研究病毒的重要性

1. 病毒會引起各種不同的病
2. 病毒是研究基因最好的對象
3. 病毒學是免疫學的始祖
4. 病毒可以作為基因治療的載體
Virus has many forms and shapes
Morphology of viruses

Icosahedral 

Helical
病 毒 的 形 狀

蛋白質

脂肪膜
Quasi­symmetry principle of icosahedral 
structure

pentamer

hexamer
 Virion structure
• Icosahedra
• Quasi-equivalent principle
• Triangulation numbers
– Capsomers C=10 x (T-1) + 12
– T=h2+hk+k2

Capsomers: The structural units of capsid

Structure of Calicivirus
製 造 病 毒 模 型 的 方 法
 Virus classification
• DNA or RNA
• Icosahedral or helical
• Enveloped or naked
 Genetic classification of viruses

• dsDNA
• ssDNA
• dsRNA
• ssRNA (+)
• ssRNA (-)
• ssRNA DNA
 The Central Dogma

DNA RNA Proteins

RNA
 One-step growth curve

• Multiplicity of infection (m.o.i.)

– Eclipse period, latent period
• Cytopathic changes (CPE)
– Syncytia, inclusion bodies
• Virus titer assays
– Plaque assays (plaque-forming units)
– Transformation assay (Focus-forming
units)
– Infectious center assay
 Viral life cycle
Adsorption
Penetration entry
Uncoating

Transcription
Translation macromolecular synthesis
Replication

Virion assembly
Maturation Exit
Release

Transcription first, then translation

(–)RNA viruses
DNA virus

Translation first, then transcription

(+)RNA viruses
 Viral entry

• Virus entry:
– Tropism: Susceptibility and permissivity
– Cellular receptors: tissue specificity

• Viral determinants of target cell specificity:

– Usually viral surface proteins

• Cellular receptors and coreceptors

 Viral penetration

• Virus-cell fusion on the plasma

membrane

• Receptor-mediated endocytosis
– pH-dependent (acidic pH)
 Replication and
transcription of DNA viruses

mRNAs transcription

α-amanitin-sensitive

Replication
 Replication and transcription of RNA
viruses
Replication (+) (+) (–) (–)

Actinomycin D-resistant
(–) (–)(+) (–)
α-amanitin-resistant
Cycloheximide-sensitive or resistant
Asymmetrical synthesis

Transcription (+) (+)(–) (+)

mRNAs

(–) (+)
 Replicative intermediate (RI) RNA

(-)(+) 3’5’
5’ 5’ 5’

RNase
5’
RF RI RF

(Replicative form)
RNA-dependent RNA polymerase (RdRP)
GDD motif
Right-handed “palm-thumb-fingers”
Share sequence and structure with other polymerases
 No proof-reading activity
“quasispecies”
High mutation frequencies
 Primer-dependent or –independent (de novo synthesis)

 Secondary structures of RNA regulates initiation and

termination
Transcription and replication of DNA viruses

Using cellular machineries- pol II and pol Ⅲ

some require virus-encoded proteins

Transcriptional cascades
early and late: divided by DNA replication

Viral promoters and enhancers

may require tissue-specific factors
 Translation of viral proteins

• Shut off cellular protein translation

– Cellular mRNA depletion or degradation
– Degradation of essential translation factors
• E.g., EIF4G degraded b poliovirus protease
• Virus-specific mechanism of translation
– IRES (Internal ribosomal entry site)
Viral assembly, maturation and release

• Burst
• Mature on the plasma membrane
• Mature on the ER or Golgi
 Genetics of virus infection

• Mutations
• Recombination
• Complementation
• Pseudotype virus particles
 Viral infection of whole animals
• Viral portal of entry (skin, respiratory tract, GI, etc)
• Regional lymph nodes

• Hematogenous (primary viremia), lymphatic and neural

spread

• Reticuloendotheial system (liver, spleen, BM, adrenal

glands)

• Spread (Secondary viremia), neural

• Target organs.
 Host defense

– Primary defense (innate immunity)

• Cytokines, chemokines, toll-like receptors,
interferons, NK cells

– Adaptive immunity
• Antibody, cell-mediated immunity
• MHC functions and expression
 Mechanisms of viral escape from host’s
defense
• Virokines
• Viroceptors
• Blocking of adaptive immunity: e.g.,
interference of MHC expression or
functions; production of decoy cytokine
receptors
• Blocking of innate immunity, e.g. interferon
• Blocking or inducing apoptosis

• Antigenic variations
 Cell Res. 16, 141-147 (2006) Feb

Interferon
induction

FADD Adapter
Caspase8, 10 TANK ?
NAP-1
J. Immunol. 176,
4520 (2006) Apr.

