Medical microbiology II

Virology
Parasitology
Mycology

Characteristics of microorganisms Sizes

General properties of viruses

High diversity in structure, genome
organization, expression, virus-host
Acellular microorganisms interactions
• Sizes:
– 20 nm Parvovirus
Viruses
– 400 nm Poxvirus
– 1000 nm Filoviruses (filamentous)

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 General properties Forms of viruses
• vegetative virus:
• Composition
– intracellular
- nucleic acid (DNA or RNA)
– replicating viral genome
- proteins
• virion (viral particle):
- lipids, carbohydrates
– extracellular
– the entire infectious unit
• Cultivation in receptive, living cells – inert in extracellular environment (no
(parasites at the genetic level) replication)
– infective
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Criteria that define a virus Classification of viruses

• viruses contain a single nucleic acid -
DNA or RNA
• inside a cell, viral replication is controlled
by the viral genome
• viruses cannot produce energy or
proteins independently from the host cell
• viruses cannot replicate by „division”
• viruses are obligate intracellular parasites
9 10
• according to nucleic acid:
– DNA viruses
– RNA viruses
• according to the virion’s morphology and
physical, chemical properties
– viral families
• antigenic structure, cellular tropism, genome
organization and replication, enzymatic
equipment – further classification

Herpes viruses

• viral family (familia):

-viridae (Herpesviridae) Herpesviridae family

• subfamilies (subfamilia):
-virinae (Betaherpesvirinae) Alpha-
herpesvirinae
Beta-
herpesvirinae
Gamma-
herpesvirinae
subfamily

• genera (genus):
-virus (Cytomegalovirus) Herpes Varicella-zoster Epstein Barr Kaposi sarcoma
Cytomegalovirus HHV7 HHV6
simplex virus1, 2 virus virus virus

• Viral strains are members of the same virus

isolated from different sources and may have
different genetic background. virus

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 DNA viruses RNA viruses
Adenoviridae Arenaviridae The structure of the virion
Hepadnaviridae Bunyaviridae
Herpesviridae Caliciviridae
Coronaviridae
Polyomaviridae
Filoviridae
Nucleic acid
Papillomaviridae
Flaviviridae
Parvoviridae Orthomyxoviridae
Poxviridae Paramyxoviridae Capsid
Picornaviridae
Reoviridae
Retroviridae Envelope
Rhabdoviridae
Togaviridae
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Virion Virion structure

• nucleic acid - limited amount of genetic
information
DNA
• encased in a protein shell = capsid or capsid nucleocapsid
naked capsid
virus
– many identical molecules of one or few proteins RNA
(capsomeres) which assemble spontaneously with no
or minimal energy consumption
• +/- envelope lipid membrane, enveloped
nucleocapsid
– derives from infected cell’s membrane glycopeptides virus

– contains virus-specific glycoproteins (spikes)

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Virion structure Nucleocapsids

1 – capsid
2 – genome
3 – capsomere
4 – nucleocapsid
5 – virion
6 – envelope
7 - glycoproteins

3
 Types of nucleocapsid symmetry
• based on the arrangement of morphologic
subunits
– cubic (icosahedral) symmetry
– helical symmetry
– complex structures
Tobacco mosaic virus
LL
Enveloped helical virus
Helical symmetry
• capsomeres are individually bound to the viral
nucleic acid, winding it into a helix → close
interaction between the nucleic acid and
proteins
• filamentous nucleocapsid
– rod shape: tobacco mosaic virus, rhabdovirus
– spherical: due to secondary coiling of
nucleocapsid
• regular (Orthomyxovirus)
• irregular (Paramyxovirus)
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Influenzavirus (EM)

25 26

Paramyxovirus (EM) Rhabdovirus

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Rhabdovirus Cubic symmetry

• icosahedral pattern
• 20 faces, 12 vertices, 30 edges
• capsomeres: pentons, hexons
• number of capsomeres is characteristic for
a given species

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 31 32

Icosahedral
Build your own capsid
virus

Packing of subunits in
picornaviruses

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 Adenovirus

37 38

Herpes simplex virus Papillomavirus

LL 39 40

Papillomavirus (electron
microscopy)

41 42

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 Poliovirus

Molecular surface of Poliovirus Type 1 Mahoney,

(X-ray crystallography)
43

Rhinovirus Complex symmetry

• some virions do not present helical or

cubic symmetry but are more complex
structures

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Bacteriophages with tail

47 48

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 HIV

Chemical composition of
viruses

LL 49 50

Nucleic acid

• single kind of nucleic acid: DNA or RNA

• Nucleic acid • 7-400 genes
• proteins, enzymes • Genome
– single stranded (ss) / double stranded (ds)
• lipids – Linear/ circular
• carbohydrates – segmented / nonsegmented
– Positive / negative polarity (RNA)

51 52

Proteins Lipids
• capsid proteins
• in the envelope
– resistant to proteolytic enzymes – protect the
genome • origin: cellular membranes in which viral
– attach to cellular receptors proteins have been integrated
– determine the symmetry type • enveloped viruses are susceptible to ether
– antigenic properties and organic solvents
• core proteins
– enzymes

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 Carbohydrates
• Envelope: glycopeptides
• encoded by the viral genome
Viral replication
• Role:
– attachment to receptors
– antigens

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Viral replication General steps in viral replication

• Viral replication occurs only in living, • Attachment

receptive cells • Penetration
• Host cell provides • Uncoating
– energy and synthetic machinery • Eclipse period
– precursors for the synthesis of viral proteins
and nucleic acids
• Morphogenesis
• Viral nucleic acid: carries the genetic • Release
information

Attachment Attachment of herpes viruses

• interaction of the virion and specific receptors on
the cell surface
– conformational homology between viral surface
structures and cell surface components (HIV-CD4,
Epstein-Barr virus - CD21, rhinovirus - ICAM)
– presence or absence of a receptor determines cell
tropism
– not all cells in a susceptible host express the
necessary receptors (cells in the CNS, intestinal tract
express receptors for poliovirus)

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 Penetration or engulfment Attachment - penetration
• the virus is taken up inside the cell by:
– endocytosis – uptake of virus particles within
endosomes
– direct penetration across the plasma membrane
– fusion of the virion envelope with the plasma
membrane - coreceptor

Attachment - penetration Penetration – fusion of the

envelope and cell membrane

Adsorbtion - attachment Uncoating

• shortly after penetration
• separation of the viral nucleic acid from the
outer structural components
– free nucleic acid (picornaviruse)
– nucleocapsid (reoviruses)
• the infectivity of the parental virus is lost at this
stage
• viruses are the only infectious agents for which
disintegration is an obligatory step during
replication

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 Uncoating Eclipse period
• a period between uncoating – appearance of
new viral particles
• synthetic phase
• essential processes:
– specific, functional mRNA transcribed from the viral
nucleic acid
– translation of mRNA
– replication of the genome
• virus-specific proteins are synthesized in a
highly organized sequence

Stages of eclipse period Stages of eclipse period

(DNA viruses) (RNA viruses)
• transcription of early mRNA
• protein synthesis
• production of early viral proteins – directly from a positive strand RNA (= mRNA)
(nonstructural) – in case of negative strand RNA, this serves as a
template for mRNA; the negative strand RNA genome
• replication of viral nucleic acid is not infectious by itself, the virus needs to carry a
polymerase to make individual mRNA for each viral
• transcription of late mRNA protein
• replication of viral nucleic acid
• production of late proteins (structural – RNA dependent RNA polymerases: synthesis of a
proteins, capsomers, enzymes ) full-length RNA to generate copies of the viral
genome

Morphogenesis – assembly of Morphogenesis

viral particles
• ∼ 3D interlocking puzzle - small “bricks” enclose the viral
genome • excess amounts of components may
• begins when the necessary pieces are synthesized
accumulate in cytoplasm – inclusion
• capsid viruses:
– procapsid (empty capsids) filled with genome bodies (diagnostic importance)
or
– assembled around the genome • low efficiency (formation of empty capsids
• helical viruses: assemble around the genome – noninfectious particles)
• enveloped viruses:
– viral glycoproteins are inserted previously in cellular membranes,
envelope is added to nucleocapsid while passing through
membranous structures

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 Release Release

• one way of the following:

– after lysis of the cell
– exocytosis
– budding – enveloped viruses (enveloped viruses are not
infective until they have acquired their envelope)

Release - budding

HIV

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 HIV replication
Cultivation of viruses

Cell cultures
Cultivation of viruses: only in
• Origin:
viable, receptive cells – human or animal
– fetal or adult
• cell cultures
– normal or tumor tissue
• embryonated egg
• primary cell cultures - monolayer
• experimental animals
• tumor cell lines
• diploid cell lines

Viral detection Embryonated eggs

• Morphological changes in cell culture • 5-14 days old embryonated egg
– cytopathic effects due to viral replication
• inoculation:
• rounded cells, dislocated cells
• cell necrosis
– Amnion sac
• multinucleated cells – Allantoic sac
– Inclusion bodies – Chorioallantoic membrane
• Cytoplasm – Yolk sac
• nucleus • Vaccine production
– malignant transformation

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 Experimental animals

Viral genetics

Viral genetics Viral genetics

• Give an understanding of: • viruses change very quickly

– pathogenesis – rapid growth – 5-10 thousands of viral
– life cycle particles/cell in 5 hours
– vaccine development – very short generation time – lots of mutants
• DNA viruses are more stable (proofreading)
– drug resistance

Changes in the viral genome Mutation

• a change in the viral nucleic acid which leads to

• by mutation changes of encoded proteins
• spontaneous mutations
• by recombination • induced mutations
– physically induced
• UV light
• X rays
– chemically induced

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 Possible phenotypic consequences
Mutation
of mutation
• conditional lethal mutants
nucleic – temperature sensitive mutants do not grow at higher
acid temperatures
• host range mutants
substitution – do not grow in all types of cells that the wild-type virus does
• drug resistance
• enzyme deficient mutants
insertion • hot mutants (better growth at higher temperatures) – less
susceptible to host fever response
• attenuated mutants – mild disease (or no symptoms at
deletion all)
– vaccine development
– pathogenesis
inversion

Recombination Recombination
classic recombination
• exchange of genes between two genomes DNA viruses
• homologous recombination
• In:
– viruses with ds DNA
– positive strand RNA
– viruses with segmented RNA genome (reassortment)

Recombination Copy choice recombination

- positive strand RNA
Vaccinia virus + strand

+ strand
Vaccinia virus –
vaccine against rabies

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 Copy choice recombination Reassortment

+ strand • recombination between viruses with

multiple segments (RNA)
• very efficient
• used in some new vaccines

Influenza virus
PB2 PB2
PB1
PA
X PB1
PA
HA
HA
NA NA
NP NP
M M
NS NS
PB2
PB1
Attenuated donor PA New virulent
virus HA strain
NA (epidemic,
NP
Resulting attenuated M pandemic)
vaccine strain: NS
Ag of the virulent strain
Virulence of the
attenuated strain

Non-segmented negative strand

Genetic reactivation
RNA viruses
• marker rescue: between the genome of
• no recombination an active virion and the genome of a virion
• no copy choice recombination that has been inactivated – certain
• no reassortment markers of the inactivated virus are
rescued and appear in the viable progeny
• least ability to exchange genetic
material
• multiplicity reactivation – when many
inactive virus particle interact in a cell to
produce a viable virus

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 Interference
• infection with 2 viruses can lead to an
inhibition of multiplication of one of the
viruses
– one virus may inhibit the ability of the second
to adsorb to the cell
– competition for components of replication
apparatus
– the first virus may induce the poduction of an
inhibitor
Complementation
Assembly:
wt N and wt M proteins
Genome: ts M or ts N
Defective interfering viruses
• decrease replication of helper viruses
(competition for viral precursors)
• may modulate wild-type infections
• may occur naturally: DI measles virus in
subacute sclerosing panencephalitis (SSPE) –
lacking maturation proteins
Complementation
• interaction at functional level, not the
nucleic acid
– one virus provides a gene product in which
the second is defective, allowing the second
to growth
Defective viruses
• lack genes necessary for complete
infectious cycle
• ‘helper’ virus provides missing function
• retroviruses (related helper)
• hepatitis delta virus (unrelated helper)
Phenotypic mixing
• a special case of complementation:
– in case of an infection with two similar viruses: the
progeny viruses acquire the phenotypic traits of
both parent viruses without modifying their
genome
• enveloped viruses: the envelope harbours
antigens of both viruses
– not necessarily closely related viruses
• naked capsid viruses: closely related viruses
(the intermixed capsid proteins must be able
to interact correctly to form an intact capsid
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 Phenotypic mixing Phenotypic mixing

Genome: unchanged
Altered host range
pseudotype – enveloped viruses
transcapsidation (phenotypic masking) – naked capsid viruses
Resistance to neutralizing antibodies

Genotypic mixing Genotypic mixing

= unstable genetic change – genetic

material from two distinct virus are
encased in the same capsid

Interactions among viruses Interactions among viruses

Non-genetic
activity Different progeny Stable Example
Type of Activity Different Genetically Example
interaction progeny stable Phenotypic active-active yes no picornavirus,
mixing adenovirus

Genetic
Genotypic mixing active-active yes no paramyxovirus
Recombination active-active yes yes influenzavirus,
poliovirus

Marker rescue active-inactive yes yes influenzavirus Complementation active-inactive no yes poxvirus

Multiplicity inactive- yes yes vacciniavirus

reactivation inactive

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 Bacteriophages with tail
Bacterial viruses
- head: icosahedral Head
Bacteriophages symmetry
- tube: contractile sheath
- base plate and fibers:
role in binding to Contractile tail
sheath
bacterial cell surface
tail fibers

Base plate

Bacteriophage with tail – electron Bacteriophage with tail – electron

microscopy microscopy

Phage infection Bacteriophages with tail

• Attachment
– tail fibers (LPS)
• Irreversible binding
through the base plate
• Penetration, uncoating

sheath contraction

nucleic acid injection

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 Bacteriophages Lysogenic conversion
• lytic (virulent) phages: • extra genes carried on the phage get
– phages multiply within the bacterial cell
– released through cell lysis
expressed in the cell
• lysogenic (temperate) phages
–genome integrates into the host genome • modify O antigens in Salmonella
– phage genome in repressed state = prophage, it is
replicated along the bacterial chromosome • toxin production in Corynebacterium
– cells carrying a prophage: lysogenic state diphtheriae
– lysogenic state can be terminated: induction → lytic cycle

Bacteriophages
• Have specific host range
– one phage – one bacterial species
– different phage susceptibility among strains of the same
species – phage typing of bacteria (Staphylococcus
aureus)
• Prophage
– when the prophage is released, bacterial DNA might be
captured in the phage genome and this can be
transferred to new bacterial cells (transduction)

Transduction – transfer of genetic

material from one bacterial cell to
another bacterial cell
Viroids

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 Viroids
– have no proteins, only nucleic acid
- infect plants
- single stranded circular RNA molecules,
intracellularly can form double stranded Prions
structures
- no virion phase
- transmitted by direct contact
- no genes, no translation
- pathogenesis not known

Prions Prion protein

• proteinaceous infectious agent, prion protein • highly conserved
(PrP) • role in the host ?
• rod shape – 10-20 nm/100-200 nm, aprox. 1000 • altered protein conformation (misfolded proteins)
assembled PrP associated with disease
• central nervous system, lymph nodes, spleen • conformation changes
– spontaneous by mutation
• coded by the host genome
– induced by infection
• no virion structure, no genome
• do not induce immune response

Prion protein Diseases

• normal prion protein: protease sensitive,
α-helix structure
• changed PrP: increased tendency for
aggregation (amyloid plaque formation),
protease resistant, β sheets
• increased resistance to heat (134°C),
detergents, disinfectants
• modified host proteins (= prion): transmit
the disease
• Unconventional slow virus diseases
• human diseases with neurological disorders:
– kuru
– Creutzfeldt-Jakob disease – CJD
– Gerstmann-Straussler-Scheinker disease
– Fatal familial insomnia
• animal diseases
– scrapie (sheep),
– bovine spongiform encephalopathy BSE (mad cow disease)
• animal – human transmission – not proven

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 Formation of PrP Prion diseases

• long incubation – several months - years

• progressively aggravating symptoms
• always fatal
+
• the brain is affected (vacuolization, decrease of
neurons, amyloid plaque formation)
• no inflammation
PrPc + PrPsc PrPsc • no antibodies
• hereditary forms

Creutzfeld-Jakob disease

• Forms
– Sporadic
– Yatrogen / acquired Pathogenesis of viral diseases
• injection, transplantation, contact with
contaminated medical devices
– Familial (hereditary)
– New variant (vCJD)
• high incidence of CJD in younger people in UK –
association with BSE epidemic?

