1. ENVR 421 Viruses: Overview
Mark Sobsey

2. Viruses Not cellular organisms Small: simple: shape:
µM diameter
simple:
nucleic acid
protein coat
(lipoprotein envelope)
shape:
spherical (icosahedral)
rod-shaped (helical)
complex

3. 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

4. Virus Composition protein coat or capsid: envelope:
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”)

5. 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

6. Taxonomy
Classify into groups based upon common physical/chemical properties
Viruses in same group often have similar biological properties
Replication
Disease
Spread
An attempt to bring order to the large number of viruses discovered in the last eighty years. It has a more practical purposes as well. All members of a group often have similar biological properties. Knowing something about the biological properties of a group sometimes tells you something about how a newly discovered member might behave. For instance, all herpesviruses have the same structure and look the same in electron micrographs. They also all become latent in their hosts and are reactivated periodically. A new member (the Brazilian Mugwump virus) would therefore also be expected to cause recurrent reactivations. Most members of the genus Flavivirus (West Nile Virus) are spread by insects. If a new virus, that has physical and chemical properties of flaviviruses, is discovered the chances are that it is spread by insects as well.

7. Classification based upon:
Genome (RNA, DNA, SS, DS etc)
Morphology of virion, envelope
Replication strategy
Serological relationships (Serotypes)
International Committee on
Taxonomy of Viruses

8. 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

9. 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

11. Virus – “Life Cycle” Attachment / Adsorption Penetration Uncoating
translocation
endocytosis
fusion
Uncoating
Biosynthesis: Replication and Transcription
segmented: monocystronic mRNA
non-segmented: polycistronic mRNA
Synthesis and assembly
Release
Maturation
virus becomes infectious
may be linked to release or may occur after the virus has been released

12. Replication of a Nonenveloped RNA Virus

13. Replication of an Enveloped Virus

14. 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)

15. 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.

16. Enteroviruses distinct animal 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

17. 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 non-human primates
Cell culture reported in 1979 in fetal rhesus monkey kidney cells

18. 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.

19. Hepatitis A Virus cause of infectious or epidemic hepatitis
replicates in liver; viral shedding: 4 weeks

20. Geographic Distribution of HAV Infection
Anti-HAV Prevalence
High
Intermediate
Low
Very Low

21. Hepatitis E Virus 32-34 nM in diameter ss(+) RNA genome
May belong to the caliciviridae family
Incubation period:
Average 40 days
Range days
Case-fatality rate:
Overall, 1%-3%
Pregnant women, 17% - 33%
Illness severity: increased with age

22. Geographic Distribution of Hepatitis E

23. 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

24. Rotaviruses

25. 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

26. 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?

27. Response of Human Volunteers to Norwalk Virus Infection via the Oral Route

28. Important Human Respiratory Viruses
Orthomyxoviruses (influenza): types A and B
Paramyxoviruses: Respiratory
Measles
Mumps
Coronaviruses
Common cold viruses
SARS
Herpesviruses
Rhinoviruses

29. Influenza Viruses Pleomorphic, spherical filamentous forms occur
nm diameter, or 20 nm diameter and 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

30. Paramyxoviruses Roughly Spherical, Pleomorphic -ssRNA genome, 17-20 kb
~200 nm diameter
-ssRNA genome, kb
Enveloped, helical nucleocapsid
Envelope is lipid bilayer with glycoprotein spikes
Includes Measles, Mumps, and RSV

31. Rhinoviruses Spherical +ssRNA genome Nonenveloped, icosahedral capsid
27-30 nm diamter
+ssRNA genome
~7.2kb
Nonenveloped, icosahedral capsid
50% of Common Cold
105 serotypes
                            

32. Coronaviruses Irregularly shaped +ssRNA genome (27-31 kb)
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

33. 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)

34. 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

35. SARS 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)
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

36. 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)

37. 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

38. Virus Detection in Cell Culture by Cytopathogenic Effects
Infected Cell Culture with CPE
Uninfected Cell Culture
From a public health and risk assessment standpoint,
microbial assays based on infectivity are the most
relevant and easily interpretable ones

39. 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 virions
many “wild-type or field viruses

40. 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

41. 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 virus particles.

42. 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+

43. 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

44. Factors Influencing Virus Infection and Illness
The probability of infection varies with:
Virus factors
Host factors
Route and site of infection and vehicle
Virus and host availability and encounters
Ingestion, inhalation, eye, skin, etc.
Water, food, droplets, aerosols
Probablility of illness from infection: 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)
Immune status
Genetics
Nutrition
behavior (personal habits) of host.

45. 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.

46. 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

47. 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
Influena vaccine for 2008 provides protection against only some circulating influenza viruses that emerged in the population; chose vaccine strains different from prevalent ones