Immune Response: Phagocytes, Pathogens, and Adaptive Response, Lecture notes of Immunology

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AAI High School Teachers Summer Research Program in Immunology:

Primer on Immunology using Dengue Fever as a Real World Application.

Extension: Detection of the Prevalence of Lyme Disease in Local Canine

Populations.

Beth Krauss, Teacher Manlius Pebble Hill School 5300 Jamesville Road Dewitt, NY 13214 Email: [email protected]

Gary Winslow, Mentor Upstate Medical University Department of Microbiology and Immunology 750 East Adams Street Syracuse, NY 13210 [email protected]

Table of Contents

• I. Science Background ........................................................................................................................
  • Primer of Immunology....................................................................................................................
    • Brief Review of the Immune System
  • Dengue Q & A ................................................................................................................................
  • Glossary of Basic Immunology Terms ..............................................................................................
• II. Student Outcomes
  • Concepts Covered: .........................................................................................................................
  • Standards Addressed:.....................................................................................................................
  • Placement in Course .......................................................................................................................
  • What students will do and technical skills learned...........................................................................
• III. Learning Objectives
• IV. Time Requirements
• V. Advance Preparation
• VI. Materials and Equipment
• VII. Student Prior Knowledge.
• VIII. Daily Unit Plans
  • Daily Lesson Plans ..........................................................................................................................
• IX. How will students demonstrate understanding?
• X. References
• XI. STUDENT SECTION
  • Video Reflection Guide – Immune System .....................................................................................
  • What are PCR, RT-PCR and Flow Cytometry? .................................................................................
  • Dengue - Is the World Cup Worth the Risk? ...................................................................................
• XII. APPENDIX
  • What are PCR, RT-PCR and Flow Cytometry? .................................................................................
  • TEACHERS ANSWERS ....................................................................................................................
  • Case Study Answers .....................................................................................................................

I. Science Background

Primer of Immunology

Brief Review of the Immune System

The immune system of animals is made up of two defense mechanisms; the Innate and Adaptive Immune Systems.

A) Innate Immune Response - Nonspecific defenses that attempt to prevent pathogens from entering the organism or quickly eliminate pathogens that do enter the organism.

1. First line of defense – barriers that prevent the pathogen from entering the organism. i. Skin Presence of normal bacteria and fungi that out-compete pathogens Acidic pH Skin Saltiness ii. Mucus membranes trap pathogens. Lysozyme- enzyme made by mucus membranes, it breaks open cell walls. Defensins – peptides made by mucus membranes that insert themselves into plasma membranes making them freely permeable to water.

2. Second line of defense is initiated when pathogens breach the skin or mucus membranes and gain access to the organism. i. Here pathogens encounter phagocytes, natural killer cells and defensive molecules such as complement and interferon proteins. ii. Inflammation stops the spread of tissue damage; it recruits molecules and other cells to the injury site to eliminate pathogens and promote tissue repair. iii. Mast cells, one of the first cells to respond to tissue damage, release several different chemicals to aid in the repair process.

• Tumor necrosis factor is a cytokine that kills target cells.
• Prostaglandins play a role in initiating the inflammatory process.
• Histamine increases blood vessel permeability to allow white blood cells and molecules access to affected tissues.

B) Adaptive Immune Response – Is initiated when the body detects the presence of an antigen. It has four key features:

i. Specificity – Each T cell and B cell is specific for one antigen. When an antigen (from a pathogen) binds to a T cell receptor and an antibody is produced by a B cell, a specific immune response is started.

ii. Diversity – To respond to the variety of pathogens the body produces many different lymphocytes, each bearing a unique antigen receptor. This is accomplished through DNA mutations of receptor proteins during cell formation in the bone marrow. It also occurs

4

when a T or B cell is activated through antigen binding causing it to divide to form clones (genetically identical cells). This is called clonal selection.

iii. Distinguishing Self from Nonself – Through clonal deletion B or T cells that have the potential of attacking one’s own body cells are eliminated through apoptosis.

iv. Immunological Memory – Once the immune system responds to a specific pathogen the system “remembers” this pathogen and can respond rapidly the next time it is found.

1. The primary immune response can take several days before B cells produce antibodies and T cells are produced with the appropriate receptors. When the B and T cells divide they make two different types of cells, effector and memory.

