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Virus Infections of Humans
Lecture 1 – Introduction 1
Basic Properties of a Virus
1
...

3
...

5
...


Small size (20-220nm)
Only visualised in EM
Inert outside host but enzyme function upon entry
Protein coat (may also have lipid membrane)
DNA or RNA, nether both
Antigens enable attachment

RNA Viruses





Genome can be single/double stranded
Positive sense: RNA acts as messenger
Negative sense: RNA synthesises complementary strand  Messenger
Segmented genome  Allows variability via reassortment

Functions of Viral Proteins
Protective coat, enzymes, antigens, attachment
...

Functions of Viral Carbohydrates
Glycosylation, influence antigens, intracellular transportation
...
g
...

b) Use embryonated hens eggs to grow influenza for vaccines
...

Propagation of Cells in Culture





Primary Cells (direct from an intact, multicellular organism) or Continuous Cell Line (tissue
culture propagated immortal cell lines)
...

Supply with growth medium and incubate at 37oC  Cells settle and grow as monolayer
...


Assays for Viruses
a) Plaque Assay
a
...
Trypan blue stains dead, infected cells as their membranes have increase
permeability
c
...
Measures all viral surface protein  Less effective
b
...

Stages of Viral Replication
1
...

3
...

5
...
Initial binding – virus may move around to find its preferred receptor
2
...
Direct – conformational changes expose fusion domains in the virus protein
b
...
Uncoating  Genome release  Replication
4
...
g
...
6bk genome
...
Chronic HCV is a significant indicator of liver infection
...
Acidification of the endosome causes the viral
glycoprotein to bind to the endosome membrane, triggering genome release
...
The viral genome is replicated in the RER  Polyprotein  Cleavage  Mature proteins
2
...
Replication occurs within membranous web  - copy RNA template  + RNA progeny
4
...
Acquisition of envelope via budding through ER membrane in a process linked to
lipoprotein synthesis
6
...

2
...

4
...

6
...
Clinoco-epidemiological Diagnosis: patient history and clinical examinations for signs and
symptoms
...
Laboratory Diagnosis

a
...
New Rapid Methods: PCR (amplify even minute viral DNA/RNA to assess viral load),
ELISA, Fluorescent antibody
Serological Testing




Acute: patient is ill and has no specific antibodies against the virus
Convalescent: patient has recovered and developed specific antibody response
Antibody titre: significant increase during simultaneous testing of paired sera  positive

Indirect ELISA (Enzyme Linked Immunosorbent Assay)
1
...

3
...


Viral antigen adsorbed to plates  Wash excess
Add sera to be tested  Wash excess  If any positive antibodies, they will bind antigen
Add conjugate (enzyme-labelled antiglobulin)  Wash excess
Add enzyme substrate  Colour change for positive sera

Also direct ELSA where the first antibodies are enzyme labelled
...
Direct Fluorescent Antibodies
a
...
However, many specific antisera must be labelled in order to test for a range of
viruses
2
...
Fluorescent dye instead coupled to an antiglobulin to e
...
IgG
b
...
Antigenic shift: new virus A appears, whose HA has no cross reactivity to the the HA of
previous strains, immunity to A develops
B
...
Antigenic drift year after year…
D
...


Diagnosis of Measles
A
...
Grown in tissue culture produce syncytia which contain virus specific protein (identified by
fluorescently labelled antibody)
C
...
ELISA to show specific IgG in serum
E
...
77% cases (from high fever)
Post-infectious encephalitis
Sub-acute sclerosing panencephalitis (SSPE): 4-5 years after infection, loss of coordination,
brain immune privileged so virus remains there and fuses the cells together to form clumps
around the capillaries

Immunisation




Live, partially attenuated (grown in non-optimal conditions  mutants)
Dead and completely unattenuated do not stimulate cellular immunity (essential)
As vaccines developed, encephalitis decreased

Measles Virus







Only one strain
Single stranded, negative RNA surrounded by a nuclear protein
Lipid envelope
HA binds red blood cells
Fusion protein allows syncytia formation

Rubella
Clinical Features of Rubella






14-21 day incubation period
Onset includes low grade fever and 2 day catarrh
Maculopapular rash which begins on the face and neck
General lymphodenopathy
Some very mild cases with no rash, many sub-clinical cases

Rubella Diagnosis
A
...
Virus grows in tissue culture showing CPE, growth neutralised by presence of specific
antibody
C
...
Specific IgM antibody during early illness
E
...

