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Phylogenetic Analyses of Lymphocystis Disease Virus of Fish using Blast and Clustal X
Dr. Md. Mosharrof Hossain Associate Professor
Department of ZoologyUniversity of Rajshahi, Bangladesh.
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Briefly LCDV research History
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Family: Iridoviridae
Genus: Lymphocystivirus
Strains/Species:
LCDV-1 (Paralichthys flexus)
LCDV-C (Paralichthys olivaceus)
LCDV-2 (Limada limanda)
LCDV-RF (Sebastes schlegeli )
What is LCDV ?
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A B
DC
LCDV virus infection4
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Objective of Research:
1. To know the biology of LCDV (a) In Vivo, (b) In vitro
2. To find the epitope of infection site
3. To know LCDV taxonomic position
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Materials and Methods
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The ExicyclerTM is a Real-Time qPCR system developed by Bioneer. The ExicyclerTM is equipped with an optical system that fits above the thermal cycler. It readily utilizes most fluorescent dyes and thus provide wide choices of excitation / emission wavelengths. It can also be used as a standard thermal cycler for general PCR reactions.
Experiment-1. PCR
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Principle of PCR
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Principle of PCR
Target DNA
Basics of PCR
Heating
95℃
Cooling
55℃
PolymerasePrimer
Extension
72℃
Cycling
Cycling
1 Cycle
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The PCR primers were designed according Kitamura et al. (2006)
Forward primer LCC-F 5´-CAA GTG TTA CTA GCG CTT T-3´
Reverse primer LCC-R 5´-ATC CCA TTG AAC CGT TCT-3´
Denaturing- 94 -1 min℃
Annealing- 54 - 1 min℃
Extension- 72 -1 min℃
Total PCR reaction mixture was 20 ㎕
A total 30 cycles
PCR Condition:
Primer design and PCR condition
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DNA transformation , cloning and sequencing:
Purified DNA
Expression in E. coli
Send for sequencing11
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Sequences of DNA for analyses 12
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LCDV Data mining
Homology inGENETYX-WIN
5.1CLUSTAL_X MEGA (NJPLOT)
DNA Sequencing analysis
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LCCR-AGCATCTTTATAACCAGAAGTATTTCCACCATTACCACCTGCTGTTATCACTGCTATTGGAGACGTCTTCAATTTAATACTGACATTGGACAAACGACCATAATTGGTAGATCCCATTGGATCTACATCCATCATATTGAGTGAATAGCAATACATGTGATAACCGGTGTCTACAGGAATAGAGCCTCCAAAATAATAAGGTTGAACCAAAGAATAATATTCACTACCCATTTCATTAAGACGAGCACTATTTTCATAAACCAAAGTAACATTTGAAATAGGATCAGCAGCAATACCCGGTAAATCGCTAGCAATTCCACCGTCAAAGATTACAGGAGAAGAACTGGTGTAATTGGATTGTATAGCTTGATAGGTAACATTACGCACACCGAAAAAAAGGATTTTAATGGCATGAGAAAATCTGATGTCAAAATTAGGACTTGGAATAGTTAGAGGTTGAAATACATGTTTAGGTGCTGTTTGTACCTGTTCTACCAAGATGTCTCTAGGTACTGTACCCATTAAACGACGTTCCTCATTGGTTACTACTACATTAGTAATCCATACTTGCACATCCTTTAAATCAGGTTTACCCCAGTCTAAATCGCCTGCTGTCAAAGGCATGATGGTAGAGTCGTTTTTATTTTGAAAGATCAATAATTCAGTCCAATCTCTCAGATGAAAAGTTAATCTTATTTCATTATAAGGCAAAGCAGCGCTGGGTAAAGCCATACCGCTATCTCGAGAAAAGAAATAAGGTAAAGGAAGTATTAACACTTTTTCAGGTAATTGACCATTGGAATCAACGGGTT
LCCF-GCTGTAGCTTATTTTGTACGAGAAACTAAACAATGTACCTGGTTCAGTAAATTACCAGTACTTTTAACACGTTGTTCTGGAACACCTAATTTTGATCAAGAATTTTCTGTCAATGTTTCTCGTGGTGGAGATTATGTACTTAATGCTTGGATGACGGTGCGTATTCCTGCTGTTAAATTGAAAACCAATAATCGTATGAACGCCAATGGTACTATCAGATGGTGTAAAAATTTATTTCATAATTTAGTTAAACAAACTTCTGTTCAATTTAATGATTTAGTTGCTCAAAAATTTGAGAGCTACTTTCTTGATTTTTGGTCCTCTTTTGGTATGTGTGGATCTAAACGTATAGGTTATGATAACATGATAGGTAATACTATTGATATGACACAACCCGTTGATTCCAATGGTCAATTACCTGAAAAAGTGTTAATACTTCCTTTACCTTATTTCTTTTCTCGAGATAGCGGTATGGCTTTACCCAGCGCTGCTTTGCCTTATAATGAAATAAGATTAACTTTTCATCTGAGAGATTGGACTGAATTATTGATCTTTCAAAATAAAAACGACTCTACCATCATGCCTTTGACAGCAGGCGATTTAGACTGGGGTAAACCTGATTTAAAGGATGTGCAAGTATGGATTACTAATGTAGTAGTAACCAATGAGGAACGTCGTTTAATGGGTACAGTACCTAGAGACATCTTGGTAGAACAGGTACAAACAGCACCTAAACATGTATTTCAACCTCTAACTATTCCAAGTCCTAATTTTGACATCAGATTTTCTCATGCCATTAAAATCC
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Iridoviruses.txt
Homology of MCP genes
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Cell Line & Virus Inoculation
Cells were seeded1.5×105 cells/ ㎖
Overnight confluence
Virus injection at 200 ㎕ /wells
Cell lines:
• FFN
• FSP
• FHM
• CHSE-214
• RTG-2
Experiment-2.Cell line infection
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M 4 8 10 12 16 CP