Effector cells – Effector B cells known as plasma B cells secrete antibodies. Effector T cells (helper T cells and cytotoxic T cells) secrete cytokines and other molecules that destroy nonself or infected cells. Memory Cells – Memory B and T cells are long lived and can divide on short notice to produce more memory and effector cells.

2. Secondary immune response occurs after the primary response to a specific antigen. Memory cells that bind to the antigen reproduce to form plasma B cells and T cells.

C) Humoral vs Cellular immune response – B cells are primarily involved with the humoral response and helper and cytotoxic T cells with the cellular response.

Flow chart of Adaptive Immune Response

Pathogen

Macrophage Engulfs Pathogen

Helper T Cell

Cytotoxic T Cell –

Attacks Infected

Cell

B Cell Cytotoxic T Cell

Plasma B Cell

Antibodies

Memory B Cell

Naïve B Cell

Humoral (Antibody-Mediated) Immunity Cellular Immunity

There are five classes of antibodies secreted by B cells. IgM – the first class of antibodies produced after the initial exposure to an antigen and is short lived. IgG – is the most abundant antibody in blood and tissue fluids. IgD – acts as cell surface receptor on B cells. IgA – provides protection for mucus membranes. IgE – present in blood, triggers release of histamines and other chemicals involved in allergic reactions.

Dengue Q & A

Question: What is dengue? Answer: Dengue is a vector borne viral disease caused by one of four different serotypes (DENV-1, DENV-2, DENV-3, DENV-4). The virus is a member of the Flaviviridae family.

Question: How is it contracted/transmitted? Answer: The most common vector is the Ades aegypti mosquito. Ades albopictus is also a carrier. When an infected mosquito bites a human, the virus can be transmitted through the fluids the mosquito inserts through its proboscis. The mosquito acquires the virus through the blood of an infected human it bites. Human to human transmission is not possible.

Question : Can I get dengue again if I have already been infected? Answer : You will have immunity to the specific virus (DENV-1, DENV-2, DENV-3, DENV-4) that you were originally infected with and, if infected again with that serotype, you will not get the disease. However, if you are infected with a different serotype you are at a greater risk of acquiring dengue hemorrhagic fever (DHF). DHF is a more severe form of dengue and can be fatal if medical treatment is not immediately sought.

Question : How can it be prevented? Answer: Because the disease is spread by mosquitoes, eradication of breeding sites is essential. Ae. aegypti lays her eggs in containers that hold water (e.g. tires, planters, water drums). However, Ae. aegypti are adaptable to varying conditions; eggs have been known to survive drought conditions. Ae. albopictus prefers cool dark areas such as home closets. The use of air conditioning and screens can prevent adults from finding their human meals.

Question: What are the disease symptoms? Answer: Many of the symptoms are very similar to malaria or the flu, they include: high fever; severe headache; pain in the joints, muscle, bone or behind the eyes; rash; and mild bleeding. The symptoms of dengue hemorrhagic fever include a fever that lasts from two to seven days with the other mentioned symptoms. As the fever declines the following symptoms may occur: vomiting, abdominal pain and breathing difficulties. This opens a 24 to 48 hour period when capillaries start to leak fluid. This increase in fluid in the body cavities can lead to the failure of the circulatory system, shock, and death. Skin hemorrhages, along with internal and external bleeding, are also possible.

Question: How is dengue treated? Answer: There is no specific treatment for the disease. If one suspects they have dengue fever, they can take analgesics, stay hydrated, rest, and speak with their doctor. If they develop more severe symptoms after the fever has subsided, they should go to a hospital. Fluid replacement therapy is appropriate at this point. Currently, there is no vaccine on the market; however the company Sanofi Pasteur has plans to register the tetravalent vaccine (protects against all 4 dengue strains) in 2015 with a target distribution time frame of late 2015.

Questions : Where has the disease been found? Answer: The four viruses originated in monkeys and spread to humans in Africa or Southeast Asia between 100 and 800 years ago. Currently, the disease can be found in Southeast Asia, the Pacific Islands, Africa, Caribbean, and the Americas.

Question: What lab tests are used to detect the virus? Answer: The test methods used is dependent upon the duration of the disease. Real time RT-PCR is used to detect the virus in patients within the first 5 days of symptoms. A positive result confirms presence of the disease and usually which viral strain is involved. A negative result in this time frame is termed indeterminate, and patients will be asked for another sample 5 days post symptoms.

