A new antiviral therapeutic
strategy for the prevention of
rotavirus infections
5th World Congress on Virology
Rebecca D Parr
Stephen F Austin State University
Rotavirus
 Double stranded RNA segmented virus in the
Reoviridae family
 Commonly infects infants and young children
 Caused severe diarrhea and dehydration.
Symptoms include: decrease in urination, dry
mouth and throat, dizziness, watery diarrhea,
vomiting and fever
 Good vaccines, but not completely protective
0.25 µm
Transmission electron micrograph shows
rotavirus infected MA104 cells eighteen
hours post infection. Final Magnification
= 57,000x Hannah N Lockwood, SFASU
Rotavirus Vaccines
 Vaccines are strain specific
 Diarrhea treatment
 Oral Rehydration salts (ORS)
 Zinc Supplements
 Intravenous fluids
 Nutrient- Rich foods
 No antiviral drug
0.25 µm
Transmission electron micrograph
shows rotavirus infected Ma104 cells
eighteen hours post infection. Final
Magnification= 52,000x Hannah N
Lockwood, SFASU
Production & Purification of the
Stilbenoids, trans-Arachidin-1 & -3
Figure analysis
3. HPLC analysis
of stilbenoids. The
X axis
time is
in minutes,
andminutes
the Y axis isand
the adsorbance
at 340nm.
A.
HPLC
of Stilbenoids.
The
X isaxis
time in
the Y axis
is the
HPLC chromatogram
ethyl acetate
extract chromatogram
of the medium of hairy
culture
of peanut
with methyl-βadsorbance
at 340of nm.
A. HPLC
ofroot
ethyl
extract
of treated
the medium
of
cyclodextrin for 72 h. Compounds: (1) arachidin-1 and (2) arachidin-3. B. HPLC chromatogram of (1) arachidin-1
hairy
root culture of peanut treated with methyl-β-cyclodextrin for 72 hours.
purified by HPCCC. C. HPLC chromatogram of (2) arachidin-3 purified by HPCCC.
Compounds: (1) arachidin-1 and (2) arachidin-3 B. HPLC chromatogram of (1)
arachidin-1 purified by HPCCC C. HPLC chromatogram of (2) arachidin-3
purified by HPCCC.
(A) One-liter peanut hairy root cultures
pre-elicitation and (B) post-elicitation
(24 hours)
Ball, et al. 2015 Adv.Virol. Volume 2015 (2015), Article ID 293524
Common Stilbenoids
 Most commonly known stilbenoid is
Resveratrol
 Biological activities include: Antioxidant,
anti-inflammatory, cardio-protective,
antiviral, anticancer and anti-aging
 Some have limited oral bioavailability
possibility due to rapid absorption and
metabolism
 Resveratrol and piceantannol prenylated
compounds are trans-arachidin-3 and
trans-arachidin-1, respectively
Cell Viability
90.0
80.0
70.0
60.0
50.0
40.0
RV
RV +
RV +
RV +
DMSO 10µM t- 20µM tRes
Res
20µM
tRes
90.0
80.0
70.0
60.0
50.0
40.0
RV
90.0
80.0
70.0
60.0
50.0
40.0
RV
Arachidin-1
100.0
RV +
RV +
RV + 20µM t-A1 Cells + Cells only
DMSO 10µM t-A120µM t-A1
DMSO
Piceantannol
100.0
Cells + Cells only
DMSO
Per Cemt Viability
Per Cent Viability
Per Cent Viability
Per Cent Viability
Resveratrol
100.0
RV +
RV +
RV + 20µM t- Cells + Cells only
DMSO 10µM t- 20µM tPa
DMSO
Pa
Pa
Arachidin-3
100.0
90.0
80.0
70.0
60.0
50.0
40.0
RV
RV +
RV +
RV +
20µM tDMSO 10µM t- 20µM tA3
A3
A3
Cells + Cells only
DMSO
Ball, et al. 2015 Adv.Virol. Volume 2015 (2015), Article ID 293524
Focus Forming Units: FFU/ml 24hpi
1.00E+09
1.00E+08
1.00E+08
1.00E+07
1.00E+07
1.00E+05
FFU/ml
1.00E+09
1.00E+04
1.00E+04
FFU.ml
1.00E+06
1.00E+06
1.00E+05
1.00E+03
1.00E+03
1.00E+02
1.00E+02
RV
RV
RV + 0.02% RV + 10µM RV + 20µM
DMSO
t-Res
t-Res
1.00E+09
1.00E+09
1.00E+08
1.00E+08
1.00E+07
FFU/ml
*
1.00E+07
FFU/ml
1.00E+06
1.00E+05
1.00E+04
RV +
RV + 10µM t- RV + 20µM t0.02%DMSO
Pa
Pa
1.00E+06
*
1.00E+05
1.00E+04
1.00E+03
1.00E+03
1.00E+02
RV
RV +
RV + 10µM t- RV + 20µM t0.02%DMSO
A3
A3
1.00E+02
RV
RV + 0.02% RV + 10µM RV + 20µM
DMSO
t-A1
t-A1
Ball, et al. 2015 Adv.Virol. 2015. ID
Plaque Forming Units: PFU/ml 24hpi
t-Res Virus Titers mean 24 hpi
1.00E+09
1.00E+09
SA114F RV plaques
in HT29.f8 cells
2.47E+08
2.10E+08
2.33E+08
3.33E+08
RV
RV+0.02%DMSO
5.67E+08
PFU/ml
PFU/ml
3.33E+08
1.00E+08
1.00E+08
1.00E+07
1.00E+07
1.00E+06
1.00E+06
1.00E+05
RV
RV+0.02%DMSO
rv+20µM t-Res
1.00E+05
tA-1 Virus Titers mean 24 hpi
rv+20µM tPA
t-A3 Virus Titers mean 24 hpi
1.00E+09
1.00E+09
2.47E+08
1.89E+08
2.10E+08
2.11E+08
1.00E+08
*
1.00E+07
4.33E+06
1.00E+06
PFU/ml
1.00E+08
PFU/ml
t-PA Virus Titers mean 24 hpi
*
3.44E+06
1.00E+07
1.00E+06
1.00E+05
RV
RV+0.02%DMSO
rv+20µM tA-1
1.00E+05
RV
RV+0.02%DMSO
rv+20µM tA-3
Ball, et al. 2015 Adv.Virol. Volume 2015 (2015), Article ID 293524
Western blot analysis of HT29.f8
cell lysates to detect RV-NSP4.
