1. Microbial diversity Today: - History of virology
- Methods of classifying viruses
- Examples of interesting viruses
- Special issues faced by viruses attacking plants
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Be sure you can compare and contrast:
- Viruses - Classification systems
- Virophage (functional, Baltimore,
- Viroids and phylogenetic)
- Virusoids - Viral relationships to mRNA
- Prions - Lytic and lysogenic paths

2. Early history of virology
No single scientist is credited with the discovery of viruses.
As germ theory took hold and evolved (1860’s and after)…
- Scientists sought to isolate causal agents of diseases
- Frequently recovered bacteria, fungi, protozoans…
- But for some diseases, no agent was found
- Many of these were later found to be viral diseases
One of the first to describe something smaller than bacteria as pathogens:
Dmitri Ivanowsky

3. Ivanowsky and the early history of virology
Ivanowsky worked on tobacco mosaic disease
In 1892…
Extracted clear juice from infected leaves
Filtered juice to remove bacteria
Placed juice on healthy plants
Observed development of symptoms
Did not know what caused disease
Subsequent work showed same phenomenon:
-1898: Beijerinck coined the term ‘virus’
1898: Frosch (foot and mouth disease in cattle)
1901: Reed (yellow fever in humans)
1930’s: viruses thought to be microbes (living)
- too tiny to see with existing microscopes

4. Crystallization of tobacco mosaic virus
Wendell Stanley (USA) in 1935…
- isolated the tobacco mosaic virus
- crystallized it (= purified it)
-method would kill cellular life
- showed that it was still active after crystallization
- later won Nobel Prize
- kicked off debate: are viruses living things?
Led to various ways of classifying viruses…
- ‘You are what you eat’:
- bacteriophage
- plant viruses
- animal viruses
Functional

5. Modern viral classification uses the viral genome
Classification system 1: Baltimore system
David Baltimore: classification scheme based on
the relationship of the viral genome
to its mRNA
To understand this requires a reminder of the central dogma
Central dogma in molecular biology:
All genetic information flows from nucleic acid to protein
(never in the reverse direction)
This is the ‘pure definition’…there’s also an operational use

6. Background: Baltimore classification of viruses
DNA
RNA
Protein
Transcription
Translation
Replication
Flow of genetic information
For viruses to replicate,
- viral genome must be copied
viral proteins must be made
Remember that proteins are made
by translation of mRNA.
Translation of viral proteins
requires virus-specific mRNA.
*Some viruses use their own
RNA genome as mRNA.
*However, most viruses must
synthesize mRNA to replicate.

7. Reminder: transcription
DNA
RNA
Protein
Transcription
Translation
Replication
Flow of genetic information
Transcription:
Process by which DNA
is unzipped by RNA polymerase,
and a complementary strand
of RNA is synthesized.
RNA is either…
- present in viral genome, or
- synthesized from viral genome

8. The Baltimore Classification
David Baltimore: classification scheme for viruses based on
relationship of viral genome to its mRNA
Viruses comprise 7 classes:
Class I ds DNA T4; pox viruses
Class II ss DNA Chicken anemia virus
Class III ds RNA Reoviruses Class IV positive-sense ss RNA Poliovirus
Class V negative-sense ss RNA Influenza virus
Class VI ss RNA using DNA intermediate Retroviruses
Class VII ds DNA using RNA intermediate Hepatitis B virus
Positive sense: viral RNA in genome has same directionality as mRNA
Negative sense: viral RNA in genome is complementary to mRNA

9. mRNA -->RT --> DNA
The Baltimore Classification
DNA
Transcription
RNA
How do viruses in each class get/have mRNA?
Translation
Protein
Class II: ss DNA
Synthesizes 2nd strand
Class III: ds RNA
Class I: ds DNA
Transcription
Class VII:
ds DNA with RNA step
mRNA -->RT --> DNA
Transcription
Transcription of (-) strand
(+) ss RNA
Reverse transcribes to make DNA
Transcription then occurs
Viral genome used directly
Transcription of (-) strand
Class VI:
(+) ss RNA retrovirus
Class IV: (+) ssRNA
Class V: (-) ss RNA

10. The Baltimore Classification is a broad classification
• Within each of Baltimore’s classes:
- orders, families, genera, species, subtypes
• A hierarchical system resembling for other living things:
Class
Order
Family
Genus
Species
Subtypes
• This is a phylogenetic classification based on analysis
of nucleotides in the viral genome
• Remember our discussion of influenza?

