Megalocytivirus | DQ198145 | South Korea | 2005 | Bastard halibut (Paralichthys olivaceus) |
Giant seaperch iridovirus (GSIV-K1) | Megalocytivirus | EU315313 | Taiwan | 2007 | Giant sea perch (Lates calcarifer) |
Barramundi perch iridovirus (BPIV-05) | Megalocytivirus | EU847416 | Taiwan | 2005 | Barramundi perch (Lates calcarifer) |
Red sea bream iridovirus | Megalocytivirus | AB109371 | Japan | 1994 | Red sea bream (Chrysophrys major) |
Red sea bream iridovirus | Megalocytivirus | AY310918 | Japan/Thailand | 2008 | Red sea bream (Chrysophrys major) |
Red sea bream iridovirus (6SB) | Megalocytivirus | AB666327 | Hong Kong | 2004 | Yellowfin sea bream (Acanthopagrus latus) |
Red seabream iridovirus (HyoDS-13) | Megalocytivirus | LC150886 | Japan | 2016 | Devil stinger (Inimicus japonicus) |
Sea bass iridovirus | Megalocytivirus | AY310917 | China | 1993 | Sea bass (Lateolabrax japonicus) |
Grouper sleepy disease iridovirus | Megalocytivirus | AB109370 | Thailand | 1993 | Brown spotted grouper (Epinephelus malabaricus) |
Grouper sleepy disease iridovirus | Megalocytivirus | AY285746 | Thailand | 1993 | Brown spotted grouper (Epinephelus malabaricus) |
Red seabream iridovirus (TGA12) | Megalocytivirus | AB666319 | Singapore | 2000 | Brown-marbled grouper (Epinephelus fuscoguttatus) |
Infectious spleen and kidney necrosis virus | Megalocytivirus | AB669096 | Southeast Asia/Japan | 2006 | Banggai cardinalfish (Pterapogon kauderni) |
Banggai cardinalfish iridovirus | Megalocytivirus | HQ857787 | USA | 2011 | Banggai cardinalfish (Pterapogon kauderni) |
Marble sleepy goby iridovirus | Megalocytivirus | HM067835 | China | 2009 | Marble goby (Oxyeleotris marmorata) |
African lampeye iridovirus | Megalocytivirus | AY285745 | Indonesia | 2005 | African lampeye (Aplocheilichthys normani) |
Megalocytivirus Sabah/RAA/2012 (HGIV67) | Megalocytivirus | JQ253369 | Malaysia | 2006 | Barramundi cod (Cromileptes altivelis) |
African lampeye iridovirus | Megalocytivirus | AB109368 | Indonesia/Japan | 1998 | African lampeye (Aplocheilichthys normani) |
Dwarf gourami iridovirus | Megalocytivirus | AY285744 | Thailand | 2002 | Dwarf gourami (Trichogaster lalius) |
Giant sea perch iridovirus (GSIV_Pt_843_05) | Megalocytivirus | JF264354 | Taiwan | 2005 | Giant sea perch (Lates calcarifer) |
Mullet iridovirus MA5/5 | Megalocytivirus | AB666343 | Singapore | 2000 | Fathead mullet (Mugil cephalus) |
Dwarf gourami iridovirus | Megalocytivirus | AY989901 | Southeast Asia | 2004 | Dwarf gourami (Trichogaster lalius) |
Infectious spleen and kidney necrosis virus (lapulapu) | Megalocytivirus | AB666339 | Philippines | 2000 | Orange-spotted grouper (Epinephelus coioides) |
Megalocytivirus Sabah/RAA/2012 (OSGIV75) | Megalocytivirus | JQ253372 | Malaysia | 2011 | Orange-spotted grouper (Epinephelus coioides) |
Murray cod iridovirus | Megalocytivirus | AY936203 | Australia | 2003 | Murray cod (Maccullochella peeli) |
Infectious spleen and kidney necrosis virus (ISKNV-QY) | Megalocytivirus | HQ317460 | China | 2009 | Mandarin fish (Siniperca chuatsi) |