MAC ELISA detects the presence of IgM antibody in the serum. The test can be used as early as 3 days post symptoms and may be able to detect the antibody as far as 90 days post symptoms. Caution should be used interpreting the results as this method can also react with other flaviviruses.

IgG ELISA is used to diagnose the presence of a past infection. To determine if the infection was primary (first exposure to dengue) or secondary (a second exposure) in nature, two samples need to be taken, one during the acute phase and one in the recovery phase. If the results are negative in the acute but positive in the convalescent phase, the patient has a primary infection. If there is a positive result in the acute phase and a fourfold increase in the convalescent phase, the infection is secondary.

Plaque Reduction and Neutralization Test is used to detect specific serotypes in the convalescent serum.

Glossary of Basic Immunology Terms

Antibody – proteins that bind to specific substances (antigens) on “non-self” cells. They are made by plasma cells in response to a pathogenic attack. Antigen – a molecule usually bound to a pathogen that stimulates the production of a specific antibody on B cells and cell division in T and B cells. B Cells (B lymphocytes) – have receptors that are specific for one antigen type. When bound to an antigen they will differentiate into B cells that produce antibodies for that specific antigen.

Complement Proteins - a set of diverse proteins that function in a cascade manner. One complement protein binds to proteins on the pathogen which alerts phagocytes to the presence of an invader. Another protein activates the inflammatory response. Other proteins are responsible for lysing the pathogen. Cytokines – signaling proteins that affect the behavior of their target cell. Immunity – the body’s ability to resist disease when invaded by a pathogen. Inflammation – damaged tissue that exhibits puffiness, warmth, and redness. Interferon – a class of cytokines produced by virally infected cells. This signal protein alerts neighboring cells increasing their ability to resist infection. Lymphocyte – type of white blood cell that is in involved in adaptive immunity; it includes B cells and T cells. Major Histocompatibility complexes (MHC) – proteins found on a host cell’s surface that hold an antigen fragment that can bind to a T cell receptor. Natural Killer Cells – a type of lymphocyte that recognizes cells that are infected or cancerous and initiates cell destruction. They are part of the innate immune system. Pathogen - organisms and viruses that can cause disease when they infect a host. Phagocyte – white blood cells that engulf pathogens, they are involved in innate and adaptive immunity (e.g. macrophage). T cells (T lymphocytes) –mature in the thymus. They have a variety of functions: activating macrophages, helping B cells produce antibodies, and killing infected cells. T cell receptors – proteins found bound in the membrane of T cells. They recognize and bind to non-self substances (antigens) bound to MHC proteins on other cells.

II. Student Outcomes

Concepts Covered:

Students will have a deeper understanding of the adaptive immune system. Through the use of virtual labs and videos, students will be familiarized with the techniques of ELISA, PCR, RT-PCR, and flow cytometry. By completing the dengue case study, students will learn about the cause, symptoms, diagnosis, and treatment of dengue fever and dengue hemorrhagic fever.

Standards Addressed:

AP Biology  Essential knowledge 2.D.4 – Plants and animals have a variety of chemical defenses against infections that affect dynamic homeostasis.  Essential knowledge 3.D.2 – Cells communicate with each other through direct contact with other cells or from a distance via chemical signaling.

Next Generation Science Standards  HS-LS1-2. Develop and use a model to illustrate the hierarchical organization of interacting systems that provide specific functions within multicellular organisms.  HS-LS1-3. Plan and conduct an investigation to provide evidence that feedback mechanisms maintain homeostasis.

Placement in Course These lessons can be incorporated in an Immunology unit or Biotechnology unit. The lessons are designed for an Honors Biology and/or AP Biology course.

What students will do and technical skills learned Students will review/learn the basics of the adaptive immune system. Through the use of cell images, students will reinforce their knowledge of the various forms of B and T cells and how they interrelate. Students will complete a virtual ELISA and PCR lab to gain knowledge of those processes along with becoming familiar with flow cytometry. By completing the dengue fever case study, students will learn about the etiology of the disease, and how to diagnosis the disease via RT-PCR, MAC and IgG ELISA.

