Virology

• Microbiology - 261• Dr. Gary Andersen• References: Black (2005). (Ch 10,

18, 21, 22, 23, 24)

Some slides taken with permission from Curtis Smith

I. General Characteristics of Virusesa. Latin word for poisonb. Tobacco Mosaic Virus (1903) 1st!c. Obligate intracellular molecular

parasites – “parasites for nucleic

acids.”d. Viruses evolved from plasmids.e. Purpose in nature is to create

genetic diversity.f. Viruses must use host cell machinery to

replicate. They are infectious particles.g. Made of DNA or RNA

h. Viral particles are NOT sensitive to traditional antibiotics.

i. Sensitive to interferon. Interferon is a chemical released by infected cells that blocks viral replication in neighboring cells.

j. Viewed only with an electron microscope.

k. Poliovirus is the smallest virus infecting humans at 30 nanometers (nm). Note: (nm = one billionth of a meter). Largest human virus is the Ebola virus at 970 nm. Ebola infects primates normally, but one strain infects humans. http://www.cellsalive.com/howbig.htm

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

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(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.

The Components of an Animal Virus

II. Types of Viral Morphology

A. 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

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B. 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.

• Budding

Viral Structure

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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

DNA Viruses

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smallpox HS1, HS2, VZV, CMV, EBV Glandular colds

warts

Hepatitis B Childhood rashes

RNA Viruses

RNA Viruses (continued)

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

• RNA Viruses

Colds SARS

Lassa fever (carried by rats) Polio

Viral darrhea, Norwalk virus syndrome

• RNA Viruses

Mumps Rabies Colorado Tick Fever

Flu

Encephalitis Ebola

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

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

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

Hepatovirus, and Rhinovirus

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

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

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

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

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

of the capsid

Bacteriophages

• Viruses that infect bacterial cells

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

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

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

Virus Replication

III. Cultivation of VirusesA. 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.

B. 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.

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

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

Plaque

4. Primary cell cultures are survive only 4-5 generations of culture passage.

5. Used to prepare some vaccines like polio.

6. Often contaminated with monkey and cow viruses.

7. Used for PhD research now, and retroviral research in the 1960’s.

Primary Cell Culture

C. Diploid Fibroblast Cell Cultures

1. Human Fetal Tissues ex. Fetal tonsil diploid fibroblasts. (Form connective tissue)

2. Grow as a monolayer.3. Last ~ 100 generations.4. Most expensive cell cultures.5. Most pure, rarely contaminated.6. Used to prepare the rabies vaccine.

Human Diploid Fibroblast Culture

Fetal tonsil cells

Tonsil cells with adenovirus

Tonsil cells with herpessimplex infection

Tissue Culturing

D. Continuous Cell Cultures(permanent cell lines)

1. Derived from human cancer cell lines; ex. HeLa cells named for Henrietta Lacks who died of cervical cancer in 1951.

2. Used for rapid growth of viruses.3. Inexpensive but often contaminated.4. Can be frozen, stored and shipped.5. Grow as a multilayer due to the absence of cell

inhibition.6. Cancer cells are immortal in vitro.7. Used for Master degree research.

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Henrietta Lacks

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

IV. VIRUSES IN VITROi.e What effect do viruses have on primary cell cultures?

A. 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.

Multinucleated cells (Herpes)(Example of cytopathic effects)

C. 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.

                                                                                                                                                                                         

Proto-oncogene Oncogene

Transformation of Normal Cells

Oncogenes

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

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V. RELATIONSHIP BETWEEN VIRUSES AND HOST CELLS IN

VIVOA. Lytic Infection – Acute infections kill host cells

and are of short duration. Acute viral infections trigger the production of interferon resulting in a self-limiting infection. Ex. Cold viruses (Action of Interferon see pg 437 and next slide)

1. Family Picornavirus – ex. Rhinovirus come in 100 antigenic variations during fall/January. 60% of all colds are caused by rhinoviruses. Size: 30 nm Type of Nucleic Acid: + SS RNA

Self-limiting Infections: Production of Interferon

Cold Viruses continued

2. Coronavirus causes late winter colds. Usually severe with viral bronchitis. 20% of all cold viruses. 60 nm/+SS-RNA

3. Adenovirus causes severe spring colds. 10% of all colds. 80 nm/DNA

4. Paramyxovirus causes summer colds. 10% of all colds. 200 nm/ -SS RNA.

Cold Viruses continuedMode of Transmission – a cold virus discussion

The M.O.T for colds!

