Analysis of MCP PCR Product - Lycoming College
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Poster #436 Genome Sequence Analysis of Largemouth Bass Virus Isolates from Smallmouth Bass Shannon Pipes and Jeff Newman Department of Biology, Lycoming College, Williamsport, PA. Abstract The viral pathogen, Largemouth Bass Virus (LMBV), is a member of the family Iridoviridae and has been known to cause large fish kills among Largemouth Bass, Micropterus salmoides. The viral pathogen is also consistently isolated from other species where diseased fish are found. Throughout the Susquehanna River Basin since 2005, Smallmouth Bass, Micropterus dolomieu, have been suffering from wide-spread disease related deaths. Even though LMBV is not clinically known to affect Smallmouth Bass, this virus is prevalent among diseased fish infected with other pathogens such as Pseudomonas aeruginosa. The question we seek to address is whether there are significant genetic differences between LMBV isolates from smallmouth bass and largemouth bass. Total DNA was isolated from preparations of LMBV derived from smallmouth bass and largemouth bass isolates. Five LMBV-specific primer sets were used to amplify fragments from each preparation for conventional Sanger sequencing and the total DNA preparations are also being analyzed using MiSeq NextGen sequence analysis. After analysis of the sequences, there does not appear to be any significant difference between the DNA sequences of the virus when it is isolated from either the largemouth bass or smallmouth bass. Further analysis using BLAST and other comparison tools will be studied to determine any differences between the sequences. Poster # 1367 Background • Isolates of LMBV were collected from smallmouth bass in the Susquehanna Watershed • Is LMBV causing disease in the smallmouth bass? • Is the smallmouth bass within the virus’s normal host range? • Are the SMB isolates different from the LMB isolates? Virus Isolates DNA Isolation (Qiagen) Qubit NextGen Full Genome Sequencing PCR for: • DNApol, Mtase • LMBVscsc, MCP MiSeq (2X300 PE) Sanger Sequence Methods Analysis of MCP PCR Product: Figure 1. Neighbor joining tree and distance matrix showing relationships among iridovirus Major Coat Protein Genes 309a LMBV Largemouth bass ranavirus major capsid protein gene Largemouth bass virus isolate LMBVPB02-30 major capsid protein gene 329Q LMBV 359 LMBV 337 LMBV 309b LMBV Largemouth bass virus MCP gene for major capsid protein Largemouth bass virus - snakehead isolate SH13 major capsid protein gene Guppy virus 6 mcp gene for major capsid protein isolate F93-20*6 Doctor fish virus ORF1 (partial) mcp and ICP 46 (partial) genes Largemouth bass ulcerative syndrome virus strain EPC060608-08 major capsid protein gene Short-finned eel ranavirus major capsid protein gene complete cds Rana tigrina ranavirus major capsid protein (MCP) gene complete cds Tiger frog virus complete genome Pike-perch iridovirus major capsid protein gene complete cds Rana catesbeiana virus XDL-2012 major capsid protein gene partial cds European catfish virus major capsid protein gene complete cds European sheatfish virus major capsid protein gene complete cds Common midwife toad ranavirus isolate Mesotriton alpestris/2008/E complete genome Chinese giant salamander iridovirus isolate CGSIV-HN1104 complete genome European sheatfish virus complete genome Andrias davidianus ranavirus isolate 2010SX complete genome 30 63 100 87 87 46 29 59 51 98 77 73 94 0.2 Analysis of DNApol PCR Product: Figure 2. Neighbor joining tree and distance matrix showing relationships among iridovirus DNA Polymerase Genes Conclusion: PCR products including MCP, DNApol, Mtase, and LMBVscsc of new isolates cluster closely with previously identified LMBV isolates. LMBV 359 Largemouth bass ranavirus DNA-dependent DNA polymerase gene partial cds LMBV 337 LMBV 329 LMBV 309a LMBV 359M Singapore grouper iridovirus complete genome