• The ability of the virus to cause disease

depends on:

– cell – virus interactions

Virus – Cell Interactions

– cell – host interactions

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 Range of virus-cell interactions 1
• cytolytic, productive infection
– host cells are termed: permissive
• non-cytolytic, productive infection
– e.g. viruses released by budding
– may lead to persistent infection (the cell and
viruses co-exist for a longer period of time)
Range of virus-cell interactions 3
• the viruses enter the cell but are not
produced by the infected cell (the virus is
maintained within the cell in the form of its
genome)
– the cells are termed: non-permissive
– latency – the cell retains its normal properties
or
– malignant transformation
Transformation
• viral DNA integrated in cellular DNA or
non-integrated or both states
• the properties of the cells are changed –
tumour cells
• DNA viruses: adenoviruses,
herpesviruses, hepadnaviruses,
papillomaviruses, poxviruses
• RNA viruses: retroviruses
• transformation: rare event (1 in 105 cells)
Range of virus-cell interactions 2
• abortive (non-productive infection)
– the viruses do not complete the replication
cycle
• viruses that cannot adsorb to cell – resistant cells
or
• cells are infected but the viruses are not produced
by the cell – the cells are termed non-permissive
Latency
• the virus is present in the form of its
genome only and there is little (mRNA, not
at protein level) or no expression of viral
genes
• the genome is maintained throughout the
cell division
• more common with DNA viruses (e.g.
herpes viruses)
Virus – Host Interactions
INFECTION
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 Type of infections
Type of infections
• acute infection: short duration infection (several
days)
• latent infection: the patient recovers from the • According to the presence of symptoms
initial infection but the virus persist (in a hidden – symptomatic
or cryptic form) and the infection may recur
• chronic infection – the virus remains detectable – asymptomatic infections
for a long period of time (asymptomatic carrier
state or result in chronic illness)
• slow virus infections: prolonged period between
the initial infection and appearance of disease
(years) – e.g. SSPE, diseases caused by
unconventional agents (prions)

145 146

Stages of a typical viral infection
• Incubation period: asymptomatic
• prodromal period: nonspecific symptoms
• specific-illness period: specific symptoms
and signs
• recovery period: the illness resolves and
the patient regains good health
• in some cases: the infection persists,
carrier state or latent infection occurs
Infections in a population
• Sporadic – few cases, no relation betweens cases
• Endemic – infections occurring at a constant rate in a
defined geographical area
• Epidemic: the number of infections increases above a
baseline
• Pandemia – spread across continents
• Morbidity – the frequency of a disease in a population
• Incidence – new cases in 100000 inhabitants
• Prevalence – all cases reported to 100000 inhabitants
• Mortality – frequency of fatal cases due to a certain
disease
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Pathogenesis Pathogenic agent

• the signs and symptoms of a viral disease result
from:
– cell killing due to viral replication – loss of function • capable of causing disease
• e.g. polio kills motor neurons – paralysis
• property of a species
– cell damage due to immune response to the viral
infection • cannot be measured
• cytotoxic T cells destroy hepatic cells infected by hepatitis A,
B and C viruses
• virus-antibody-complement complexes deposited in tissues

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 Virulence Pathogenesis involves:
• transmission of the virus and its entry into
• ability to cause disease
the host
• property of a viral strain, measurable
• replication of the virus and damage to cells
• may increase or decrease
• spread of the virus
• viruses that have lost their ability to cause
• immune response as
a disease: attenuated viruses
– host defense
(vaccinology)
– cause of damage in certain viral infections
• persistence of the virus in some cases

Transmission Portal of entry

• person-to-person spread (horizontal transmission) • Skin
– direct transfer of respiratory secretions, saliva, blood, semen – Papillomaviruses – injuries
– Flaviviridae – transmitted by vectors
– indirectly by contamination of water, food
– Rabies virus – bite of an infected animal
– blood: transfusion or by sharing needles – Hepatitis B, C, D viruses, HIV – needles (blood)
• vertical transmission (between mother and offspring, • Respiratory tract – respiratory secretions
– Influenzavirus
perinatal infections) – Rhinovirus
– transplacental – in utero across the placenta – Respiratory syncytial virus
– at the time of delivery – Herpes viruses (herpes simplex virus 1, Epstein-Barr virus, cytomegalovirus,
varicella-zoster virus)
– during breast feeding – Measles virus
– Mumps virus
• animal to human transmission (zoonotic diseases) – Rubella virus
– directly from the animal reservoir (bite) – Hantavirus
– insect vectors (e.g. mosquito, tick) transfer the virus from the – Adenovirus
reservoir animal to human

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Portal of entry Localized or disseminated infections

• gastrointestinal tract – viruses resistant to acid pH, bile salts
– infections localized on the gastrointestinal tract (adenovirus, rotavirus)
• localized to the portal of entry
– systemic infection (Hepatitis A virus, enteric viruses, poliovirus)
– anus: portal of entry for some viruses (HIV, human papillomavirus) – common cold – involvement of the upper respiratory
• Genital tract – sexual intercourse tract
– Papillomavirus
– Hepatitis B virus • Influenzavirus
– HIV
– Herpes simplex virus 2 • Rhinovirus
• conjunctiva – adenovirus • Coronavirus
• transplacental
– Cytmegalovirus – Rotavirus - enteric disease -
– Rubella
– Herpes simplex virus – papilloma viruses: warts, cervical cancer
– Varicella-zoster virus
–
–
Hepatitis B virus
HIV
• spread systematically through the body
– Parvovirus B19
– e.g. poliovirus

155 156

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 Systematic infections Tropism
• spread through:
– bloodstream (viraemia), lymphatic system • range of host cells that can be infected by
– nerves a virus
– epithelial cells
• receptors
• role of macrophages
– inactivate the virus
• coreceptors
or
– the virus can multiply inside the macrophage and the
infected macrophages may distribute the viruses in the
body (HIV)

157 158

Elimination of viruses from the

body (viral shedding)
• skin lesions – Herpes simplex virus, papillomaviruses
• respiratory tract secretions
• saliva – mumps virus, Epstein-Barr virus, rabies
• faeces – enteric viruses, Rotavirus, hepatitis A virus
Immunity in viral infections
• urine – adenoviruses,
• genital secretions – HIV, hepatitis A, B viruses,
papillomavirus, herpes simplex virus 2
• milk – human T-cell lymphotropic virus 1 (HTLV-1), HIV,
cytomegalovirus
• blood – hepatitis B, C virus, cytomegalovirus, Epstein-
Barr virus, HIV, HTLV-1, arborviruses

Host defense against viruses The response to viral infections

• protection of the individual • Innate immunity

• elimination of viruses – Interferons
– Natural killer (NK) cells
– many infections are subclinical (asymptomatic) – Macrophages, dendritic cells
or autolimited • Acquired (adaptive) immunity
on the other hand – Humoral immunity
– virus-induced immunity may have – Cellular immunity
immunopathological consequences

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 Interferons (IFN) Biological effect of IFN
• factors produced by cells in response to viral • interact with cell surface receptors
infections that protect other cells of the same • after binding: up-regulation of some genes, down-
species from from attack by a wide range of regulation of others →
viruses – inhibit viral replication
• types: • inhibit attachment and uncoating
– α - 20 subtypes – produced by periferal blood • early viral transcription
mononuclear cells as a response to the presence of • viral translation
viruses (used also as therapeutic agent) • protein synthesis
– β - produced by fibrobalsts and epithelial cells as a • budding
response to the presence of viruses – activate host defense mechanisms
– γ - produced by T-lymphocytes, NK cells in response • enhancement of MHC-encoded molecules
• control of B cell response
to a specific antigen – (immune interferon)
• enhancement of cytotoxicity of immune effector cells (macrophages,
neutrophils, T cells, NK cells)

163

Effects of IFN INTERFERON

• induces resistance against viral infections

in the neighbouring cells
• IFN appears after a few hours from
infection
• important role in limiting the extension of
the infection

165 166

INTERFERON INTERFERON

antiviral state antiviral state

antiviral state antiviral state antiviral state antiviral state

antiviral state antiviral state

167 168

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 INTERFERON Natural killer (NK) cells
• distinct functional population of lymphocytes
• “natural”: active without prior exposure to the
antiviral state
virus, are not enhanced by exposure and are not
specific for any virus
• recognize and destroy cells infected by viruses
• they can kill without antibody, but antibodies
antiviral state antiviral state enhance their effectiveness (ADCC – antibody-
dependent cellular cytotoxicity)

antiviral state
169 170

NK cells Macrophages
• Functions:
– phagocytosis – role in limiting viraemia
– antigen presentation (limited role for viruses)
– cytokine production (IL1, TNF)
• Some viruses can replicate in
macrophages (cytomegalovirus, HIV)

Dendritic cells Humoral immunity

 antibodies (Ab) – bind to extracellular viral
• antigen presenting cells (important for
viruses)
• they present viral antigens through MHCI
and II – activation of both CD4+ and CD8+
T lymphocytes
• in the skin and mucosal epithelium
epitopes (inefficient against latent viruses and
those that spread directly from cells to cells)
 on virions
 Ab-s block binding to the host cell (stop attachment,
penetration)
 IgG, IgM – in serum, body fluids
 IgA – on mucosal surfaces
 uncoating may be inhibited
 aggregation of virions (phagocytosis)
 complement: opsonization or lysis of enveloped viruses
 on the surface of infected cells
 cell lysis with the aid of complement system, NK
 Antibodies can modulate or stripping antigens from the
cell surface to avoid cell destruction by other
mechanisms
174

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 Efficiency of humoral immunity
• depends on whether the virus passes through
the bloodstream to reach its target organ
– polio:
• intestinal wall – blood – spinal cord
• small amount of Ab: neutralize virions in the bloodstream
– influenzavirus:
• target organ: at the portal of entry
• large amounts of circulating antibodies relatively inefficient
• antibody must be present in the mucous secretions
• implications in vaccine strategy: live attenuated vaccines
(intranasal application) – efficient immune response

Cell-mediated immunity Cellular immunity

• destruction of infected cells
– before progeny viruses are released: termination of
infection
– condition: the immune system must recognize the
infected cell
• Ag-s processed in the infected cell – bound to MHC I -
expressed at the cell surface = recognition unit for cytotoxic T
lymphocytes (TC) CD8+
– induce apoptosis
• virus specific proteins, glycoproteins inserted in cellular
membrane during viral replication can act as signals
indicating the presence of the virus in the cell – antibody
binds to them – the cell is destroyed by ADCC (effector cells:
Fc receptor)

177

Strategies to avoid immune

response Antiviral drugs
• infection of the cells of the immune system
– rubella, mumps, measles, herpesviruses, HIV
• temporary immune deficiency against unrelated antigens and • the number of antiviral drugs is very small
sometimes to the infecting virus (predisposing to bacterial
infections) – difficulty to obtain selective toxicity
• permanent depression of immunity to unrelated antigens and to the
infecting virus (HIV) – intense replication in incubation period when
• antigenic variation
no signs of infections are present
• the production of antigens at sites that are inaccessible
to the immune system (e.g. viruses hiding inside the cell, – latent infections: drugs are inefficient
or viruses passing from one cell to another)
• latent infection – the virus remains in the cell and allows – drug-resistant mutants
no viral antigen expression on the cell membrane
(herpesviruses)

180

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 Inhibitors of viral nucleic acid
Inhibition of early events
synthesis
• Inhibitors of herpesviruses
• amantadine – Acyclovir
• Herpes simplex virus 1,2,
– prevents replication of influenza A virus by • Varicella-zoster virus
– Ganciclovir
inhibiting uncoating • cytomegalovirus
• Inhibitors of HIV
– Azidothymidine (AZT)
• effective against DNA synthesis by reverse transcriptase
– Dideoxyinosine
– Stavudine
– Lamivudine
– etc.
• Inhibitors of other viruses
– Ribavirin
• respiratory syncitial virus infections
• influenza B

Inhibitors of viral protein synthesis Inhibition of release of viruses

• Interferon
– treatment of chronic hepatitis B, C infections
– pegylated interferon (interferon alpha
conjugated to polyethylene glycol) – longer
half-life, once/week instead of 3 doses/week
• Zanamivir and oseltamivir (Tamiflu)
– influenza virus A and B
– inhibit the neuraminidase of influenza virus –
limit the spread of viruses to neighbouring
cells

Prevention of viral infections Active immunity

• Immunization • antigen stimulus - antibody formation

– active (attenuated/inactivated virus or viral • needs time to develop
antigens/subunit vaccines)
– passive (preformed antibodies)
• efficient and in most cases durable
• Objective • booster doses
– prevention
– therapy