11. Classification of the influenza virus - from lecture 2
Influenza A virus
Genomic classification (Baltimore):
Class V: Negative sense, single stranded RNA
Taxonomic classification:
Family: Orthomyxoviridae
Three genera:
Influenzavirus A (mammals, birds)
One species: Influenza A virus
Influenzavirus B (humans, seals)
Influenzavirus C (humans, pigs)
Influenza A virus has many subtypes:
• 8 RNA modules encoding for 11 proteins, including
hemagglutinin (HA; protein) and neuraminidase (NA; enzyme)
• HA and NA interact with host cells during infection
• Different subtypes are given numbers, e.g., H1N1, H5N1
Hemagglutinin (binding)
Neuraminidase
(budding)

12. Coronavirus (Coronaviridae)
• Many species; some cause the common cold* in humans
• One -- SARS Coronavirus -- causes Severe Acute Respiratory Syndrome
• SARS was first reported in Asia in 2003
- Rapidly spread to at least 37 countries via human vectors
- 8,098 known cases in outbreak
10% mortality rate for young adults
>50% mortality in elderly patients (>65y old)
Last human case in nature: 2003
but not ‘eradicated’
Natural reservoir unknown
In many animals (palm civets,
domestic cats, etc.)
Bats are likely reservoir
Class IV: (+) ss RNA virus
Enveloped animal virus

13. Orthopoxvirus (Poxviridae)
• One species, Vaccinia virus, causes smallpox
• May have coevolved with humans for >10,000 years*
• First likely outbreak recorded in humans: Greece, in 430 BC
- Possibly infected Ramses V (died in 1160 BC)
• Indian civilizations had early inoculation methods in 1000 BC (variolation)
- Chinese civilizations used inoculation methods in 10th and 16th centuries
- British experimentation with cowpox inoculations in 18th century
• Mortality (in old world, historical period) typically 20-60% (80% in children)
… but up to 90% of adults in New World when colonized by Europeans
• Killed up to 400,000 Europeans/year in the last years of the 18th century
…and up to million deaths in the 20th century
- the major cause of blindness in European history
• Highly stable in the environment (due to DNA genome?)
- Proximity to infected person can cause infection
- Historically used in biowarfare
• Eradicated in nature in 1979/1980; threat still exists
Class 1: ds DNA
Enveloped animal virus

14. Another role for Vaccinia virus
• Therapeutic treatment as an oncolytic virus
- Specifically targets and destroys tumor cells
• Historical observation: remission in many cancers after vaccination
or viral illness
• 20th century: purposeful viral infection to inhibit cancer
- used poliovirus, adenovirus, and others
- insufficient technology to engineer viruses
- led to unrestrained illness, morbidity…
- …or an immune response that destroyed virus
• 2005: First engineered oncolytic virus: modified adenovirus into H101
- did not specifically infect cancer cells, but preferentially killed them
- used in conjunction with chemotherapy
- injected directly into a tumor and resulting fever is suppressed
- doubled short-term response rates to treatment

15. Hantavirus (Bunyaviridae)
• One of five genera in the Bunyaviridae
- Four are transmitted by insects
- Hantavirus is transmitted by rodents (aerosolized excreta, bites)
• 1993: newly recognized species connected with HCPS
(Hantavirus cardiopulmonary syndrome)
- Common in 8 western states of the US
- Also known from Panama, Argentina, and other western nations
• Other strains cause hemorrhagic fevers in Asia
Class V: (-) ss RNA virus
Enveloped animal virus
First documented
outbreak in US:
>70% mortality

16. Hantavirus Hantavirus and hosts Similar phylogenies:
• Long co-evolutionary history
with New World mice
- not a recent introduction
to the Americas
• Hinted that many species
of mice would have their
own hantaviruses, even
if not discovered yet
• Surveys led to discovery of
25 strains…some even before
disease outbreaks occurred
• Helped researchers develop
“predictive treatments”
• Hypothetical Calomys example

17. Flavivirus (Flaviviridae)
• One species, West Nile Virus, causes disease of same name
• Major outbreak in New York City, 1999
- Thought to be the virus responsible for St. Louis encephalitis
- But birds were dying (not normal for SLE)
• Phylogenetic analysis: not closely related to SLE
- closely related to old world strains of WNV
- provided basic biology data needed
for treatment, eradication
Class IV
(+) ss RNA
Enveloped
animal
virus