III. Learning Objectives

AP Biology–  LO 2.29 The student can create representations and models to describe immune responses.  LO 2.30 The student can create representations or models to describe nonspecific/specific immune defenses in plants and animals.  LO 3.34 The student is able to construct explanations of cell communication through cell to cell direct contact or through chemical signaling.  LO 3.35 The student is able to create representations that depict how cell to cell communication occurs by direct contact or from a distance through chemical signaling.

IV. Time Requirements

This unit can be completed in three to four 50-minute blocks. Day 2 may require 2 blocks of time depending in the student’s computer familiarity and the number of techniques assigned.

V. Advance Preparation

Copying Student handouts Reproduce cell images (Note: Laminate the copies for repeated use.)

VI. Materials and Equipment

 Cell images – laminated or on card stock for use with multiple classes over multiple years. One set per student.  Handouts for technology. One set per student.  Computer access. One computer per student if possible.  Dengue fever Case Study. One set per student.

VII. Student Prior Knowledge.

Students should have a basic knowledge of the function of the human immune system. They should have the ability to successfully navigate virtual labs.

VIII. Daily Unit Plans

Daily Lesson Plans General overview of unit - The unit is designed to introduce or reinforce the concepts of the adaptive immune system to students using dengue fever as a real-life application.

Assign the video and reflection guide (see Student Section) to be completed prior to the start of the unit. http://www.bozemanscience.com/immune-system

Day 1 – Teacher will review or introduce (depending on the class level) the components of the adaptive immune system; how different cell types interrelate and the general schemata of how the human body responds to a pathogenic insult.

Activity -could be used Day 1 as reinforcement or Day 2 as formative assessment. Each student will be given a set of images that contain the different cell types of the adaptive immune system. The images are located in the Student Section. Students will first be asked to separate them into different cell types (e.g. B and T cells). Then, using the cell images students will create a flow chart representing how the immune system responds to an exposure to a pathogen for the first time. Next, they will arrange the cells as if this is the second exposure to the pathogen. This is an individual activity; each student will do this independently.

Day 2 – Students will be introduced to the techniques used in the immunology, medicine, and/or research arenas. Each student should have access to a computer and the internet.

Activities that could be used include: a) ELISA This simulation guides the students through the ELISA technique from set-up through data analysis. The Immunology Virtual lab simulation and student worksheet from HHMI are located here: http://www.hhmi.org/biointeractive/immunology-virtual-lab

b) PCR This short video on PCR (1:27 minutes) from the DNA Learning Center at Cold Spring Harbor briefly describes the process of PCR (Polymerase Chain Reaction). https://www.youtube.com/watch?v=2KoLnIwoZKU The associated worksheet is located in the Student Section and the answers are found in the Appendix.

This virtual lab takes students through the laboratory experience of performing a PCR. http://learn.genetics.utah.edu/content/labs/pcr/ The associated worksheet is located in the Student Section and the answers are found in the Appendix.

c) Reverse Transcription PCR This is a general written overview of the RT-PCR technique from Davidson College. http://www.bio.davidson.edu/people/Kabernd/seminar/2002/method/lowry/RTPCR .htm The associated worksheet is located in the Student Section and the answers are found in the Appendix.

d) Flow Cytometry A basic introduction to the technique of Flow Cytometry. http://www.biotechnologyforums.com/thread-2185.html The associated worksheet is located in the Student Section and the answers are found in the Appendix.

Additional Resources that may be of use:

ELISA

 MAC ELISA step by step directions. This is highly detailed information suitable for accelerated AP Biology students. http://www.diatek.in/inbios/DENV%20Detect%20IgM%20ELISA.pdf  IgM CAPTURE ELISA step by step directions. This is highly detailed information suitable for accelerated AP Biology students. http://www.inbios.com/cms/file/900106- 04%20IVD%20DENV%20Detect%20IgM%20Capture%20ELISA%20insert%281%29.pdf RT-PCR  This animation steps the viewer through the basic steps of RT-PCR. http://www.bio.davidson.edu/courses/immunology/flash/rt_pcr.html There are no associated written assignments with this animation.

DAY 3 – Dengue Fever Case Study Students will work in small groups of 2-3. The case study focuses on the principles of immunology as told through a person who may have been bitten by a mosquito carrying the dengue virus. Students will act as the health care worker trying to determine if the person has contracted dengue fever. Questions will be interspersed throughout the case study.