185

31
 Live vaccines
• attenuated viruses
• greater and longer-lasting response compared
to inactivated viral vaccines
• the virus multiplies in the host, prolonged
antigenic stimulus
• production of IgA and IgG + cellular immunity
• contraindicated in case of immune deficiency,
pregnant women
• possibility to reverse to virulent form
• excretion of vaccine virus and transmission to
non-immune contact is possible
Inactive vaccines (killed viruses)
• shorter duration of immunity, lower
effectiveness compared to live vaccines
• IgG, weak or no cellular immune response
• reversion to virulent form is not possible
• excretion of vaccine virus and
transmission to non-immune contact is not
possible

Subunit vaccines Vaccination

• administered preexposure
• resemble in properties the inactivated • exception in case of infections with long
vaccines incubation period:
• no viral replication – rabies
– HBV

Vaccines Passive immunity

• active (live, attenuated) • naturally acquired: maternal IgGs are passed
– poliomyelitis, measles, mumps, rubella, through the placenta to the fetus
adenovirus, yellow fever, rotavirus, • artificially acquired: administration of preformed
influenza antibodies – immune globulins
• inactivated Characteristics:
– poliomyelitis, rabies, influenza, hepatitis • immediate protection
A, encephalitis viruses • short period of duration
• subunit Passive-active immunity is induced by giving both
– HBs Ag (hepatitis B virus surface immune globulins and vaccine (rabies, hepatitis
antigen), influenza B virus - HBV)

191

32
 Immune globulins against: Herd immunity
• Rabies • sufficiently large percentage of the
– administered at the bite site + intramuscularly population is immunized so that an
• HBV unimmunized person is protected
– for those exposed to infection by needle-stick
• prevention of transmission
– neonates born from HBV carrier mother
• Varicella-zoster
– for immunecompromised persons
• Hepatitis A virus
• measles

193

Universal System of Virus

Taxonomy
• viral family (familia) – according to viral
Viral pathogens and associated morphology:
diseases -viridae (Herpesviridae)
• subfamilies (subfamilia):
-virinae (Betaherpesvirinae)
• genera (genus) – physicochemical or
serological differences:
-virus (Cytomegalovirus)

International Committee on RNA viruses

DNA viruses
Taxonomy of Viruses Adenoviridae Arenaviridae
Bunyaviridae
• 4000 animal and plant viruses Hepadnaviridae
Caliciviridae
Herpesviridae
– 56 families Coronaviridae
Polyomaviridae Filoviridae
– 9 subfamilies Papillomaviridae Flaviviridae
– 233 genera Parvoviridae Orthomyxoviridae
– hundreds unassigned Poxviridae Paramyxoviridae
Picornaviridae
• 24 families – viruses that infect humans
Reoviridae
and animals Retroviridae
Rhabdoviridae
Togaviridae

33
 DNA viruses RNA viruses
• ds DNa • ss DNA • positive ss RNA • negative ss RNA
– enveloped – enveloped – enveloped – enveloped
– Herpesviridae – Parvoviridae • Togaviridae • Ortho-, Paramyxoviridae
– Hepadnaviridae • Flaviviridae • Rhabdoviridae
– unenveloped • Coronaviridae • Bunyaviridae
• circular DNA • Retroviridae • Arenaviridae
– Papovaviridae – unenveloped • Filoviridae
• linear DNA • Picornaviridae • ds RNA
– Adenoviridae • Caliciviridae
– unenveloped
– Poxviridae
• Reoviridae

Adenoviruses Adenovirus

• virion: cubic symmetry - icosahedron

• DNA double stranded, linear
• core proteins, small peptides
• capsid: 252 capsomeres, 12 pentons, 240
hexons
• fibers - antigens
• diameter: 70-90 nm

• human adenoviruses: 6 groups (A-F)

Diseases associated with

Pathogenesis
• adenoviruses infect and replicate in
epithelial cells of the respiratory tract, eye,
gastrointestinal tract, urinary tract, urinary
bladder, liver
• some viruses (grup C) persist as latent
infections many years in adenoids and
tonsils
adenoviruses
• Upper respiratory tract infections
– pharyngitis, common cold
• Lower respiratory tract infections - pneumonia
• Eye infections
– pharyngoconjunctival fever
• outbreaks, “swimming pool conjunctivitis”
– epidemic keratoconjunctivitis
• mainly in adults, contagious
• opacities on the cornea
• Gastroenteritis
• Other diseases
– acute haemorrhagic cystitis (in children, especially boys)
– encephalitis, meningoencephalitis
– infections in transplant recipients, patients with acquired
immunodefifiencies

34
 Classification
• Alphaherpesvirinae
– Simplexvirus
• Herpes simplex virus 1,2 (HHV1,2)
– Herpes B virus (simiae)
Human herpesviruses (HHV) – Varicellovirus
• Varicella-zoster virus (VZV/ HHV3)
• Betaherpesvirinae
– Cytomegalovirus
• Human cytomegalovirus (CMV/ HHV5)
– Roseolovirus
• Human herpesvirus 6,7 (HHV6,7)
• Gammaherpesvirinae
• Epstein-Barr virus (EBV / HHV4)
• Human herpesvirus 8 (KSHV / HHV8)

Structure Replication
• herpesviruses are indistinguishable by electron microscopy • Penetration by fusion with plasma
• genome: DNA, double stranded, linear, 120-230 kbp, 70-
200 proteins are encoded, reiterated sequences membrane
• capsid: 162 capsomeres, cubic symmetry • Nuclear site of replication
• envelope: derived from the nuclear membrane, contains
viral glycoprotein spikes • Capsids assemble in nucleus and bud
• tegument: an amorphous structure between the capsid and through nuclear membrane
envelope
• size: with envelope – 150-200 nm, without envelope: 100
nm

Herpesvirus - capside

35
 Genome Overview of herpesvirus diseases

• Latent infections
– primary infection (with or without symptoms)
– latency
– reactivation
• Malignancies
– Epstein-Barr virus
– Kaposi sarcoma virus
• Tropism
– epithelial cells
– neurons
– lymphocytes

Herpes simplex viruses Herpes simplex viruses

• types 1 and 2 (serologically distinguishable)
• differ in their mode of transmission:
– HSV 1 by contact (involving saliva)
– HSV 2 by sexual contact or vertically (mother to
infant)
• Pathogenesis: HSV causes cytolytic infections,
necrosis of the infected cell + inflammation
– intranuclear inclusion bodies
– cell-to-cell spread
• Primary infections
– usually mild infections, frequently asymptomatic
– portal of entry: mucosa or broken skin
• HSV1 oropharynx
• HSV2 genital mucosa
• Invasion of nerve endings – dorsal root ganglia –
latency
• HSV1 – trigeminal ganglia
• HSV2 – sacral ganglia
• Recurrent infections
– due to reactivations, triggered by different factors
• 5-10%: frequent clinical reactivation
• most individuals reactivation is clinically asymptomatic (virus shed!)

HSV1 latency Clinical findings

• Oropharyngeal disease
– primary infection:
• usually asymptomatic
• symptoms may occur in
– young children: gingivostomatitis,
– >5 ys old: pharyngitis
– recurrent infection: herpes labialis (cold sore)
• cluster of vesicles (usually at the border of the lip)
• pain
• healing without scarring
– factors inducing reactivation: immune deficiency,
stress, effort, sunlight, hormonal changes, fever

36
 Herpetic gingivostomatitis Cold sore, herpes labialis

Keratoconjunctivitis Keratoconjunctivitis
• HSV1
• corneal lesions
• lesions of the conjunctival epithelium
• recurrent keratitis: visual impairment,
blindness

Genital herpes 2/3

• usually caused by HSV2
• primary genital herpes
– can be severe
– vesiculoulcerative lesions of the penis/cervix, vulva,
vagina
of the acquisitions of genital
– painful
– fever, malaise, dysuria, inguinal lymphadenopathy
herpes come from clinically
• recurrences are milder asymptomatic partners
• some recurrences may be asymptomatic
(asymptomatic shedding of virus –
transmission!)

37
 Genital herpes Skin infections
• infection through cuts or abrasions
• herpetic whitlow – on fingers
– dentists, hospital personnel, thumb-sucking
children
• herpes gladiatorum (wrestling, rugby)

Herpetic whitlow Encephalitis

• HSV1
• sporadic encephalitis
• high mortality rate/residual neurological
defects
• half of the patients due to primary
infection, the rest due to recurrent infection

Neonatal herpes Neonatal herpes

• usually HSV2
• acquired in utero, during birth (75%) or
after
• severe, generalized disease (50%
mortality rate)
– lesions on skin, eye, mouth
– encephalitis
– involvement of organs

38
HHV3, Varicella-Zoster Virus (VZV) Pathogenesis
• Initial replication: respiratory tract
– epithelial cells (VZV can spread cell-to-cell)
• Primary infection: varicella (chickenpox) – the virus is spread by viremia to skin → lesions in
successive crops
• Latency: dorsal root or cranial nerve
• latency
ganglia
• recurrence: in case of depression of cell-
• Reactivation: herpes zoster (shingle) mediated immunity and other mechanisms of
viral activation;
– the virus replicates and is released along the neural
pathways to the skin, causing a vescular rash along
the dermatome

Varicella Varicella – papulovesicular rash

“dewdrop on a rose petal”

Zoster Zoster

39
HHV4 (Epstein-Barr Virus, EBV)
• “B-lymphocyte parasite”
• Outcomes of EBV infections:
– replication in B cells and epithelial cells
permissive for infection
– latent infection of B cells
– stimulation and immortalization of B cells
Immunity
• first antibodies: anti-VCA, anti-MA antibodies
• later: anti-EA antibodies
• after resolution of infection: anti-EBNA
antibodies
• T-cells: essential for limiting the infection
• infectious mononucleosis: results from a “war”
between infected B cells and protective T cells
– lymphocytosis (atypical mononuclear cells) results
from the activation and proliferation of T cells
– swelling of lymph glands, spleen, liver (T cell
proliferation)
Disease mechanisms of Epstein-
Barr Virus
• virus initiates infection of oral epithelia
• spreads to B cells in lymphatic tissue
• productive infection (B cell, epithelia)
– growth of B cells
• T cell response:
– kill and limit B cell outgrowth
• symptoms of infectious mononucleosis
– promote latency in B cells
EBV antigens
• different groups or proteins are expressed for
the different types of infections
– permissive cells: early genes are activated + lytic
cycle
• viral proteins: viral capsid antigen (VCA), membrane antigen
(MA), early antigen (EA)
– nonpermissive infection: some early antigens are
expressed
• Epstein-Barr nuclear antigens (EBNA), latent proteins (LP) –
essential for maintaining the infection and immortalization
• latent membrane proteins (LMP) – oncogene-like activity,
stimulate the growth of and immortalize B cells
• latency in memory B cells
Pathogenesis
• overactive immune response: infectious
mononucleosis
• lack of effective immune response:
lymphoma
• B-cell mitogen: B cell proliferation
Epidemiology
• EBV: transmitted in saliva
• 90% of EBV infected people intermittently
shed the virus for life
• children can acquire the virus in early life
– subclinical disease
• adolescents, young adults: “the kissing
disease”
• 70% of the population: seropositive by the
age of 30
40
 Diseases
• Infectious mononucleosis
– heterophile antibody positive
• EBV induced lymphoproliferative diseases
• people lacking T cell immunity
• transplant recipients
• AIDS
– Burkitt – lymphoma: B cell lymphoma of the jaw and
face
• children living in malarial regions of Africa
– Hodgkin’s lymphoma
– Nasopharyngeal carcinoma (epithelial cells)
Heterophile Antibody
• EBV induces polyclonal proliferation of B
cells
• One of the antibodies produced (heterophil
antibody) is able to agglutinate sheep red
blood cells – Paul-Bunnell test
– EBV monucleosis is heterophile antibody
positive
– CMV mono is heterophile antibody negative

EBV mononucleosis Burkitt lymphoma

Disease mechanism of CMV

HHV5 (Cytomegalovirus, CMV)
infection
• acquired from: blood, tissue, body
• common human pathogen
secretions
• most common viral cause of congenital
defects • productive infection of epithelial cells
• opportunistic pathogen in • latency in T cells, macrophages, other
immunocompromised patients cells
• cell-mediated immunity is required for
resolution and contributes to symptoms

41
 Diseases Microcephaly
• Heterophile antibody negative infectious
mononucleosis
• immun ocompromised patients: - pneumonia,
encephalitis, colitis, disseminated disease
• congenital infections – risk is especially high for
infants born to mothers who underwent primary
infection during their pregnancy
– cytomegalic inclusion disease (microcephaly,
hepatosplenomegaly, rash), hearing loss
• perinatal infections – no clinically evident
infection in full-term infants
– pre-term infants: hepatitis, pneumonia

HHV6 Roseola
• Exanthema subitum (roseola)
• Viral replication: CD4+ lymphocytes
• Receptor: other than CD4

HHV8 (Kaposi Sarcoma

HHV7
Herpesvirus, KSHV)
• no association with disease mucocutaneous neoplasm

• persists in salivary glands

• receptor: CD4, - HIV antagonism

42
 Human papillomavirus (HPV) Viral replication
• more than 100 serotypes • steps in viral replication are in parallel with the
• genome: DNA differentiation of the epithelium
• cubical symmetry – infection of cells in the basal layer
• no envelope – early genes: stimulate cell growth – thickening of the
• E1-7 regulatory (early) basal and the prickle cell layer (stratum spinosum)
proteins – as the basal cells differentiate – transcription of viral
• L1-2 – structural (late) genes
capsid proteins – terminally differentiated upper layer cells: expression
of late genes
– the virus is shed with the dead cells of the outer layer

HPV Diseases
• Narrow host range
• Oncogenic virus
• Tropism: • warts - benign proliferation of the skin
– squamous epithelial cells of the skin (cutaneous HPV - warts)
– mucous membranes (mucosal HPV - genital, oral, conjunctival
• benign head and neck tumours (HPV6, 11)
papillomas) – oral papillomas
– laryngeal papillomas
• Transmission
– direct contact • anogenital warts
– sexual intercourse
– during birth
– condylomata acuminata (HPV6, 11)

Warts Oral papilloma

43
 Anogenital warts Cervical dysplasia and cancer
• HPV 16, 18 – 70%
• in most cases (90 %): HPV infection is cleared in 1-2
years;
• cytological changes – koilocytes (detected by
Papanicolau smears)
• first changes detected by microscopy: dysplasia (regress
in 40-70%)
• cervical cancer develops through progressive cellular
changes: mild - CIN I (cervical intraepithelial neoplasia),
moderate - CIN II to severe lesions – CIN III, carcinoma
in situ (1-4 years)
• untreated CIN II/III can progress in invasive cancer

Cervical cancer

Normal Pap smear Abnormal Pap smear

Koilocytes: HPV infected epithelial cells – hyperchromatic nuclei, perinuclear

clear zone

Oncogenity Role of oncoproteins

• different according to serotype: • E5 protein
- low risk HPV (6, 11) – activates the receptors of growth factors
(EGF)
- high risk HPV (16, 18)
• E6 protein
Malignant transformation: due to early – binds p53 tumor suppressor protein
proteins encoded by E5, E6, E7 genes
• E7 protein
– binds Rb tumor suppressor protein

44
Development of cervical cancer • Co-factors in the development of cervical cancer
– genetic background
– smoking
– immune status

• Risk factors:
– sexual activity
– age at first intercourse
– oral contraceptive use
– occurrence of other sexually transmitted diseases

HPV infection and cervical cancer

Prophylaxis
vs age
Screening:
– cytology (Papanicolau smear)
– colposcopy,
– detection and genotyping of HPV (PCR, hybrydization)

Cervical cancer is a curable disease when detected

early!

