Introduction to Virology

Oumer Ali (MD, MSc)

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 are

obligate intracellular parasites.

cannot be observed with a light microscope
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Differences between cells and viruses

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.

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 the following steps: Attachment Penetration Uncoating of the viral genome Synthesis of early proteins involved in genome replication Synthesis of late proteins (structural components 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 viruses obtain their envelope. is preceded by the insertion of virus-specific glycoproteins into the membranes of a host

cell.
occurs most frequently at the plasma membrane, but also occurs at other

membranes.
confers infectivity to enveloped viruses
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Introduction to viral Diagnostics

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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. 4. Antigen detection immunofluorescence, ELISA etc. 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 Vesicle Fluid Rotavirus, Adenovirus 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 is

coated 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

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
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Serology
Serologic diagnosis is based on a greater-than-fourfold rise in IgG antibody concentration when acute- and convalescent-phase

serum 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 antibody between 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 clinical

symptoms 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.
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Problems with Serology

Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react nonspecifically 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

Note that during reinfection, IgM may be absent or present at a low level transiently
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ELISA
Direct ELISA

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
Indirect ELISA The indirect system is similar to the direct system in that antigen 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 antibodies linked to enzyme. Such antibodies are produced against the immunoglobulins of the species in which the detecting anti-bodies are produced and are termed antispecies conjugates After an incubation period and washing, a substrate system is added and color is allowed to develop Indirect 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

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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 flourescent

microscope

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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

PCR was the first nucleic acid amplification technique to be described and, the most widely used molecular diagnostic technique in clinical virology. PCR is a process by which a portion of viral nucleic acid (DNA or RNA) is replicated a million times or more.

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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 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 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 techniques are their sensitivity specificity Safety These technique do not require isolation of the infectious agent and can be performed on chemically fixed or inactivated samples or extracts.
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