Additional Resources Associated with Dengue:  General Information World Health Organization. Dengue and severe Dengue (2013). Centers for Disease Control. Dengue (2013). Nature. http://www.nature.com/scitable/topicpage/current-dengue-fever-research- You Tube. https://www.youtube.com/watch?v=0PUsdv1kDTc The New York Times. http://www.nytimes.com/health/guides/disease/dengue-hemorrhagic- fever/overview.html

 Prevention This Blog discusses the use of genetically modified mosquitoes to control their population size. http://www.nature.com/scitable/blog/viruses101/are_modified_mosquitoes_the_future

Campbell discusses the world wide spread of dengue and current attempts to prevent the spread. Campbell, C. "If You're Not Worried About Dengue Fever, Here's Why You Should Be." Time. November 18, 2013.

IX. How will students demonstrate understanding?

 Successful arrangement of cell images according to directions.  Successfully answering the questions regarding technology and completion of the virtual labs.  Successful completion of the case study.

X. References

Clinic Staff, Mayo. "Dengue Fever." Mayo Clinic. N.p., 19 Sept. 2014. Web. <http://www.mayoclinic.org/diseases-conditions/dengue-fever/basics/definition/con- 20032868 >.

"Dengue." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 26 Mar. 2015. Web. http://www.cdc.gov/Dengue/.

"Dengue Guidelines for Diagnosis, Treatment, Prevention and Control." (2009): 1-160. Dengue. World Health Organization, 2009. Web. http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf.

DeSalle, Rob, and Michael R. Heithaus. Holt Biology. Orlando, Fla: Holt, Rhinehart and Winston,

2008. Print

Garcia-Navarro, Lourdes. "Ready, Set, Spray! Brazil Battles Dengue Ahead Of The World Cup." NPR. NPR, 29 May 2014. Web. <http://www.npr.org/blogs/health/2014/05/29/316668643/ready-set-spray-brazil-battles- dengue-ahead-of-the-world-cup>.

Hillis, David M. Principles of Life. Sunderland, MA: Sinauer Associates, 2012. Print

Murphy, Kenneth, Paul Travers, Mark Walport, and Charles Janeway. Janeway's Immunobiology. New York: Garland Science, 2008. Print.

Reece, Jane B, and Neil A. Campbell. Biology. Boston: Pearson Learning Solutions, 2008. Print.

XI. STUDENT SECTION

This section contains:

• Video Reflection Guide
• Immune Cell Images
• What are PCR, RT-PCR and Flow Cytometry?
• Case Study – Dengue Fever

Video Reflection Guide – Immune System

Name_________________________________________________________ Block__________

Respond to the following questions after watching the Immune System Video. http://www.bozemanscience.com/immune-system

Brief Summary “In this video presentation I learned…”

The main points “Specifically…” (bullet points are appropriate here)

What you did not understand well “I am still confused about…”

Optional “I was really excited to learn about….” OR “I would like to learn more about…”

Innate and Adaptive Cells of the Immune system

What are PCR, RT-PCR and Flow Cytometry?

Name____________________________________________________ Block________________

I. The following video from the DNA Learning Center at Cold Spring Harbor, explains

the steps that take place in the typical PCR reaction.

https://www.youtube.com/watch?v=2KoLnIwoZKU

1. PCR uses repeated cycles of what to make many copies of DNA?

2. What causes the DNA to denature into single strands?

3. What is added to the DNA once the temperature has been decreased and then been raised slightly?

4. What is the function of the primer and Taq Polymerase?

5. How many cycles are needed before the desired DNA sequence starts to accumulate?

II. Virtual PCR Lab from Learn.Genetics the Genetics Science Learning Center at

University of Utah Health Sciences.

http://learn.genetics.utah.edu/content/labs/pcr/

Define the following vocabulary words located on the home page. Primer –

DNA Polymerase –

Nucleotide –

Continue on to the PCR Virtual lab and answer the following questions.

