Introduction
Materials and methods
Viruses
Virus name | Fish species | Size (g) | Year | Accession no. | |
---|---|---|---|---|---|
MCP | ATPase | ||||
GGIV/Pt/1691/01 | Giant grouper (Epinephelus lanceolatus) | 9.4 | 2001 | JF264367 | JF264227 |
GGIV/Pt/1241/01 | Giant grouper (Epinephelus lanceolatus) | 2.2 | 2001 | JF264359 | JF264219 |
GGIV/Pt/1348/01 | Giant grouper (Epinephelus lanceolatus) | 8.2 | 2001 | JF264363 | JF264223 |
CSIV/Pt/2431/02 | Crimson snapper (Lutjanus erythropterus) | 3.3 | 2002 | JF264357 | JF264217 |
LMBIV/Kh/460/03 | Largemouth bass (Micropterus salmoides) | 5.3 | 2003 | JF264364 | JF264224 |
GGIV/Tn/337/04 | Giant grouper (Epinephelus lanceolatus) | 51.3 | 2004 | JF264360 | JF264220 |
GGIV/Cy/346/04 | Giant grouper (Epinephelus lanceolatus) | 7.4 | 2004 | JF264366 | JF264226 |
OSGIV/Pt/403/04 | Orange-spotted grouper (Epinephelus coioides) | 3.5 | 2004 | JF264365 | JF264225 |
OSGIV/Ph/617/04 | Orange-spotted grouper (Epinephelus coioides) | 11.6 | 2004 | JF264361 | JF264221 |
OSGIV/Tn/352/05 | Orange-spotted grouper (Epinephelus coioides) | 4.2 | 2005 | JF264358 | JF264218 |
SSBIV/Pt/703/05 | Silver sea bream (Rhabdosargus sarba) |
a
| 2005 | JF264356 | JF264216 |
GSIV/Pt/836/05 | Giant seaperch (Lates calcarifer) | 200 | 2005 | JF264350 | JF264210 |
GSIV/Pt/843/05 | Giant seaperch (Lates calcarifer) | 5 | 2005 | JF264354 | JF264214 |
GGIV/Pt/36/06 | Giant grouper (Epinephelus lanceolatus) | 10 | 2006 | JF264347 | JF264207 |
GGIV/Pt/48/06 | Giant grouper (Epinephelus lanceolatus) | 10.3 | 2006 | JF264351 | JF264211 |
GGIV/Pt/96/06 | Giant grouper (Epinephelus lanceolatus) |
a
| 2006 | JF264355 | JF264215 |
GSIV/Pt/113/06 | Giant seaperch (Lates calcarifer) | 2.2 | 2006 | JF264353 | JF264213 |
OSGIV/Pt/141/06 | Orange-spotted grouper (Epinephelus coioides) | 2.2 | 2006 | JF264345 | JF264205 |
RBIV/Tp/45/08 | Rock beam (Oplegnathus fasciatus) | 2.6 | 2008 | JF264352 | JF264212 |
OSGIV/Pt/308/08 | Orange-spotted grouper (Epinephelus coioides) | 4.7 | 2008 | JF264349 | JF264209 |
GSIV/Pt/327/08 | Giant seaperch (Lates calcarifer) | 6.8 | 2008 | JF264346 | JF264206 |
GSIV/Pt/610/08 | Giant seaperch (Lates calcarifer) | 52 | 2008 | JF264362 | JF264222 |
MGIV/Nt/546/09 | Marble goby (Oxyeleotris marmorata) | 24 | 2009 | JF264348 | JF264208 |
DNA extraction and primer design for PCR
Virus genus | Primer | Sequence | Product (bp) |
---|---|---|---|
Ranavirus
| RMCP-F | 5′-ATGACTTGTACAACGGGT-3′ | 1392 |
RMCP-R | 5′-TTACAAGATAGGGAACCCCAT-3′ | ||
RATPase-F | 5′-TGTGTTAAAATGTCTCAATA-3′ | 972 | |
RATPase-R | 5′-CGAATCATGGAAAGCACCGCAACC-3′ | ||
Megalocytivirus
| MMCP-Fa
| 5′-ATGTCTGCRATCTCAGGTG-3′ | 1362 |
MMCP-Rb
| 5′-TYACAGGATAGGGAAGCCTG-3′ | ||
MATPase-Fc
| 5′-ATGGAAATCMAAGAGTTGTCCYTG-3′ | 720 | |
MATPase-R | 5′-TTACRCCACGCCAGCCTTGTA-3′ |
PCR amplification, TA cloning and sequencing
Phylogenetic analysis
