Genetic system: Virus

1. Principles of Virology: Molecular Biology,

Pathogenesis and Control of Animal Viruses,
ASM Press, Washington DC 2004
2. Fields Virology, Fifth Edition, 2007

Why study virus?

Genetic systems for understanding cell
biology due to co-evolutionary relationship
with hosts--- basic research
Relevant with human/animal/crop health--medical and economical
Powerful molecular tools--- biotech

History and Discovery of Virus

1580-1350 B.C.

Virus Structure

Poliovirus
Enterovirus
Rotavirus

Influenza Virus
HIV
Dengue Virus

Size of Virus

Baltimore classification

Virus Taxonomy

NIH Guidelines
Classification of Biohazardous Agents by Risk Group (RG)
Risk Group 1 (RG1): Agents that are not associated with disease in healthy adult humans
Adeno-associated virus (AAV) types 1 through 4
Murine Retroviral Vectors
Risk Group 2 (RG2): Agents that are associated with human disease which is rarely serious and
for which preventive or therapeutic interventions are often available
Adenoviruses;
Flaviviruses- Dengue virus
Hepatitis A, B, C, D, and E viruses;
Herpesviruses - except Herpesvirus simiae (Monkey B virus
Orthomyxoviruses--Influenza viruses types A, B, and C;
Papovaviruses --human papilloma viruses;
Paramyxoviruses--Measles virus--Mumps virus-Parainfluenza viruses types 1, 2, 3, and 4--Respiratory syncytial virus;
Parvoviruses;
Picornaviruses--Coxsackie viruses A/B-Echoviruses --Polioviruses--Rhinoviruses;
Poxviruses - all types except Monkeypox virus
Amphotropic and xenotropic strains of murine leukemia virus

Risk Group 3 (RG3): Agents that are associated with serious or lethal human disease for which
preventive or therapeutic interventions may be available (high individual risk but low community risk)
Alphaviruses (Togaviruses) - Group A Arboviruses --Semliki Forest virus
Bunyaviruses--Hantaviruses including Hantaan virus--Rift Valley fever virus
Flaviviruses (Togaviruses) - Group B Arboviruses--Japanese encephalitis virus--Yellow fever virus
Poxviruses--Monkeypox virus
Prions--Transmissible spongioform encephalopathies agents (Creutzfeldt-Jacob disease; kuru agents)
Retroviruses --HIV-- HTLV --SIV
Rhabdoviruses--Vesicular stomatitis virus
Risk Group 4 (RG4): Agents that are likely to cause serious or lethal human disease for which
preventive or therapeutic interventions are not usually available (high individual risk and high
community risk)
Arenaviruses--Guanarito virus--Lassa virus--Junin virus--Machupo virus--Sabia
Bunyaviruses (Nairovirus)--Crimean-Congo hemorrhagic fever virus
Filoviruses --Ebola virus--Marburg virus
Flaviruses (Togaviruses) - Group B Arboviruses --Tick-borne encephalitis virus complex
Herpesviruses (alpha) --Herpesvirus simiae (Herpes B or Monkey B virus)
Paramyxoviruses--Equine morbillivirus
Hemorrhagic fever agents and viruses as yet undefined

Virus Cultivation, Detection and

Genetics
Virus Cultivation:
Cell cultures
Virus growth in cell cultures
Growing virus stocks
Virus Detection
Measurement of infectious units
Nucleic acid detection
Virus Genetics
One-step growth curve vs. multiple round infections
M.O.I. / Burst size/
Genetic mutation analysis

Growing virus stocks

Embryonated eggs
Cell lines

 Embryonated eggs
Cell line establishment

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Virus cultures and Virus-induced cytopathic effect

(CPE)

Host cells
Primary cells

Infected cells

immortalized cells

transformed cells
Syncytia

Choosing a culture system for animal viruses

Culture system

Advantages

Disadvantages

Animal

Natural infection;
Native host

Cost of upkeep is
expensive. Large
variation between
individuals even if
inbred. Need large
numbers

Organ, e.g. pieces

of brain, gut or
trachea

Natural infection.
Fewer animals needed,
Tissue specificity

Multiple cell types

exist, Unnatural since
isolated.

Cell

Can be cloned therefore

Very unnatural since
variation between
cells change when
individuals is minimal,
cultured
Best for biochemical
studies as the environment
can be controlled;
Can be immortal,

Virus Detection
VV particle count

EM

3-D Deep-Etch EM

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CryoEM
Atomic force
Microscopy

Microsc Microanal 11 (supp 3), 2005

CryoEM

CryoEM of Virion Structure

SFV-E1/E2

TBEV

DV

Pletnev, S.V. et al, Cell 105: 127- 136 (2001)

Ferienghi, I. et al, Mol. Cell 7: 593-602 (2001)
Kuhn, RJ et al, Cell 108: 717-725 (2002)

Virus Detection
Measurement of infectious units: Virus titer assays
1. Plaque assays
Plaque: a clear zone of cell lysis due to an initial infection event
Plaque formation unit (PFU): the amount of the virus that is required
to form a plaque
Dilutions: 101, 102 105,
PFU:

106

TMTC ..(68, 72), (7, 9)

Volumn used :1 ml per plate

Virus Concentration
= (68+72)/ 1ml x 105
= 7x106 PFU/ml

2. Fluorescence focus assay

- when Ab is available

4. End-point dilution assay

One ID50 Unit = the amount of

viruses required to infect 50% cells
3. Transformation focus assay
- For oncogenic viruses

