Download Viral Pathogens

ENVR 195
Viruses:
Introduction and Overview
Chip Simmons
And
Mark Sobsey
Viruses
• Not cellular organisms
• Small:
– 0.02-0.3 µM diameter
• simple:
– nucleic acid
– protein coat
– (lipoprotein envelope)
• shape:
– spherical (icosahedral)
– rod-shaped (helical)
– complex
Virus Composition
• nucleic acid:
– RNA or DNA
– double- or single-stranded
– one piece or multiple,
genetically distinct pieces
• represent separate
genes
• some have multiple
copies of same gene
– linear, circular or
circular+supercoiled
Virus Composition
• protein coat or capsid:
– contains one or more distinct proteins; multiple
copies of each
– proteins arranged in a stable array to form capsid
– some proteins on virus surface are glycosylated
• envelope:
– usually derived from cell membrane
• lipid bilayer with inserted virus-specific proteins
(peplomers)
• acquired from cell upon exiting (“budding”)
Virus Replication and Infectivity
• no biological activity outside of host cells/or host
organisms
• obligate intracellular parasites; active only in host cell
• recruit host cells biosynthetic machinery and building
blocks to make new viruses
• typically produce 1000s to 100,000s per infected cell
• often destroy (lyse) the host cell as a result of infection
– some viruses: host cell survives to shed viruses over time
• productive infection
– other viruses: host cell survives and is transformed by
presence of virus genes
• tumor viruses
Taxonomy
• Classify into groups based upon common
physical/chemical properties
• Viruses in same group often have similar
biological properties
– Replication
– Disease
– Spread
Classification based upon:
•
•
•
•
Genome (RNA, DNA, SS, DS etc)
Morphology of virion, envelope
Replication strategy
Serological relationships (Serotypes)
International Committee on
Taxonomy of Viruses
Important Definitions For
Virus Replication
• Virion – a virus particle; the virus nucleic acid
surrounded by a protein coat and sometimes, a
lipoprotein envelope
• Messenger RNA (mRNA) – an RNA molecule
transcribed from DNA that contains the genetic
material necessary to encode a particular protein
• Plus-strand (+) nucleic acid – an RNA or DNA
strand that has the same sense as the mRNA of a
virus (can act as mRNA > make viral proteins)
• Minus- strand (-) nucleic acid – an RNA or DNA
strand that has the opposite sense
(complementary) of the mRNA of a virus
The Central Dogma of Molecular Biology
Transcription
Replication
DNA
RNA (mRNA)
Translation
Protein
Transcription is carried out by RNA polymerase
Translation is carried out by ribosomes in the cell
Replication is carried out by DNA polymerase
Reverse transcriptase copies RNA to DNA
Virus Replication Strategies Based on Nucleic Acid:
The Baltimore Classification
Virus – “Life Cycle”
1. Attachment / Adsorption
2. Penetration
1. translocation
2. endocytosis
3. fusion
3. Uncoating
4. Biosynthesis: Replication and Transcription
•
•
segmented: monocystronic mRNA
non-segmented: polycistronic mRNA
5. Synthesis and assembly
6. Release
7. Maturation
•
•
virus becomes infectious
may be linked to release or may occur after the virus has
been released
Non-segmented Viruses:
Polycystronic mRNA
Replication of a Nonenveloped RNA Virus
Replication of an enveloped Virus
Infection of the cell
Release by lysis of cell
(cytopathic)
OR by budding (without
death of cell, non-cytopathic)
Viruses and the Environment
• non-enveloped viruses are most persistent in the
environment than enveloped viruses
– protein coat confers stability and resistance to stressors
• enteric viruses are important for environmental health
– transmitted by direct and indirect contact, fecally
contaminated water, food, fomites and air.
– Most enteric viruses are nonenveloped
• respiratory viruses also important
– transmitted by direct and indirect contact, air and fomites
(some by water and food, too)
– some respiratory viruses are nonenveloped (rhinoviruses and
adenoviruses)
– others are enveloped (influenza viruses and coronaviruses)
Important Human Enteric Viruses (150+)
Viruses/Groups
Animals Reservoirs?
