presented by: kunqian li mentor: dr. xiaoyong bao ph.d. utmb galveston pediatrics independent study...

The Most Interesting VirusIn the World

Presented By: Kunqian Li

Mentor: Dr. Xiaoyong Bao Ph.D.

UTMB Galveston

Pediatrics

Independent Study Mentorship: HST – Fall 2014

ISM Teacher: Mrs. Click

DISCLAIMER: SOME INFORMATION IN THIS PRESENTATION COMES FROM OUTSIDE SOURCES

The Scientific Research of

Human Metapneumovirus

What is Independent Study Mentorship (ISM)?

This course is a project-based learning experience at the professional level.

Provides opportunities for an in depth study in a particular curricular area.

Students must:Document 3-5 hours of work each weekSubmit weekly logs of documentationMeet with mentor on a regular basisPresent a final project

A little bit about my mentor

Dr. Xiaoyong Bao Ph.D

-Assistant Professor of Pediatrics in the

Division of Clinical and Experimental

Immunology and Infectious Disease

-Ph.D. in Cellular Physiology and

Molecular Biophysics from UTMB

-Research focuses on host-virus

interaction, with the goal to generate

suitable vaccine candidates to prevent

paramyxovirus infection

"UTMB Department of Pediatrics." Welcome. N.p., n.d. Web. 12 Oct. 2014.

The University of Texas Medical Branch

• Established in 1891

• More than 70 major buildings, more than 2,500 students and more than

1,000 faculty.

• UTMB’s mission is to improve health for the people of Texas and around

the world.

"The University of Texas Medical Branch at Galveston." Welcome to UTMB Health. N.p., n.d. Web. 12 Oct. 2014.

What is Human Metapneumovirus (hMPV)?

Identified in 2001 in the Netherlands

Can cause upper and lower respiratory tract infections in people of all ages

Most people with hMPV have mild symptoms but some can have severe illness including pneumonia and a flare-up of asthma

More serious in children under the age of 1 and in the elderly"Human Metapneumovirus - American Lung Association."

American Lung Association. N.p., n.d. Web. 12 Oct. 2014.

"American Society for MicrobiologyClinical Microbiology Reviews." Human Metapneumovirus: Lessons

Learned over the First Decade. N.p., n.d. Web. 12 Oct. 2014.

Purpose of This ProjectResearch hMPV and the effects it can have

Educate audience on respiratory viruses

Discover unknown information that will lead to vaccines

Pictures taken from Microsoft Office Clipart

RED

Current Research on hMPVhMPV is a single negative-stranded RNA

enveloped virus

hMPV is second to RSV as the most common cause of respiratory tract infection

hMPV lacking the M2-2 Protein made it a promising vaccine candidate

hMPV is the first identified human pathogen belonging to the genus “Metapneumovirus in the Pneumovirinae subfamily of the Paramyxoviridae family”

"Human Metapneumovirus." - MicrobeWiki. N.p., n.d. Web. 07 Dec. 2014. <https://microbewiki.kenyon.edu/index.php/Human_Metapneumovirus>.

Current Research on hMPV microRNA and small-interfering RNA both suppress gene

expression by complementary binding to target mRNAs

Northern blots and real-time polymerase chain reactions techniques are used to investigate hMPV

hMPV leads to significant changes in miRNAs in human airway epithelial cells

hMPV infection does not induce tRFs

In a CDC test, hMPV was detected in 6% of children

“From 2003-2009 the average annual rate of hMPV hospitalization was 1/1000 children less than five years old”

"Human Metapneumovirus." - MicrobeWiki. N.p., n.d. Web. 07 Dec. 2014. <https://microbewiki.kenyon.edu/index.php/Human_Metapneumovirus>.

Survey To gather more information on the subject at

hand, a survey was conducted through Survey Monkey

Here are the questions to my survey:

Are you male or female?

What is your age?

Have you taken healthcare related courses?

Have you heard of Human Metapneumovirus or Respiratory Syncytial Virus?

Which of the symptoms have you suffered from in the past 12 weeks?

High fever

Severe cough

Difficulty breathing

Rapid breathing

Wheezing

Vomiting

Diarrhea

None

How many times do you wash your hands per day?

How long do you wash your hands for?

What do you sneeze into?

Have you shared food and/or eating utensils in the past 2 weeks?

How often do you touch your eyes, mouth, or nose?

Results of the SurveyI had hypothesized that:

Health care professionals are more aware of hMPV because they are more exposed to viruses

40% of all participants exhibit at least two risk factors or severe symptoms of hMPV

Women are taking better precautionary measures in preventing hMPV infection than men are due to their natural maternal instinct

Results ContinuedMost people are unaware of the virus regardless

of education background

Has taken healthcare related courses

Hasn’t taken healthcare related courses

Results ContinuedOn average, females wash their hands longer

than men

FemalesMales

Results ContinuedMales tend to touch their face more

Females Males

Online Weeblyhttp://kunqianli.weebly.com

Laboratory Procedures Dr. Bao observing the

growth of cells in minimum essential medium

45 wells

3 time groups (6,15, 24 hours)

5 different hMPV variables with a mock (control)

3 wells for each variable

Cell Infection

Inoculate Cells

Dispose of serum

Introduce serum mixed with hMPV variables

Observe Cells under microscope after appropriate time

A few of the laboratory equipment necessary in cell infection

96 Well PlateElectronic Pipette

Fixing 96 – Well Plate1. Wash with 150 microliters of (DPBSx1)

for 3 times

2. Fix solution:

(10ml of Methanol +667microliters of Hydrogen Peroxide 30wt), mix well.

