Virology

1. Virology • Microbiology - 261 • Dr. Gary Andersen • References: Black (2005). (Ch 10, 18, 21, 22, 23, 24) Some slides taken with permission from Curtis Smith

2. General Characteristics of Viruses • Latin word for poison • Tobacco Mosaic Virus (1903) 1st! • Obligate intracellular molecular parasites – “parasites for nucleic acids.” • Viruses evolved from plasmids. • Purpose in nature is to create genetic diversity. • Viruses must use host cell machinery to replicate. They are infectious particles. • Made of DNA or RNA

3. Viral particles are NOT sensitive to traditional antibiotics. • Sensitive to interferon. Interferon is a chemical released by infected cells that blocks viral replication in neighboring cells. • Viewed only with an electron microscope. • Poliovirus is the smallest virus infecting humans at 30 nanometers (nm). Note: (nm = one billionth of a meter). Largest human virus is the Ebolavirus at 970 nm. Ebola infects primates normally, but one strain infects humans. http://www.cellsalive.com/howbig.htm

4. Viral sizes and shapes: Variations in shapes and sizes of viruses compared with a bacterial cell, an animal cell, and a eukaryotic ribosome

6. (K. continued) Vaccinia virus is a genetically engineered cross between cowpox and smallpox viruses. It is avirulent in humans and used to vaccinate against smallpox.Smallpox was declared eliminated from the earth in 1979. (1798 Jenner; 11th Century India & China vaccinations) • Viruses are referred to as filterable agents. They can be filtered from tissue fluids using a 0.2 micrometer millipore filter. N. It is estimated that 10% of all humans cancer possesses a viral etiology. Seven cancer (oncogenic) viruses have been discovered in humans, but many have been found in animals.

7. The Components of an Animal Virus

8. II. Types of Viral Morphology • Naked – Consists of only the nucleocapsid which is a protein head (capsid) that encloses DNA or RNA. Generally these viruses cause lysis of the host cell. 1. Icosahedral (20 sided) 2. Helical

10. ENVELOPED - Consists of a nucleocapsid plus a phospholipid bilayer (envelope). The phospholipid bilayer may contain sugar proteins called glycoproteins. If the sugars stick out of the envelope they are called spikes. Enveloped viruses are usually not lytic. They exit the host cell by a process known as budding. (pg 284-285 and next slide). 1. Nucleocapsid can be icosahedral, 2. Nucleocapsid can be helical. C. COMPLEX – ex. Ebola and Smallpox, or Vaccinia.

11. Budding

12. Viral Structure F 08

13. Some Types of Viruses • DNA Viruses (6 families) below are 5. • Pox viruses – smallpox, cowpox • Herpes viruses – HSV1, HSV2, Varicella zoster virus VZV (chickenpox shingles), Epstein-Barr Virus EBV • Adenoviruses – glandular infections, colds • Papilloviruses- Human papillovius HPV (warts) • Hepadnaviruses – Hepatitis B

14. DNA Viruses

15. smallpox HS1, HS2, VZV, CMV, EBV Glandular colds warts Hepatitis B Childhood rashes

16. RNA Viruses

17. RNA Viruses (continued)

18. Some Types of Viruses 2 p. 270-275 Black • RNA Viruses (10-14 families) (five important groups are listed below) • Naked • Picornaviruses – Polio, rhinoviruses (colds), Hepatitis A (short term hepatitis) • Enveloped • Flaviviruses – West Nile fever, Hepatitis C • Orthomyxoviruses – Flu • Retroviruses – Human Immunodeficiency Viruses HIV, Human T-cell leukemia virus HTLV • Coronaviruses – Colds, bronchitis, Severe acute respiratory syndrome SARS

19. RNA Viruses Colds SARS Polio Lassa fever (carried by rats) Viral darrhea, Norwalk virus syndrome

20. RNA Viruses Mumps Rabies Colorado Tick Fever Flu Encephalitis Ebola

21. General Properties of RNA Viruses • Many ssRNA viruses contain positive (+) sense RNA, and during an infection acts like mRNA and can be translated by host’s ribosomes • Other ssRNA viruses have negative (-) sense RNA and the RNA acts as a template during transcription to make a complementary (+) sense mRNA • Negative (-) sense RNA must carry an RNA polymerase within the virion

22. Coronaviridae: Coronavirus got their name (corona, Latin for “crown”)

23. Picornaviruses: very small, naked, polyhedral, (+) sense RNA viruses. They include the Enterovirus, Hepatovirus, and Rhinovirus

24. Retroviruses: are enveloped viruses that have two complete copies of (+) sense RNA. They also contain the enzyme reverse transcriptase, which uses the viral RNA to form a complementary strand of DNA, which is then replicated to form a dsDNA

