in situ hybridization for tilv · in - situ . hybridization . inside. site “in position” or...
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Session 2Ha Thanh Dong1,2 & Kathy F.J. Tang3,4
In situ hybridization for TiLV1King Mongkut’s University of Technology Thonburi; 2Fish Health Platform, Centex Shrimp (Mahidol University/BIOTEC); 3Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shangdong, P.R. China; 4Tucson, Arizona 85719, United States of America.
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In - situ hybridization
inside site“in position” or “in place”
“Basically it involves formation of a hybrid molecule between an endogenous single-stranded RNA or DNA in the cell and a complementary single-stranded RNA or DNA probe”Gall (2016) Methods 98:4-9
a hybrid molecule
TiLV RNA in the cellTiLV specific probe
3’ 5’
5’ 3’
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In situ hybridization (ISH)
Purpose
To detect and confirm the presence of TiLV (through its nucleic acid) in the tissues and histopathological lesions.
To identify tissue tropisms of TiLV
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Development of TiLV-specific ISH probe
TiLV-infected tilapia
Extraction of total RNA
RT-PCR amplification of 415 bp genomic segment-3
clone a TiLV genomic fragment into a plasmid
PCR labeling with digoxigenin-11-dUTP
DIG-labeled TiLV probe
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Sequence of a 415-bpderived from genome segment 3 of TiLV cloned in pGEM-T. (used primer Nested ext-1 & ME1)
DNA probes preparation
• Probes were prepared using DIG-labeling Mix (Roche, Germany)
• Plasmid pGEM-415 bp was used as a template in the labeling reaction
• 282-bp fragment derived from IMNV was employed as an unrelated negative probe
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6
1. Labeling DNA probe
2. Denaturation of DNA probe
3. Hybridization
4. Color development
5. Result interpretation
Principle of in situ hybridization (ISH)
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1. React
2. Detect
3. Visualize
Non-Radioactive DetectionBased on Digoxigenin-label
Target DNA or RNAGene Probe
Anti-Digoxigenin Antibody
Color detection system
Digoxigenin Label
Alkaline Phosphatase
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In Situ Hybridization Incubator
Temperature control Trough for water to maintain humidity Rack to hold slides Cover to maintain temperature & humidity
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Unrelated probe TiLV-specific probe
Liver
Kidney
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Unrelated probe TiLV-specific probe
Brain
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Unrelated probe TiLV-specific probe
Gills
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H&E Unrelated probe Specific-probe
Example of ISKNV (1)
Taken in conjunction with outbreaks described in Dong et al. 2017 Fish & Shellfish Immunology 68: 65-73
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• ISKNV-specific probe (517 bp)
• Kidney (A, B), liver and pancreas (C) and brain (D).
• Strong signals were observed in the epithelial cells (Epi), of hematopoietic tissue (Hpt) and the glomerulus (Glo) of the kidney (A).
Example of ISKNV (2)
Dong et al. 2017 Fish & Shellfish Immunology 68: 65-73
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Applications of ISH in the study of shrimp viruses
1. 3 major penaeid species, P. vannmei, P. stylirostris, P. monodon, are susceptible to IMNV infection
2.Prove that yellow head virus has a positive sense RNA genome
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IMNV: non-enveloped ds RNA virus
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In situ hybridization
(DIG-IMNV probe)
(DIG-AP Ab)
84oC, 10 min
dsRNA genome
Colorimetric development
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H&E staining and ISH detection of IMNV in skeletal muscle of L. vannamei
H&E
ISH
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Experimental infection
P. vannamei, P. stylirostris, P. monodon
Injection with an IMNV inoculum
4 wks
ISH: all positive for IMNV infection
Appearance of clinical signs and mortality
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Susceptibility of penaeid shrimp to IMNV infection
Laboratory infections to species of P.vannamei, P. stylirostris, P. monodon
20
P. vannamei P. stylirostris P. monodon
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Yellow head virus44 nm x 173 nm
Was suspected to be a negative-sense RNA virus?
Rhabdovirus70 nm x 180 nm
With a single, negative-sense, RNA genome
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ISH to YHV-infected shrimp using single-stranded DIG-RNA probes
ISH with a anti-sense DIG-RNA probe: Positive reaction
Probe with a DIG-sense RNA:Negative reaction
The result indicated that YHV has a positive-sense RNA genome
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pGEM-T Easy vector
T7 promoter
PstI
YHV cDNA insert
YHV DIG-RNA probe
RNA Pol
In vitro transcribed DIG-RNA probe
Linearized with SmaI
In vitro transcription with DIG-UTP ( ) using T7 RNA polymerase
PCR using YHV primers containing T7 (or SP, T3) promoter sequence
T7 Promoter
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Thank you for your attention!