Enteroviruses:
no
(polios, echos*, coxsackies*, etc.)
Hepatitis A virus
no
Hepatitis E virus
pigs?; rats?
Reoviruses
yes
Rotaviruses
yes**
Adenoviruses*
yes**
Caliciviruses*:
Norwalk, Snow Mountain, etc.
Cattle?
Swine?
Astroviruses
uncertain
*On EPA’s candidate contaminants list (CCL)
**Humans & animals infected by different ones; but not always.
Enteroviruses
• Icosahedral shape
• ~27-30 nm diameter
• single-stranded +sense RNA
– about 7,500 nucleotides
• icosahedral protein coat (capsid)
– 4 capsid proteins: VP1, VP2, VP3,
VP4 (all cleaved from VP0)
• >71 antigenically distinct human types
–
–
–
–
polioviruses (3 types)
coxsackie B viruses (6 types)
coxsackie A viruses (23 types)
echoviruses (31 types)
• distinct animal enteroviruses
Hepatitis A Virus (HAV)
•
•
•
•
•
icosahedral
27 nM in diameter
non-enveloped capsid
ss(+) RNA genome
At least 3 major structural
polypeptides
• Single serotype
• Infects humans and nonhuman primates
• Cell culture reported in
1979 in fetal rhesus monkey
kidney cells
Hepatitis A Virus (HAV)
• Fecal-oral route of exposure
• Incubation period: 2-6 weeks
• Serious debilitating disease (general infection): fever,
abdominal pain, headache, jaundice, nausea, diarrhea
• Fecally excreted at concentrations up to 106 infectious
units per gram (>109 virions per gram)
• Infectious at relatively low doses
• Persistent in feces, sewage, soil and water and on foods
and environmental surfaces for weeks to months,
depending on temperature and other environmental
factors.
• Heat resistant: requires >60oC for rapid inactivation.
Hepatitis A Virus
cause of infectious or epidemic hepatitis
replicates in liver; viral shedding: 4 weeks
Geographic Distribution of HAV
Infection
Anti-HAV Prevalence
High
Intermediate
Low
Very Low
Hepatitis E Virus
• 32-34 nM in diameter
• ss(+) RNA genome
• May belong to the
caliciviridae family
• Incubation period:
– Average 40 days
– Range 15-60 days
• Case-fatality rate:
– Overall, 1%-3%
– Pregnant women, 17% - 33%
• Illness severity: increased
with age
Geographic Distribution of Hepatitis E
Reovirus and Rotaviruses
•
•
•
•
~spherical; icosahedral
~75-80 nm diameter
double-layered capsid
nucleic acid:
– double-stranded RNA
– 11 segments (rota)
– 10 segments (reo)
– electropherotypes
• 7 Groups (A-G) by VP6
– Subgroups, serotypes
• Group A most important in
humans (children)
• Group C causes sporadic illness
• Group B has caused large
outbreaks (adults), rare
Rotaviruses
ADENOVIRUSES:
•
•
•
•
icosahedral
~80 nm diameter
double-stranded, linear DNA
protein coat; at least 10 proteins
– Hexons, pentons, minor
polypeptides
– attachment fibers with knobs
• At leat 41 human adenoviruses
– types 1-39 mostly respiratory
• but fecally shed
– types 40 and 41 are enteric
• Often the most prevalent viruses
in treated sewage
– resistance to treatment?
• Highly resistant to UV radiation
• Distinct animal adenoviruses
Noroviruses and Other Caliciviruses
•Icosahedral
• “structured”; cup-like surface morphology
• 30-35 nm diameter
• ss(+) RNA, ~7.7 KB
•1 major capsid polypeptide, ~60 kD
• minor protein, ~30 kD
• 4 major HuNOV groups
•(G1 and G2 most prevalent
• Sappoviruses; genetically distinct human enteric caliciviruses
• NoVs and other caliciviruses are genetically diverse/variable
• No culture of human NoVs, (except in humans and maybe chimps)
• Distinct animal caliciviruses & noroviruses
•some genetically similar to human NoVs
•cross-species transmission?
