My Internship Experience At The Food Animal Health Research Program

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My Internship Experience At The Food Animal
Health Research Program

• By Mital Pandya
• Summer Research Student
• The Ohio State University
• Advisor Dr. Wick

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Background

• Ohio Agriculture Research and Development Center
  (OARDC) located in Wooster, Ohio
• ATI Apartments
• 8.00/hour paid internship
• I worked for Dr. Linda Saif, her areas of
  expertise include Animal immunology, Animal
  Virology, Diseases/swine (enteric), Enteric
  diseases of cattle, Enteric diseases of swine,
  Immunology

Dr.Linda Saif
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My Responsibilities

• My Research Project
• General Lab Work
• Autoclave room
• Cleaning
• Paperwork

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Introduction

• First recognized in 1969 in calves and later in
  1973 in humans, rotavirus is the most common
  cause of severe diarrhea among children,
  resulting in the hospitalization of approximately
  55,000 children each year in the United States
  and the death of over 600,000 children annually
  worldwide
• An effective rotavirus vaccine could save the
  lives of approximately 1,400 children per day

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Background
Rotaviruses are nonenveloped, triple-shelled
viruses. They belong to the Reoviridae family.
Rotavirus has a wheel-like shape under an
electron microscope, which earned it the name of
rota virus

• The virus genome is composed of 11 segments
  of double-stranded RNA, which encode for six
  structural and six nonstructural proteins. The
  virus is stable in the environment

ds RNA segments
Proteins VP1 VP2 VP3 VP4 NSP1 VP6 NSP2 NSP3
VP7 NSP4 NSP5
1 2 3 4 6 7 8 9 10 11
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(European Molecular Biology Organization,
www.nature.com)
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Background

• A recent study in gnotobiotic pigs showed that
  the virulent (Vir) Wa strain human rotavirus
  (HRV) and the attenuated (Att) Wa strain HRV have
  different replication efficacy (Azevedo, et al.,
  2005. J. Virol)
• The AttHRV replicated more effectively in the
  respiratory tract than in the intestine the
  VirHRV replicated to higher titers in the
  intestine than in the respiratory tract

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Objectives of My Project

• To compare the physiochemical properties of
  VirHRV and AttHRV
• Compare effects of different pH and temperature
  on virus stability and in-vitro replication
  between the AttHRV and the VirHRV
• Compare effects of trypsin and pancreatin on
  virus replication of the AttHRV and the VirHRV

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Approaches of My Study

• To compile a growth curve at 27ºC, 33ºC and 37ºC
  temperatures for AttHRV and VirHRV
• Verify the stability of AttHRV and VirHRV after
  exposures to pHs 3 and 5

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Materials and Methods

• MA 104 Cells
• Cell culture immunofluorescence assay (CCIF) for
  VirHRV and AttHRV
• Enzyme linked immunoabsorbant Assay (ELISA)

Un-infected MA104 Cells
HRV-infected MA104 cells detected by CCIF
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Resistance to Temperature
4oC
AttHRV Stock
Tested by CCIF, ELISA
30 minutes
37oC
56oC
4oC
30 minutes
Tested by CCIF, ELISA
37oC
VirHRV Stock
56oC
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Resistance to pH
Not treated
AttHRV Stock
pH adjusted to 7.2 with NaOH
30 minutes At 37oC
pH 5.0
pH 3.0
Not treated
pH adjusted to 7.2 with NaOH
30 minutes At 37oC
VirHRV Stock
pH 5.0
pH 3.0
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Influence of trypsin and pancreatin in the growth
of VirHRV and AttHRV by CCIF
Trypsin
37oC and 33oC
AttHRV Stock
Pancreatin
CCIF
Trypsin
VirHRV Stock
37oC and 33oC
Pancreatin
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Effects of temperature on titers (FFU) of AttHRV
and VirHRV
Temperature
Type of Virus
37oC
4oC
56oC
AttHRV
2.8x105 7.0x105
3.0x106 1.0x106
2.6x106 1.65x106
1.2x106
2.0x106
1.1x106
VirHRV

• Summary
• Titers of VirHRV reduced slightly after
  treatment
• with high (560C) temperatures compared to
  temperatures of 4oC and 370C

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Effects of pH on titers (FFU) of AttHRV and
VirHRV
pH
Type of Virus
pH 3
pH 5
Control (pH7.2)
2.2x106 7.0x106
AttHRV
2.0x106 9.5x104
6.5x103 1.1x103
2.5x106 4.0x105
1.2x106 7.2x105
4.8x105 4.8x104
VirHRV

• Summary
• Titers of both AttHRV and VirHRV reduced more
• after treatment with pH 3 compared to pH 5

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Virus titers (FFU) after cell-culture passage at
different temperatures measured by CCIF

• Summary
• VirHRV had higher titer at 370C than at 330C
  after first passage in MA104 cells
• AttHRV had higher titer at 330C than at 370C
  after first passage in MA104 cells

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Trypsin Pancreatin 33oC
Trypsin Pancreatin 37oC

• Summary
• AttHRV trypsin treatment in 370C showed to be
  more efficient in virus replication
• VirHRV there was no difference between trypsin
  and pancreatin treatments with the highest titers
  being reached at temperature of 37oC

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Virus titers after cell-culture passage at
different temperatures measured by ELISA

• Summary
• VirHRV extremely decreased titers after first
  passage in MA104 cells at 370C or 330C
• AttHRV showed no difference in the virus titer
  after four passages at 330C or 370C in MA104
  cells

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Preliminary Conclusions

• AttHRV and VirHRV did not differ significantly in
  their resistance to treatment with low pH or low
  or high temperatures
• Titers of AttHRV and VirHRV were equally affected
  (reduced) by treatment with low pH (pH 3.0)
• Low temperature (330C) did not negatively affect
  the replication of AttHRV in cell culture, but
  reduced the replication of VirHRV after one
  passage

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Future Studies

• Complete and repeat all the experiments
• Passage AttHRV and VirHRV in cell culture for at
  least 5 times at different temperatures

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My Experience Future Goals

• Highly Recommended for anyone who is interested
  in research
• Hands on Experience
• Graduate School for Public Health

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Acknowledgements

• Advisors Dr. Lijuan Yuan Dr.Wick
• Dr. Linda Saif
• Dr. Marli Azevedo
• Dr. Ana Gonzalez
• Lab 113 Family

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Any Questions?