Vaccination – VLP (virus like particles), L1, L2

Gardasil (6, 11, 16, 18)
Cervarix (16, 18)

Poxviridae Variola
• the largest and most complex viruses
• diseases are characterized by: rash,
proliferative lesions
• Variola virus: smallpox
• Molluscipoxvirus: molluscum
contagiosum – benign epidermal
tumour
– spread by direct and indirect contact
– sexually transmitted disease

45
 Molluscum contagiosum Viral morphology
• complex structure

• virion: brick-shaped or ellipsoid

• envelope: covered by tubular elements

• core: large viral genome (ds linear DNA)

• between the core and envelope: 2 lateral bodies

(unknown function)

Parvoviruses
• Parvovirus B19 causes
– erythema infectiosum (slapped cheek syndrome)
– aplastic anemia
– fetal infections
• Viral morphology:
– very small (22 nm)
– single-stranded DNA genome
– icosahedral symmetry
– nonenveloped virus

Pathogenesis and immunity Transmission

• Parvovirus B19 infects
– red blood cell precursors in the bone marrow • respiratory tract
(accounts for anemia)
• transplacental transmission
– endothelial cells (rash)
• blood transfusion
• lifelong immunity against reinfection

46
 Orthomyxoviridae
RNA viruses
• Influenza viruses
• associated with respiratory tract diseases
of humans and animals (mammals and
birds)
• myxovirus: these viruses interact with
mucins, glycoproteins on the surface of the
cells (myxa=mucous)

Orthomyxoviridae Structure
influenzavirus A, B, C • genome: 8 segments,
negative polarity ssRNA
• type A: • Proteins PB1, PB2, PA:
– humans, swine, aquatic birds, chickens, ducks, horses, polymerase components
seals
• nucleoproteins (NP) –
• type B:
– only humans
attached to the genome
• type C: • M – matrix protein (M1, M2)
– humans (swine) – associated with the
envelope
• 100 nm, helical symmetry • envelope: glycoproteins
• spherical/oval hemagglutinin (HA) and
neuraminidase (NA)

ORTHOMYXOVIRUS Viral replication

HA - hemagglutinin

NA - neuraminidase • binding of HA to cell receptors (sialic acid structures)

• internalized into a coated vesicle – endosome
nucleocapsid (RNA + • fusion of viral envelope and the endosome membrane -
NP protein) nucleocapsid is released into the cytoplasm
• nucleus: -ssRNA transcribed to mRNA
lipid membrane • translation to proteins
• HA, NA, M: inserted into cell membrane
polimerase components
(PB1, PB2, PA) • RNA replication in the nucleus – transported in the
cytoplasm; assembly with NP to form nucleocapsids
• genome segments are enveloped – budding through the
M1 cell membrane where HA, NA, M had been previously
inserted
Types A, B, C: according to NP, M1 proteins
Subtypes: HN

47
 Antigenic structure Hemagglutinin (HA)
• core antigens: • spike-shaped – elongated stalk capped by a
– nucleoproteins (A, B, C), RNA dependent RNA large globule
polymerase • binds the virion to the surface of the
– stable
susceptible cells
• outer antigens: glycoproteins (HA, NA)
• major antigen – antibodies directed against
H, N antigens
– H: 1-15
HA are neutralizing (protective)
– N: 1-9 • variability – continuous evolution of new
– subtypes: H1N1, H3N2, H5N1 strains
– changing structure

Neuraminidase (NA) Antigenic drift

• spike-shaped – slender stalk capped by • minor antigenic changes
a box-shaped head
• function: • point mutation in HA, NA genes, resulting in
– sialidase – facilitates release of virus amino-acid changes in the proteins
particles from infected cells during the
budding process
• preexisting antibodies cannot neutralize the
– prevents aggregation of virions
changed antigens
• antibodies formed against NA are not
fully protective
• sporadic illnesses, outbreaks (1-3 yearly)

Antigenic shift Pathogenesis

• major changes due to reassortment of genomes
among different strains (including animal strains)
• “new” HA or NA proteins, new subtypes
• only with the influenza A virus
• infrequent event (on average every 10 years)
• pandemics
• human-to-human transmission
• portal of entry: upper respiratory tract – the virus first
targets and destroys mucus-secreting and ciliated cells →
loss of primary defense system.
• NA cleaves sialic acid residues of the mucus → access to
cellular receptors
• cell-to-cell spread – lower respiratory tract –
desquamation of bronchial or alveolar epithelium to the
basement membrane
• promotes bacterial adhesion to the epithelial cells
• inflammatory cell response
• recovery: associated with interferon + cell-mediated
immune response
– classic flue symptomes (fever, malaise, headache, myalgia) are
due to interferon release

48
 Pathogenesis Disease
Antibody
Antibody Future
protection
T-cellT-cell • Influenza – respiratory tract infection
response
response
– self-limited in persons with intact immunity
Interferon
– most frequent complication: pneumonia
• elderly
• infants
Desquamation
Infection Replication of epithelial Influenza • chronic underlying diseases
cells – other complications: myocarditis, pericarditis,
encephalitis
Bacterial pneumonia secondary bacterial infection
– Reye’s syndrome mainly in children, adolescents:
primary viral pneumonia acute encephalopathy + hepatic disorders (salicylate
use)
CNS, muscle involvement

Characteristics of influenza Immunity

• humoral immunity: subtype-specific
A B C – antibodies against HA and NA
Severity of the
+++ ++ +
– HA antibodies decrease the chance of
disease infection
Animal reservoir yes no no
– antibodies against NA decrease the severity
Pandemics yes no no of the illness and the capacity to transmit the
Epidemics yes yes no
disease
Antigenic – antibodies: in serum, nasal secretions (IgA)
shift, drift drift drift
changes
• cellular immunity: type-specific

Epidemiology Pandemics
• annual outbreaks caused by
– influenza A: 1-3 yearly
• 1890: H2N2
– influenza B: 3-6 yearly • 1900: H3N8
• 3-5 million cases of severe illness, 250000-
500000 deaths worldwide • 1918-19: Spanish flu (H1N1 swine-like)
• Northern Hemisphere: January – April • 1957-58: Asian flu (H2N2)
• epidemics developed across continents:
pandemics (exclusively influenza A) • 1968-69: Hong Kong flu (H3N2)
• the size and the extent of an outbreak depends • 1977: H3N2 and H1N1
on the virulence of the new subtype/type and the
immunity in the population • 1990: H3N2 and H1N1

49
 Spanish flu Transmission
• Airborn respiratory droplets
• Contagion precedes symptomes

• human-to-human, animal-to-human
• reservoir: birds (in birds the infection is
usually asymptomatic)
• wild birds – domestic birds, animals –
humans

Transmission

• Bird – human: difficult (different receptors)

• Swine: both receptors are present (bird

and human), it can be infected by both bird
and human influenza viruses - in case of a
double infection of the swine reassortment
can occur (antigenic shift)

Diagnosis Treatment, prevention

• rapid tests (point-of-care tests based on • Amantadin (influenza A)
antigen detection) • Oseltamivir (Tamiflu) – NA inhibitor
• immunofluorescent assay – 2-4 hours • Zanamivir
• RT-PCR – 2-4 hours • vaccine
– inactivated virus (developed on embryonated egg) -
• culture: eggs, cell culture (3-10 days) trivalent
• serology – 2 weeks (seroconversion) – H, N split vaccine,
– live attenuated vaccine
• symptoms + epidemiological data

50
 2009 seasonal influenza vaccine
• Both TIV (Trivalent Inactivated Vaccine)
and LAIV (live attenuated influenza
vaccine)
– A/Brisbane/59/2007 – H1N1-like
– A/Brisbane/10/2007 – H3N2
– B/Brisbane/60/2008
Vaccines
• TIV (trivalent inactivated vaccine)
– Fluzone – Sanofi Pasteur
• regular
• high dose (>65 year old) – contains four times the amount of
antigen, provides stronger immune response
• intradermal (18-64 years old) – requires less antigen,
provides the same immune response as the regular one
– since 2011-2012 season
– Fluvirin – Novartis
– Fluarix – GSK
– Fluluval – GSK
• LAIV – live attenuated influenza vaccine
– FluMist - MedImmune

2011-2012 seasonal flu vaccine

• has the same composition as in the
previous year
• revaccination is recommended even for
those who received the vaccine in the
previous year, as immunity to influenza
declines after one year

2009 Influenza A H1N1

1998
PB2 bird
PB1 A/H3N2
PA bird
HA swine
NP swine
NA Asian swine
M Asian swine
NS swine
1918
1918
1918

51
 H1N1 in post-pandemic period
• WHO - Director-General's opening statement at
virtual press conference (10 August 2010):
„The world is no longer in phase 6 of influenza
pandemic alert. We are now moving into the post-
pandemic period. The new H1N1 virus has largely run
its course.”
„As we enter the post-pandemic period, this does not
mean that the H1N1 virus has gone away. Based on
experience with past pandemics, we expect the H1N1
virus to take on the behaviour of a seasonal influenza
virus and continue to circulate for some years to
come.”
2009 influenza
• 4572 deaths related to laboratory-
confirmed influenza in the WHO/Europe
region (5,1 deaths/1000000 population)
• Clinical consultation and influenza
positivity rates observed during 2009/2010
pandemic have been comparable with
recent influenza seasons.
• The age profile of persons at risk for worse
outcome differed (younger age group)

Avian influenza outbreak (H5N1)

• 1996 – China – Guangdong – geese
• 1997 – Hong-Kong – several cases of animal
disease
• 2003 February – first human diseases

CDC

H5N1 Paramyxoviridae
• Human illness • Paramyxovirus
– 335 cases, 206 deaths – Parainfluenza
– Mumps
– 2007: 72 cases, 48 deaths
• Morbillivirus
• Pneumovirus –
Respiratory Syncytial
Virus

52
 Morphology

• genome: - ss RNA, not segmented

• enveloped
• helical nucleocapsid: pleomorphic
• proteins:
– NP – nucleocapsid
– HN – hemagglutinin neuraminidase
– F glycoprotein – fusion, syncitium formation
– M – matrix protein
• antigenically stable

Parainfluenza viruses Mumps virus

• Parotitis (mumps)
• ubiquitous, infections are common • Pathogenesis:
– replication in salivary glands
• respiratory tract disease – viremia
– spread to the pancreas, testes, ovaries,
– usually mild coldlike symptoms kidneys, CNS
– in infants: laryngotracheobronchitis (croup) • Immunity: life-long (antibodies against HN)
• immunity: type-specific, short-lived • Prevention: attenuated virus
– mono-, bi- or trivalent vaccines
– trivalent vaccine: MMR (measles, mumps,
rubella)

Morbillivirus – measles virus Koplik’s spot

• Measles
– incubation 10-12 days
– high fever, cough, conjunctivitis,
– maculopapular rash
– Koplik’s spot (typical mucous membrane lesion, on the
buccal mucosa across from the molars)
• Pathogenesis:
– inhalation of droplets
– replication in the cells of respiratory tract, spread to
lymphatic system → viremia
– rash is caused by immune T cells directed against
infected endothelial cells lining small blood vessels

53
 Measles Measles

• Complications
– pneumonia, otitis media
– bacterial infections
– encephalitis
– SSPE (subacute sclerosing panencephalitis)
• Prevention
– attenuated viral vaccine
• monovalent vaccine
• trivaccine: MMR (measles, mumps, rubella)

Respiratory syncytial virus Togaviridae

• lower respiratory tract disease, Rubella virus
– from common cold to pneumonia – genome: + ss RNA
– icosahedral symmetry
• most common cause of fatal acute – envelope –
respiratory tract disease in infants and glycoprotein spikes
young children (bronchiolitis)
• it infects almost everyone by the age of 4
ys, reinfection occur throughout the life • rubella = little red
• “German measles”

Pathogenesis Rubella

• postnatal infection:
– 12-21 days incubation
– upper respiratory tract – local lymph nodes -
The lesions are less intense and
lymphadenopathy have not coalesced, as usually occurs in
measles.
– viraemia
– rash: maculopapular: face – body

54
• Congenital infection
– the virus can pass the placenta and replicate Prevention:
in the fetus Vaccine - MMR
– malformation
• heart
• eyes
• brain (deafness, mental retardation)

Picornaviruses causing
Picornaviridae human disease
• smallest viruses of vertebrates (28 nm) GENUS Virus
• + ss RNA Enterovirus poliovirus
Coxsackie A, B virus
• icosahedral symmetry
echovirus
• no envelope (naked virion) enterovirus (68-71)
Hepatovirus Hepatitis-A-virus
Rhinovirus rhinovirus
Cardiovirus encephalomyocarditis-virus
Aphtovirus Foot-and-mouth disease virus

Poliovirus Poliovirus 1, 2, 3
• types1, 2, 3
– portal of entry – digestive tract
– disseminated infection (90% of infections:
asymptomatic)
– poliomyelitis – most severe outcome of the
infection
• CNS - anterior horn cells of the spinal cord and
the motor cortex of the brain

55
 Polio

• Epidemiology
– fecal-oral transmission
– before vaccination: outbreaks
– 2002: wild type polio-free Europe
• Prevention: vaccination
– IPV – Salk – inactivated virus (parenteral)
– OPV – Sabin – attenuated virus (oral)

Coxsackie A, B viruses

• Transmission: fecal-oral, droplets

• Pathogenesis:
– A : skin – mucosa - herpangina
– B: heart, liver pacreas
– both: meningitis

56
 Rhinoviruses Caliciviridae

• common cold • Calix = cup

• only upper respiratory • + ss RNA
tract
• Icosahedral symmetry
• Immunity: type-specific
• No envelope
• Norovirus (formerly
Norwalk-like viruses)
• Sapovirus (Sapporo-like
viruses)

Disease caused by noroviruses Norovirus epidemics

• Kaplan criteria (when microbiological
• gastroenteritis (school-aged children, diagnosis is not feasible)
adults) – a mean illness duration of 12 to 60 hours,
• fecal-oral transmission – a mean incubation period of 24 to 48 hours,
– highly contagious – more than 50% of people with vomiting
• incubation: 24-48 hours – no bacterial agent found
• resistant (chlorinated tapwater)