Infant is very infectious
Rarely lives past 3-4 years, if so then the child would need institutionalisation

Rubella Vaccine



Passage virus in tissue culture under stress  Clones which induce immunity but not
symptoms
Must be live as immunity dependent on a cell mediated response

Interferon



Produced by host cells against viruses (lags after virus)
Inhibits virus replication by inducing ribonucleases (RNAse L destroys all RNA in the cell) and
protein kinases (PKR phosphorylates the translation initiation factor EIF2A, inhibiting viral
protein synthesis)

Lecture 5 – Human Herpes Virus
Classification




Alpha: wide host range (HHV-1,2,3)
Beta: slow growing, T cell host (HHV-5,6,7)
Gamma: B cell host (HHV-4,8)

Diseases





Herpes simplex virus type-1 and type-2  oropharyngeal herpes and genital herpes
respecitively; also encephalitis in the elderly and encephalitis in the newbords respectively
Varicella-Zoster virus type-3  chickenpox and shingles
Epstein-Barr virus type 4  glandular fever, Hodgkin’s lymphoma, Burkitt’s lymphoma
Human Cytomegalovirus type 5  cytomegalic inclusion disease

Herpes Virus Particle




Nucleocapsid containing double stranded DNA
Tegument – protein rich region containing ICPO, ICP4 and VP16
Lipid envelope

The HSV Genome




Linear, 152kbp
Replication via rolling circle in the nuclear
Generates long HSV progeny concatemers which are cleaved and packaged into viral
particles

Major HSV-I Proteins



22 tegument proteins in total, 4 are structural and another 4 alter the environment
The most important proteins include:
o VP16: an alpha-TIF (transcription initiation factor)
o ICPO and ICP4: immediate early proteins
o vhs (virion host shut off): shuts down host cell protein synthesis

Immediate Early Proteins





ICP4: essential for transcription
ICP0: transcriptional activator, ubiquitin ligase degrades host cell repressors of HSV
transcription
ICP22: deregulates host cell cycle
ICP27: regulates gene expression

Stages in Lytic Replication
1
...
VP16 binds HCF-I  + Oct-I  Rearrangement of HSV DNA  Complex can bind TAATGARAT
3
...
Translation of HSV immediate early genes (ICP0 and host RNAPII)
HSVI Entry Receptors





gB – fusogen for cell entry
gD – fusion trigger
gH + gL – complex, fusion regulator
Receptors for each on host cell

Progression to Cell Nucleus
1
...
Nucleocapsid and remaining tegument proteins transported to nuclear membrane via MTs
3
...
Viral genome rearranged into nucleosomal arrays to mimic host cell chromatin
HSV Proteins against Host Metabolism





vhs: accelerates decay of host cell mRNA, disrupts pre-existing polysomes
ICP0: E3 ubiquitin ligase which degrades host cell repressors of HSV transcription
ICP27: inhibits host pre-mRNA splicing
Immediate early proteins change host metabolism from host proteins  viral proteins

Gene Cascade in Lytic Infection
1
...

3
...


Immediate early genes: induced by VP16, responsible for synthesis of 5 proteins
Early genes: expression not stimulated by DNA replication
Late-I genes: low levels of expression in absence, but greatly stimulated by DNA replication
Late-II genes: not expressed in absence of DNA replication

Entire process takes 8-13 hours in fully permissive cultures
Chromatin and HSV DNA Replication





Nucleosome: 8 histones with DNA wrapped around outside and a HI molecule to stabilise
HAT + HCF (co-activator)  Modifies chromatin  Increases gene expression via binding to
an activator e
...
VP16
Modification involves acetylation and methylation of nucleosome core
Relaxes connections between histones and DNA to expose control regions of promoters
close to enhancers

Interactions between HSC and Host



Causes cell arrest in G1 (favourable for viral replication)
Inhibits apoptosis prior to viral release

HSV Release
1
...
Nucleocapsid enters cytoplasm and proteins here form the tegument
3
...
Association of glycoproteins + nucleocapsid + tegument
5
...