18. Generalized process of viral infection in animal cell
Replication of an animal virus
- Adsorption to receptors on cell surface
- Penetration of cell membrane and uncoating
- Replication and synthesis of viral nucleic acid, protein
- Export of proteins to cell surface
- Re-enveloping in cell membrane material from host
- Release

19. T-4 like viruses: Myoviridae
• Most famous species: T4 phage
• Bacteriophage: virus attacking bacteria
- Complex virus
- Attacks E. coli
- ds DNA virus (Class I)
- Lytic, virulent phage
Replication of T4:
- Adsorption to bacterial cell wall
- Penetration and injection of genome
- Capsid remains outside! Only genomic material enters
- Replication and synthesis of viral nucleic acid, protein
- Assembly and packaging of new virions
- Cell lysis and release of virions
Movie

20. T-4 like viruses: Myoviridae
Given the ability of some bacteriophages to kill specific bacterial cells,
can bacteriophage be used to combat bacterial diseases?
Phage therapy
- Explored extensively from ’s (> 800 scientific papers)
- Not particularly successful
- Relatively little was known about viruses and bacteria
- Still debated today
Pros: Should be highly specific and inexpensive
Cons: Concerns about the ability of viruses and bacteria to evolve
- Phage-resistant bacteria may explore in numbers
- Could viruses switch hosts and wipe out beneficial bacteria?

21. Lambda-like viruses (Syphoviridae)
• Best known species: lambda phage:
- Class I, ds DNA
- Capable of both the lytic and lysogenic pathways
- lytic: cell breaks apart and dies, liberating virions (T4)
- lysogenic: cell does not die
- viral DNA integrated into host genome
- viral genome replicated with host genome
Phage lambda

22. Lysogenic pathway of Lambda phage Can be induced to then initiate
lytic pathway

23. Plant viruses - a diverse group
Plant viruses also must get their genomes across cell walls
- But cell walls of plants are very thick (>10 µm)
- Too thick for viruses to penetrate on their own
Plant viruses rely on…
- Mechanical injury that breaches cell wall (cutting)
- Injection into cells by sucking insects (e.g., aphids)
Movement via a fungal vector
Movement via pollen
Also transmitted via seeds, cuttings, and grafting

24. Plant viruses transmitted by mechanical injury
Example: pepper mild mottle virus (PMMV)
Class IV, (+) ss RNA virus
Family unknown; genus Tobamovirus
Related to and resembles tobacco mosaic virus
Infects pepper plants
- Transmitted by hand clippers
Healthy and PMMV-infected red peppers

25. Plant viruses transmitted by sucking insects
• Many plant viruses are transmitted by aphids, whiteflies, scale insects…
• Example: soybean mosaic virus (SMV)
- Class IV, (+) ss RNA virus
Potyviridae, Potyvirus
- infects soybeans
- transmitted by soybean aphids
Soybean aphid
Piercing mouthparts

26. Plant viruses vectored by fungi
Appressorium: specialized fungal structure for penetrating plant tissues
Fungal
spore
Leaf
surface
Fungal vectors transmit…
- lettuce big vein virus: Class V, (-) ss RNA virus: Varicovirus
- cucumber necrosis virus: Class V, (-) ss RNA virus: Tombusviridae
and many others

27. Plant viruses vectored by pollination
• Example: Bushy dwarf virus of raspberry
• Class IV, (+) ss RNA virus
• Family unknown; Idaeovirus
- virions travel in pollen grains
- moved about by pollinators
Pollen grains of raspberry
Honeybee on raspberry flower

28. Viral biodiversity
• The most abundant biological entity in aquatic systems
• 1 teaspoon of sea water: 1 million virus particles
• Greatest component of marine biomass
• Most are bacteriophage
- Destroy ca. 20% of oceanic biomass each day
- Limit effects of harmful algal blooms (HABs)

29. Hypotheses for the origin of viruses
Primordial hypothesis
- RNA viruses present since the beginning of life
- Common ancestor with cellular life is ancient
- Ancestral lineage diverged into (a) viruses and (b) cellular life
- Viruses co-evolved with cellular life
Bacteria
Archaea
Eucarya
RNA
viruses
Current estimated age of LUCA
Primordial hypothesis: estimated LUCA