Coronaviridae Coronavirus

• + ss RNA
• helical symmetry
• envelope – glycoproteins (E1-3)
• E2 spikes – solar corona-like
appearance

57
 Disease Reoviridae

• ds RNA, segmented
• common cold
• cubical symmetry
• SARS (severe acute respiratory
• double-layered capsid
syndrome)
• no envelope
• gastroenteritis

Rotavirus (Rota = wheel)

• common agents
of infantile
diarrhea

Pathogenesis Epidemiology
– portal of entry: digestive tract • ubiquitous
– target cells: enterocytes • only infants and small children have
– shortening and blunting of of the microvilli symptomatic disease

– diarrhea
– lactose intolerance
– viremia: respiratory tract

58
 Arboviruses Roboviruses
Arthropode borne Rodent borne
• Bunyaviridae
– Togaviridae – Alphaviruses – encephalitis Hantavirus genus
– Flaviviridae – yellow fever, tick encephalitis, nephritis, hemorrhagic fever
Dengue- fever
– Bunyaviridae – encephalitis, haemorrhagic
fever

Filoviridae
• Arenaviridae: arena = sand • filamentous
• haemorrhagic fever
Haemorrhagic fever • transmitted from human-
– Lassa - Nigéria to-human
– Junin – Argentina • Marburg, 1967
– Machupo –Bolívia • Ebola, 1976 Zair,
2003 Congo

Morphology Replication

– helical symmetry
– negative, ss RNA
– size: 900 nm/80 nm
– proteins, polymerase, glycoprotein
– envelope

59
 Haemorrhagic fever Rhabdoviridae
• Lyssa virus – agent of rabies
• sudden onset with high fever, malaise, – negative ss RNA
myalgia, sore throat – helical symmetry
• diarrhea, vomiting, rash – polymerase
• liver and kidney dysfunction, hemorrhages – envelope - spikes
– appearance: bullet

Rhabdovirus Pathogenesis
• bite of an animal
• the virus infects the nerve endings
• days to months – progress to the central
nervous system, then disseminates to
highly innervated sites: skin, salivary
glands, retina, cornea, renal parenchyma,
etc.
• fatal disease

Hepatitis viruses

• heterogeneous group of viruses

Hepatitis viruses • main target organ: the liver
– inflammation of the liver
• fever, gastrointestinal symptoms, jaundice
• regardless of the virus type: identical
histopathological lesions during acute disease

60
 Hepatitis A Virus (HAV)
Hepatitis viruses
Fecal-
Fecal-oral
A E transmssion

Hepatitis viruses NANB

B D C
Parenteral
F, G, transmission
? others

HEPATITIS A VIRUS (HAV) HAV

• Picornaviridae (enterovirus)
– genus: Hepatovirus
• genome: +ss, linear RNA
• icosahedral symmetry
• no envelope
• one serotype

Pathogenesis Pathogenesis (2)

• fecal-oral spread • hepatocytes: viral replication without direct
– HAV is ingested cytopathic effect
– enters the blood stream through the oropharynx or the linen
of intestines • new virions are released into the bile → stool
– → liver
• interferon limits bacterial replication
• short viraemia • NK cells, cytotoxic T lymphocytes: lysis of the
– 2 weeks before symptoms – few days after infected cells
• antibodies, complement and antibody-
• close contact favors transmission
dependent cellular cytotoxicity facilitates the
clearance of the virus
• icterus (jaundice) results from liver damage

61
 Disease HAV infection
• Incubation: 3-4 weeks Symptoms
• acute disease HAV antibodies

• no chronic hepatitis, no chronic carrier state ALT

• dark urine, pale feces, elevated transaminase
levels

Titer
• spontaneous resolution in 2-4 weeks HAV
in feces
• symptomatic/asymptomatic form:
HAV IgM
– children: 1/12
– adults: 1/3
• letality: 0,1%
0 1 2 3
Months 4 5 6 1 2
2 4

Immunity Epidemiology
• neutralizing antibodies (IgM, IgG) • worldwide occurrence
• IgM titer: peak level during the sixth week
• outbreaks (summer and autumn peaks)
of infection
• IgG antibodies persist lifelong – protection • source of infection: the infected person (most often:
asymptomatic)
against reinfection
• IgA: saliva, gastrointestinal mucosa - not • Common route of spread: fecal-oral
• Also possible through blood, saliva
neutralizing • Rarely: by sexual activity (anal, oral contact)
• cellular immunity

Geographic spread of HAV Prevention

• Vaccination – inactivated virus

• Passive immunization – human Ig

Seroprevalence
High
Medium
Low
Very low

62
 Diagnosis
• Serology - ELISA
– IgM – acute infection
– IgG – lifelong persistence
HEPATITIS B VIRUS (HBV)
• Hepadnaviridae
• Partially double-stranded circular DNA (a
complete negative strand and an
incomplete positive strand)
• Icosahedral symmetry
• Envelope - HBsAg (surface antigen)
Hepatitis B Virus (HBV)
Serum: 3 morphologic forms
• Whole virion (Dane): spherical – 42 nm
– Outer surface - envelope – HBsAg
– Inner part: nucleocapsid core – HBcAg
• Small spherical particles - 20-22 nm - made up
exclusively of HBsAg (empty particles)
– Not infectious
• Filamentous forms - 50-230 nm – resulted from
the aggregation of the small spherical particles
63
 HBV genome

• S gene – encodes HBsAg

– S region - stable
– pre-S1, pre-S2 – unstable (mutations during chronic
infection lead to evasion of immune response)
• C gene – encodes HBcAg and HBeAg
• P gene – polymerase (may function also as a reverse
transcriptase and ribonuclease)
• X gene – X protein – transcriptional transactivator

HBV proteins

• HBsAg - HB surface antigen

• HBcAg - HB core antigen
• HBeAg - HB e antigen
• HBxAg - HB x antigen
• DNA polymerase

Diagnostic markers found in

Pathogenesis
serum
• Source of infectious virions: blood, saliva, semen,
milk, vaginal and menstrual secretions, amniotic fluid
• Target cells: hepatocytes
• Viral replication starts within 3 days from infection – • HBs Ag ⇒ anti-HBs antibodies
symptoms appear later (after 45 days) • Hbe Ag ⇒ anti-HBe antibodies
• No direct cytopathic effect
• Cell-mediated immunity and inflammation are • Anti-HBc antibodies
responsible for causing the symptoms and clearing • DNA-polymerase
the infected cells
• Antibodies against HBsAg are protective • HBV-DNA
• Immune complexes formed between antibodies and
HBsAg may lead to hypersensitivity reactions –
vasculitis, rash, renal diseases, arthralgia

64
 HBV

Antigens in hepatocytes • Incubation: 45-180 days

(liver biopsy specimens) • Acute/chronic
• Carrier state
HBsAg • Symptomatic/asymptomatic infections
HBcAg • Oncogenic virus
HBxAg

Acute HBV infection

Complications of HBV infection
Symptoms

HBeAg anti-HBe

anti-HBc
• chronic hepatitis (5-10%)
Titer
• cirrhosis
HBsAg anti-HBc IgM anti-HBs
• hepatocellular carcinoma (HCC)

0 4 8 12 16 20 24 28 32 36 52 100

Weeks

Chronic HBV infection HBV infections in relation to age at the

100 infection 100
Acute Chronic
Chronic infection (%)

(6 months) (years)
Symptomatic infection (%)

80 80
HBeAg anti-HBe
HBsAg
60 60
anti-HBc Chronic infection
Titer
40 40

20 20
anti-HBc IgM

Symptomatic infection
0 0
1-6 months 7-12 months 1-4 years adults
0 4 8 12 16 20 24 28 32 36 52 Years
Weeks
Age at the infection

65
Geographic distribution of HBV infection HBV
• 300.000.000 infected people
• 2.000.000 deaths/year due to HBV
infections
• 700.000 deaths due to hepatocellular
carcinoma

HBsAg prevalence
≥8% - high
2-7% - medium
<2 - low

Transmission
• Parenteral spread
Romania – Blood, blood products (transfusion, needle
sharing, acupuncture, piercing, tattooing) –
- HBsAg prevalence: medium 0.0001 ml plasma
- carrier state - 7% of the population – Sexual contact (semen, saliva, vaginal
secretions) – more efficient transmission than in
case of HIV – 30%
– Perinatal route

Prevention Hepatitis C virus (HCV)

•Flaviviridae
• Hepacivirus
• Vaccination – recombinant HBs vaccine
• Infects only humans and chimpanzees
• Passive immunization – HB Ig
• genome: ss RNA
• icosahedral symmetry – C protein
• envelope
• spikes

66
 HCV HCV genome

• positive polarity ss linear RNA which encodes:

– core protein,
– 2 envelope glycoproteins (E1, E2) – variable structure
– 4 nonstructural proteins – NS
• 6 major genotypes (clades)
– Differ in their worldwide distribution
– Romania: 1b

HCV infection Acute HCV infection

anti-
HCV
• Incubation: 2-26 weeks symptoms +/-
• Transmission: parenteral
• acute/chronic HCV RNA
Titer

• Acute disease: mild (15-25%)

• Chronic disease: ≈75-85%
• No direct cytopathic effect
ALT
• Cell-mediated immunity responsible for tissue
damage
• Cancer development (5%) Normal

• Extrahepatic manifestations 0 1 2 3 4 5 6 1 2 3 4
Months Years
• Vasculitis, renal impairment
ALT – alanin aminotransferase

Chronic HCV infection Epidemiology

anti-
HCV • Most HCV-infected patients have a history of parenteral exposure to
Symptoms+/- the virus
– Intravenous drug users (IDU) – seroconversion in this group: 20%
yearly
HCV RNA • Most of them have chronic hepatitis
– Blood transfusion (before 1991)
Titer

– Recipients of plasma products

– Transplant recipients
– Haemodialysis patients
ALT – Health-care workers (needles-tick injuries)
– Tattoing and acupuncture
• Sexual contact:
– little evidence
Normal – Rarely (monogamous relation: the chance of transmission by sex 0-
0.6%),
0 1 2 3 4 5 6 1 2 3 4 – increased risk of transmission in case of HCV/HIV coinfection
Months Years • Mother-to-child transmission: 3-10% of infected mothers
– At birth

67
 Epidemiology HCV in Romania
• HCV incidence in Romania is the highest
in European Union
• 44.5 mortalities per 10 000 inhabitants
related to HCV
• 1 058 000 estimated cases (2007)

Prevention Hepatitis D (Delta) virus

δ agent
• Screening of blood donors HBsAg
• Standard precautions
• No vaccine
• Experimental: recombinant envelope
proteins

RNA

Hepatitis D virus (HDV) HDV

• Deltavirus
• envelope (HBsAg)
• genome: circular negative sense ss RNA –
very small

68
 HBV - HDV coinfection
HDV infection Symptoms

ALT
• Transmission: parenteral
anti-
• Coinfection HBs

Titer
anti-HDV IgM
–Severe acute disease
• Superinfection HDV RNA
–Chronic disease
HBsAg
–Unfavorable prognosis anti-HDV

– Cytotoxcity and liver damage Time

HBV - HDV superinfection Geographic spread of HDV

Icterus

Symptoms

anti-HDV
ALT
Titer

Taiwan
Pacific Islands

HDV RNA HDV prevalence

high
HBsAg
medium
low
anti-HDV IgM very low
no data
Time

Hepatitis E virus
Prevention
• HBV-HDV coinfection
- Prevention of HBV infection by
vaccination
• HBV-HDV superinfection
- Education, standard precautions

69
 Hepatitis E virus (HEV) HEV infection

• Caliciviridae • Incubation: 15-60 days

• Hepevirus • Transmission: fecal-oral
• No envelope • acute
• High mortality in pregnant women
• genome: positive sense ss RNA
(20%)

HEV infection
Geographical distribution of HEV
Symptoms

ALT
anti-HEV IgG

anti-HEV IgM
Titer

Virus in feces

0 1 2 3 4 5 6 7 8 9 1 1 1 1
0 1 2 3
Weeks
Weeks

HIV
Human immunodeficiency virus • Retroviridae
• Lentivirus
– slow viruses associated with neurologic and
immunosuppressive diseases
(HIV) – life-long infections
– cannot be eliminated from the organism
• HIV-1 – discovered in 1983/1984
– groups, subtypes (Ro. group M, subtype F)
• HIV-2 – 1986 (40% homology with HIV-1)
– Less transmittable, mostly confined to West Afria

70
 HIV structure HIV

• Size: 100 nm
• complex symmetry
• Capsid – polipeptidic antigens
• Envelope - glycoproteins, lipids
• Genome – 2 copies of positive, single
stranded, linear RNA molecules

HIV genome
– 3 groups of genes
• structural genes
• transcriptional activator genes
• accessory genes
Structural genes
• gag (group specific antigen)
– CA (capsid) – p24
– MA (matrix)
– NC (nucleocapsid)
• pol (polymerase)
– RT (reverse transcriptase)
– IN (integrase)
– PR (protease)
• env (envelope)
– SU (surface) – gp120
– TM (transmembrane) – gp41

Transcriptional activator genes Accessory genes

• encoded proteins are essential in viral
replication
• tat (trans-activator of viral transcription)
– enhances replication
• rev (regulator of viral expression)
– RNA transport
• nef (negative factor)
– reduces cell surface CD4 expression
– required to maintain high viral loads
– essential for the progression to AIDS (strains
lacking nef – long-term survivors)
• vif (virion infectivity factor)
– promotes assembly and maturation
• vpr (viral protein regulatory) – vpx in HIV2
– gives the virus growth advantage
• vpu (viral protein unknown)

71
 HIV genome Target cells, tropism
• Receptor: CD4+ T-lymphocytes, monocytes,
macrophages
• Co-receptor: chemokine receptor: CCR5 or CXCR4

– initially: gp120 binds preferentially to CD4 expressed by cells of

the macrophage lineage and to CCR5 on macrophages (M-
tropic)
– later during infection: gp 120 binds to CD4 on T lymphocytes
and to CXCR4 (fusion) (T-tropic)

• Receptors: adhesion
• Coreceptors: fusion

Viral attachment Chemokine receptors

• Persons genetically deficient in CCR5:

resistant against HIV infection
• ∼ 10% European population: CCR5
mutations
– selective pressure from the bubonic plague
(?)