2
...

4
...

1
...
Invade local nerve ending of trigeminal and superior cervical ganglia, or sacral ganglia,
respectively
3
...
Frequency of reactivation can vary significantly, triggered by stress, hormones, UV, dental
surgery and cranial trauma
Factors in the Establishment and Maintenance of HSVI Latency
1
...

3
...

5
...
Chromatin: histone methylation so HSV DNA in a heterochromatic state
7
...
HSVI CD8 cells maintain latency via IFNy/granzyme B secretion
Establishment of Latency
1
...
Circularisation of HSV genome (probably through host DNA repair mechanisms)
3
...
HSV genome maintained as heterochromatin due to low HCF-I, Oct-1, VP16
...
5 (replication regulator)




Function as epigenetic regulators (chromatin modifiers)
Anti-apoptotic ability

Maintenance of Latency
1
...
NGF-TrkA produces signals that suppress HSV-I reactivation via mTORCI (cellular stress
sensor kinase)
3
...
A transient interruption in the mTORCI pathway can reactive HSV-I in mouse ganglia
Reactivation
Must involve chromatin reorganisation and high IE expression to overcome miRNAs and host cell
response
...
Viral DNA moves from the neurons to
the epithelial cells and the lytic cycle starts
...
Quasispecies  Antibodies against the dominant strain exert a selection pressure
2
...
Repeat of 1 with a new dominant strain
4
...

Development of Innate Immunity
1
...
Soluble molecules neutralise virions e
...
complement and IgM
b
...
Early Intracellular Factors
a
...
IFNa/B response of infected cells primes nearby cells into an antiviral state via
cytokines, also recruits NK cells, macrophages and neutrophils
Toll-like Receptors



10 in humans, located on cell membranes and endosomes, sense foreign nucleic acids,
lipoproteins and LPS
Activates NF-kB and IFN transcription

Cellular DNA Sensors






IFI16 and cGAS
Located in the cytosol and binds foreign dsDNA  AMP and GMP dimerization  cGAMP
CGAMP binds STING  Phosphorylation of IRF3 (via TBK1)
IRF3 enters the nucleus  IFN transcription  Inflammation genes
STING also aids lysosome formation to degrade the pathogen

Hepatitis C NS3/4A Protein
RIG-I (hepatocytes) + dsRNA  IFR3  NF-Kb  ISG expression
NS3/4A blocks the activity of RIG-I

Viruses and Dendritic Cells
Antigen contact  Maturation of DCs  Migrate to T cell zones of lymphoid organs  Upregulate
co-regulator molecules (via cytokines)  Prime naïve T helper cells
mCD83 is marker of DC maturity and enables strong upregulation of T cell activity
...
CD8 cell  Cytotoxic granules  Fas surface receptor on infected host
2
...
BAX family proteins  Insert into mitochondria  Releases cytochrome c  Forms a
complex with apoptosis protease activating factor-1 (APAF-1)
4
...
Activates caspase 3 and effector molecules  Apoptosis

Apoptosome regulatory proteins are also released from the mitochondria and remove inhibition of
the apoptosome pathway
...
7K: inhibits apoptosis induce by TNFa, probably via binding caspase 8
E1B-55K: bind p53 in order to prevent transcription of apoptotic proteins

Complement and HSV





30 serum proteins that operate in a cascade  MAC
Bind to the Fc region of antibodies bound to antigen  Opsonisation
HSV expresses gE and gI on its envelope which forms a dimer and expresses as an FcR 
Complement unable to bind Fc
gC-1 also binds and inhibits C3b

Viral modulation of host MHC antigens and of complement demonstrates the interconnection of
these mechanisms
...

