Laboratory Activity 5: Avian-Human Hybrid Flu Virus

Materials:

quart-size plastic bag

pint-size plastic bag

2 plastic novelty eggs shells that come apart

8 paper clips and 2 small rubber bands, all of one color

8 paper clips and 2 small rubber bands, all of one color (different color from first set)

tape or small labels

2 film containers

Procedure:

Assemblage of Lung Cell.

Pint-size plastic bag - lung cell nuclear membrane

Quart-size plastic bag - lung cell plasma membrane

Assemblage of Avian Influenza A Virus

8 red paper clips - used to represent the viral RNA strands

2 red paper clips - surface glycoproteins

Plastic egg - core

Plastic egg inserted to specimen container - serve viral envelope

Assemblage of Human Influenza A Virus.

 8 blue paper clips - used to represent the viral RNA strands

2 of the blue paper clips - surface glycoproteins

All of the paper clips were placed inside the plastic egg (core). The plastic egg was
inserted into the specimen container (viral envelope) to make the human influenza A
virus.

Construction of an Avian-Human Hybrid Flu Virus Model.

16 paper clips from both of the assembled virus models were mixed.

Without looking, eight RNA strands were removed from the nucleus.

4 blue and 4 paper clips were randomly picked.

8 RNA strands were placed inside a plastic egg (core).

The core was inserted into a specimen container (viral envelope) to make a new virus
that could potentially infect another cell.

Specimen container was closed with one blue rubber band and one red rubber band
sticking out its rim - represent the surface glycoproteins of the hybrid virus model.

Concepts:

Antibody

- also known as an immunoglobulin, is a Y-shaped structure which consists of four polypeptides

— two heavy chains and two light chains.

Heavy Chains

- large polypeptide subunit of an antibody; also known as IgH.

- In human genome, the IgH gene loci are on chromosome 14.

- IgH allow for antigen-specific binding and subsequent activation of B lymphocytes.

- can bind antigens despite having only VH domains

Light Chains

- small polypeptide subunit of an antibody; also known as IgL.

- IgL ensure the expression and secretion of functional antibodies and contribute to antigen
binding by increasing the variability of the antibodies

Domains

- folded functional units

Each light chain consists of one variable domain (VL) and one constant domain (CL).
 Each heavy chain has one variable domain (VH) and three or four constant domains
(CH1, CH2, CH3, CH4).

Disulfide Bonds

- functional group present in some proteins. It describes the covalent connection of two thiol
groups, usually of cysteines, in the form of R–S–S–R'.

Gene Segment

- involved in the early stages of pattern formation that define repeated units

Fragmant Crystallizable Fragment (FC)

- the tail region of an antibody that interacts with cell surface receptors called Fc receptors and
some proteins of the complement system.

- allows antibodies to activate the immune system.

Fragment Antigen Binding Fragment (FAB)

- region on an antibody that binds to antigens.

- composed of one constant and one variable domain of each of the heavy and the light chain.

Key Notes:

Influenza A viruses (IAVs)

are the only influenza viruses known to cause pandemics, or worldwide outbreaks of flu
disease

made up of eight single-stranded viral RNA segments contained in separate viral

ribonucleoprotein (vRNP) complexes that are packaged together into a single virus
particle.

When influenza A viruses from two different species infect the same person or animal at the
same time, the segmented genome specifically enables reassortment, or the mixing of genes, to
create a new virus

Reassortment between established human IAVs and IAVs in the animal reservoir can result in
the emergence of pandemic influenza strains against which the human population has little
immunity.

The vRNAs are transcribed and replicated inside the nucleus by the heterotrimeric viral RNA-
dependent RNA polymerase.
The replication of the influenza genome consists of two steps:

1. transcription of complementary RNA (cRNA)

2. transcription of new vRNA copies using the cRNAs as templates

The majority of those who contract influenza A are humans, and it typically affects the

nose, throat, and lung cells.

The influenza virus infects the respiratory epithelial cells that line the lower (to the alveoli) and
upper respiratory tracts, including the nasal passages.

The development of alveolar disease depends on early interactions between the influenza virus,
the alveolar macrophages that reside in the lungs'airways, and the epithelial lining.

Uninfected people can contract Influenza A by:

touching contaminated surfaces

inhaling viruses from infected individuals' coughs or sneezes

Laboratory Activity 6: Antibody Structure
Materials:

2 whole pipe cleaners (chenille stems) of the same color (heavy chain

segments)

1 pipe cleaner (chenille stem) cut into 2 pieces (light chain segments)

1 “sparkle” pipe cleaner (chenille stem) cut into 4 pieces (disulfide bonds)

Container of colored beads to use as VDJ segments

Assembly of the model

Prepare 3 colors of chenile stems

2 chenile stems (paired) - serve as the heavy chain.

1 chenile stem and cut it in half (positioned beside the heavy chain) - represents the light
chain

1 chenile stem and cut it to 4 equal parts.

2 shortened chenile stems - ties the middle and lower part of the structure.

2 tie the heavy chain and light chain together, one stem per side.

Bet the structure in order to form a “Y” shape.

Assemblage of DVJ.

3 pairs of beads - inserted through the heavy chain (equilaterally distant apart)

3 pair of beads of another color - placed at the end of each light chain.

Key Notes:

Antibody molecule

composed of 4 polypeptides

with 2 heavy chains and 2 light chains that join together to form a "Y" shaped
molecule.