HIV – receptor, coreceptor HIV replication

72
 CD4 antigen CD4 antigen
Co-receptor Co-receptor

CD4+ cell CD4+ cell

HIV gp120 to CD4 and co-receptor

HIV CD4 + Matrix

cell
gp4
1
Co-receptor

gp4 CD4
1 antigen

gp120

Gp120 binds to CD4 and co-receptor

Reverse
transcriptase

Protease
Integrase

tRNA

Conformational change
gp41 embeds into the cells membrane

73
 Fusion
Fusion

The nucleocapsid is inside

the cytoplasm

The nucleocapsid enters the cell

Nuclear
membrane

74
 RT

protease

integrase

RNA
HIV components enter
the nucleus
tRNA

Pathogenesis
• major determinant in pathogenesis:
tropism for CD4 + cells
• infection – viruses reach the lymph nodes
(within 2 days) – CD4+ T lymphocytes are
infected
• macrophages: major reservoir and means
of distribution of HIV
• continuous replication of the virus –
release of the virus and infected T cells
into the blood
Pathogenesis 2
• the virus can be detected in the blood and
circulating lymphocytes by 5 days
• as the immune system responds: p24 and
viral RNA decreases
– by 6-12 months:
• p24 becomes undetectable
• RNA level stabilizes (temporary increases in RNA
levels during intercurrent infections, immunizations
and pregnancy)

75
 Pathogenesis 3
• only a small fraction of CD4+ cells are
productively infected
• many infected cells are killed, a fraction survive,
revert to a resting memory state
→ long-term latent reservoir
• when exposed to antigen (foreign antigens):
activation – release of virions
• the proportion of infected cells and the level of
circulating virus increase as the infection
progresses
Pathogenesis 4
• reduction of the number of CD4+ T cells
– direct cytolysis – the ability of HIV to kill the cell
correlates with the amount of CD4 molecules
expressed by the cell (macrophages express less
than T lymphocytes)
• lysis of infected cells
• syncytia formation
• induction of apoptosis
• increased permeability of the plasma membrane
– cytotoxic T-cell immune cytolysis

Pathogenesis 5
• immune response: restricts viral infection
but contributes to pathogenesis
– neutralizing antibodies: against gp120
– antibody-coated virus: still infectious – is
taken up by macrophages
– cytotoxic T lymphocytes (CTL) – can kill
infected cells
• BUT: they require activation by CD4+ cells, CTL
number decreases with CD4+ cell number –
disease progression

76
 Untreated infected persons develop
HIV – escaping immune control
AIDS:
• rapid mutation – alteration of antigenicity • 5% within 3 years
– escape antibody clearance and the targeted • 20-25% by 6-7 years
killing of the CD4+ cell
• 5-10% each year
– several different viral genomes are present at
any time during an infection • <5% asymptomatic for more than 10 years
– regions of the envelope glycoproteins are the • 2% asymptomatic for more than 12 years
most variable

Laboratory markers associated with

Markers of HIV infection
progression of HIV infection
• nr of CD4 + lymphocytes
• increasing proportion of infected CD4+
cells HIV infection
AIDS
Serological window
• increasing viral load (HIV RNA copy
number) HIV RNA
anti GP120
antibodies

• isolation of virus in culture: rapid growth, P24

anti P24
antibodies
P24
syncytium formation)
• loss of antibody against p24
• loss of cutaneous hypersensitivity mo Year
s

RNA load / CD4 lymphocyte count The course of HIV infection

• primary infection
• clinical latency
• symptoms
– ARC (AIDS-related complex)
– AIDS
• opportunisitc infections
• tumours
• neurological disease

77
 Primary infection Clinical latency
• 2-4 weeks after infection • years
• influenza- or mononucleosis-like disease – • continuous replication of the virus in
generalized lymphadenopathy – the lymphoid tissue
symptoms disappear spontaneously after 2-3 • increased turn-over of CD4+ cells –
weeks replacement of destroyed cells → balance
• immune response – seroconversion (3
months –serological window)
• number of CD4+ cells decreases

ARC – AIDS-related complex AIDS – indicator diseases

• opportunistic infections
– protozoal: Toxoplasma gondii, Isospora belli, Cryptosporidium
parvum
– fungal: Candida albicans, Cryptococcus neoformans,
• CD4 + cells < 500/µl Pneumocystis jiroveci
– bacterial: Mycobacterium avium-intracellulare, Listeria
• insidious onset - non-specific symptoms monocytogenes, Mycobacterium tuberculosis, Slamonella
sepsis, pyogenic bacterial infections
– malaise, fever, fatigue, diarrhea, night sweat, – viral: cytomegalovirus, herpes simplex virus, Epstein-Barr virus,
weight loss, lymphadenopathy adenovirus
• opportunistic neoplasias
– Kaposi sarcoma (HHV8), B-cell lymphoma (EBV), anogenital
carcinoma (HPV)
• HIV encephalopathy
• HIV wasting syndrome

Prevention Control measures

• to maintain a lifestyle that minimizes or
• vaccines against HIV
– under development
eliminates the risk
– different stages of testing • blood donor testing
– recombinant viral proteins – likely candidates
– gene therapy approach: “intracellular immunization” – • health education
genetically alter target cells to become resistant to – general population
HIV
– difficulties in vaccine development: – HIV infected persons
• rapid mutations of the HIV
• lack of an appropriate animal model (chimps develop only
viraemia and antibodies but no immunodeficiency)

78
 Treatment Transmission
• Antiviral drugs
– reverse transcriptase inhibitors • high titers of HIV: blood, semen
– protease inhibitors
– fusion inhibitors
• sexual contact
• HAART (1996) – highly active antiretroviral
• blood products
treatment
– combination therapy
• vertical transmission
– prolongs life – does not cure

Sexual contact Blood and blood products

• Hetero-, bi- homosexual
– heterosexual 90%
– both sexes are equally affected
– sex workers – high risk
• less efficient transmission than HBV
– 0,1-0,2% for vaginal intercourse
– 0,1-0,3% for anal intercourse (receptive)
• oral sex - transmission has been documented, but not a
major route
• the presence of other sexually transmitted disease
increases the risk of transmission
• Cocaine use increases the risk of transmission in case of
oral sex
• most efficient route of transmission
• chance of transmission 90%
• IDU – needle sharing, tattoo
• transfusion (1 case/600000)
• nosocomial infection
• health care workers: at risk for HIV infection from
accidental needlesticks or cuts (seroconversion
occurs in less than 1% of those exposed to HIV-
positive blood)

Mother-to-infant transmission Bodily fluids which shed HIV

• with a role in transmission
• 13-40% in untreated women – blood
– in utero – genital secretions
– milk
– during birth
– breastfeeding
• without a role in transmission
– saliva
• high maternal viral load: risk factor for – urine
transmission – tears
– sweat
– CSF

79
 Romania

2005 2006 2007

141 (first 6
New cases 490 391
months)

General data on HIV/AIDS in

HIV Romania
Romania at 31.12.2008
Total AIDS cases 11311
1200
AIDS – children 7739
living 3414 1000
Death 4167 800
Lost from records 158
600
AIDS adults 3572
living 2226 400
Death 1235 200
Lost from records 111
0
Total HIV infected 4322 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002

children 2041
adults 2281

80
Cumulative HIV cases – adults and children Cumulative HIV cases (1992 – 2008) on children
(1992 – 2008) by counties (0-14 years at diagnosis date) by counties
yellow < 50 CASES yellow < 50 CASES
green 50-100 CASES green 50-100 CASES
blue 101-500 CASES blue 101-500 CASES
red > 500 CASES red > 500 CASES

HIV - Romania HIV – Mures county

• 50% of new cases: young people (15-29 years
old)
• route of infection: • total number of cases: 500
– sexual >78%
• living with AIDS: 200
– vertical >5%
– IDU 2%
• increasing risk – mainly in Bucharest and surroundings
• 2007-2009: 3-5 cases annually
• 2010: 12 cases
• 2011 – first 9 months: 62 cases

Classification of parasites
Parasites

Parasitology Protozoa Metazoa (Helminths)

Sarcodina Sporozoa Mastigophora Ciliata Platyhelminthes Nemathelminthes

Protozoa (amoebas) (sporozoans) (flagellates) (ciliates) (Flatworms) Roundworms

Helminths
Arthropods Trematoda (flukes)

Cestoda (tapeworms)

81
 Protozoa Protozoa
• eukaryotes • amoebas • Sporozoa
– Entamoeba – Isospora
– nucleus, necleolus, cytoplasm, – Naegleria – Cryptococcus
– special organelles for movement – Acanthamoeba – Plasmodium
– sexual and asexual reproduction • Mastigophora – – Toxoplasma
flagellates • Ciliata
• Sizes: 2-3 – 60-80 µm – Chilomastix – Balantidium
• Forms – Giardia
– Dientamoeba
– Vegetative form – trophozoite – active form
– Trichomonas
– Cysts – Trypanosoma
– Leishmania
488

Intestinal protozoa Entamoeba histolytica

• Intestinal tract • Ameoba
– Entamoeba histolytica • Disease: amebic dysentery and liver
– Giardia lamblia abscess (mostly in the tropics)
– Cryptosporidium • Life cycle:
– 3 stages:
• motile ameba (trophozoite) – found in intestinal
and extraintestinal lesions, diarrheal stool
• intermediate precyst
• nonmotile cyst – non-diarrheal stool,

Morphology
Entamoeba histolytica
• Trophozoite (Entamoeba dysenteriae)
dysenteriae)
– single nucleus with an even lining of peripheral
chromatin and a prominent central nucleolus
– cytoplasm: hyalin outer margin and granular intern
region
– pseudopodes (finger-like)
– engulfed erythrocytes (diagnostic importance!)
• Cyst:
– four nuclei
– thick wall
– chromatoidal bodies with round ends
492

82
 Entamoeba histolytica E. histolytica
(Entamoeba dysenteriae)
dysenteriae) ingestion of the cyst

excystation

replication (8 metacysts
metacysts)

coecum (ulcers)
ulcers)

colon (encystatio
(encystation
n)

494

Abscess formation due to

E. histolytica infection

495 496

Pathogenesis Flask-shaped ulcer

• ingested cyst transforms to trophozoites in the
duodenum
• disease results when the amoebas invade the
colonic epithelium
• proteolytic enzymes: necrosis
• “flask-shaped” ulcers
• progression into submucosa: invasion of the
portal circulation – extraintestinal disease (the
liver is the most frequently affected organ)

497

83
 Liver abscess Intestinal ulcerative lesions

Clinical findings Laboratory diagnosis

• dysentery – bloody, mucus-containing diarrhea,
lower abdominal discomfort, flatulence, • finding the trophozoites in diarrheal stool
tenesmus or the cysts in formed stool – wet mount
• Liver abscess • important criteria for the differential
– right-upper-quadrant pain diagnosis (differentiation from
– weight loss nonpathogenic entamoebas!)
– fever – aspect of nucleus
– tender, enlarged liver
– cyst size ad number of nuclei
– aspiration of liver abscess: brownish-yellow pus
• serology – invasive amebiasis
502

Epidemiology Giardia lamblia

• flagellate
• cysts – ingested through contaminated • Disease: giardiasis
water, food, vegetables
• Life cycle:
• asymptomatic cyst passers – main source
– 2 stages:
of contamination • trophozoite
• cyst

504

84
 Morphology Giardia lamblia
• trophozoite
– pear-shaped
– 2 nuclei
– 2 axostyles (rod-like supporting organelles)
– 4 pairs of flagella
– suction disk
– dancing motion (falling leaf motility)
• cyst
– 4 nuclei
– several internal fibers

506

Arturo Gonzalez, CINVESTAV, Mexico. Dr. Stanley L. Erlandsen

www-medlib.med.utah.edu

507 508
S.J. Upton

G. lamblia
- ingestion of cysts
- excystation - pH < 3
- attachment to intestinal
epithelium
-multiplication (binary
fission)
- encystation in the colon

509 510

85
 M.Lisci MD and G.Cera MD:

511 512

kleintiermedizin.ch

G. lamblia trophozoites Clinical findings

• nonbloody, foul smelling diarrhea

• nausea, anorexia, flatulence, abdominal
pains
• symptoms may persist for weeks, months

513

Epidemiology Cryptosporidium parvum

• sporozoan
– occurs worldwide
– ingestion of fecally contaminated water or
• disease: cryptosporidiosis
contaminated food – main symptom: diarrhea
– outbreaks can develop – severe in immuncompromised host
• life cycle:
– oocysts → sporozoites → trophozoites
(schizont) → micro- and macrogametes
(sexuate replication) → zygote → oocysts

515

86
 Pathogenesis, epidemiology Cryptosporidium parvum
• fecal-oral transmission of oocysts
• pathogenesis of diarrhea: unknown
• outbreaks of cryptosporidia: inadequate
purification of drinking water

518

Cryptosporidium parvum Urogenital protozoa

• Trichomonas vaginalis

519

Trichomonas vaginalis
• flagellate
• disease: trichomoniasis
• morphology: S.J. Upton

– pear-shaped organism
– one nucleus
– 4 anterior flagella
– the fifth flagellum delimits an undulating membrane
– axostyl
– no cyst form

521 522

87
 Trichomonas vaginalis T. vaginalis

524

Trichomonas vaginalis

• transmitted by sexual contact

• primary locations: vagina – prostate

• worldwide occurrence

525 526

Clinical findings Tissue protozoa

• Women: watery, foul-smelling vaginal
discharge + itching, burning
• Men: usually asymptomatic
– 10%: urethritis
• Toxoplasma gondii
• sporozoan
• infects a wide range of animals and birds but
does not cause disease in them
• final host: cat and its relatives
– sexual stage: fusion of gametes results in oocysts
– oocysts pass out through feces
– oocyst – contain 2 sporocysts – contain 4 sporozoits
each -

528

88
 T. gondii Life cycle
• intermediate host: animals, humans
– asexual reproduction
– ingested oocysts release sporozoites – circulate in
the body – invade different cells (especially
macrophages) → trophozoites, multiply, break out,
spread in the organism
• rapidly multiplying cells (tachyzoites) - responsible of acute
stage disease
– slowly multiplying toxoplasma cells (bradyzoites) –
form tissue cysts (pseudocysts) – responsible for
chronic stage disease
• pseudocysts are infective
– ingested by a cat – sexual reproduction
– ingested by other animal or humans – asexual reproduction

530

Morphology
• Trophozoites
– boat shaped thin-walled cells
– stain with Giemsa’s stain
• Cysts: cells filled with trophozoites

531

Trophozoites Pseudocyst

533 534

89
 Trophozoites of T. gondii Pathogenesis
• Tachizoite: destructs the infected cell
• usually asymptomatic – mononucleosis-like
symptoms may develop
• immune response
• congenital infections in case of the infection of
the mother during pregnancy:
– stillbirth, eye lesions, intracerebral calcifications,
psychomotor disturbances, hydrocephaly,
microcephaly
– prenatal toxoplasmosis is a major cause of blindness
535

Congenital toxoplasmosis Laboratory diagnosis

• infection during first semester: abortion, • detection of pseudocysts, trophozoites
major central nervous system anomalies • serology
• second-, third-trimester infections: • screening of pregnant women
– less severe damage
– clinical manifestation may be delayed (even
beyond childhood)