Response of Human Volunteers to Norwalk Virus
Infection via the Oral Route
Important Human Respiratory Viruses
• Orthomyxoviruses (influenza): types A and B
• Paramyxoviruses: Respiratory
– Measles
– Mumps
• Coronaviruses
– Common cold viruses
– SARS
• Herpesviruses
• Rhinoviruses
Influenza Viruses
• Pleomorphic, spherical
filamentous forms occur
– 50-120 nm diameter, or 20 nm
diameter and 200-300 nm long
• Segmented, linear -ssRNA genome
– 7 to 8 segments
• Enveloped, filamentous
nucleocapsids
– Envelope is lipid bilayer with
~500 spikes (Hs & Ns)
• Hemagglutinin and neuraminidase
Causes influenza – “the flu”
Animal reservoirs of influenza
viruses that “jump” to humans or
co-nfect animals, usually pigs,
along with human strains to
create new strains that are highly
transmissable and virulent
Paramyxoviruses
• Roughly Spherical,
Pleomorphic
– ~200 nm diameter
• -ssRNA genome, 17-20
kb
• Enveloped, helical
nucleocapsid
– Envelope is lipid bilayer
with glycoprotein spikes
• Includes Measles,
Mumps, and RSV
Rhinoviruses
• Spherical
– 27-30 nm diamter
• +ssRNA genome
– ~7.2kb
• Nonenveloped,
icosahedral capsid
• 50% of Common
Cold
• 105 serotypes
Coronaviruses
• Irregularly shaped
– 60-220 nm diameter
• +ssRNA genome (27-31 kb)
• Enveloped particles with loosely
wound nucleocapsid
– characteristic “club-shaped”
peplomers
• 10 % of Common cold
• Severe Acute Respiratory
Syndrome - SARS
SARS - Coronaviruses
• Discovered in March, 2003
• pleomorphic, enveloped particles
– 60 and 130 nm
• Short incubation (2 – 7 days)
• Complete sequence known
– five major ORFs
• Very distinct group; not related to
other HuCo-Vs
• Concensus genotype, but strain
variability (epid studies)
– High rate of RNA-RNA
recombination
• More environmentally stable than
other HuCo-Vs
• Zoonotic pathogen? (civets)
SARS HuCo-V Phylogenetic Tree
SARS: Clinical Detection
• Up to 109 particles per mL in
sputum
• Detected in nasopharyngeal
aspirates by RT-PCR in 32% at
initial presentation (mean 3.2
days after onset of illness) and
in 68% at day 14
• Detected in 97% of patient’s
stools and 42% of urine
samples two weeks after the
onset of illness
SARS
Symptoms:
• high fever (>100.4°F; >38.0°C).
Other symptoms: headache,
malaise, and body aches.
• Some people also have mild
respiratory symptoms at outset.
• 10 percent to 20 percent of
patients have diarrhea.
• After 2 to 7 days, SARS patients
may develop a dry cough.
• Most patients develop
pneumonia.
Source: Initially certain mammals in
SE Asia (esp. China): palm civet
cat; recent evidence in bats
Spread
close person-to-person contact.
• respiratory droplets (droplet
spread) from coughs or sneezes.
– propelled a short distance (generally
up to 3 feet) through the air and
deposited on the mucous membranes
of the mouth, nose, or eyes of
persons who are nearby.
• Also spread by fomites (person
touches a surface or object
contaminated with infectious
droplets and then touches his or
her mouth, nose, or eye(s).