Viral Evasion against Acquired Immune Mechanisms





Intracellular location, spread (via cell fusion), and virus assembly
Latency  Down regulation of viral proteins
Antigenic variability
Infection of immune system cells  Immunosuppression

Virus Variability
Glycosylation of Virus Envelope Proteins



Carbohydrates can mask the epitope of viral proteins
HIV-I contains 28-30 potential N-linked glycosylation sites

Antigenic Variability




~100 genetically stable rhinoviruses serotypes known
Infection with one does not provide immunity for another
Only infect the upper respiratory tract so there is little stimulus of the immune response

There is also antigenic shift/drift and formation of quasispecies
...

2
...

4
...
1% children, 2
...

HAV grows slowly in culture with CPEs and can be detected via EM in faecal samples with addition of
the specific antibody (agglutination for diagnosis)
...

There is an IgM and IgG response to HAV and the virus remains in faeces for ~6 weeks
...

HAV Structure




Picornavirus
Positive, single stranded RNA
Capsid of 60 copies of VP1,2,3

Hepatitis B
1
...
Incubation: 6-26 weeks (average 10 weeks)

3
...
Illness: similar to HAV + serum sickness and glomerulonephritis due to Ab complexes,
fatalities 1-10%
5
...
5-2
...
Transmission: same as HBV but less infectious, main route appears to be parenteral but
sexual transmission also occurs
HCV was declared as a public health problem by WHO and 1-3% of the world’s population has
chronic HCV (70% of all chronic hepatitis infections)
...
There are six major phenotypes which all mutate and
therefore there is no vaccine
...

HCV Structure


Positive, single stranded RNA (HAV)




Genome associated with a highly conserved core protein  Nucleocapsid
Envelope proteins not highly conserved

HCV Prevalence in England



0
...
There is a high potential for cross-species transfer between avian and
mammalian species
...

Cross Species Transmission of Type A Influenza
Cells lining the intestinal tract infected in wild/domesticated birds  Virus excreted in their faeces
(normally LPAI)  Contamination of water during migrations  Infection routes for other birds

Genetic analysis of type A influenza strains infecting pigs, horse, seals and whales suggest derivation
from birds
...

Pathogenicity in Non-Human Hosts





Avian serotypes of influenza A cause no disease in wild birds
Therefore it is probably that HPAI strains evolve following transmission into domestic birds
HPAI strain may arise from mutations that adapts the strain to a new host
HPAI in domestic fowl has a mortality rate approaching 100%

Factors Affecting Transmission to Humans
1
...

3
...

5
...
Host HA cleavage via intracellular endoproteases in most cells
a
...
HA cleavage site sequence in multiple, basic amino acids
a
...
Phenotypic changes in PB2 protein so cause an enhanced systemic reaction
4
...
Induce proinflammatory proteins
6
...

2
...

4
...
Symptoms of infected animals
include the head/tail being down, extreme salivation and paralysis
...
However, vectors can
include all animals (except birds)
...

Rabies in Humans






Incubation period: 2 months – 2 years, generally the further the bite is from the brain, the
longer the incubation period
Days 1-4: irritation at entry site, fever
Days 4-6: localised paralysis, hydrophobia
Days 6-7: convulsions
Days 8-10: death via convulsions or cardiovascular collapse (only two humans ever known to
survive and were shown to have no cardiovascular decline)

Rabies Diagnosis
1
...
Virus present in saliva and brain: rabies recapitulated in experimental animals with Negri
bodies in the brain at post-mortem
3
...

You must be vaccinated to enter countries where the disease is dominant
...

Mitosis  G0  Rests OR Mitosis via G1  Mutation  Apoptosis
Activation of an oncogene or deletion of an anti-oncogene can result in cell proliferation, and
inhibition of apoptosis and differentiation
...
There are many different types of RNA virus which can cause many
different types of tumour which can occur in many different species
...

2
...

4
...