Epidemiology, prevention Helminths

• domestic cats have been icriminated in • Platyhelminthes • Nemathelminthes
transmission of T. gondii to humans – Enterobius
– Trematodes
– Trichuris
• prevention: • Fasciola
– Ascaris
• Schistosoma
– avoidance of contact with cat feces • Paragonimus – Toxocara
– proper handling of raw meet – Ancylostoma
– Cestodes –
– Necator
• Hymenolepis
• Taenia
– Strongyloides
• Echinococcus – Trichinella
• Dipylidium – Wuchereria
• Diphyllobotrium
540

90
 General properties
Cestoda
• Morphology – segmented parasites
– head – scolex
• suckers
Taenia group (giant and minute tapeworms) • anterior rings of hooks
Hymenolepis group – neck
Diphyllobotrium group (fish tapeworm) – body – strobila
• ribbon-like chain of segments (proglottids)
• each segment contains male and female reproductive organs
(hermaphroditic)
• no digestive system (nutrients are absorbed directly from the
gut lumen)

General properties Taenia solium

• Life cycle • “pork tapeworm”
– adult: intestine of the final host • final host: human
– larvae: develop in the tissues of intermediate • intermediate host: swine
hosts
• Morphology
– adult: 2-5 m;
• scolex: 4 suckers + 1 crown of hooks
– eggs: oncosphere (six-hooked embryo)

Taenia solium T. solium

scolex

adult in intestinal lumen

gravid proglottids

91
 Life cycle T. solium life cycle
• humans ingest pork containing small bladder-
like larvae (cysticerci) → release of larval
worms, attach to gut wall → adult
multisegmented worms (3 months) – egg-filled
terminal segments are eliminated with feces →
eggs are eaten by pigs → muscular larvae
(cysticerci)
• particularity: eggs can also infect humans, affect
various organs (brain, eyes) → cysticercosis

Pathogenesis Tania saginata

• “beef tapeworm”
• tapeworm in gut causes little damage • final host: human
• cysticerci – lesions (mainly in the brain) • intermediate host: cows
• Morphology
– adult: 8-10 m;
• scolex: 4 suckers, no hooks
– eggs: oncosphere (six-hooked or hexacanth
embryo)

Life cycle Adult T. saginata

• similar to T. solium

• cysticercosis does not develop

92
 T. saginata Taenia egg

• yellow-brown
• round to oval
• 30-40 µm
Proglottid
Scolex
• thick radially striated
shell
• oncosphere with
visible hooks

554

Taenia solium
Taenia saginata Echinococcus granulosus
• “dog tapeworm”, hydatid worm
• minute, three-segmented tapeworm
• Final host: dog, other carnivores
• Intermediate hosts: herbivores, humans
• Life cycle:
– herbivores ingest eggs – in the gut oncospheres are released –
spread to organs (especially liver) – the larvae form huge, fluid-
filled cysts (hydatid cyst) in which thousands of scoleces form
– dogs are infected when they ingest internal organs of herbivores
containing hydatid cyst
– humans are infected from dog feces – hydatic cysts can form in
different organs - hydatidosis

555 556

Echinococcus granulosus Hymenolepis nana

Adult worm
protoscolecses in hydatic cyst
Histopathology of hydatic cyst:
-fibrous outer memrane
-hyaline membrane
-capsules filled with protoscolecses
• “dwarf tapeworm” – 3-5 cm
• eggs are directly infectious, no intermediate host
is required (although they may have
intermediate hosts – beetles – in which
cysticercoids develop)
• fecal-oral transmission
• self-infection
• internal autoinfection
• infection is frequently asymptomatic
• children are affected usually (day care centers)

93
 Hymenolepis nana
Hymenolepis nana egg

• 40-60 - 30-50 µm

• 2 membranes,
Scolex: armed rostellum, 4 suckers filaments between
them

Hymenolepis nana adults • oncosphere

(hexacanth embryo)
560

Diphyllobotrium latum Diphyllobotrium latum

• “fish tapeworm” – 13 m long
• Final host: humans
• Intermediate hosts: crustacean and freshwater fish
• Humans are infected when they eat undercooked or raw
fish containing the larval forms
• Tapeworm in human gut causes little damage proglottids
– vitamin B12 deficiency – megaloblastic anaemia and neurologic
manifestations
• higher prevalence in northern hemisphere: Alaska, The scolex is elongated, 1 mm by 3 mm,
Canada, Russian Federation, Baltic countries, with two shallow, longitudinal grooves.
Scandinavian region

rosette-shaped uterus
at the center of each proglottid

Diphyllobotrium latum egg Operculated eggs of D. latum

• operculated eggs,
• 58-75 by 40-50 µm
• a small knob is
visible at the
opposite site of the
operculum

563

94
 Diphyllobotrium latum life cycle

Nemathelminthes
(Nematodes)

565

General properties Intestinal nematodes

• Cylindrical, unsegmented body • Enterobius (pinworm)
• More developed than flatworms • Ascaris (giant roundworm) transmitted by
• Separate sexes • Trichuris (whipworm)
ingestion of eggs

– the female is usually larger than the male

– the male typically has a coiled tail and spicule (male
genital organ) • Strongyloides (small roundworm)
• Well developed digestive tract • Necator and Ankylostoma
(hookworms) transmitted by
ingestion of larvae
• Intestinal nematodes • Trichinella spiralis
• Tissue nematodes

Tissue nematodes Disease caused by nematodes

• Filarial worms
– Wuchereria • as a result of the presence of adult worms
– Onchocerca • certain nematodes (larval forms) migrate
– Loa through tissues (Ascaris, Strongyloides,
• transmitted from person to person by Trichinella, hookworms) → increase of
mosquito bites or flies eosinophils (eosinophilia)

95
 Ascaris lumbricoides Life cycle
• the largest intestinal nematode (20-35 cm)
• life cycle:
– fertilized eliminated eggs become infectious
after 2 weeks spent on soil (larva formation) -
geohelminth
– the ingested egg releases the larva →
penetrates the duodenal wall → blood stream
→ liver → heart → lung → alveoli of the lung
→ coughed up, swallowed → small intestine:
maturation to adult form

Ascaris lumbricoides Egg production

1. both male and female are present:
– fertilized eggs
2. only female is present:
Female - unfertilized eggs
Male

3. only male adult is present:

- no eggs are produced

Tangled mass of Ascaris lumbricoides Spicule of male Ascaris

lumbricoides.

A. lumbricoides egg containing

Ascaris lumbricoides fertilized egg larva
• oval
• average size 55-75µm.
• thick shell – knobby-coated,
bile stained
• content: fertilized ovum.
– there is a new-moon shaped
clear space at the each end
inside the shell.

576

96
 A. lumbricoides egg containing Ascaris lumbricoides unfertilized
larva egg

• longer and slender than a

fertilized egg.
– content: granules

577 578

Clinical disease Epidemiology

• infection may be asymptomatic • Ascaris is the most common helminth
• occlusion worldwide
• perforation of the intestine – peritonitis • poor sanitation – human feces used as
• pulmonary migration of larvae: pneumonitis fertilizer
– hypersensitivity
• contaminated food and drinking water
– eosinophilia
• migration of adult forms due to fever, drugs,
anesthetics can lead to serious tissue damage

Trichuris trichiura Trichuris trichiura

• whipworm – it resembles the handle and
lash of a whip
• has the same epidemiology as Ascaris
• Life cycle:
– ingested eggs hatch into a larval worm in the
small intestine → cecum → penetrate the
mucosa → adult (3 months from the moment
of infection) → eggs
– eggs passed into the soil mature in 3 weeks
(geohelminth)
582

97
 Trichuris trichiura egg Life cycle
• dark bile staining
• lemon shape
• polar plugs in the
eggshell

583 584

Clinical disease Enterobius vermicularis

• related to the intensity of the worm burden
• abdominal pain and bloody diarrhea,
weakness, weight loss
• secondary bacterial infection may occur
(the heads of the worms are deeply
inserted in the intestinal mucosa)
• pinworm
• infection: confined to humans, most
frequently encountered in children
• Life cycle:
– ingested eggs hatch in the small intestine →
larvae differentiate in adults → migrate in the
colon, mating → the female migrates to the
anus and releases eggs onto the perianal skin
→ within 6 hours larvae are formed in the
eggs and become infectious

Clinical findings E. vermicularis adult forms

• perianal pruritus
• scratching predisposes to secondary
bacterial infection

• Diagnosis:
– eggs are not found in the stool
– scotch tape technique

98
 Enterobius vermicularis eggs
• elongated and
flattened on one side
• thick colorless shell,
• 50-60 / 20-30 mm
589
Epidemiology
• World-wide distribution with the highest
prevalence in temperate and cold climates
and in crowded conditions.
Trichinella spiralis larva in muscle
Life cycle
590
Trichinella spiralis
• disease: trichinosis
• Adult: lives in the small intestine of flesh-
eating animals
• Larva: encysted in striated muscles of
carnivorous and omnivorous mammals
• Most affected domestic animal: swine
T. spiralis female and male
99
 Life cycle
• ingestion of meat containing encysted
larvae → in the small intestine the larvae
develop into adults → females produce
larvae (vivipar) → blood stream → larvae
coil in striated muscles
• most commonly invaded muscles:
– muscles of eye, tongue, deltoid, pectoral and
intercostal muscles, diaphragm,
gastrocnemius muscle

• Epidemiology Clinical findings

– worldwide
• Clinical findings
– clinical presentation depends on the tissue burden
and location of larvae
• 10 larvae/g of tissue: asymptomatic
• at least 100 larvae/g of tissue: significant disease
• 1000-5000 larvae/g of tissue: very serious disease
– flue-like symptoms, diarrhea, fever
– eosinophilia, muscle pain, periorbital oedema
– “splinter” haemorrhages beneath the nails
– severe neurologic symptoms
– lethal trichinosis results in case of myocarditis, “splinter” haemorrhages periorbital oedema
encephalitis and pneumonitis combine

Tropical parasitic infections

• Protozoal infections • Helminthic infections
– malaria – human hookworm
– leishmaniasis disease
Tropical parasitic infections – trypanosomiasis – strongyloidiasis
– amebiasis – filariasis
– onchocerciasis
– dracunculiasis
– schistosomiasis

100
 Plasmodium species Plasmodium – life cycle
• asexual cycle:
• P. vivax, P. malariae, P. ovale, P. – schizogony in humans
falciparum • sexual cycle:
• disease: malaria – gametogony (production of gametes) in
• infect red blood cells and tissue (liver, humans – but to complete the sexual cycle
kidney, brain) the gametocytes must be taken up by
mosquitos
– sporogony (production of sporozoites) in
mosquito
601

Life cycle in humans

• sporozoites from the saliva of a female
Anopheles enter the bloodstream
– extraerythrocytic phase:
• hepatocytes – merozoites (asexual progeny) → erythrocytes
– erythrocytic phase
• multiplication in red cells – release – infect new red blood
cells
• release in synchronous pattern
– 3 days P. malariae
– 2 days for others
• some merozoites form gametes – must be sucked up by
Anopheles

Anopheles

Merozoite
• erythrocytes
Sporozoite
• salivary glands
• hepatocytes
Ookinete
• epithelial cells –
Anopheles gut

101
 cold stage
Clinical findings • feeling of intense cold
• vigorous shivering, rigor
• lasts 15-60 min

• incubation period
– exoerythrocytic phase + at least one
erythrocytic cycle
• 10-15 days – P. vivax, P. falciparum
• 28 days – P. malariae
• symptoms:
– severe chills, high fever, sweating + other
symptoms

hot stage sweating stage

• intense heat • profuse sweating
• dry burning skin • declining temperature
• throbbing headache • exhausted, weak → sleep
• lasts 2-6 hours • lasts 2-4 hours

Disease Disease evolution

• P. vivax – “benign tertian malaria” • exoerythrocytic cycles coexist with erythrocytic
– cycle of paroxysms every 48 hours
• P. ovale - “benign tertian or oval malaria” cycles
– similar to P. vivax • P. ovale, P. vivax: nongrowing resting forms
• P. malariae – “quartan malaria” (hypnozoites) after clearance from the peripheral
– periodicity of 72 hours
• P. falciparum – “malignant tertian malaria” blood
– quotidian (daily) attacks at beginning – then tertian attacks – the – breaking of hypnozoites → release of merozoites →
most serious form of malaria
– involvement of the brain – cerebral malaria (may result in coma
red cell infection = relapse
and death) – periodic relapses – up to 5 years
– kidney involvement – blackwater fever (may result in acute renal
failure) • P. malariae: in some cases long persistence due
– liver involvement to cryptic erythrocytic infection = recrudescence

102
 Laboratory diagnosis Thin film
• microscopic examination
– thick film (a method of concentration)
– thin film of blood – species identification

– specimens can be collected anytime, preferably

midway between paroxysms

• serology
– for epidemiological survey
– screening blood donors

Thick film

Plasmodium
falciparum
Plasmodium falciparum

103
 Plasmodium
vivax Plasmodium
vivax

Plasmodium vivax Plasmodium

vivax

• enlarged erythrocyte
• Schüffner’s dots
• ‘ameboid’ trophozoite
• many merozoites in mature segmenter

Plasmodium Plasmodium malariae

ovale

104
 Plasmodium Plasmodium
malariae malariae

Epidemiology Treatment
• Transmission:
– through mosquito bite • supportive measures
– rarely: congenital acquisition, blood transfusion
– often seasonal (rainy season) • chemotherapy
• endemic in Africa, Asia, Oceania, South America
• P. vivax
– chloroquine, primaquine
– most prevalent
– tropics, subtropics, temperate regions
• P. ovale
– tropical Africa (often more prevalent than P. vivax), Asia, South America
• P. malariae • chemoprophylaxis
– less prevalent
• P. falciparum
– tropical, subtropical regions

Hemoflagellates Trypanosoma

• Trypanosoma
• African: Trypanosoma brucei rhodesiense,
Trypanosoma brucei gambiense
• Leishmania
– sleeping disease (vector: tsetse fly)
• American: Trypanosoma cruzi
– Chagas’ disease (vector: triatoma)

105
 Morphology Trypanosoma in blood smear
• in the blood: trypomastigotes
– elongated body
– longitudinal lateral undulating membrane
– flagellum that borders the free edge of the membrane
– at the anterior pole of the cell: free whip-like
extension
• other developmental forms
– leishmanial intracellular stage - amastigote
– flagellated extracellular stage without undulating
membrane – promastigote
– elongated extracellular stage with short undulating
membrane - epimastigote

Pathogenesis Vectors of trypanosomas

• entry: bite of:
– tsetse fly (Trypanosoma brucei rhodesiense, Trypanosoma
brucei gambiense)
• multiplication at the site of entry: primary lesion (induration,
swelling) – chancre
→ lymph nodes → bloodstream → CNS
• sleeping sickness: lethargy, inability to eat, wasting,
unconsciousness, death
– triatoma – kissing bug (T. cruzi - Chagas’disease) – chagoma:
erythematous and indurated lesion at the site of entry
• edema around the eyes (Romana’s sign)
• migration to other tissues (cardiac muscle, brain, liver, etc) tsetse fly (Glossina sp) –
vector of Trypanosoma – Triatoma sp – vector of T. cruzi
• acute infection: fever, chills, myalgia, malaise, fatigue sleeping disease – Chagas disease
• most severe disease in children (<5 ys old) – CNS involvement

Sleeping sickness Chagas’ disease – Romana’s sign

chancre at the site of tsetse bite

106
 Diagnosis Leishmania
• blood, CSF, lymph node aspirates, bone marrow
• L. tropica - cutaneous leishmaniasis (oriental
• microscopy sore)
– thick, thin films • L. brasiliensis - mucocutaneous leishmaniasis
• culture (espundia)
• animal inoculation • L. donovani - visceral leishmaniasis (kala-azar)
• serology
• morphology: identical
• xenodiagnosis (Chagas’ disease) – laboratory – in mammals: intracellular nonflagellated amastigotes
triatoma bugs are fed on the patient – their feces (macrophages)
are examined in 7-10 days • vector: sandfly

Sandfly Pathogenesis
• L. tropica
– dermal lesion at the site of entry
– oriental sore, Delhi sore, etc.
• L. brasiliensis (Amazonian South America)
– nasopharyngeal leishmaniasis
– slow-growing extensive lesions
– erosion, destruction of nasal septum and surrounding regions
• L. donovani: inoculation → RES (liver, bone marrow,
spleen, lymph nodes), marked spleen, liver hyperplasia
– growing weakness
Phlebotomus - vector of visceral Leishmaniosis – irregular fever
– untreated is fatal

Mucocutaneous leishmaniasis (L.