• might spread more broadly
through the air (airborne spread)
Assay Methods for Viruses
• Electron Microscopy (EM) and Immune EM
– Insensitive (>1,000,000 particles/ml)
– OK for clinical but not environmental virology
• Animal Infectivity
– Slow, cumbersome, expensive, ethical considerations
• Culture or infectivity
– Now widely used in environmental virology
– Cytopathogenic effects
– Growth, but no cytopathogenic effects
• detect viral antigens or nucleic acids
• Immunoassays
– insensitive for direct detection
– Amplify viruses in cell cultures
• Nucleic acid assays
– insensitive for direct detection by hybridization
– Amplify in cell culture or in vitro (PCR or RT-PCR)
Virus Infectivity and Infectivity Assays
• Viruses differ in their their human infectivity
• Enteric viruses and some respiratory viruses are
generally infectious at low doses
– As little as one cell culture or animal infectious dose has a high
probability of infecting an exposed human
• Many enteric viruses in environmental samples do not
cause cytopathogenic effects (CPE)
– will not be detected by microscopic examination
– require additional methods to detect their presence
• immunochemical methods
– detect antigens in infected cells
• nucleic acid methods
– nucleic acid hybridization
– nucleic acid amplification
Virus Detection in Cell Culture by Cytopathogenic Effects
Uninfected Cell Culture
Infected Cell Culture with CPE
From a public health and risk assessment standpoint,
microbial assays based on infectivity are the most
relevant and easily interpretable ones
Quantifying Human Virus Infectivity is a Challenge
• Some infect only humans
• Some infect certain experimental animals, too
• Some infect experimental animals and cell cultures
• Different ratios of infectivity to virions (particles)
– 1 infectious unit ~ 1 virus particles
• some bacteriophages
– 1 infectious unit ~100 virions:
• some cell culture adapted viruses
– 1 infectious unit ~10,000-100,000 virions
• many “wild-type or field viruses
Progress in Virus Detection in Cell Culture
• Some viruses (some enteroviruses, adenoviruses,
rotaviruses, astroviruses and hepatitis A virus) grow poorly
or slowly in cell cultures and produce little or no CPE.
• Greater detection with additional analytical techniques:
– Viral antigens
• Immunofluorescence assays, enzyme immunoassays,
radioimmunoassays, etc.
– Viral nucleic acid assays: hybridization and/or amplification
• Combined cell culture + RT-PCR demonstrates
presence of greater numbers of infectious viruses
than CPE alone
– Post-disinfection, more viruses are detected than by CPE
Challenges in Assaying Viral Infectivity
• Host susceptibility and variability in host
susceptibility
– Type of host cells and cell culture assay methods:
• Plaque (enumerative) assays
• Quantal (liquid culture) assays
– Quantal assay endpoints:
» Cytopathogenic effects: visible changes in cells
» Immunodetection or nucleic acid detection
– Animal bioassays: used if no cell culture assay available
• Virus titer often increases with serial passages in hosts
Estimating Viral Dose:
Relationship of Infectivity to Virus Particle Count
• As little a one or a few intact, functional virus particles
are capable of causing infection in a susceptible host.
• Ratios of virus particles to infectious units are highly
variable and are subject to change:
– Passage of viruses in susceptible host cells reduces the
ratio of virus particles to infectious units
• rotavirus:
initial ratio: ~50,000 virus particles/infect. unit
– after cell culture passage: ~100 particles/infect. Unit
• Norwalk Virus appears to be infectious at doses
corresponding to as little as 10-100 virus particles.
–
Emerging Microbial Indicators of Fecal
Contamination for Enteric Viruses
• Somatic and F+ (male-specific)
coliphages may be useful indicators of
enteric viruses in water, wastewater and
other fecally contaminated samples.
– Plentiful in raw sewage
– Reduced less effectively than are
conventional indicator bacteria by
sewage treatment processes.
– Superficially resemble enteric viruses
(F+ coliphages)
– Easy, rapid and economical to detect
and quantify by reliable methods
somatic
F+
Localized Infections
1.
Organism enters the body and reaches target site of
infection
2.
Organism adheres to or enters host cells and multiplies at
site of infection
3.
Infection spreads within the site (e.g., respiratory tract;
intestines)
4.
Symptoms of illness appear
5.
Organism does not spread through the lymphatic system or
reach the bloodstream
6.