Virus | Genus | Origin | MCP accession no. | ATPase accession no. | References |
---|---|---|---|---|---|
SGIV |
Ranavirus
| Singapore | AAS18087 | AY521625 | [25] |
GIV |
Ranavirus
| Taiwan | AAV91066 | AY666015 | [30] |
FV3 |
Ranavirus
| USA | AAT09750 | AY548484 | [28] |
TFV |
Ranavirus
| China | AAL77814 | AF389451 | [10] |
ATV |
Ranavirus
| North America | AAP33191 | AY150217 | [13] |
TRBIV |
Megalocytivirus
| China | AY590687 | AY608684 | [22] |
DGIV |
Megalocytivirus
| Southeast Asia | AY285744 | AY319288 | [26] |
ISKNV |
Megalocytivirus
| China | AAL98730 | AF371960 | [9] |
OSGIV |
Megalocytivirus
| China | AAX82316 | AY894343 | [17] |
RSIV |
Megalocytivirus
| Japan | AY310918 | AB007367 | [16] |
FLIV |
Megalocytivirus
| Korea | AY633992 | N/A | [6] |
Genotype and host | Virus strain/nucleotide sequence maximum identity (%) (number of isolates) | Total number of fish species | ||
---|---|---|---|---|
GIVa
| ISKNVb
| RSIVc
| ||
GGIV (Giant grouper) | 97.9–100 (n = 5) | 94.9 | 99.6–99.9 (n = 3) | 8 |
OSGIV (Orange-spotted grouper) | 98.2–100 (n = 3) | 99.9 (n = 2) | 95.1 | 5 |
GSIV (Giant seaperch) | 98.6d (n = 1) | 99.5–99.8d (n = 3) | 99.7 (n = 1) | 5 |
CSIV (Crimson snapper) | 97.9d (n = 1) | 51.8 | 50.8 | 1 |
SSBIV (Silver sea bream) | 51.8 | 99.9 (n = 1) | 95.1 | 1 |
RBIV (Rock bream) | 51.3 | 92.9 | 93.3 (n = 1) | 1 |
MGIV (Marble goby) | 51.7 | 94.2 | 97.9d (n = 1) | 1 |
LMBIV (Largemouth bass) | 97.9 (n = 1) | 52 | 51.3 | 1 |
Total number of isolates | 11 | 6 | 6 |
Results
Phylogenetic analysis of MCP
Phylogenetic analysis of ATPase
Identity analysis using MCP
Analysis of genetic mutations in MCP
Isolate name | No. of nucleotide substitutions | No. of amino acid substitutions | Non-synonymous substitution rate (Ka) | Synonymous substitution rate (Ks) | Omega value (Ka/Ks) |
---|---|---|---|---|---|
OSGIV/Ph/617/04 | 3 | 0 | 0 | 0.009 | 0 |
OSGIV/Pt/403/04 | 4 | 1 | 0.001 | 0.009 | 0.111 |
OSGIV/Tn/352/05 | 3 | 0 | 0 | 0.009 | 0 |
OSGIV/Pt/141/06 | 2 | 0 | 0 | 0.003 | 0 |
OSGIV/Pt/308/08 | 2 | 0 | 0 | 0.003 | 0 |
GGIV/Pt/1348/01 | 1 | 0 | 0 | 0.003 | 0 |
GGIV/Pt/1691/01 | 1 | 0 | 0 | 0.003 | 0 |
GGIV/Pt/1241/01 | 2 | 0 | 0 | 0.006 | 0 |
GGIV/Tn/337/04 | 0 | 0 | 0 | 0 | 0 |
GGIV/Cy/346/04 | 2 | 2 | 0.002 | 0 | 0.002 |
GGIV/Pt/36/06 | 3 | 1 | 0.001 | 0.006 | 0.166 |
GGIV/Pt/48/06 | 3 | 1 | 0.001 | 0.006 | 0.166 |
GGIV/Pt/96/06 | 5 | 1 | 0.001 | 0.012 | 0.083 |
GSIV/Pt/843/05 | 3 | 0 | 0 | 0.006 | 0 |
GSIV/Pt/836/05 | 4 | 2 | 0.002 | 0.003 | 0.666 |
GSIV/Pt/113/06 | 7 | 2 | 0.003 | 0.006 | 0.5 |
GSIV/Pt/327/08 | 4 | 2 | 0.002 | 0.006 | 0.333 |
GSIV/Pt/610/08 | 11 | 1 | 0.001 | 0.030 | 0.033 |
CSIV/Pt/2431/02 | 8 | 4 | 0.004 | 0.012 | 0.333 |
LMBIV/Kn/460/03 | 9 | 1 | 0.001 | 0.024 | 0.041 |
SSBIV/Pt/703/05 | 2 | 0 | 0 | 0.003 | 0 |
RBIV/Tp/45/08 | 45 | 8 | 0.010 | 0.106 | 0.094 |
MGIV/Nt/546/09 | 29 | 2 | 0.006 | 0.062 | 0.096 |