Dwarf gourami iridovirus DGA4/6K | Megalocytivirus | AB666344 | Singapore | 2000 | Dwarf gourami (Trichogaster lalius) |
Giant sea perch iridovirus (GSIV_Pt_836_05) | Megalocytivirus | JF264350 | Taiwan | 2005 | Giant sea perch (Lates calcarifer) |
Sea perch iridovirus (CH-1) | Megalocytivirus | HM067603 | South Korea | 2010 | Sea perch (Lateolabrax sp.) |
Turbot iridovirus isolate (R-603) | Megalocytivirus | HM596017 | China | 2009 | Turbot (Scophthalmus maximus) |
Turbot reddish body iridovirus | Megalocytivirus | AY590687 | China | 2002 | Turbot (Scophthalmus maximus) |
Korean flounder iridovirus (FLIV-MI) | Megalocytivirus | AY633982 | Korea | 2005 | Olive flounder (Paralichthys olivaceus) |
Olive flounder iridovirus | Megalocytivirus | AY661546 | Korea | 2004 | Olive flounder (Paralichthys olivaceus) |
Korean flounder iridovirus (FLIV-JJY) | Megalocytivirus | AY633990 | Korea | 2005 | Olive flounder (Paralichthys olivaceus) |
Korean flounder iridovirus (FLIV-WD2) | Megalocytivirus | AY633985 | Korea | 2005 | Olive flounder (Paralichthys olivaceus) |
Olive flounder iridovirus (OFLIV-1) | Megalocytivirus | EU276417 | Korea | 2007 | Olive flounder (Paralichthys olivaceus) |
Turbot reddish body iridovirus (case3) | Megalocytivirus | KX354221 | USA | 1991 | Oscar (Astronotus ocellatus) |
Rock bream iridovirus (RBIV_Tp_45_08) | Megalocytivirus | JF264352 | Taiwan | 2008 | Rock bream (Oplegnathus fasciatus) |
Turbot reddish body iridovirus (case1) | Megalocytivirus | KX354223 | Canada/Southeast Asia | 1986 | Angelfish (Pterophyllum scalare) |
Turbot reddish body iridovirus (case2) | Megalocytivirus | KX354222 | Australia/ Singapore | 1988 | Dwarf gourami (Trichogaster lalius) |
Threespine stickleback iridovirus | Megalocytivirus | HQ857785 | Canada | 2012 | Threespine stickleback (Gasterosteus aculeatus) |
Tiger frog virus | Ranavirus | AF389451 | China | 2002 | Tiger frog (Rana tigrina rugulosa) |
Isolate | OIE Primer Sets a | Universal Primer Set b | Primer Sets Respectively Specific to c | hn-TRBIV d | |||
1-F/1-R | 4-F/4-R | RSIV/ISKNV/TRBIV | RSIV | ISKNV | TRBIV | ||
GSPIV/TW1 | – | – | – | – | – | – | + |
GSPIV/TW2 | – | – | + | – | – | + | + |
GSPIV/TW3 | – | – | – | – | – | – | + |
GSPIV/TW4 | – | – | + | – | – | + | + |
GSPIV/TW5 | – | – | + | – | – | + | + |
GSPIV/TW6 | – | – | – | – | – | – | + |
GSPIV/TW7 | – | – | + | – | – | + | + |
GSPIV/TW8 | – | – | + | – | – | + | + |
GSPIV/TW9 | – | – | + | – | – | + | + |
GSPIV/TW10 | – | – | – | – | – | – | + |
GSPIV/TW11 | – | – | + | – | – | + | + |
GSPIV/TW12 | – | – | – | – | – | – | – |
GSPIV/TW13 | – | – | + | – | – | + | + |
GSPIV/TW14 | – | – | – | – | – | – | + |
GSPIV/TW15 | – | – | – | – | – | – | + |
GSPIV/TW16 | – | – | – | – | – | – | + |
GSPIV/TW17 | – | – | + | – | – | + | + |
GSPIV/TW18 | – | – | – | – | – | – | + |
GSPIV/TW19 | – | – | – | – | – | – | + |
GSPIV/TW20 | – | – | – | – | – | – | + |
GSPIV/TW21 | – | – | + | – | – | + | + |
GSPIV/TW22 | – | – | + | – | – | + | + |