Each end of the molecule carries out different tasks:

1. one binds to a variety of antigens (i.e. Fab fragment)

2. the other binds to a limited number of effector molecules and cells (i.e. Fc region).

Three globular regions form a Y structure

Two antigen-binding sites - found in the two arms of the Y end region which are tethered
by a flexible hinge region.

Chains are joined together by a disulfide bond so that each heavy chain is linked to a
light chain and the 2 heavy chains are also linked together.

The 2 heavy chains (green) and 2 light chains (yellow) are identical in any
immunoglobulin molecule.

2 identical antigen-binding sites in an antibody molecule - enables the antibody to bind

simultaneously to two identical structures

Effector function of the antibody molecule is defined by the structure of the heavy
chains.

The tail region of the antibody, Fc region, binds with surface cell receptors and thus
called Fc receptors.

Fc receptors - also permit the antibodies to activate the immunity system.

T and B cell receptors - key cells of the adaptive immune response, share the same
mechanism and that is called the V(D)J recombination.

V(D)J recombination:

V = variability

D = diversity

J = joining

Process of V(D)J recombination results in a numerously diverse set of

antibodies/immunoglobulins and T cell receptors found on B cells and T cells,
Laboratory Activity 7: Antibody Structure
1. Which group of symptoms would you use to determine if a patient represented a “case?”
Why would you not want to include a symptom such as “sore throat” in your definition?

Malaise, myalgia, fever, chills, headache. If a patient had three of these 5 symptoms then they
would be considered a “case.” These are the most prevalent of the many different syptoms.
Sore throat was only reported in about 25% of the patients, so would not be a good indicator of
the illness.

2. What is the attack rate for the employees at the time of your arrival on Saturday?

(91 / 100) x 100% = 91%

3. What information must you have to determine an attack rate for the visitors? Why is it much
easier to determine the attack rate for the employees than for the visitors?

You must know how many total visitors there were during the time in question and know how
many of them became ill. The identities of the employees was fairly easy to determine, however,
tracking down who exactly were visitors to the health department would be much more difficult
to determine.

4. What is the one thing that all cases appear to have in common?

All of the people who became ill were, at one time, present in the health department building.

5. Formulate a hypothesis as to the source of the illness. Back it up with evidence that you
have collected.

The illness is caused by something in the building itself. It could be a pathogen such as a virus
or bacterium, or it may be a toxin produced within the building. The fact that it affects all types
of people in the same way, regardless of age or race may indicate this. In addition, you can rule
out water or food borne disease, as there seemed to be no relationship between what was eaten
or drank and the likelihood of becoming ill. Also, you can rule out the dust clouds as a source of
the illness, as workers in other nearby buildings were unaffected.

6. What does this information tell you about the transmission of the agent causing the
outbreak?

It is not transmitted from one person to another.

7. How would you go about identifying visitors to the health center?

You could look through any sign-in sheets in the various departments or clinics, check local
doctor’s offices and clinics to see if anyone has had similar symptoms. You could also make
public announcements on radio and TV to encourage people to contact you if they were in the
building

8. Calculate the attack rate for visitors to the health department. (show work)

(47 / 170) x 100% = 27.6%

9. Why do you suppose that the attack rate is considerably lower for visitors than it is for
employees?

The employees were exposed to the conditions in the building for a longer period of time than
the visitors.

10. Are the epidemic curves for the employees and visitors the same? Give a possible
explanation for this.

No. The number of visitor cases peaked around Wednesday of the first week while those for the
employees peaked on Tuesday. This may be due to the fact that there were fewer visitors than
employees exposed at the beginning of the week. In addition, it was subsequently discovered
that being in the building early in the morning, just when the A/C was turned on, greatly
increased one’s risk of contracting the illness. During the second week, more visitors than
employees became ill. By this time most of the employees had recovered and were back at
work, immune from any further infections.

11. What evidence points to this suspicion?

You must be in the building to become ill. It is not transmitted person-to-person. People become
ill sometime after being in the building.

12. About how long does it take someone to come down with symptoms after being exposed?

12 to 24 hours.

13. Based upon this information alone, which would you suspect as the culprit?

Influenza or Shigellosis.

14. However, based on other evidence you have gathered, you choose to rule these out.
Explain why.

Influenza is transmitted person-to-person, shigellosis is water- or food-borne and there was no

relationship between food and water and rate of attack.

15. If your suspicions are correct, where would you expect the smoke to go?

It would move from the condensing system into the cool air system. It could leak through the
main ducts or could exit the vent on the roof and enter the fresh air intake because they were so
close to one another.

16. What types of samples would you want to take to try to identify the organism responsible
for the outbreak?

From the paitents you would want to collect blood, sputum, urine, stool. You also would collect
water from the condensing unit and possibly from the water supply to the building to rule out
water-borne pathogens.

17. Suppose you had some laboratory animals, such as guinea pigs, available for testing the
air conditioning system.
18. Describe an experiment that you could perform to determine if the air conditioning system
in the health department building is really responsible. Be sure to include replication and
controls in your design.

Groups of animals should be exposed to some of the following conditions:

i. In the offices near the A/C vent.

ii. In the basement.

iii. In the other buildings on the campus.(control)

iv. In cages away from the health dept. building but sprayed with water from the
condensing unit.

v. In cages away from the health dept. building but sprayed with distilled water. (control)

vi. In cages away from the health dept. building but sprayed with water from the
condensing unit that had been autoclaved to kill any bacteria.