3. Add 100microliters of fixing solution to each well, then incubate for 30 minutes

4. Wash after the 30 min incubation for 3 more times

5. Add 100microliters of (DPBSx1) to each well

6. Store in 4°C

A549 Cells Cells located in the basal layer of the epidermis

The primary targeting cells of hMPV are airway epithelial cells

A549 Cells can serve as an airway epithelial cell model for hMPV infection.

Evidence that A549 Cells can be used successfully in this study

X, Bao, Liu T, Spetch L, Kolli D, Garofalo R.P, and Casola A. "Images in This Article." NIH Public Access. U.S. National Library of Medicine, 25 July 2007. Web. 12 Oct. 2014.

Sandwich ELISAIn order to identify the plaques growing on the

cells, the Sandwich ELISA method was employed

Pros to this method:High specificity, since two antibodies are used the

antigen/analyte is specifically captured and detected

Suitable for complex samples, since the antigen does not require purification prior to measurement

Flexibility and sensitivity, since both direct and indirect detection methods can be used

"Sandwich ELISA, Highly Sensitive." Sandwich ELISA. N.p., n.d. Web. 07 Dec. 2014. <http://www.elisa-antibody.com/ELISA-Introduction/ELISA-types/sandwich-elisa>.

Data Gathered at the Lab

Final Product

• Poster board

displaying data

gathered over a

specific time

period

• Focus on RANTES

and IL-8

RANTES A cytokine that is a member of the interleukin-8 superfamily of

cytokines. RANTES is a protein. It is a selective attractant for memory

T lymphocytes and monocytes. It binds to CCR5, a coreceptor of HIV.

RANTES is an acronym for Regulated on Activation, Normal T

Expressed and Secreted. It is also known as CCL5.

"RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted), Inflammation, Obesity, and the Metabolic Syndrome." RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted), Inflammation, Obesity, and the Metabolic Syndrome. N.p., n.d. Web. 07 Dec. 2014. <http://circ.ahajournals.org/content/115/8/946.full>.

Interleukin-8 Interleukin 8 (IL-8) or CXCL8 is a chemokine produced by

macrophages and other cell types such a epithelial cells.

Through a chain of biochemical reactions, IL-8 is secreted and is an important mediator of the immune reaction in the innate immune system response.

IL-8, also known as neutrophil chemotactic factor, has two primary functions: It induces chemotaxis in target cells causing them to migrate

towards the site of infection. It induces phagocytosis once those cells arrive.

"Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 07 Dec. 2014. <http://www.ncbi.nlm.nih.gov/pubmed/7657403>.

Significance of this Data

WT 15 Hrs Mult 1 15 Hrs Mult 2 15 Hrs0

1000

2000

3000

4000

5000

6000

3119.8

4816.94

3901.413666.86

4761.534308.8

3397.14

4928.735293.72

IL-8 (54)Obs Conc

Trial 1Trial 2Trial 3

WT 15 Hrs Mult 1 15 Hrs Mult 2 15 Hrs0

50

100

150

200

250

33.08

138.86

194.26

51.95

135.25

178.1

59.8

135.04

216.46

RANTES (37)Obs Conc

Trial 1Trial 2Trial 3

ConclusionThis semester has been one of the most

influential semesters I’ve had in high school

Much has been learned through hands-on experiences in a professional field

My knowledge on hMPV has increased tremendously

Picture from Microsoft Office Clipart

QuestionsHelpful Sources

kunqianli.weebly.comArticle published by mentor

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2386556/

Contact Information832-738-9166kunqianli96@gmail.com

Thank You

Special thanks to:My outstanding mentor:

Dr. Xiaoyong Bao

And of CourseMy wonderful ISM teacher:

Mrs. ClickMy Parents:

George Li and Congying WangMy generous and terrific evaluators:

Mr. Thomas and Mr. MayberryAnd all those who supported me this

semester

Works Cited "American Society for MicrobiologyClinical Microbiology Reviews." Human Metapneumovirus:

Lessons Learned over the First Decade. N.p., n.d. Web. 12 Oct. 2014.

"Human Metapneumovirus - American Lung Association." American Lung Association. N.p., n.d. Web. 12 Oct. 2014.

"Human Metapneumovirus." - MicrobeWiki. N.p., n.d. Web. 07 Dec. 2014. <https://microbewiki.kenyon.edu/ index.php/Human_Metapneumovirus>.

"RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted), Inflammation, Obesity, and the Metabolic Syndrome." RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted), Inflammation, Obesity, and the Metabolic Syndrome. N.p., n.d. Web. 07 Dec. 2014. <http:// circ.ahajournals.org/content/115/8/946.full>.

"Respiratory Syncytial Virus Infection of Human Lung Endothelial Cells Enhances Selectively Intercellular Adhesion Molecule-1 Expression." Respiratory Syncytial Virus Infection of Human Lung Endothelial Cells Enhances Selectively Intercellular Adhesion Molecule-1 Expression. N.p., n.d. Web. 07 Dec. 2014. <http://www.jimmunol.org/content/174/11/7359.full>.

"Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 07 Dec. 2014. <http://www.ncbi.nlm.nih.gov/pubmed/7657403>.

"Sandwich ELISA, Highly Sensitive." Sandwich ELISA. N.p., n.d. Web. 07 Dec. 2014. <http://www.elisa- antibody.com/ELISA-Introduction/ELISA-types/sandwich-elisa>.

"The University of Texas Medical Branch at Galveston." Welcome to UTMB Health. N.p., n.d. Web. 12 Oct. 2014.

"UTMB Department of Pediatrics." Welcome. N.p., n.d. Web. 12 Oct. 2014.

X, Bao, Liu T, Spetch L, Kolli D, Garofalo R.P, and Casola A. "Images in This Article." NIH Public Access. U.S. National Library of Medicine, 25 July 2007. Web. 12 Oct. 2014.