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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.

mshzool@yahoo.com

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Briefly LCDV research History

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

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

Sequences of DNA for analyses 12

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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15

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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

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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