6
...
If the oncogene is away from its normal control site, this may result in cell proliferation
Rat Sarcoma Virus contains an extra SRC, which when integrated into a host genome, signals for cell
division
...

RNA retroviruses are found in 20-25% of breast cancers and have also been found in prostate and
bowel cancers
...

Retoviruses in Adult T cell Leukaemia/Lymphoma (ATLL)





Proliferation of primitive T cells resulting in large pleomorphic lymphocytes 
Immunodeficiency
Symptoms include lymphadenopathy and skin rashes, with a 50% death rate 9 months from
diagnosis
Human T-lymphotrophic virus 1 (HTLV-1)
Related to other conditions such as Sezary’s syndrome – same or related virus?

Incidence of HTLV-1



20-40% Japan  Immigration  20% Hawaii
40-70% Isolated African tribes  Slave trade  10-20% Caribbean and Florida

HTLV-1 Structure




ENV, GAG, and POL structural genes
5’ LTR virus promoter
3’ LTR downstream promoter  Random integration could activate an oncogene

The virus is not only amplified by replication, but also via reinfection and intracellular retrotransposition
...

Cancer Induction by HTLV-1
1
...
Viral RNA transcribed into DNA and integrated into host genome

3
...
Accidental integration of viral terminal transactivator near an oncogene  Accelerated cell
division
5
...

Action of RAS




RAS causes GTP to revert back to GDP
Therefore if RAS is mutated, GTP is permanently switched on
This causes a continuous production of cytokines  More GTP  Cell grows

Lecture 13 – DNA Viruses and Cancer
There are many different types of DNA viruses, which can cause many different types of tumour,
which can occur in many different species
...

2
...

4
...

6
...
EBV from Glandular fever multiplies in the respiratory tract
2
...
Monocytes cause amplification of viral gene products

4
...
Cell rapidly divide  Translocation of Myc on 8 next to IgH on 14
6
...
Integration of X sequence  Cell proliferation
2
...
Regeneration of hepatocytes  Cell proliferation
Sequence of Events to Liver Cancer
1
...

3
...

5
...

2
...

4
...


Attachment, entry and uncoating
RT and genome integration
Latency
Transcription and splicing of viral RNA
Assembly of new virions and release from cell

Cell Attachment and Entry
1
...

3
...


V3 loops of gp120 binds CD4  gp120 conformational change
High conserved domain exposed in gp120  Binds CCR5/CXCR4 chemokine receptor
Fusion domain also exposed on gp41 (TM)  HIV fuse with host cell membrane
Viral particles uncoated immediately upon entry

Synthesis of HIV-1 DNA
1
...

3
...

5
...

2
...

4
...

2
...

4
...

6
...

2
...

4
...
The p6 protein ensures efficient separation of the viral envelope from the cell membrane +
recruits cellular factors to the budding site
6
...
1% of all CD4 cells in the host, it is still a major barrier to
eliminating infection

Overview of HIV-1 Pathogensis






Virus present in CD4 cells and macrophages in both a latent and replicative form
Control of the infection by immune defences, especially CD8 cells
Overtime, there is a loss of control due to virus variability
There is a loss of T cells via virus and host destruction
Development of AIDs and the appearance of opportunistic infections

Mechanisms of HIV-1 Variability
Recombination


Following infection with two different subtypes, genomic recombination may occur prior to
viral integration



These recombinant strains create much antigenic diversity and several have spread in
epidemics

Quasispecies




HIV-1 has a high mutation rate (no proof reading enzyme) and after a few rounds of
replication, huge numbers of variants are generated
A quasispecies consists of closely-related but distinct viral genomes defined by a master
sequence and a mutant spectrum
Results in the appearance of escape mutation, changes in virulence and host range, and
resistance to antiviral agents

Glycosylation




The HIV envelope contains 28-30 potential N-linked glycosylation sites
The position of the glycans modulate antibody neutralisation specificity
Removal of N-linked glycans in the V3 loops increases sensitivity

Lecture 15 – Type 1 Human Immunodeficiency Virus, Immunology and Control
Stages of Infection
1
...
Acute seroconversion associated with transient immunosuppression and a fall in
CD4 cells
b
...
After seroconversion, CD4 rises to normal levels and the viral load falls
2
...
Lasts many years, progressive loss of CD4 cells
b
...
Acquired Immunodeficiency (AIDs)
a
...
Initial sexual exposure  HIV-1 targets DCs and macrophages (via Siglec-1 and DC-SIGN) in
the mucosal epithelial tissues
2
...
HIV-1 activates T cells and large numbers of CD4 cells are infected (via DC-SIGN)  Rapid
HIV-1 spread

4
...