25. Rhabdoviruses: Another (-) sense RNA virus group consists of medium-sized, enveloped viruses. The capsid is helical and makes the virus nearly rod or bullet-shaped. Contain an RNA-dependent RNA polymerase that uses the (-) sense strand to form a (+) sense strand that serves as a mRNA and template for synthesis of new viral RNA

26. Orthomyxoviruses: medium-sized, enveloped, (-) sense that vary in shape from spherical to helical. Their genome is segmented into eight pieces

27. Reoviruses: have a naked, polyhedral capsid. They are medium-sized dsRNA and replicate in the cytoplasm. Ingestion of only 10 rotavirus particles is sufficient to cause infection and diarrhea

28. Computer-generated model of a human rhinovirus, cause of the common cold. The colors represent different capsomeres of the capsid

29. Bacteriophages • Viruses that infect bacterial cells

30. Bacteriophages: Structure and electron micrograph of a T-even (T4) bacteriophage

31. Bacteriophage: DNA normally is packaged into the phage head. Osmotic lysis has released DNA from phage, showing the large amount of DNA that must be packaged into a phage

32. Replication of a virulent bacteriophage: A virulent phage undergoes a lytic cycle to produce new phage particles within a bacterial cell. Cell lysis releases new phage particles that can infect more bacteria

33. Virus Replication

34. III. Cultivation of Viruses • Embryonated chicken eggs . 1. Provides a cheap, sterile environment. 2. Used to prepare the influenza virus vaccine (do not take if allergic to eggs!) Clear zone is called plaque. • Primary Cell Culture – 1. Homogenous collections of adult eukaryotic cells. (infected with virus, then look for plaque. See pg 281) ex. Monkey kidney cells (MKC) 2. Fetal calf serum – nutrient broth used to culture eukaryotic cells 3. Primary cell cultures die when the bottom of the dish is covered due to “programmed” cell inhibition. Grow as a monolayer.

35. Viral culture in eggs: Some viruses, such as influenza viruses, are grown in embryonated chicken eggs

36. Plaque assay: The number of bacteriophages in a sample is assayed by spreading the sample out over a “lawn” of solid bacterial growth Plaque

37. Primary cell cultures are survive only 4-5 generations of culture passage. • Used to prepare some vaccines like polio. • Often contaminated with monkey and cow viruses. • Used for PhD research now, and retroviral research in the 1960’s.

38. Primary Cell Culture

39. C. Diploid Fibroblast Cell Cultures • Human Fetal Tissues ex. Fetal tonsil diploid fibroblasts. (Form connective tissue) • Grow as a monolayer. • Last ~ 100 generations. • Most expensive cell cultures. • Most pure, rarely contaminated. • Used to prepare the rabies vaccine.

40. Human Diploid Fibroblast Culture

41. Fetal tonsil cells Tonsil cells with adenovirus Tonsil cells with herpes simplex infection

42. Tissue Culturing

43. Continuous Cell Cultures(permanent cell lines) • Derived from human cancer cell lines; ex. HeLa cells named for Henrietta Lacks who died of cervical cancer in 1951. • Used for rapid growth of viruses. • Inexpensive but often contaminated. • Can be frozen, stored and shipped. • Grow as a multilayer due to the absence of cell inhibition. • Cancer cells are immortal in vitro. • Used for Master degree research. GA sp07

44. Henrietta Lacks http://www.news.harvard.edu/gazette/2001/07.19/04-filmmaker.html

45. VIRUSES IN VITROi.e What effect do viruses have on primary cell cultures? • Productive Response – ex. Cold viruses. 1. Lytic cycle – kills cells during burst and releases many naked virus particles; or from budding enveloped viruses released in large numbers. B. Cytopathic Effect – ex. Herpesviruses 1. Lysogenic cycle – virus integrates into the chromosome. Host cells are not killed but their appearance is altered.

46. Multinucleated cells (Herpes)(Example of cytopathic effects)

47. Transformation - A few viruses transform primary cell cultures into continuous cell lines. These are cancer causing viruses = oncogenic viruses. Oncogenic = mass generating. ~ 10% of all cancer is believed to be caused by viruses. Oncogenic viruses carry oncogenes which trigger cells to divide rapidly and lose cell inhibition. The same genes found in oncogenic viruses are found in normal human cells. In human cells, these genes are called protooncogenes (potential cancer causing genes). Protooncogenes regulate embryogenesis, but are “turned off” after the first trimester. Note that human cancer cells are wild growing, unregulated embryonic-like tissue.

48. Proto-oncogene  Oncogene

49. Transformation of Normal Cells

50. Oncogenes http://cancerquest.emory.edu/index.cfm?page=261 Web site with analogy GA F07