1. What is the importance of PCR?

2. Where can DNA samples come from for PCR reactions?

3. To start a PCR reaction what do you need to add to the PCR tube besides the DNA?

4. What is the name of the machine that can heat and cool the PCR tube?

5. What happens to DNA at 95 0 C?

6. Once the DNA Polymerase locates the primers what does it do?

7. By the end of cycle three, how many copies of the DNA fragment you targeted are there?

III. You now have an understanding of the basic principles of PCR. Read the following

short article on Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and

answer the following questions.

http://www.bio.davidson.edu/people/Kabernd/seminar/2002/method/lowry/RTPCR.htm

1. What nucleic acid is detected by this technique?

2. If that nucleic acid is present in the sample being tested, what does it mean for a specific gene that it is associated with and its product?

3. What are the steps in the “RT” portion of RT-PCR?

IV. What is Flow Cytometry?

Read the information on the Flow Cytometry and answer the accompanying questions. http://www.biotechnologyforums.com/thread-2185.html

1. What is the function of a flow cytometer?

2. What is the basis for the separation of different cell types?

3. Explain two uses for this technique.

Dengue - Is the World Cup Worth the Risk?

Introduction:

October 30, 2007 – Brazil has been elected as the host nation for the 2014 FIFA World Cup. Fast forward to August 20, 2013.

Chris J., an enthusiastic soccer fan, is anxiously awaiting the online ticket sales of the 2014 FIFA World Cup to open up. Chris is a Ph.D. student in the Microbiology and Immunology Department at Upstate Medical University. He spends his summers in Brazil researching the mosquito dengue fever vectors, Aedes aegypti and Aedes albopictus. Next summer, while collecting mosquitoes from the City of Natal, he is hoping to see the U.S. play a soccer match in the Arena das Dunas. He will be taking Drew, an undergraduate research assistant, with him to help with collecting the mosquitos and he wants to go to the match as well.

As their June departure date draws closer, Drew excitedly approaches Chris. “Chris I heard a story on NPR this morning about dengue fever and how Brazil is having an epidemic this year. Here is the transcript of the interview.”

Ready, Set, Spray! Brazil Battles Dengue Ahead Of The World Cup

Transcript: http://www.npr.org/templates/transcript/transcript.php?storyId=

Drew: “In another news article it said that the cities of Natal, Fortaleza, and Recife have the greatest risk of dengue fever. We are headed to Natal. I’m nervous that I will be bitten by a mosquito carrying one of the dengue viruses?”

Answer these questions based on the NPR interview.

1. How is the dengue infection spread?

2. How many different strains of dengue are there?

3. How is poor water infrastructure contributing to the spread of dengue?

4. If you were Drew would you be concerned about contracting dengue fever? Support your answer.

Background

Chris: “While I can’t guarantee that you will not be exposed to dengue while we are in Brazil, here is what you need to know about the disease and its transmission.”

Question: What is dengue? Answer: Dengue is a vector borne viral disease caused by one of four different serotypes (DENV-1, DENV-2, DENV-3, DENV-4). The virus is a member of the Flaviviridae family.

Question: How is it contracted/transmitted? Answer: The most common vector is the Ades aegypti mosquito, Ades albopictus is also a carrier. When an infected mosquito bites a human, the virus can be transmitted through the fluids the mosquito inserts through its proboscis. The mosquito acquires the virus through the blood of an infected human it bites. Human to human transmission is not possible.

Question: Can I get dengue again if I have already been infected once? Answer: You will have immunity to the specific virus (DENV-1, DENV-2, DENV-3, DENV-4) that you were originally infected with and, if infected again with that serotype, you will not get the disease. However, if you are infected with a different serotype you are at a greater risk of acquiring dengue hemorrhagic fever (DHF). DHF is a more severe form of dengue and can be fatal if medical treatment is not immediately sought.

Question : How can it be prevented? Answer: As the disease is spread by mosquitoes, eradication of breeding sites is essential. Ae. aegypti lays her eggs in containers that hold water (e.g. tires, planters, water drums). However, Ae. aegypti are highly adaptable to varying conditions; eggs have been known to survive drought conditions. Ae. albopictus prefers cool dark areas such as home closets. The use of air conditioning and screens can prevent adults from finding their human meals.

Question: What are the disease symptoms? Answer: Many of the symptoms are very similar to malaria or the flu, they include: high fever; severe headache; pain in the joints, muscle, bone or behind the eyes; rash; and mild bleeding. The symptoms of dengue hemorrhagic fever include a fever that lasts from two to seven days with the other symptoms mentioned above. As the fever declines the following symptoms may occur: vomiting, abdominal pain and breathing difficulties. This opens a 24 to 48 hour period when capillaries start to leak fluid. This increase in fluid in the body cavities can lead to the failure of the circulatory system, shock, and death. Skin hemorrhages, along with internal and external bleeding, are also possible.

