UV Influence on Viral Infection Alec DiVito Central Catholic High School.

UV Influence on Viral Infection

Alec DiVitoCentral Catholic High School

Problem

• Does UV light affect viral infectivity?

Electromagnetic Spectrum

• The Group of all types of radiation. Including: radio waves, microwaves, infrared, visible, ultraviolet, X-rays, and gamma rays.

UV Light Rays

• Ultraviolet (UV) rays are light rays that have shorter wavelengths than visible light. They range from 150nm – 300nm.

• Greater energy than visible light yields a greater risk to life.

• They are naturally given off by the sun, but most are absorbed by the ozone layer.

Effects of UV Light

• The UV light waves that reach Earth can lead to many problems in a vast number of species.

• Humans – Heavy exposure to UV light without protection can lead to skin cancer and photokeratitis.

• Used for sterilization• Thought to interfere with many important

biological molecules including DNA.• Increased mutation rate of DNA

Direct DNA Damage

• Can occur when DNA directly absorbs the UV-B-photon.

• Due to the excellent photochemical properties of DNA this nature-made molecule is damaged only by a tiny fraction of the absorbed photons.

• In DNA this internal conversion is extremely fast - and therefore efficient.

Viruses

• Small infectious agent that can replicate only inside the living cells of organisms.

• Particles consist of two primary parts: proteins and nucleic acids.

• Ubiquitous, they hold the largest biomass in the world.

• UV light from the sun may be altering interaction between viruses and their hosts.

T2 Bacteriophage

• A virus that infects E. coli.• Like all viruses, T-Even phages cannot

reproduce extracellularly. They need a host cell in which to replicate their genetic material.

• Can quickly turn an E. coli cell into a T2-producing factory that releases phages when the cell ruptures.

• The first phages that were studied in detail included seven that commonly infect E. coli.

Lytic Cycle

• Viruses of the lytic cycle are called virulent viruses. • Six-stage cycle.

Escherichia Coli

• Large and diverse group of bacteria commonly found in the intestine of most mammals. Has been the most studied type of bacteria in biological research.

• The majority are harmless, but some can cause harmful, and even fatal, illnesses.

• One of the most commonly used host cells for viral infectivity experiments.

E. Coli B

• A large amount of the recent studies in radiation bacteriology and allied fields is concerned with the detailed behavior of cultures of Escherichia coli B.

• E. coli B is a bacterial strain that has been maintained for many years, mainly in laboratories concerned with bacteriophage synthesis and behavior.

• It is characterized by its unusual sensitivity to several harmful agents, including the T series of coli bacteriophages.

Purpose

• To assess the effects of UV light on viral infectivity efficiency.

Experimental Measurement and Interpretation

• The effects of UV light on Viral infectivity was assessed by the growth of plaques within a confluent E. coli B lawn grown on an LB agar plate.

Hypothesis

• UV light will reduce viral infectivity efficiency.

Null Hypothesis• The UV light will not significantly affect viral

infectivity.

Materials• LB (Luria Broth)

– 1% tryptone– 0.5% yeast extract– 1% NaCl

• Microtubes• Micropipettes + Tips• Incubator• UV hood• Gloves + Safety glasses• LB agar plates• E. coli B• T2 Bacteriophage• Thermometer• Side-arm flask

• Klett Spectrophotometer• Sterile dilution fluid

– 10 mM KH2PO4

– 10 mM K2HPO4

– 1 mM MgSO4

– 0.1 mM CaCl2

– 100 mM NaCl• Macropipettes• Top agar

– 10 g tryptone– 5 g yeast extract– 5 g NaCl– 7 g agar

Procedure

1. E. coli B was grown in a slide arm flask to a reading of 60 – 100 K.U. (mid-log phase).

2. Virus was diluted from 109 to 103 by serial dilution and SDF.3. 1 mL of phage was added to 5 microtubes and were

exposed to 0, 20, 40, 80, and 120 seconds of UV light in a Laminar Flow Hood.

4. 0.1 mL virus, 0.4 mL E. coli, and 2.5 mL top agar were then mixed in sterile 15mL conical tubes and immediately poured onto LB agar plates.

5. Plates were incubated overnight at 37˚ C.6. Plaques were counted and recorded.

UV Radiation Effects on Viral InfectivityP-Value = 2.35E-26

UV Radiation Effects on Viral Infectivity

0 Seconds 20 Seconds 40 Seconds 80 Seconds 120 Seconds0

10

20

30

40

50

60

70

80

90

100

UV Radiation

Plaq

ues

LD50 ≈ 50 Seconds UV Radiation

Dunnett’s Test

UV Exposure T-Value Interpretation

20 Seconds 3.12 Significant

40 Seconds 5.29 Significant

80 Seconds 9.90 Significant

120 Seconds 11.38 Significant

T-Critical = 3.02

Conclusion

• The null hypothesis can be rejected for every group. The hypothesis can be accepted.

• The variable appeared to effect viral infectivity efficiency.

• The Dunnett’s test shows that the of each of the individual results compared to the control are significant.

• The virus was in some way altered resulting in less efficient infection.

Limitations/Extensions

• Synchronize the exact times of plating.• Sequence the actual virus to see if the viral DNA

was mutated or proteins were damaged and this was the cause of less efficient infection.

• Increase the sample size.• Utilize different viruses.• Investigate other viruses and their bacterial

hosts.• Utilize various other types of electromagnetic

radiation.

Works Cited

Betsey, Tom. Microbiology Demystified. New York: Wagner, 2005. Print.

Chung, C. T. "PNAS." PNAS. Web. Oct. 2009.

Clark, David. Molecular Biology Simple and Fun. New York: Warner, 2007. Print.

"Cloning and Transformation." Web. 5 Nov. 2009.

Ferguson, L.R., ed. "Mutation Research." Fundamental and Molecular Mechanisms of

Mutagenesis 12.1 (2007): 1+. Print.

"Honors Biology 1." Personal interview. 3 Nov. 2009.

"Nutrigenomics." ScienceDirect - Home. Ed. L.R. Ferguson. Elsevier. Web. 05 Nov.

2009. <http://www.ScienceDirect.com>.

"X-Rays." NASA Science. Ed. Ruth Netting. National Aeronautics and Space

Administration. Web. 04 Jan. 2010.

<http://science.hq.nasa.gov/kids/imagers/ems/xrays.html>.