Cutaneous leishmaniasis
brasiliensis)

107
 Kala-azar Diagnosis
• lymph node aspirates, scrapings, biopsies
• microscopic examination
• culture
• serology

Epidemiology Helminthic infections

• L. donovani – in most tropical, subtropical – human hookworm disease
countries – strongyloidiasis
– distribution related to prevalence of sandflies – filariasis
– reservoirs: – onchocerciasis
• Mediterranean littoral area, middle Asia, south
America: domestic and wild canids – dracunculiasis
• Sudan: various wild carnivores and rodents – loiasis
• kala-azar forms of India, Kenya: no reservoirs – schistosomiasis
• Oriental sore: Mediterranean region, North
Africa, Middle and Near East

Human hookworm disease Ancylostoma duodenale

• Nematodes: Ancylostoma duodenale (Old World
hookworm) & Necator americanus (New World
hookworm)
• Intestinal nematode
• Life cycle:
– larvae penetrate the skin → blood → lungs →alveoli
→ trachea: swallowed → become adults in the small
intestine
• attach to walls
– via cutting plates (Necator)
– via teeth (Ancylostoma)
• eggs passed in feces → rhabditiform larvae (noninfectious)
→ filariform larvae (infectious)

108
 Necator americanus Transmission and epidemiology

• filariform larvae in soil penetrate skin of

feet – walking barefooted predisposes to
infection
• humans are the only hosts
• worldwide, in tropical area

Pathogenesis and clinical finding Laboratory diagnosis

• major damage: loss of blood at the site of
attachment in the small intestine • detecting eggs in the stool
• anticoagulant made by the worm → the • occult blood in the stool is frequent
blood oozes from the site of attachment • eosinophilia
• weakness, microcytic anaemia, pallor
• at the site of entry of larvae: “ground itch”
• pneumonia with eosinophilia during
migration of larvae

Prevention Strongyloides stercoralis

• nematode
• strongyloidiasis
• life cycle
1. in humans: penetration of the skin (feet) by infectious (filariform)
larvae → lung → alveoli → bronchi, trachea → swallowed → in
• proper disposal of sewage the small intestine they transform into adults → penetrate the
mucosa → eggs;
• wearing shoes • eggs hatch in the mucosa forming rhabditoid larvae → feces;
• some larvae molt in to form filarial larvae (infectious) which penetrate
the intestinal wall → lungs … (autoinfection)
• larvae → warm soil: adult male and female
2. in soil:
• adult male and female mate:
• egg →larvae → adults
• filarial larvae: in contact with human skin: penetrate – initiate the parasitic
cycle in humans

109
 Strongyloides stercoralis Pathogenesis and clinical findings
• asymptomatic cases
• inflammation of the intestinal mucosa →
Tail of filariform larva watery diarrhea
larva in lung Filariform larva
– massive autoinfection: sepsis due to mucosal
injury
• pneumonitis (during migration of larvae)
• “ground itch”

free living female

• Epidemiology
Wuchereria bancrofti
– primarily in the tropics, especially Southeast
• filariasis
Asia
• tissue nematode
• Laboratory diagnosis
• vector: mosquito
– detecting the larvae in the stool
• Life cycle: bite of female mosquito → larvae in
– eosinophilia wound → migrate to the lymphatic system (in the
• Prevention arms, legs, groin) → adults: mate after 3 to 12
– proper disposal of sewage months microfilariae (larvae) → blood →
ingested by mosquitoes → after several stages
– wearing shoes
of development infective larvae are formed

Wuchereria bancrofti Pathogenesis, disease

• adult worms cause inflammation → blocks

lymphatic vessels → edema →
elephantiasis (massive edema of the legs)
• microfilariae do not cause symptoms
• chronic, repeated infection required for
symptoms to occur
• tourists, who typically are infected only
once, do not develop elephantiasis

110
 Elephantiasis Transmission, epidemiology
• transmitted by female mosquitoes:
Anopheles, Culex (depending on
geographic area)
– tropical regions of Africa, Asia, Latin America
– 200-300 million people are affected

Onchocerca volvulus
• Diagnosis • tissue nematodes
– thick blood smears • onchocerciasis (river blindness)
• Prevention • vector: blackfly
– mosquito control • Life cycle: bite of female blackfly in
– protective clothing, mosquito netting, subcutaneous tissue → adult worms (skin
repellents nodules) → females produce microfilariae
→ interstitial fluid → ingested by blackflies
→ infective larvae

Microfilariae Pathogenesis
• microfilariae migrate in subcutaneous
tissue, concentrate in eye: blindness
• adult worms: inflammatory nodules in skin
• scaly dermatitis “lizard skin” (thick, dry,
scaling skin)
• loss of subcutaneous elastic fibers:
“hanging groin” (inguinal region)

111
 Onchocerca volvulus Epidemiology
• Africa, Central America
• major cause of blindness
• river blindness: blackflies develop in rivers
• infection rate: >80% in edemic area
keratic precipitates and microfilaria
onchocerca nodule on inside surface of cornea
(adult curled up within)

Laboratory diagnosis Treatment

• biopsy of affected skin: microfilariae • surgical removal of skin nodules to
• examination of blood: not useful eliminate adult worms and stop production
• serology not useful of microfilariae
• antiparasitic drugs

Dracunculus medinensis Pathogenesis

• nematodes • adult female produces a substance that
• “little dragon of Medina”
causes inflammation, blistering and
• guinea fire worm – dracunculiasis
• Life cycle: tiny crustaceans (Cyclops sp)
ulceration of the skin (usually lower
containing infective larvae are swallowed in extremities)
drinking water → larvae released in small
intestine → retroperitoneal space where they
become adults and mate → the female migrates
to subcutaneous tissue (meter-long) → skin
ulceration → release larvae → water →
crustaceans eat larvae → molt to form infectious
larvae

112
 Dracunculus medinensis Loa loa
• Loiasis
• Tissue nematode
• Life cycle: bite of deer fly (mango fly) →
infective larvae crawl in the skin → adults
→ migrate subcutaneously; females
produce microfilariae → blood; ingested by
deer flies → infective larvae;

Transmission, epidemiology Pathogenesis

• hipersensitivity to adult worms causes
• humans are the only definitive hosts
swelling in the skin
• no animal reservoir
• adult worm seen crawling across
• endemic in central and west Africa conjunctivas
• laboratory diagnosis: microfilariae
detected from blood

Loa loa Deer fly

Microfilaria of Loa loa

113
 Schistosoma spp.
• schistosomiasis
• trematode (blood fluke)
• adults exist as 2 sexes but are attached to each other
• intravascular parasites approx. 1 cm
• life cycle: humans are infected by cercariae penetrating
skin → larvae → blood vessels → liver → adults →
portal vein: mesenteric venules (S. mansoni, S.
japonicum) or bladder venules (S. haematobium) →
eggs penetrate the gut or bladder wall → excreted →
hatch in fresh water → ciliated larvae

Schistosoma spp. eggs Transmission, epidemiology

• humans are definitive hosts
• snails: intermediate hosts
• endemic in tropical areas:
– S. mansoni: Africa and Latin America
– S. haematobium: Africa and Middle East
– S. japonicum: Asia

S. mansoni; S. haematobium; S. japonicum

Pathogenesis Laboratory diagnosis

• penetration of the intact skin by larvae - • Eggs visible in feces, urine
dermatitis
• eosinophilia
• S. mansoni damages the colon (inferior
mesenteric venules)
• S. japonicum damages the small intestine
(superior mesenteric venules);
• S. hematobium damages the bladder
• eggs in tissue induce inflammation, granulomas,
fibrosis, obstruction (in liver, spleen)

114
 • Fungi (yeasts and molds): eukaryotic
organisms
– nucleus, nucleolus
– cytoplasm: mitochondria and endoplasmic
Mycology reticulum are present
– cell membrane: contains ergosterol in contrast to
human cell membrane which contains
cholesterol
– cell wall – chitin = long chains of N-acetyl-
glucose-amin (NAG)
– may have capsule

686

Yeasts and molds Metabolism

• Yeasts: • Yeasts – aerobic, facultative anaerobic
– grow as single cells • molds – obligate aerobic
– reproduce by budding
– Gram-positive staining • require preformed organic source of carbon
• Molds: (association with decaying matter)
– grow as long filaments (hyphae) • Vitamins
• septate (transversal walls) • pH 5,5-7,2
• non-septate (multinucleated)
– produce mat (mycellium) • optimal temperature for growth: 20-30°C
• dimorphic fungi • moist environment
– molds in the environment at room temperature • growth: slower
– yeasts in human tissues at body temperature
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Replication of fungi Virulence factors

• Asexual – all fungi • toxins
– fission
– Candida albicans – exotoxin production –
• conidia - asexual spore formation (in case of fungi spores
are not only for survival but also for reproduction)
its role is unknown in pathogenesis
• budding – Aspergillus genus – exotoxins (aflatoxin,
• fragmentation of hyphae ochratoxin, clavacin, gliotoxin)
• Sexual – most fungi • others
– diploid status is transient – capsule (Cryptococcus neoformans)
– cell wall composition (Histoplasma capsulatum,
– donor cell’s nucleus enters recipient cell – fusion –
Aspergillus fumigatus, Candida albicans)
meiosis
– invasiveness – enzymes
– sexual spores (zygospores, ascospores, basidiospores)
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 Risk factors for fungal disease Mycoses
• Systemic mycoses
– coccidioidomycosis
– Histoplasma
• underlying diseases – tumours, diabetes – Blastomycosis
mellitus, immunosuppression • Opportunistic mycoses
– Candida
• trauma – burns – Cryptococcus
– Aspergillus
• medical interventions – catheters, corticosteroid – Mucor, Rhizopus
– Fusarium
therapy, antibiotics, cytostatic therapy – Pneumocystis
• alcoholics, drug addicts • Cutaneous, subcutaneous mycoses
– dermatophytoses
• medical personnel working in laboratories, – Malassezia furfur
– others
dermatology

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Coccidioides immitis Histoplasma capsulatum

Arthrospores
In the lung: spherule
Mold phase - macroconidia Yeast form in the
containing endospores
lungs

• Habitat
– widely distributed
– present in the normal flora
• Morphology
– oval/round yeast cells – blastospores (3-5 µm)
Candida genus – pseudohyphae – elongated yeast cells that
visually resemble hyphae but are not true hypae
– Gram-positive
C. albicans – Candida albicans
Non-albicans species: • Chlamydospores – on special culture media

C. krusei, C. parakrusei, C. tropicalis, C. pseudotropicalis, • Cultivation

C. glabrata, C. stellatoidea, C. lusitaniae, C. rugosa – Sabouraud agar
– large, butter-like colonies

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 Candida albicans Disease
• candidiasis/candidosis
• in the presence of risk factors
• in persons with normal immunity – local factors that allow
infection (disturbances of the balance of normal flora) –
benign infections
– oropharynx (soor, glossitis, gingivitis, stomatitis,
angulus infectiosus oris, atrophic candidiasis, others)
– genitalia (vulvo-vaginitis – favored by high pH, diabetes,
use of antibiotics, balanitis)
yeast cells and chlamydospore – skin, nails – when repeatedly immersed in water
Gram stain
pseudohyphae (wet mount) – organs (urinary, respiratory tract, CNS, disseminated)

698

Disease Diagnosis
• underlying diseases, immunosuppression – more severe
candidiasis, dissemination
• cultivation and identification to species
– disseminated oralis candidiasis level is important to guide therapy
– oesophagitis • non-albicans species are emerging and
– pneumonia involved more and more often in infections
– nephritis
– endocarditis
– sepsis
• Intravenous drug users, indwelling catheters, hyperalimentation also
predispose to disseminated candidiasis

699

Cryptococcus – negative staining –

Cryptococcus neoformans
large capsule
• widely disseminated
• soil containing bird droppings (birds are not infected)
• oval budding yeast – polymorphism, wide polysaccharide
capsid
• Infection: inhalation of yeast cells – lung infection is
usually asymptomatic
• impaired cellular immunity (especially AIDS):
disseminated infection, meningitis
• subcutaneous nodules are often seen in disseminated
infection

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 Aspergillus infections Aspergillus spp.
• Molds – widely distributed in the environment
• A. flavus, A. fumigatus
• Disease
– skin – burns, traumatic wounds, external ear
– pulmonary aspergillosis – neutropenic patients –
aspergilloma (fungus ball) – severe disease, high
mortality
– allergic bronchopulmonary aspergillosis – asthmatic
symptoms

703

Dermatophytes
Penicillium
• infections of skin structures
• only superficial keratinized structures are infected
• spread by direct contact,
• chronic infections – warm, humid areas of body
(athlete’s foot, jock itch); ringworm lesions; broken hair,
thickened broken nails;
– tinea capitis, corporis, pedis, barbae, cruris, unguium, manum
• scrapings of skin or nail placed in 10% KOH on a glass
slide show hyphae under microscope
• dermatophytid – hypersensitivity reactions

706

Microsporum

• Epidermophyton floccosum
• Microsporum spp.
• Trichophyton spp.

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 Athlete’s foot Tinea capitis

Dermatophytoses (Tinea)

ringworm lesions

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