1. introduction to virology

7/27/2019 1. Introduction to Virology

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Introduction to Virology

Oumer Ali (MD, MSc)

1



Nature of Viruses

Virus particles are called virions.

are composed of either RNA or DNA that is encased

in a protein coat called a capsid.

are either naked or enveloped, depending on

whether the capsid is surrounded by a lipoprotein

envelope.

replicate only in living cells and therefore areobligate intracellular parasites.

cannot be observed with a light microscope

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Differences between cells and viruses

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The viral genome

• may be single-stranded or double-stranded, linear or

circular, and segmented or nonsegmented.

• Single-stranded viral RNA genomes are further

subdivided into those of positive polarity , and those of

a negative polarity or are antisense (that is,

complementary to messenger RNA which cannot

therefore be used directly as a template for protein

synthesis

•

is used as one criterion for viral classification.• is associated with viral-specific enzymes, other

proteins within the virion, or both

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The viral capsid

is composed of structural units called

capsomers, which are aggregates of

viral-specific polypeptides.

has a symmetry that is classified as

• helical

• icosahedral (a 20-sided polygon),

is used as a criterion for viral

classification.

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Figure Nucleocapsid of a helical virus.

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The viral capsid

serves four functions:

– As protection of the viral genome

– As the site of receptors necessary for naked viruses

to initiate infection – As the stimulus for antibody production

– As the site of antigenic determinants important in

some serologic tests

The viral nucleocapsid:

– refers to the capsid and enclosed viral genome.

– is identical to the virion in naked viruses

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The viral envelope

The viral envelope

– surrounds the nucleocapsid of enveloped

viruses.

– is composed of viral-specific glycoproteins and

host-cell derived lipids and lipoproteins.

– contains molecules that are necessary for

enveloped viruses to initiate infection,

– act as a stimulus for antibody production,

– and serve as antigens in serologic tests.

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Characteristics Used to Define Virus Families,

Genera, and Species

Viruses are divided into related groups, or

families, and, sometimes into subfamilies

based on:

1) type and structure of the viral nucleic acid,

2) the strategy used in its replication,

3) type of symmetry of the virus capsid (helical

versus icosahedral), and

4) presence or absence of a lipid envelope.

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Characteristics Used to Define Virus

Families, Genera, and Species

Within a virus family, differences in additional specific

properties, such as host range, serologic reactions,

amino acid sequences of viral proteins, degree of nucleic

acid homology, among others, form the basis for division

into genera (singular = genus) and species (Figure ).

Species of the same virus isolated from different

geographic locations may differ from each other in

nucleotide sequence. In this case, they are referred to as

strains of the same species.

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Figure Classification of viruses:

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Viral replication

occurs only in living cells.

involves many host-cell enzymes and

functions.

may be incomplete in some cells (abortive

infection)

may lead to the death of the host cell (virulent

viruses)

or may occur without apparent damage to the

host cell (moderate viruses).

is similar for all viruses in a specific family.

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Replication process

has a sequential pattern that includes thefollowing steps:

Attachment

Penetration

Uncoating of the viral genome

Synthesis of early proteins involved ingenome replication

Synthesis of late proteins (structuralcomponents of the virion)

Assembly

Release

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Attachment

involves the interaction of viral receptors

and specific host-cell receptor sites.

plays an important role in viral pathogenesis,

determining viral cell tropism.

may be inhibited by antibodies (neutralizing

antibodies) against viral receptors or cellular receptor sites.

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Penetration

can occur by a cellular mechanism called

receptor-mediated endocytosis

can involve the fusion of the virus envelope

with the plasma membrane of the host cell.

Uncoating

refers to the separation of the capsid from

the viral genome.

results in the loss of virion infectivity

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Budding

is the process by which enveloped virusesobtain their envelope.

is preceded by the insertion of virus-specific

glycoproteins into the membranes of a hostcell.

occurs most frequently at the plasma

membrane, but also occurs at other membranes.

confers infectivity to enveloped viruses

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Diagnostic Methods in Virology

Direct Examination

Indirect Examination (Virus Isolation)

Serology

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1. Electron Microscopy morphology / immune

electron microscopy

2. Light microscopy histological appearance - e.g.

inclusion bodies

3. Antigen detection immunofluorescence, ELISA etc.

4. Molecular techniques for the direct detection of

viral genomes

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Electron Microscopy

106 virus particles per ml required for visualization, 50,000 -

60,000 magnification normally used.

Viruses may be detected in the following specimens.

Faeces Rotavirus, Adenovirus

Vesicle Fluid HSV

VZV

Skin scrapings papillomavirus,

molluscum contagiosum

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Electron Microscopy

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Problems with Electron Microscopy

Expensive equipment

Expensive maintenance

Require experienced observer

Sensitivity often low

Requires at least 105 to 106 / ml particles in

preparation

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Immune Electron Microscopy

The sensitivity and specificity of EM may be enhanced by

immune electron microscopy. There are two variants:-

Classical Immune electron microscopy (IEM) - the sample is

treated with specific anti-sera before being put up for EM.

Viral particles present will be agglutinated and thus congregate

together by the antibody.

Solid phase immune electron microscopy (SPIEM) - the grid iscoated with specific anti-sera. Virus particles present in the

sample will be absorbed onto the grid by the antibody – easily

observed

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Light Microscopy

• Histological changes in infected cells

• Viral inclusion bodies are basically collections

of replicating virus particles either in the

nucleus or cytoplasm.