Antibodies and HIV-1






Early destruction of CD4 cells delays maturation of antibody response
Mostly IgG but they have little neutralising activity
Antibodies play only a minor role
HIV-1 escapes antibodies via their high degree of gp120 variability and only a transient
exposure of neutralising epitopes during entry (CCR5)
Also less than 0
...
CD4/8 via direct killing
a
...
HIV tat protein
a
...
Dysregulates inflammatory cytokine and cytokine receptor expression
c
...
Destruction of lymph nodes
a
...
HIV nef protein
a
...
Syncytium formation
6
...
HIV reactivation via activation of infected CD4 cells via interaction with APCs
b
...
g
...
Suspected by shepherds when new sheep introduced into the flock
2
...
Inoculation of brain tissue from sheep who died of scrapie  Induces scrapie
Isolating the Causative Agent






Sporadic incidence (no pattern of spread)
Failure to isolate causative bacteria/virus
No pathology suggesting bacteria/virus
Only abnormality in cell loss and holes in brain structure i
...
spongiform encephalitis
Similar features in Creutzfeldt-Jakob disease (CJD)

Properties of Scrapie Agent




Resistant to 100oC for 30 minutes, and 121oC for 20 minutes
Also resistant to UV, DNase and RNase, and chloroform
Sensitive to pH <2 or >10, sodium lauryl sulphate, and 1210oC for 2 hours

Creutzfeldt - Jakob disease











1 million cases per year in the UK
Onset age 50-70 years
10-15% cases familial, with most occurring in Libyan Jewish families
Long and variable incubation period
Prodromal symptoms include fatigue and weight loss
Onset includes dementia and ataxia
100% death rate at 6-24 months from onset (occasionally up to 5 years)
No evidence of infection by conventional organisms
All tissues normal except brain where there is neurone loss, spongiform encephalitis, and
increased number of astrocytes and amyloid plaques
...

FOR
One CJD case in recipient of liver transplant and
blood products
Four cases of CJD in recipients of blood
transfusions

AGAINST
No CJD cases amongst 35 blood recipients from a
donor who later developed CJD
No CJD in haemophiliacs after repeated
treatments

Accounts for 0
...


Kuru






Incubation period less than 7 years
Early symptoms include fatigue and loss of balance
Onset brings dementia, tremors and ataxia
100% death rate in less than 2 years from onset
Pathology same as CJD and scrapie

Kuru Pathology





Adults have a higher rate of death than children, and women have a higher rate than men
Occurs in cannibal tribes in the Highlands of Papau New Guinea, where majority is carried
out by women and children whilst adult men live independently
Thought initial feed was infected with CJD
The disease mostly disappeared when cannibalism ended

Origin of Prion Protein Disease








Resembles no other infectious agent
Physical properties suggest that it may be a protein
Biochemical techniques and animal models indicated an abnormal protein  Prion
No DNA or RNA
Amyloid fold consisting of tightly packed beta-sheets (stability and extended half-life)
There are a number of codons that can mutate to form prions, can also be inherited or come
from intestine/cuts/surgery that travel to the brain via nerves
Prions form a template for directing normal protein to fold into prion form

Knockout mice deprived of abnormal gene  No natural protein and no CJD (even after inoculation
of large quantities of infected brain)
Model mice must have natural and prion protein  Increases concentration of prion protein due to
extended half-life  Cell death and prion spread
Bovine Spongiform Encephalitis
Young people were developing CJD, thought to be from eating infected cattle

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