FADD: Fas-associated death domain

 Interferon action (antiviral function)

Inactive eIF2α Degrade RNA

Int. Rev. Immunol. 17, 53-73 (1998)

 Patterns of viral infections
• Acute infection
• Persistent infection
• Latent infection
• Slow virus infection
Patterns of viral infection

Acute infection
(SARS)

Persistent infection
(LCMV)

Latent, reactivating
infection (HSV)

Slow virus infection

(measles SSPE)
 Influenza virus

QuickTimeª and aTIFF (Uncompressed) decompressorare ne

Influenza virus

N (Neuraminidase)

H (Hemaggutinin)

PB1
PB2
PA
HA
NP
NA
Lipid bilayer

NS
M
RNA M1

8
M2 (Ion channel)
4 5 6 7
1 2 3

PB1
PB2 Transcriptase
PA complex
NP

NS2
 Influenza virus proteins
Hemagglutinin (H): 
Binds to cellular receptor (Determines the 
host species)
The major viral antigen
Induces protective antibody

Neuraminidase (N): 
Helps virus spread through the body
Induces semi­protective antibodies

Other proteins (e.g. PB2­ 1997 avian flu):
Viral replication
NS1 inhibits interferon ptroduction
Sialic acid as the receptor for influenza
viruses

(From Principles of Virology, Flint et al., 2000)

 Two major antigens: hemagglutinin (H)
and neuraminidase (N).

16 major H subtypes (H 1-16)

9 major N subtypes (N 1-9)

Only 3 H (1-3) and 2 N (1, 2) are found

to infect humans (H1N1, H3N2). The
rest (H5N1 etc.) are mainly avian and
porcine viruses.
Viral RNA

 Eight RNA segments

 Every particle contains one of each RNA

segment. Each RNA segment is
packaged into each virus particle
independently.

 “RNA reassortment” -- High frequency

genetic exchanges between different
virus strains.
RNA Reassortment

1
2

“poison” 3 “poison”
H1 H1
4 H5 Anti-avian H5 Ab
5
6

7
8

Avian Huma Reassortant

H5N1 n Human virus
H1N1
 Antigenic Shifts

Wholesale change of the entire H

or N due to reassortment
between a human and an animal
(particularly avian) virus in an
intermediate host (pig).

The cause of pandemics in 1957

and 1968.
 Antigenic Drift
 Single amino acid changes in the H
or N genes (or other genes)

 Causes of annual flu outbreaks and

may have caused 1918 Spanish flu

 Causing appearance of pandemic

strain from avian flu virus?
The origin of 3 pandemic
flu
• Spanish flu (1918): derived from an avian flu (H1N1).
The origin of this virus not known

• Asian flu (1958): “RNA reassortment (H2N2)”

– H, N and PB1 reassorted

• Hong Kong flu (1968): “RNA reaoosrtment (H3N2)”

– H3, PB1 reassorted
 The revival of 1918 Spanish flu

• Formalin-preserved lung tissue: RNA used for

PCR. A small stretch of flu RNA determined
• RNA from an Alaskan body in permafrost
• Complete the whole viral RNA sequence
(2005)
• Chemically synthesized the whole genome and
transfect into cells
• Eureka! A live 1918 flu virus was reborn (2005)
• Showed high virulence of this virus in mice
Why is the 1918 virus
so virulent?
• H: easily infects animals (Cleavage
sites)

• N: helps virus spread

• PB1: helps viral replication

• NS1?: Inhibits interferon production?

Two mechanisms for the appearance of
pandemic flu

• RNA reassortment
– Simultaneious infection of two virus
strains (Antigenic Shift), acquiring the
human H protein
• Accumulation of RNA mutations to make
an avian H protein (I.e. H5N1) capable of
infecting human cells
 How can an avian virus
mutate into a human virus?
• May need more than 25 mutations?

H: 1 or 2 amino acids
PA: 4 or 5 amino acids?
PB1: 1 amino acid?
PB2: 2 amino acids?
 anti-flu drugs

• Anti-M2. Amantadine and

Rimantadine: Most of H5N1 are
resistant to amantadine

• Anti-Neuraminidase. Relenza
(Zanamivir) and Tamiflu
(Osteltamivir)
The predominant Influenza virus
strains
(trivalent vaccine)

B
H3N2
H1N2
H2N2
A
H5N1
H1N1 H1N1

1918 40 50 60 70 80 90 2000
 Viral virulence of influenza virus

• Hemagglutinin
• Neuraminidase
• PB1 (?)
• NS1 (?)

• Why is H5N1 virulent for chickens?

– Hemagglutinin cleavage?