Direct Contact:

1. Nasal secretions

2. 1 meter aerosol droplet.

Indirect contact: from surfaces that cold viruses may survive on for up to 3 hours.

http://www.engr.psu.edu/ae/iec/abe/topics/epidemiology.asp Web site with Cold transmission rate charts and graphs.

B. Persistent Chronic Infections

Viruses that multiply at low levels and normally do not kill host cells. These are usually budding viruses.

Ex. Hepatitis B Virus (HBV) – “serum hep” produces only 10 “virus particles” per cell. The result is that there are many carriers for HBV thereby setting up a chronic persistent infection of the liver.

C. PERSISTENT “SLOW WASTING” INFECTIONS

These viral infections kill cells very slowly.

Ex. Family Retrovirus. Retroviruses are lysogenic, they integrate into the human chromosome. Reverse Transcriptase is an enzyme that converts RNA into DNA! Ex. HIV (Human Immunodeficiency Virus) which infects and slowly kills a specialized lymphocyte called the T – helper cell.

http://www.clunet.edu/BioDev/omm/hivrt/hivrt.htm Reverse Transcriptase Model

HIV Virus with reverse transcriptase

HIV RNA is transcribed into DNA

Transcribed DNA enters nucleus and is in turn transcribed back

into RNA

RNA Translated into viral proteins

Summary of Stages of Viral Infection - HIVAbsorption

Penetration

Replication

Assembly

Release

D. LATENT VIRUS INFECTIONS

Herpesvirus are dormant in host cells without adverse effects. When the host experiences environmental stress (fever, hypothermia, sunburn, lack of sleep, menses, or psychological) the virus is triggered to excise from the chromosome and starts replicating.

Examples of Herpesviruses1. HSV-I - Herpes Simplex Virus Type I causes Oral Herpes.

Cold Sores. Dormant or latent in the trigeminal (head, facial) nerve.

• HSV-II - Genital Herpes resides in the sacral ganglia (lower back).

• VZV – Varicella Zoster Virus causes chicken pox in children. Dormant in the thoracic ganglia. Shingles in the thoracic region. Only occurs in people who have low immunity to VZV.

• CMV – Cytomegalovirus. Dormant in parotid gland. Causes birth defects if during pregnancy. Rare mononucleosis.

• EBV - Epstein-Barr Virus – classic mononucleosis. Dormant in the parotid gland (salivary). Causal or casual for non-Hodgkin’s lymphoma?

• HHV-6 – Human Herpes Virus Type 6 – infant chest rash – “6th Disease”, dormant in parotid gland, associated with MS, and as an opportunist with AIDS.

• HHV – 8 - probably more rare, opportunist with AIDS in Africa. Kaposi’s sarcoma (connective skin cancer)

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Herpes Simplex I & IIHSV I “cold sores”

Genital Herpes ImagesLinkHSV II “genital herpes”

E. Oncogenesis (the 5th way viruses affect host cells in vivo)

There are seven examples of oncogenic viruses in humans (Epstein Barr Virus (EBV), Hepatitis B Virus (HBV), Human Papaloma Virus (HPV), Human T-cell Lymphotrophic Virus-1 (HTLV-1), HTLV-2, HTLV-4 & Human Herpes Virus-8 (HHV-8). We will detail the first 3 of these.

Normal Cells vs Tumor (Transformed) Cells

• Normal cells– Grow in monolayer– Attach to one

another and glass.– Limited

generations– Do not form

tumors when injected into hosts

• Tumor cells– Growth unorganized– Attach less firmly to

surfaces– Multiply indefinitely– Form tumors when

injected into hosts

1. Epstein-Barr Virus (EBV)pp 706-707

a. African Jaw Tumor discovered by Denis Burkitt in 1958 in West Africa. Also, known as Burkitt’s lymphoma. A cancer of the B – lymphocytes.

b. Tony Epstein and Yvonne Barr isolated EBV from cell free tumor filtrates in 1964.

c. The course of EBV pathogenesis depends on the patients age at time of first exposure; environmental conditions (geography); and genetic predisposition.