30. Hypotheses for the origin of viruses
Escaped transcript hypothesis
- Viruses arose as genetic elements that escaped from cells
- May be the source of DNA viruses and retroviruses
- Unclear as to which domain they come from
- No real data as yet
Bacteria
Archaea
Eucarya
DNA viruses
and retroviruses ?
RNA
viruses?
No change in estimated age of LUCA

31. Hypotheses for the origin of viruses
Regressive hypothesis
- Viruses arose from bacteria that have lost functions
- Molecular data give no support for this hypothesis
Bacteria
Archaea
Eucarya
Viruses
No change in estimated age of LUCA

32. Amoebae, Mimivirus, and Sputnik virophage
Acanthamoeba polyphaga mimivirus (APMV, or ‘mimivirus’)
- Class I, ds DNA; Mimiviridae
- Inhabits amoebae
- Largest capsid diameter of all known viruses
- Visible under light microscope
- capsid: 400nm in diameter
- fibrils extend total diameter to 600nm
- naked
- Large genome: 1.2 million bases
- larger than ca. 30 tiny cellular genomes
- contains genes for sugar, lipid, AA metabolism
- unique suits of metabolic genes
- Originally thought to be a bacterium (Gram stain)
- due to unique lipids in capsid

33. Amoebae, Mimivirus, and Sputnik virophage
Mimivirus interacts closely with a subviral agent:
Virophage called Sputnik
- 50 nm in diameter
Class 1, dsDNA virus
But special:
- Can’t replicate when alone in the amoeba
Grows rapidly in amoeba infected with mimivirus
- Uses the mimivirus/host interaction as a factory for replication
Deleterious to mimivirus: abnormal capsid assembly
- Decreases mimiviral load by 70% after 24 hours
Beneficial to amoeba:
- Reduces amoeba lysis by threefold after 24 hours
- Evidence for horizontal gene transfer

34. Amoebae, Mimivirus, and Sputnik virophage
Acanthamoeba polyphaga mimivirus (APMV, or ‘mimivirus’)
- Class I, ds DNA; Mimiviridae
- Inhabits amoebae
- Largest capsid diameter of all known viruses
- Visible under light microscope
- capsid: 400nm in diameter
- fibrils extend total diameter to 600nm
- naked
- Large genome: 1.2 million bases
- larger than ca. 30 tiny cellular genomes
- contains genes for sugar, lipid, AA metabolism
- unique suits of metabolic genes
- Originally thought to be a bacterium (Gram stain)
- due to unique lipids in capsid

35. Amoebae, Mimivirus, and Sputnik virophage
Mimivirus interacts closely with a subviral agent:
Virophage called Sputnik
- 50 nm in diameter
Class 1, dsDNA virus
But special:
- Can’t replicate when alone in the amoeba
Grows rapidly in amoeba infected with mimivirus
- Uses the mimivirus/host interaction as a factory for replication
Deleterious to mimivirus: abnormal capsid assembly
- Decreases mimiviral load by 70% after 24 hours
Beneficial to amoeba:
- Reduces amoeba lysis by threefold after 24 hours
- Evidence for horizontal gene transfer

36. Viroids Viroids: small, circular, single-stranded RNA molecules
- very small genomes ( bases)
- tightly folded over so they appear double-stranded
- discovered in 1971 by Theodor Diener
Ted Diener Potatoes: healthy (L), and with PTSVd
(potato tuber spindle viroid)

37. Viroids Viroids: small, circular, single-stranded RNA molecules
- very small genomes ( bases)
- tightly folded over so they appear double-stranded
- discovered in 1971 by Theodor Diener
Viroids differ from viruses in several key ways:
- viroids are naked genetic material
- genome contains no protein-coding genes
- viroids have no protein coats
- viroid genome is tiny, compared to viruses:
- smallest virus capable of infection: 2000 bases
- viroids never have DNA genomes -- only RNA
Often called ‘subviral agents’

38. Viroids Many are pathogens of plants…
- cause about 15 known diseases; some nonpathogenic
- often transmitted by seed or pollen
Cadang-cadang disease
in coconut palms
Cachexia in citrus
Avocado sunblotch
Apple scar-
skin disease

39. Viroids Two families of viroids recognized
- within those families, about 33 ‘species’
Family 1: Pospiviroidae (poss-pee-vi-roid-ee-ee)
Pathogenic
domain
Variable
domain
Left terminal
domain
Conserved
central domain
Right terminal
domain
Virulence
Classification