1347bp
PCR detection of LCDV
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Basics of PCR
1 Cycle
2 Cycle
3 Cycle
N Cycle
?Ideal graph
Real graph
Principle of PCR
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Principle of PCR
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Immunofluorescence TestProtocol
FFN Cells Seeded in 6-well round plate at 2 x 104 cells/chamber
Virus inoculation in the wells
Washing cells with PBS
MAbs treatment for 1h at 37°
Microscopy observation
Cells Stained with FITC (conjugate) for 1h at 37℃
Washing with PBS
Washing cells with PBS
Mounted with glycerol
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A
0
20
40
60
80
100
0 10 20 30 40 50 60
Days post challenge
Cu
mu
lati
ve L
CD
-in
cid
ence
(%
)
LCDV 10℃Cont. 10℃
B
0
20
40
60
80
100
0 10 20 30 40 50 60
Days post challenge
Cu
mu
lati
ve L
CD
-in
cid
ence
(%
)
LCDV 20℃
Cont. 20℃
C
0
20
40
60
80
100
0 5 10 15 20 25 30 35 40 45 50 55 60
Days post change
Cu
mu
lati
ve L
CD
-in
cid
ence
(%
)
LCDV 30℃
Cont. 30℃
LCDV In vivo infection
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0
20
40
60
80
100
0 5 10 15 20 25 30 35 40 45Days post change of rearing temperature
Cum
ulat
ive
LCD
-inci
denc
e(%
)
10℃=> 20℃ quickly
10℃=> 20℃ gradually
10℃=> 10℃ control
LCDV in vivo infection in changing temperature
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Rearing water
temperature
Days post challenge
Detected LCDV dose by PCR (PCR-U mg-tissue-1 * )
Fin Skin Spleen Kidney Brain Intestine
10℃35 10-3 10-3 - - - -
60 10-3 10-3 - - - -
20℃35 10-6 10-6 - - - -
60 10-6 10-6 - - - -
30℃ 35 10-3 10-3 - - - -
10℃=> 20℃ 60+45 10-6 10-6 - - - -
20℃=>10℃ 60+45 10-6 10-6 - - - -
LCDV infection in changing temperature and DNA copies
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0
1
2
3
4
5
6
7
4 8 10 12 16
Days post inoculation
Vir
us
infe
ctiv
ity(
logT
CID
50 m
l-1)
LCDV in vitro infection in cell lines
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A
G HE
B C D
F
Mock 4 Days 8 Days 12 Days
Fig.4b. Cytopathic effect morphology at the indicated times (upper panel, A,B,C,D) and immunofluorescence of lymphocystis disease virus infected FFN cells (lower panel, E,F,G,H)(×200, Scale bar 50µm). The infected cells showing strong antigen specific fluorescence (arrows) stained with MAbs and secondary antibody FITC goat anti-mouse IgG (Sigma, USA).
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Lymphocystivirus RanavirusMegalocytivi
rusG-I G-II G-III G-IV G-V G-VI EHNVSGIV/
GIV
G-I (European
flounder)100
78.6-
78.981.3 79.7 78.9 80.9 52
54.7-
54.952.6-54.2
G-II (Japanese
flounder)
99.6-
100
85.0-
85.2
90.1-
90.3
86.3-
86.7
84.3-
84.9
51.1-
51.7
56.5-
56.951-52.6
G-III (Rockfish) 100 85.8 84.9 86.5 51.754.7-
55.241.1-52.8
G-IV (Sea bass) 100 88.2 84.4 5255.6-
55.944.2-53.6
G-V (Painted
glassfish)100 83.9 53.8
56.6-
56.752.7-54.1
G-VI (Gourami) 100 50.656.8-
57.151.6-53.2
EHNV 10069.5-
69.956-57.4
SGIV/GIV 98.3 51.5-53.1
Megalocytivirus 93.0-100
LCDV genotyping and Homology of MCP gene
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J F03ShinJ i
FL
IV-E
J
AL IV
RB
IV-K
OR-T
Y2
0.05RanavirusIridovirus
Lymphocystivirus
Megalocytivirus
G-IV
1000
1000
1000
1000
1000
1000
1000
1000
Molecular phylogenetic tree for the relationship among 63 isolates of lymphocystiviruses and other iridoviruses based on the nucleotide sequence of the MCP gene. Bootstrap value 1000. 35
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Conclusions:
1. LCDV is an opportunistic pathogen that persistently exists in the flounder epidermis at low temperature and outbreaks at suitable temperature.
2. LCDV multiply in the optimum temperature at 20 when the fish have ℃healthy condition for virus persistence.
3. FFN is susceptible to LCDV, and LCDV is organ- specific both in in vivo & in vitro infections and multiply in fibroblast cells.
4. LCDV isolates from different habitats has the specific viral protein expression patterns and common antigenecity. These antigenic proteins enzymatic activity may help to find an epitope for vaccine preparation.
These research published in1. Hossain M. et al. 2008. Journal of Fish Diseases 31(6): 473–479, doi: 10.1111/j.1365-2761.2008.00917.x (IF: 1.697 )
2. Hossain M. et al. 2009. Journal of Fish Diseases 32(8): 699–703, doi: 10.1111/j.1365-2761.2009.01048.x (IF: 1.697)
3. Hossain M. et al. 2011. Journal of Fish Pathology, 24(2): 47-51.
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