Question: How is dengue treated? Answer: There is no specific treatment for the disease. If one suspects they have dengue fever, they can take analgesics, stay hydrated, rest, and speak with their doctor. If they develop more severe symptoms after the fever has subsided, they should go to a hospital. Fluid replacement therapy is appropriate at this point. Currently, there is no vaccine on the market; however, the company Sanofi Pasteur has plans to register the tetravalent vaccine (protects against all 4 dengue strains) in 2015 with a target distribution time frame of late 2015.

Questions : Where has the disease been found? Answer: The four viruses originated in monkeys and spread to humans in Africa or Southeast Asia between 100 and 800 years ago. Currently, the disease can be found in Southeast Asia, the Pacific Islands, Africa, Caribbean, and the Americas.

Question: What lab tests are used to detect the virus? Answer: The test methods used is dependent upon the duration of the disease. Real time RT-PCR is used to detect the virus in patients within the first 5 days of symptoms. A positive result confirms presence of the disease and usually which of the viral strains is involved. A negative result in this time frame is termed indeterminate, and patients will be asked for another sample post 5 days of symptoms.

MAC ELISA detects the presence of IgM antibody in the serum. The test can be used as early as three days post symptoms and may be able to detect the antibody as far as 90 days post symptoms. Caution should be used interpreting the results as this method can also react with other flaviviruses.

IgG ELISA is used to diagnose the presence of a past infection. To determine if the infection was primary (first exposure to dengue) or secondary (a second exposure) in nature, two samples need to be taken one during the acute phase and one in the convalescent phase. If the results are negative in the acute but positive in the convalescent phase, the patient has a primary infection. If there is a positive result in the acute phase and a fourfold increase in the convalescent phase, the infection is secondary.

Plaque Reduction and Neutralization Test is used to detect specific serotypes in convalescent serum.

Drew: “Thank you Chris for all of that information. I feel better about my chances of contracting dengue now.”

Answer the following questions based on the above information.

6. What are the symptoms of dengue fever and dengue hemorrhagic fever?

7. What tests can be used to confirm the disease?

The story continues: Chris and Drew travel to Brazil

Chris and Drew organize the supplies needed for their research in Natal, Brazil. Mindful of the continued news articles about the threat of dengue, they pack appropriate clothing and repellents to deter mosquitoes. On June 1, 2014, they leave Syracuse for Brazil.

Their research went well; they were able to sample adult mosquito populations as well as collecting eggs from a variety of sources. While it was winter in Brazil mosquitoes were still plentiful. On June 16 th, the day before they were to leave Natal, they went to the Arena das Dunas to watch the US vs Ghana soccer match. The US won 2-1, what a great way to end their trip!

Symptoms?

Chris and Drew had a non-eventful trip back to Syracuse on the 17 th. Chris was feeling a bit “off.” He was tired and a bit achy when he got back, but put it down to a hectic few weeks. He went into the lab on the 18 th, by mid-day he had a throbbing headache. He was also hot to the touch and felt like he was coming down with the flu. He spoke with his advisor who sent him to the Upstate Emergency Room.

7. Why did the advisor send Chris to the E.R.?

In the E.R., Dr. Court took Chris’ medical history and vital signs. Chris mentioned to her that he may have been exposed to dengue. Hearing this information, Dr. Court asked for a consult from Dr. Matthew from the Infectious Disease Department. Chris explained to Dr. Matthew that he had just returned from Natal, Brazil, an area that had a high risk of dengue infection. In fact, he had been collecting the species of mosquito, Ae. aegypti and Ae. albopictus, which are the preferred vectors of the disease.

After confirming Chris’ symptoms, Dr. Matthew ordered the following tests to be run: Real Time RT PCR, MAC ELISA, and IgG ELISA. He also filled out the dengue Case Investigation Report. Dr. Matthew explained to Chris that a positive Real Time RT-PCR result would indicate the presence of the virus in his blood. It was not unusual for this particular test to come back negative as the virus is only present for 5 days post infection. The MAC ELISA reports on the presence of the IgM antibody, an antibody that the body makes during the initial phase of an infection which shows up approximately 5 days post fever. A positive MAC ELISA indicates that the patient has recently been exposed to the dengue virus. To be effective the IgG ELISA must be done twice on serum samples collected 14 days apart. If the first sample collected during the early days of infection is negative, but the second sample is positive, then the infection is a primary or first exposure dengue infection. If the first sample is positive and the second sample is positive with a fourfold increase in the IgG titer, it is a secondary exposure to another DENV serotype.