40. Viroids Two families of viroids recognized
- within those families, about 33 ‘species’
Family 1: Pospiviroidae (poss-pee-vi-roid-ee-ee)
Replicate in an asymmetric fashion using host cell’s
RNA polymerase, RNase, and RNA ligase:
1. viroid RNA serves as template (+) to make (-) strand
2. host RNA pol makes new (+) strands from that product
3. host RNase cleaves (+) strands into viroid units
4. units ligated to form new virioids

41. Viroids Two families of viroids recognized
- within those families, about 33 ‘species’
Family 1: Pospiviroidae (poss-pee-vi-roid-ee-ee)
Replicate in an asymmetric fashion using host cell’s
RNA polymerase, RNase, and RNA ligase:
Replicate in host nucleus

42. Pospiviroidae replication
In the nucleus!
Rolling-circle replication
Synthesis of (+) strand
Host RNA polymerase
Cleavage into viroid units
Host RNase
Circularization
Host RNA ligase

43. Avsunviroidae Family 2: Avsunviroidae (av-sun-vi-roid-ee-ee)
- Lack the central, conserved domain (see illustration)
- Possess ribozyme activity
- Replicate in a symmetric fashion without host products
- May replicate in plant chloroplasts
1. viroid RNA serves as template (+) to make (-) strand
2. (-) strand is cleaved by ribozyme activity
3. (-) strand circularizes
4. long (+) strand is synthesized from each (-) circle
5. (+) strand is cleaved by ribozyme activity
6. units ligated to form new viroids

44. Avsunviroidae replication
In the chloroplasts!
Rolling-circle replication
(-) strand cleaved by ribozyme activity
(-)
(-) strands circularize
- - -
Cleavage into viroid units *by ribozyme activity*
long, (+) strand synthesized
from each by ‘reverse’ rolling
Circularization

45. And then… Virusoids Virusoids resemble viroids in several ways:
• Both are circular, single-stranded RNAs
• Both have only a few hundred nucleotides in their genomes
• Both include plant pathogens
…but virusoids are unique:
• Depend on plant viruses to replicate (‘helper’ viruses)
• Genome encodes only structural proteins
• Often worsen symptoms of the virus on which they depend
Also known as ‘satellite RNAs’

46. And last but not least…Prions
“Prion” is an abbreviated form of proteinaceous infective particle
Prion: minute, infectious agent composed entirely of protein
- no nucleic acid present
How do prions persist and replicate if lacking nucleic acid?
- Host cell encodes a protein similar to a prion
- Prion modifies host protein during or after protein synthesis
- Changes folding pattern
- Causes loss of function of host protein
- More prion-type proteins are then produced
Causal agent of neurological diseases in mammals:
- spongiform encephalopathy (‘spongy brain disease’)
- example: bovine SE, or mad cow disease

47. What are prions? Prions pose a special challenge…
-resistant to denaturation by protease, heat, radiation
This helped lead to their discovery:
-treatment of materials with UV radiation destroys nucleic acids…
-but Alper and Griffith (1960’s) showed that:
-infected materials remained virulent after UV treatment
-a non-nucleic acid ‘form of life’?
Mysterious until 1982:
Stanley Prusiner purified infectious material
-showed it was primarily protein
-coined the term (catchy!)
-received Nobel Prize in Medicine in 1997

49. SC

50. BSE: 190,000 confirmed cases (light + dark green on map)
nvCJD: 213 confirmed cases (3 in the US) (dark green) SC

51. BSE…what else? Chronic wasting disease
Infects members of the deer family (Cervidae)
Typified by chronic weight loss until death
Proteins can be excreted
Contaminated forage completes transmission
Kuru disease
First noted in the early 1900s in New Guinea
“laughing sickness” and “shaking sickness”
Especially common in eastern tribes
- cultural history of mortuary cannibalism
- especially favored kuru victims as food

52. BSE…what else? Fatal filial insomnia (FFI)
Exceptionally rare: 50 families worldwide
Autosomal-dominant inherited disease
Adult-onset (age 30-50)
Degradation of the thalamus
Four stages:
First four months: insomnia, with panic attacks
Next five months: hallucinations and further panic attacks
Three months: absolute insomnia; severe weight loss
Remaining months (1-6 months): dementia; coma; death.