Grouper iridovirus complete genome Rana grylio iridovirus complete genome Soft-shelled turtle iridovirus complete genome Frog virus 3 isolate SSME complete genome Tiger frog virus complete genome Common midwife toad ranavirus partial pol gene for DNA polymerase Regina ranavirus clone PstI-1.6 RSIV DNA polymerase-like protein gene partial cds European sheatfish virus complete genome Epizootic haematopoietic necrosis virus complete genome Common midwife toad ranavirus isolate Mesotriton alpestris/2008/E complete genome Chinese giant salamander iridovirus isolate CGSIV-HN1104 complete genome Andrias davidianus ranavirus isolate 2010SX complete genome Andrias davidianus ranavirus isolate 1201 complete genome Ambystoma tigrinum stebbensi virus complete genome 11 12 48 54 66 100 100 99 45 61 47 5 5 86 0.1 Acknowledgements: Funding provided by PA Fish and Boat Commission and CWI NextGen Access through GCAT-SEEK References L. Iwanowicz , C. Densmore , C. Hahn , P. McAllister & J. Odenkirk (2013) Identification of Largemouth Bass Virus in the Introduced Northern Snakehead Inhabiting the Chesapeake Bay Watershed, Journal of Aquatic Animal Health, 25:3, 191-196. Table 1. Whole Genome Sequencing Stats Conclusions • 1 st genome sequences of LMBV • No obvious differences observed in LMBV from SMB Figure 4. LMBV gene comparison 329 (LMB) 309 (SMB) 337 (SMB) 359 (SMB) reads 2,009,727 1,384,503 1,376,857 3,551,237 coverage 1350x 1040x 1650x 3025x contigs 6 6 4 12 genome size 98,613 98,032 98,625 101,410 annotated genes (RAST) 105 107 107 108 Figure 3. Comparison of LMBV to closest Iridovirus genome (from redfin perch Accession FJ433873) Figure 5. ANI Comparison: (A) Comparison of LMBV329 isolated from Largemouth Bass and LMBV337 isolated from Smallmouth Bass (B) Comparison of closest Iridovirus genome (Accession FJ433873) from Redfin Perch and LMBV329 isolated from Largemouth Bass (A) (C) Comparison of closest Iridovirus genome (Accession FJ433873) from Redfin Perch and LMBV337 isolated from Smallmouth Bass (B) (C)
Transcript of Analysis of MCP PCR Product - Lycoming College
Poster #436 Genome Sequence Analysis of Largemouth Bass Virus Isolates from Smallmouth BassShannon Pipes and Jeff Newman
Department of Biology, Lycoming College, Williamsport, PA.
AbstractThe viral pathogen, Largemouth Bass Virus (LMBV), is a member of the family Iridoviridae and has been known to cause large fish kills among Largemouth Bass, Micropterus salmoides. The viral pathogen is also consistently isolated from other species where diseased fish are found. Throughout the Susquehanna River Basin since 2005, Smallmouth Bass, Micropterus dolomieu, have been suffering from wide-spread disease related deaths. Even though LMBV is not clinically known to affect Smallmouth Bass, this virus is prevalent among diseased fish infected with other pathogens such as Pseudomonas aeruginosa. The question we seek to address is whether there are significant genetic differences between LMBV isolates from smallmouth bass and largemouth bass. Total DNA was isolated from preparations of LMBV derived from smallmouth bass and largemouth bass isolates. Five LMBV-specific primer sets were used to amplify fragments from each preparation for conventional Sanger sequencing and the total DNA preparations are also being analyzed using MiSeq NextGen sequence analysis. After analysis of the sequences, there does not appear to be any significant difference between the DNA sequences of the virus when it is isolated from either the largemouth bass or smallmouth bass. Further analysis using BLAST and other comparison tools will be studied to determine any differences between the sequences.
Poster # 1367
Background• Isolates of LMBV were collected from smallmouth bass in the
Susquehanna Watershed• Is LMBV causing disease in the smallmouth bass?• Is the smallmouth bass within the virus’s normal host range?• Are the SMB isolates different from the LMB isolates?