55kD-
Multimeric forms NSP4
40kDDiglycosylated NSP4
35kD-
Monoglycosylated NSP4
25kD-
Unglycosylated NSP4
Cleavage fragments
15kD1
2
3
4
HT29.f8 cell lysates
5
Ball, et al. 2015 Adv.Virol. Volume 2015 (2015), Article ID 293524
Five micrograms of HT29.f8 cell
lysates were separated on a 12.5%
SDS-PAGE,
electroblotted
onto
nitrocellulose membranes, probed
with rabbit anti-NSP4150-175 peptidespecific and goat anti-rabbit HRPconjugated IgG, visualized with Super
Signal® West Pico chemiluminescent
substrate
(Pierce)
followed
by
exposure to Kodak X-OMAT film.
Lane 1) RV-infected HT29.f8 cells and
lane 2) RV-infected HT29.f8 cells with
0.02% DMSO, respectively, show
cleavage fragments, unglycosylated,
mono, diglycosylated and multimeric
forms of NSP4. Lane 3) RV-infected
with 20µM t-A1 and Lane 4) RVinfected with 20µM t-A3 only shows
the diglycosylated form of NSP4.
Lane 5) HT29.f8 with no virus showed
no NSP4 banding pattern.
 t-A1 & 3 binds to Cannabinoid Receptor 1 (CBR1) and
Cannabinoid Receptor 2 (CBR2). Brents, L. et al. 2012. Xenobiotica 42(2):139-156.
Cannabinoid Receptors 1 & 2 on HT29.f8 and MA104 cells
Rabbit anti-CNR-2
Rabbit anti-CNR-1
10µg
5µg
2.5µg
1.25µg
A
B
C
A
B
C
Cell lysates A-uninfected HT29.f8; B-uninfected
MA104; C-RV-infected MA104/HT29.f8 cell lysates were
added to nitrocellulose membranes, probed with a
1:1000 dilution of rabbit anti-CNR1/2 antibodies from
Antibodies Online (Atlanta, GA) and reactive bands
were visualized by the addition and excitation of goat
anti-rabbit
antibodies
Alexa
Fluor®546
(Life
Technologies) using the Typhoon 8600 laser scanner.
Cannabinoid Receptors 1 & 2
 Classical
cognate receptors for all
types of agonist
 Distribution
through out the GI
tract; predominantly in the enteric
nervous system
 CBR1/2
are upregulated during
intestinal inflammation
http://www.doping-prevention.sp.tum.de/ substances- andmethods/cannabinoids /cannabinoids.html
Cannabinoid Receptors 1 & 2

Shown to exert apoptotic actions in
several cancer lines

CBR1 mediates apoptotic effects
including
1.
Inhibition of RAS-MAPK and PI3KAKT pathways
2.
Downregulation of cAMPdependent PKA pathway
3.
Stimulation synthesis of proapoptotic lipid mediator ceramide
Quantification of Transcripts for Caspases
Total RNA was extracted at 8 hours post RV infection of HT29.f8 cells and used to make
cDNA to detect Caspases 3, 7, 8, 9, & 10 using qRT-PCR
QNANO SYSTEM FROM IZON
TUNABLE RESISTIVE PULSE SENSING TECHNOLOGY (TRPS)
2-D gel analyses of HT29.f8 cell lysates
Acknowledgement
Stephen F Austin State University
 Rebecca D Parr
 Hannah N Lockwood
 Caleb Witcher
 Ravaen Slay
 Macie Mattila
 Ron Havner
Texas A&M University
 Judith M Ball
Arkansas State University
 Fabricio Medina-Bolivar