Warning! Graphic Picture next slide

Effects of African Jaw Tumor(Burkett’s Lymphoma)

d. mode of transmission is by direct contact with saliva by kissing or sharing a drinking vessel.

e. Pathology depends on geographic location. In West-Central Africans and East Africans, 90% of the population is EBV + by age 5. 20% secrete EBV in saliva. There are no symptoms. These children are immunized and protected for life by natural antibodies. However, in those children who are exposed to malaria and survive prior to age 5, many develop Burkitt’s lymphoma at the ~ age of 7. Plasmodium vivax, a protozoan parasite is therefore a cofactor in causing B cell cancer!

f. In coastal areas of SE Asia and North Africa, there is no Plasmodium vivax. In this case only older men have high antibody titers to EBV.

These men have long term exposure to high quantities of nitrates used to cure fish, and a tendency to develop nasopharyngeal carcinoma (and sometimes abdominal masses). In SE Asia, this cancer kills ~ 100,000 elderly males per year. Nitrates are therefore an environmental cofactor in causing cancer!

g. In the Western hemisphere, Europe and Australia, people are exposed to EBV between the ages of 15-25. 15% get infectious mononucleosis. Brief initial symptoms are flu-like (lymphadenopathy (swollen lymph glands) and headaches) within 3-5 days. Clinically there is B-cell leukopenia (low white blood cell count). The infection is usually self-limited and symptoms disappear in one week. However, EBV is latent in the parotid (salivary) glands.

h. If EBV is not self limited, it means the person does NOT produce enough interferon (see pg 460). A more SEVERE infection surfaces 4-7 weeks later. Symptoms include extreme fatigue and splenomegaly (spleen enlargement). Clinical symptoms include B cell leukocytosis (increased count). The B cells appear abnormally large and secrete incomplete antibodies. Exercise may result in spleen rupture. Eventually abnormal cells and symptoms disappear. EBV is an infection of the cardiovascular system and/or lymphatics in Europe, Australia, and the west hemisphere.

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2. HPV: Human Papilloma Virus p.559-560a. DNA virus. Family Papovavirus (40 strains

infect humans). An STD! b. Pathology: “genital warts” / genital

condylomata strains 6 & 11. Warts are soft pink cauliflower like growths (benign tumors). These occur on the external genitalia, vagina, on the cervix or rectum. Most cases have no symptoms, virus is “self limited.” 13 strains associated with cervical cancer. HPV + does not always mean cervical, vulvular or penile cancer. Prostate cancer? Note: smoking is the second most common risk factor for abnormal pap smears.

Genital Warts (male)

Genital Warts (female)

c. HSV-II is a an environmental cofactor with HPV in predisposing women to cervical cancer.

d. Epidemiology; 20 million infected in the U.S., ~ 5.5 million HPV + cases each year. 14,000 new cases of cervical cancer each year, and HPV is present 99.7% of the time. Most common STD in the U.S. Most common risk for abnormal pap smear. Transmitted by sexual intercourse, via abrasions, and perinatally. Rarely by shared swim suits, towels, or gymnastic equipment.

e. Treatment: remove warts with imiquimod cream which stimulates the immune system, podophyllin or trichloroacetic acid, laser or cryotherapy.

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3. HBV (Serum Hepatitis B Virus)a. DNA enveloped virus, 42 nm.

b. Persistent Chronic Infection – Pathology

c. a. Incubation period 4-26 wks.

d. b. 50% no symptoms infection self limited (eliminated)

e. c. 50% get fever, loss of appetite, joint pains, clay stools, dark urine, and jaundice from a viral infection of the liver (hepatitis)

f. d. 90% of those who get hepatitis symptoms recover without reoccurrence.

g. e. 5-10 % become chronic active carriers.

Jaundice

HBV - Cirrhosis of the Liver

Cirrhotic LiverNormal Liver

HBV Course of Infection

h. Epidemiology: a) ~ 4,000 deaths and , 150,000 –

320,000 new cases annually, 1.2 million chronic cases in U.S.; 200-300 million cases globally.

b) In SE Asia & tropical Africa, liver cancer rate is higher due to long term exposure to spoiled grain products. Aspergillus flavus, a grain mold, produces a toxic byproduct during fermentation called aflatoxin. Aflatoxin is an environmental cofactor for liver cancer.

i. Modes of Transmissiona) IV (shared needles) drug useb) semen (55%) use condoms!

c) perinatal (in birth canal)

d) blood transfusion is rare

e) special risk of contamination from dried biological fluids ex. feces, blood & urine. Greatest risk to ER nurses, pathology staff, in dialysis wards and for morticians.

j. Prophylaxis: Recombinant yeast vaccine. A genetically engineered yeast cell with antigen from HBV envelope on the surface. Note, 29% not immune after the vaccine.

k. Treatment: alpha interferon & chemotherapy.

