Download Lecture 3 Virology - GG Andersen Consulting

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 Viruses
a.
b.
c.
d.
e.
f.
g.
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
h.
i.
j.
k.
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 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
•
(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)
L. 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
•
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
F 08
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
•
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)
Viral darrhea, Norwalk virus syndrome
Polio
•
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 RNAdependent 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.
A.
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.
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 45 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 herpes
simplex 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.
GA sp07
Henrietta Lacks
http://www.news.harvard.edu/gazette/2001/07.19/04-filmmaker.html
IV. VIRUSES IN VITRO
i.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
GA F07
V. RELATIONSHIP BETWEEN
VIRUSES AND HOST CELLS IN
VIVO
A. 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/+SSRNA
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 continued
Mode 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 - HIV
Absorption
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 Herpesviruses
1.
2.
3.
4.
5.
6.
7.
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 nonHodgkin’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)
•
•
Herpes Simplex I & II
HSV I “cold sores”
HSV II “genital herpes”
Genital Herpes ImagesLink
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), HTLV2, 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.
GA F06
2. HPV: Human Papilloma Virus p.559560
a. 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.
sp07
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
Normal Liver
Cirrhotic Liver
HBV Course of Infection
h. Epidemiology:
~ 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.
a)
i. Modes of Transmission
a)
b)
c)
d)
e)
IV (shared needles) drug use
semen (55%) use condoms!
perinatal (in birth canal)
blood transfusion is rare
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, 4070 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
A.
B.
C.
D.
p.631
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.
MOT: contact transmission (1 meter zone)
Incubation: 1-2 days.
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
Diagram of Flu Virus
•
Mediates attachment
To the cell receptors.
Types (A, B &C)
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.
2.
3.
4.
5.
6.
Treat symptoms and rely on natural immune system to
do its job.
Stimulate host immune response. (Flu-mist)
Bar penetration of virus into host cell.
Stop budding process and prevent viral particles from
exiting the cell and infecting other cells. (Tamiflu blocks
neuraminidase)
Block the transcription and translation of viral RNA or
DNA. (analog nucleotides) (ex Cyclovir and AZT)
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 / CreutzfeldtJakob Disease
• West Nile Virus
• Ebola
• Herpes viruses 6, 7, 8