Volumn used :1 ml per plate

Virus Concentration = 105 ID50/ml

Virus Detection
Measurement of virus particles and their components
Electron microscopy
Particle-to-PFU ratio: Low ratio = High infectivity
Bacteria phage (2 ) <<< Mammalian virus ( 10-1000)

Virus Detection
Measurement of virus from clinical samples

1. Clinical samples difficult to obtain---> Sensitive

2. Emergency ---> Quick
3. Specificity ---> Multiple criteria
1. - immunological
2. - morphological EM
3. - genetic evidence (PCR)
4. - recover etiological agents

Virus Detection
Measurement of viral nucleic acids: sensitive
Real-Time RT-PCR

Virus Detection

Determination of Serum DV titers in experimental monkeys infected with DV

Virus Detection
Measurement of virus from clinical samples

1. Clinical samples difficult to obtain---> Sensitive

2. Emergency ---> Quick
3. Specificity ---> Multiple criteria
1. - immunological
2. - morphological EM
3. - genetic evidence (PCR)
4. - recover etiological agents

2003- An Emerging Infectious Disease in

North America
35 confirmed cases
in
Wisconsin,
Indiana,
Illinois,
Missouri,
Kansas, and
Ohio;

http://www.cdc.gov/ncidod/monkeypox

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Serology

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PCR genome

Plaque assay

Monkeypoxvirus transmission
from animals to human
April 9, 2003
Ghana shipment
Arrival at US
Gambian giantpouched rats

Dormice

Early June

Prairie dog

By July 8

Human
35 confirmed cases
in
Wisconsin,
Indiana,
Illinois,
Missouri,
Kansas, and
Ohio;

Monkeypoxvirus transmission in
humans in Africa

Virology

The extent of virus infection is determined by

Multiplicity of Infection (MOI)
MOI= The ratio of virus titer to cell number
at the beginning of infection
Complete infection in one round: 5-10
Multiple rounds of infections: 0.01-0.05

One-Step Growth Curve of Virus

M.O.I. =5-10 or 5-6 ID50
Synchronized growth
Burst size= the yield of infectious viruses per cell
Non-enveloped
Bacteria
Enveloped

12-24h

30

n1
.

..

n2

21
22

nn

2n

Genetic isolation of mutant viruses

Temperature sensitivity (ts)
39C

31C

Complementation assays to
sort mutants into different
complementation groups
39C

39C

39C

39C
Marker rescue

Genetic isolation of mutant viruses

Drug resistance

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In vivo host selection

Different virus mutants affecting

viral virulence
Mutations impair virus replication in the host --essential
genes
Mutations impair virus virulence but do not alter normal virus
replication in assay cells- nonessential genes or regulatory
elements
-Mutations of viral proteins involved in host defense
antagonism: virokines, anti-apoptosis, down regulate immune
response
-Mutations impair virus trafficking or spread in the hosts
Host factors and environmental changes provide selection
pressure for mutant generation.

Spanish Flu in 1918

The Influenza Pandemic of 1918-19 and How It Affected the USA

Timeline of human influenza in

20th century

How the Flu Virus Can Change Antigenic Drift" and "Shift"

Antigenic drift.
Slow and mild changes on H and N genes
Cause the lack of immune protection from different strains
why people can get the flu more than one time.
protected from flu need to get a flu shot every year.

How the Flu Virus Can Change Antigenic Drift" and "Shift"

Antigenic shift."

abrupt, major change only occurred in type A influenza

results in a new influenza A subtype.
happens only occasionally.

Host Cell
Avian
Swine

SA

Human

Avian flu (H5) vs HK/99 (H9)

Avian
Swine

Human

Avian
Swine

Human

Reverse genetics of Flu Vaccine

(6+2) Preparation

Viral Vectors
Components for viral vectors
Promoter
Packaging limit
Virulence in vitro and in vivo

DNA vector
Vaccinia virus
Baculovirus
Adenovirus

RNA vector
- Retrovirus/ lentivirus
- Alphavirus

Applications of Viral Vectors

Introduction of genes into cells of interest
Overexpression of proteins for functional,
structural and biological studies
Preventive vaccines
Therapeutic tools as in vivo vehicles

Retro Viral Vectors

Drawback:
Low titers, unstable
Species barrier
Replicating cells
Integration hot spots

Baculoviral Vectors
Polyherin promoter
Inclusion- phenotype
Incomplete CHO
Protein production
Cheaper and feasible

Vaccinia Viral Vector

1st generation vector:

Tk locus
Constitutive
P7.5/ p11K

Adeno viral vector

Propagation on 293 cells (E1A+)
Adenoeasy E. coli system
Host range wide

Example: lentiviral vectors making

shRNA (National RNAi core)
Structure of VSV-G-Pseudotyped Lentivirus

Modified from http://www.washington.edu/alumni/columns/dec00/cells4.html

Example: using lentivirus making

shRNA (National RNAi core)

Psi
R-U5 signal

shRNA
U3-R

U6 promoter Puro

http://www.accessexcellence.org/RC/VL/GG/retrovirus.html

Example using lentivirus making

shRNA (National RNAi core)
Procedures:
Day1: seed cells
Day2: co-transfect
Day3: re-fresh media
Day4: harvest viruses/
re-add media
Day5: harvest viruses

Q: Sort these mutants into different complementation groups

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The End