Infection subsides due to host defenses (e.g., immunity)
7.
Agent eliminated from the body; infected cells replaced;
"cured"
Generalized/Systemic/Disseminated Infections
1.
2.
3.
4.
5.
6.
7.
Organism enters the body and reaches target site of initial
infection
Organism adheres to or enters host cells and multiplies at
initial site of infection
Infection spreads within site and to other sites via tissues,
lymphatic system, bloodstream (bacteremia, viremia, etc.)
and possibly other routes
Symptoms of illness may appear
Organisms infect other organs, tissues and cells; more
spread via bloodstream
Symptoms of illness become severe
Host defenses eliminate organisms leading to cure or
disease continues, possibly leading to irreversible damage or
death
Virus Infections:
Some Important Viruses Cause Localized
Infections and Others Systemic Infections
Enteric Viruses:
• Localized:
–
–
–
–
caliciviruses
astroviruses
adenoviruses
rotaviruses
• Generalized:
– enteroviruses
– hepatitis A and E
viruses
Respiratory Viruses:
• Localized:
–
–
–
–
rhinoviruses
coronaviruses
Orthomyxoviruses(Flu)
paramyxoviruses
• Generalized:
– herpesviruses
– measles
– mumps
Factors Influencing Virus Infection and Illness
• The probability of infection varies with:
– Virus factors
– Host factors
– Route and site of infection and vehicle
• Ingestion, inhalation, eye, skin, etc.
• Water, food, droplets, aerosols
• The probablility of illness from infection is high (>50%) for
many enteric viruses
– Varies with age of host and with type of virus:
• Some: high rates of illness in infants and children
• Others: high rates of illness in adults
– Varies with health status: “sensitive populations”
• Elderly: high risk of illness with adverse outcomes
• Immune compromised: high risk of chronic, lethal illness
• Pregnancy: high risk of illness and death (ex: HEV)
Role of Immunity in Virus Infections:
Generalized/Systemic/Disseminated Infections
• Immunity against generalized/systemic/disseminated
infection is usually lifelong, unless immune system is
severely compromised
• Localized (e.g., gastrointestinal) re-infection is possible
• Hepatitis A and E and many enteroviruses cause
systemic/generalized/disseminated infections
– Typically, immunity against severe illness is long-term and
probably lifelong.
• Proof of concept: live, oral poliovirus vaccine and poliomyelitis
eradication; susceptibles are newborns and infants
• Antigenic changes in viruses may overcome long-term
immunity and increase risks of re-infection or illness.
Role of Immunity in Virus Infections:
Localized Infections:
• Immunity to infection is usually short-term and transient
– Gut (secretory or IgA) immunity wanes over time
• Proof of concept: live, oral rotavirus vaccine:
– immunity declines over time and reinfection with “wild”
type rotaviruses occurs
• Repeated localized (e.g., gastrointestinal) re-infection is
possible
• Rotaviruses, caliciviruses, adenoviruses and some
enteroviruses, cause localized infections
Role of Selection of New Viral Strains in
Susceptibility to Infection and Illness
• Antigenic changes in viruses overcome immunity,
increasing risks of re-infection or illness
– Antigenically different strains of viruses appear and are selected
for over time and space
– Constant selection of new strains (by antigenic shift and drift)
– Partly driven by “herd” immunity and genetic recombination,
reassortment and point mutations
• Antigenic Shift:
– Major change in virus genetic composition by gene substitution
or replacement (e.g., reassortment)
• Antigenic Drift:
– Minor changes in virus genetic composition, often by mutation
involving specific codons in existing genes (point mutations)
• A single point mutation can greatly alter virus virulence
Factors Influencing Exposure and Infection:
Host Factors and Host Susceptibility
• Opportunities for host exposure
– transmission routes
– host availability
• Susceptibility factors
– Dosage (quantity) and "quality" of infectious
organisms, including their "virulence";
– age
– immunity
– nutritional status
– immunocompetence and health status,
– genetics
– behavior (personal habits) of host.