8. If a patient tested positive for a DENV serotype during a Real Time RT PCR test does the patient have dengue? Why or why not?

RUNNING THE TESTS – WHAT WILL THEY REVEAL?

Dr. Matthew drew a blood sample to run the Real Time RT PCR, MAC ELISA, and IgG ELISA tests. He sent the sample over to the lab. Dr. Matthew told Chris that he would have the results back

in a day. In the meantime, he should go home making sure that he drinks fluids and, if needed, take acetaminophen.

Chris went to see Dr. Matthew the next day. Dr. Matthew had received the lab reports back. The Real Time RT PCR was negative; it showed no viral RNA in the blood sample. As the virus is only present in the blood for the first five days after infection, this was not an unexpected result. The MAC ELISA, testing for the presence of IgM, was positive for DENV-2. The IgG ELISA was negative for DENV. Dr. Matthew told Chris that it was likely that he has been infected with DENV-2. The negative IgG ELISA result indicates that this probably is a primary not secondary infection. That determination will be made when you come back in 14 days for another blood draw for the second IgG ELISA.

15 days later – Chris now fully recovered from his symptoms returns to Dr. Matthew’s office for the results of his second IgG ELISA test. Dr. Matthew tells Chris that his second IgG ELISA was positive. The official diagnosis is “recent probable primary dengue infection.”

9. Explain what Chris’ final diagnosis is.

The follow up

Dr. Matthew asked Chris if he remembers being bitten by a mosquito. Chris said he couldn’t remember being bitten. About 6 days before they left Natal, he and Drew were sampling in an area of the city that had significantly more pools of stagnant water than the other areas that they had sampled. It seemed as if there were clouds of mosquitoes hovering around some of their sampling spots.

Dr. Matthew asked if Chris would be going back to Brazil to gather more samples in the future; reminding him that an encounter with the DENV-1, 3 or 4 serotypes could lead to dengue hemorrhagic fever and potential death. The body’s immune system will produce antigens to the specific DENV serotype it was first exposed to but not to all the serotypes. Dr. Matthew reminded Chris of the symptoms to be aware of: fever lasting 2-7 days during which time the general dengue symptoms will be present. After the fever subsides, he may experience vomiting, abdominal pain and difficulty breathing. This opens a 24-48 hour window where medical attention must be sought as fluids may accumulate in the abdomen and lungs leading to failure of the circulatory system, shock, and death.

This was Chris’ last collecting trip in Brazil, he will be now spending his time in the states analyzing his data and writing his dissertation. Citizens of Puerto Rico, U.S. Virgin Islands, Samoa, and Guam are in areas endemic for the virus. While there have been cases of dengue reported in the continental US, these were acquired by travelers when they were outside of the country. Because contact between the Aedes mosquito and inhabitants of the continental US is infrequent the probability of a secondary infection is rare. As long as Chris takes precautions when in areas known to harbor dengue, he will not sustain a secondary infection.

10. If you were Chris would you be concerned about a secondary dengue infection? Why or why not?

EXTENSION

1. Students can read about the dengue virus on the Protein Data Bank web page. Using Jmol a student can manipulate an envelope protein with antibodies bound and the complete Dengue Virus with antibodies attached. http://www.rcsb.org/pdb/101/motm.do?momID=103

2. Students can construct a paper model of the dengue Virus. http://www.rcsb.org/pdb/education_discussion/educational_resources/dengue_virus_3Dmode l.pdf

XII. APPENDIX

Virus

Macrophage

engulfing a virus

Humoral (Antibody- Mediated Immunity

Immune Cell Types

Cytotoxic T Cell

Helper T Cell

Plasma B Cell

with antibodies

Memory B Cell

B Cell

Naïve B Cell

Memory B Cell

(Duplicate)

Cytotoxic T Cell

attacking an

infected cell

Cellular Immunity