GSPIV/TW23 | – | – | – | – | – | – | + |
GSPIV/TW24 | – | – | – | – | – | – | + |
GSPIV/TW25 | – | – | + | – | – | + | + |
GSPIV/TW26 | – | – | + | – | – | + | + |
GSPIV/TW27 | – | – | – | – | – | – | + |
GSPIV/TW28 | – | – | + | – | – | + | + |
GSPIV/TW29 | – | – | + | – | – | + | + |
GSPIV/TW30 | – | – | + | – | – | + | + |
GSPIV/TW31 | – | – | – | – | – | – | + |
GSPIV/TW32 | – | – | – | – | – | – | + |
GSPIV/TW33 | – | – | + | – | – | + | + |
GSPIV/TW34 | – | – | + | – | – | + | + |
GSPIV/TW35 | – | – | + | – | – | + | + |
GSPIV/TW36 | – | – | + | – | + | – | – |
GSPIV/TW37 | – | – | – | – | – | – | – |
GSPIV/TW38 | – | – | – | – | – | – | + |
GSPIV/TW39 | + | + | + | + | – | – | – |
Virus Isolate a | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1. MH237932 | 100.00 | |||||||||||||||
2. MH237933 | 98.01 | 100.00 | ||||||||||||||
3. MH237934 | 99.85 | 98.01 | 100.00 | |||||||||||||
4. MH237935 | 97.86 | 99.85 | 97.86 | 100.00 | ||||||||||||
5. MH237936 | 97.78 | 99.77 | 97.78 | 99.62 | 100.00 | |||||||||||
6. MH237937 | 97.78 | 99.77 | 97.78 | 99.62 | 99.54 | 100.00 | ||||||||||
7. MH237938 | 98.01 | 100.00 | 98.01 | 99.85 | 99.77 | 99.77 | 100.00 | |||||||||
8. MH237939 | 97.71 | 99.69 | 97.71 | 99.54 | 99.46 | 99.46 | 99.69 | 100.00 | ||||||||
9. MH237940 | 97.86 | 99.85 | 97.86 | 99.69 | 99.62 | 99.62 | 99.85 | 99.54 | 100.00 | |||||||
10. MH237941 | 97.94 | 99.92 | 97.94 | 99.77 | 99.69 | 99.69 | 99.92 | 99.62 | 99.77 | 100.00 | ||||||
11. MH237942 | 97.86 | 99.85 | 97.86 | 99.69 | 99.62 | 99.62 | 99.85 | 99.54 | 99.69 | 99.77 | 100.00 | |||||
12. MH237943 | 97.86 | 99.85 | 97.86 | 99.69 | 99.62 | 99.62 | 99.85 | 99.54 | 99.69 | 99.77 | 99.69 | 100.00 | ||||
13. JF264352 | 99.85 | 98.01 | 99.85 | 97.86 | 97.78 | 97.78 | 98.01 | 97.71 | 97.86 | 97.94 | 97.86 | 97.86 | 100.00 | |||
14. KX354221 | 99.85 | 98.01 | 99.85 | 97.86 | 97.78 | 97.78 | 98.01 | 97.71 | 97.86 | 97.94 | 97.86 | 97.86 | 99.93 | 100.00 | ||
15. KX354222 | 99.85 | 98.01 | 99.85 | 97.86 | 97.78 | 97.78 | 98.01 | 97.71 | 97.86 | 97.94 | 97.86 | 97.86 | 99.93 | 100.00 | 100.00 | |
16. KX354223 | 99.85 | 98.01 | 99.85 | 97.86 | 97.78 | 97.78 | 98.01 | 97.71 | 97.86 | 97.94 | 97.86 | 97.86 | 99.93 | 100.00 | 100.00 | 100.00 |
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Tsai, J.-M.; Huang, S.-L.; Yang, C.-D. PCR Detection and Phylogenetic Analysis of Megalocytivirus Isolates in Farmed Giant Sea Perch Lates calcarifer in Southern Taiwan. Viruses 2020, 12, 681. https://doi.org/10.3390/v12060681
Tsai J-M, Huang S-L, Yang C-D. PCR Detection and Phylogenetic Analysis of Megalocytivirus Isolates in Farmed Giant Sea Perch Lates calcarifer in Southern Taiwan. Viruses. 2020; 12(6):681. https://doi.org/10.3390/v12060681
Chicago/Turabian StyleTsai, Jia-Ming, Song-Lang Huang, and Chung-Da Yang. 2020. "PCR Detection and Phylogenetic Analysis of Megalocytivirus Isolates in Farmed Giant Sea Perch Lates calcarifer in Southern Taiwan" Viruses 12, no. 6: 681. https://doi.org/10.3390/v12060681