Virus Isolates
DNA Isolation (Qiagen)
Qubit NextGen
Full Genome Sequencing
PCR for:• DNApol, Mtase• LMBVscsc, MCP
MiSeq(2X300 PE)
Sanger Sequence
Methods
Analysis of MCP PCR Product:
Figure 1. Neighbor joining tree and distance matrix showing relationships amongiridovirus Major Coat Protein Genes
309a LMBVLargemouth bass ranavirus major capsid protein gene
Largemouth bass virus isolate LMBVPB02-30 major capsid protein gene329Q LMBV359 LMBV337 LMBV309b LMBV
Largemouth bass virus MCP gene for major capsid proteinLargemouth bass virus - snakehead isolate SH13 major capsid protein gene
Guppy virus 6 mcp gene for major capsid protein isolate F93-20*6Doctor fish virus ORF1 (partial) mcp and ICP 46 (partial) genes
Largemouth bass ulcerative syndrome virus strain EPC060608-08 major capsid protein geneShort-finned eel ranavirus major capsid protein gene complete cdsRana tigrina ranavirus major capsid protein (MCP) gene complete cdsTiger frog virus complete genomePike-perch iridovirus major capsid protein gene complete cdsRana catesbeiana virus XDL-2012 major capsid protein gene partial cdsEuropean catfish virus major capsid protein gene complete cdsEuropean sheatfish virus major capsid protein gene complete cds
Common midwife toad ranavirus isolate Mesotriton alpestris/2008/E complete genomeChinese giant salamander iridovirus isolate CGSIV-HN1104 complete genome
European sheatfish virus complete genomeAndrias davidianus ranavirus isolate 2010SX complete genome30
63100
87
87
46
29
59
51
98
77
73
94
0.2
Analysis of DNApol PCR Product:
Figure 2. Neighbor joining tree and distance matrix showing relationships amongiridovirus DNA Polymerase Genes
Conclusion: PCR products including MCP, DNApol, Mtase, and LMBVscsc of new isolates cluster closely with previously identified LMBV isolates.
LMBV 359Largemouth bass ranavirus DNA-dependent DNA polymerase gene partial cdsLMBV 337LMBV 329LMBV 309aLMBV 359M
Singapore grouper iridovirus complete genomeGrouper iridovirus complete genome
Rana grylio iridovirus complete genomeSoft-shelled turtle iridovirus complete genomeFrog virus 3 isolate SSME complete genome
Tiger frog virus complete genomeCommon midwife toad ranavirus partial pol gene for DNA polymerase
Regina ranavirus clone PstI-1.6 RSIV DNA polymerase-like protein gene partial cdsEuropean sheatfish virus complete genomeEpizootic haematopoietic necrosis virus complete genomeCommon midwife toad ranavirus isolate Mesotriton alpestris/2008/E complete genomeChinese giant salamander iridovirus isolate CGSIV-HN1104 complete genomeAndrias davidianus ranavirus isolate 2010SX complete genomeAndrias davidianus ranavirus isolate 1201 complete genome
Ambystoma tigrinum stebbensi virus complete genome1112
4854
66
100
100
99
45
614755
86
0.1
Acknowledgements:Funding provided by PA Fish and Boat Commission and CWINextGen Access through GCAT-SEEKReferencesL. Iwanowicz , C. Densmore , C. Hahn , P. McAllister & J. Odenkirk (2013) Identification of Largemouth Bass Virus in the Introduced Northern Snakehead Inhabiting the Chesapeake Bay Watershed, Journal of Aquatic Animal Health, 25:3, 191-196.
Table 1. Whole Genome Sequencing Stats
Conclusions• 1st genome sequences of LMBV• No obvious differences observed in
LMBV from SMB
Figure 4. LMBV gene comparison
329 (LMB) 309 (SMB) 337 (SMB) 359 (SMB)
reads 2,009,727 1,384,503 1,376,857 3,551,237
coverage 1350x 1040x 1650x 3025x
contigs 6 6 4 12
genome size 98,613 98,032 98,625 101,410
annotatedgenes (RAST) 105 107 107 108
Figure 3. Comparison of LMBV to closestIridovirus genome (from redfin perchAccession FJ433873)
Figure 5. ANI Comparison:
(A) Comparison of LMBV329 isolated from Largemouth Bass and LMBV337 isolated from Smallmouth Bass
(B) Comparison of closest Iridovirus genome (Accession FJ433873) from Redfin Perch and LMBV329 isolated from Largemouth Bass
(A)
(C) Comparison of closest Iridovirus genome (Accession FJ433873) from Redfin Perch and LMBV337 isolated from Smallmouth Bass
(B)
(C)