• Though not sensitive or specific , they are

useful adjunct in the diagnosis

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Inclusion bodies

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Inclusion bodies

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Identification of virus

Effect on cell culture

CPE

Inclusion body formation

Giant cell formation

Neutralization with specific serum

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Cytopathic Effect

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Effect of enterovirus 71 and HSV in cell culture: note the

ballooning of cells. (Virology Laboratory, Yale-New

Haven Hospital, Linda Standard, University of Cape

Town) Cytopathic



Serology

Serologic diagnosis is based

on a greater-than-fourfold rise in IgG antibody

concentration when acute- and convalescent-phaseserum samples are analyzed at the same time.

A simultaneous fall in IgM antibody confirms recent

primary viral infection

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Serology

Criteria for diagnosing Primary Infection

• 4 fold or more increase in titer of IgG or total antibody

between acute and convalescent sera

• Presence of IgM

• Seroconversion

• A single high titer of IgG (or total antibody) - very unreliable

Criteria for diagnosing Re-infection

•4 fold or more increase in titer of IgG or total antibodybetween acute and convalescent sera

• Absence or slight increase in IgM

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Usefulness of Serological Results

How useful a serological result is depends on the individual virus.

1) For viruses such as rubella and hepatitis A, the onset of clinicalsymptoms coincide with the development of antibodies. The detection

of IgM or rising titers of IgG in the serum of the patient would indicate

active disease.

2) Many viruses often produce clinical disease before the appearance of

antibodies such as respiratory and diarrhea viruses. So in this case,

any serological diagnosis would be retrospective and therefore will not

be that useful.

3) There are also viruses which produce clinical disease months or years

after Seroconversion e.g. HIV and rabies. In the case of these viruses,

the mere presence of antibody is sufficient to make a definitive

diagnosis.

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Problems with Serology

Long period of time required for diagnosis for

paired acute and convalescent sera.

Mild local infections such as HSV genitalis may not

produce a detectable humoral immune response.

Extensive antigenic cross-reactivity between related

viruses e.g. HSV and VZV, Japanese B encephalitis

and Dengue, may lead to false positive results.

immunocompromised patients often give a reduced

or absent humoral immune response.34



Problems with Serology

Patients with infectious mononucleosis and those with

connective tissue diseases such as SLE may react non-

specifically giving a false positive result.

Patients given blood or blood products may give a

false positive result due to the transfer of antibody.

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Typical Serological Profile After Acute

Infection

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Note that during reinfection, IgM may be absent or

present at a low level transiently



ELISA

Direct EL ISA

Direct ELISA can be regarded as the simplest form

of the ELISA

Antigen is attached to the solid phase by passive

adsorption.

After washing, enzyme-labeled antibodies are

added.

After an incubation period and washing, a substrate

system is added and color is allowed to develop

Not commonly used

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Direct ELISA

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ELISA

Ind irect ELISA

The indirect system is similar to the direct system in thatantigen is directly attached to the solid phase

and targeted by added antibodies (detecting antibodies).

However, these added antibodies are not labeled

with enzyme but are themselves targeted by antibodieslinked to enzyme.

Such antibodies are produced against theimmunoglobulins of the species in which the detectinganti-bodies are produced and are termed antispeciesconjugates

After an incubation period and washing, a substratesystem is added and color is allowed to develop

Ind irect ELISA is widely used in diagnosis

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INDIRECT ELISA

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Microplate ELISA for HIV antibody: coloured wells indicate reactivity



Western blots

Western blots can confirm the presence of

antibody to multiple specific viral proteins

simultaneously.

The proteins are separated by size and

transferred to an inert membrane, where they

are incubated with serum antibodies.

Western blots are inherently difficult to

quantitate

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Western blots

HIV-1 Western Blot Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate Sample C: Positive

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IMMUNOFLOURESCENCE

Flourescent dies can be covalently

linked to antibody molecules and made

visible by UV light in the flourescentmicroscope

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IMMUNOFLOURESCENCE

The direct fluorescent antibody (DFA) test

The DFA test is used to detect antigen in

the patient.

The labeled antibody directly interacts with

unknown antigen

The sample could be any tissue suspected

of infection with a specific organism.

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IMMUNOFLOURESCENCE

The indirect fluorescent antibody (IFA) test

The IFA test is used to detect antibody in the patient

Known antigen is attached to the slide

The patients serum (unlabelled) is added

Flourescent labeled antihuman antibody added &

examined by UV microscopy

If the test Ag is fluorescent following these steps,then the patient had antibody against this antigen

in their serum.

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POLYMERASE CHAIN REACTION

Detection of this amplified product (amplicon) indicates that the

sample is positive for the target virus .

It is based on an enzymatic reaction involving the use of

synthetic oligonucleotides flanking the target nucleic

sequence of interest.

These oligonucleotides act as primers for the thermo stable Taq

polymerase. Repeated cycles (usually 25 to 40)

denaturation of the template DNA (at 94oC)

annealing of primers to their complementary

sequences (50oC

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PCR -principles

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PCR ti



PCR reactions Thermostable DNA polymerase is used for the PCR.

– This enzyme, which is isolated from thermophilic

bacteria, like for example Thermus aquaticus ,

– does not denature at high temperature and remains

active through the entire PCR reaction.

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PCR ti



PCR reactions To perform PCR we just need to mix in the reaction tube:

1. DNA containing the gene to be amplified

2. Primers complementary to the flanking regions of the gene

3. Thermostable DNA polymerase

4. Nucleoside triphosphates (dATP, dGTP, dCTP, dTTP) and

then place the reaction mixture in the PCR machine that can

rapidly change the temperature of the reaction mixture

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Molecular methods

The advantage of molecular techniquesare

their sensitivity

specificitySafety

These technique do not require

isolation of the infectious agent and canbe performed on chemically fixed orinactivated samples or extracts.

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1. introduction to virology

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