Hepatitis C Virus (HCV)

A. Characteristics: Family Flavivirus, 40-70 nm, + SS-RNA, enveloped, icosahedral.

B. Epidemiology: est. 4 million infected in U.S., expecting 12 million by 2010. ~10,000 deaths/yr currently. Leading cause of liver transplants. 170 million cases globally.

C. Pathology – persistent chronic infection

1. 60-70% have no symptoms at first. 30-40% experience jaundice and abdominal pain in the acute phase.

2. 85% of infected that experience acute phase develop chronic hep. Some asymptomatics take 20 years to

develop symptoms of liver damage. Result is many carriers. Overall 50% progress to chronic hep!

3. MOT and Treatment same as HBV.

Orthomyxovirus – influenza virus p.631

A. 8 segments of -SS-RNA, enveloped & helical, 100 nm. Annual mutations in the H or N spike genes results in antigenic drift. The immune system therefore fails to recognize last year’s strain of the virus and a new case of influenza results annually. Major changes in spike genes result in antigen shift (6 times in the 20th century), and are due to recombination of viral segments with swine or chicken influenza viruses. Ex. The 1918 flu epidemic.

B. MOT: contact transmission (1 meter zone)C. Incubation: 1-2 days.D. Symptoms: chills, fever, headache, cough, muscle

aches, NOTE: diarrhea is the result of a disruption of normal microbiota. Self limited gone in 1 wk.

1918 Flu Pandemic

1918 Flu Pandemic

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Types (A, B &C)

Mediates attachmentTo the cell receptors.

Diagram of Flu Virus

Flu Virus Classification• Virus Classification - On the basis of their

nucleocapsid and M protein antigens, the influenza viruses are divided into 3 distinct immunological types. (A, B, and C) Influenza A viruses also occur in pigs, birds, and horses. However, only man is infected by influenza B and C. The antigenic differences of the haemagglutinin and the neuraminidase antigens of influenza A viruses provide the basis of their classification into subtypes. eg. A/Hong Kong/1/68 (H3N2) signifies an influenza A virus isolated from a patient in 1968, and of subtype H3N2.

E. Pathogenesis: Invades oropharyngeal cells, disrupts mucociliary escalator, depresses immune system. Secondary bacterial respiratory infections result in serious complications. Ex. HIB (Haemophilus Influenza Type B)

F. Epidemiology: 20% of U.S. infected each year. ~30,000 die, ~ 100,000 hospitalized.

1918 Pandemic: 20-100 million dead

President Woodrow Wilson – Did the flu alter political history at the Treaty of Versailles?

Treatment of Viral Infections 1. Treat symptoms and rely on natural immune system to

do its job.2. Stimulate host immune response. (Flu-mist)3. Bar penetration of virus into host cell.4. Stop budding process and prevent viral particles from

exiting the cell and infecting other cells. (Tamiflu blocks neuraminidase)

5. Block the transcription and translation of viral RNA or DNA. (analog nucleotides) (ex Cyclovir and AZT)

6. Prevent the maturation of viral particles.

Viral infections are difficult to treat because they utilize the host genetic machinery and have no real metabolism to block.

Five Ways of Detecting Viral Disease

• Examination of symptoms

• Examine cells for morphological changes (cytopathic effects)

• Screening for antibodies

• Genetic analysis - Polymerase chain reaction (PCR)

• Introduce into cell cultures (look for plaque) or animals and look for symptoms.

Prions

• Proteinaceous infectious particles thought to be self-replicating. Problem: Only Nucleic acids do that! Hmmmm……

• Found in sheep (scrapie)• Found in cattle - Mad Cow Disease, Bovine

Spongiform Encephalopathy• Found in people - Kuru / Creutzfeldt-Jakob

Disease

Emerging Viral Infections

• HIV• Hepatitis C• Hantaviruses• Bovine Spongiform Encephalopathy / Creutzfeldt-

Jakob Disease• West Nile Virus• Ebola• Herpes viruses 6, 7, 8