Section II - Virology By Dr. Kareem Lilo
Examples of Cytopathic Effects of Viral Infection
• Nuclear shrinking (pyknosis) • Margination and breaking of chromosomes
• Proliferation of nuclear membrane • Rounding up and detachment of cultured cells
• Vacuoles in cytoplasm • Inclusion bodies
Tissue Culture Infectious Dose: TCID50 Measure cytopathic effects other than lysis
Concentration of virus it takes to produce cytopathic effect (CPE) in 50% of the dishes of cells
infected with virus (Table 2-2)(Figure 2-11)
Table( 2-2) Example of endpoint dilution data
Figure 2-10 example at cytopathic effect A-Inclusion bodies , BSyncytia
Section II - Virology By Dr. Kareem Lilo
Plaque assays Lytic viruses only
• Steps Serial dilution of virion-containing solution
• Create tissue culture plates
• Overlay with agar—prevents diffusion
• Each plaque represents 1 PFU (Plaque Forming Unit)(Figure 2-12)
Figure 2-12 plaque neutralization assay
Section II - Virology By Dr. Kareem Lilo
• A single virus infective dose can cause an area of cell destruction
• Movement of virus within cell is restricted by an agar overlay
• This causes areas of localized destruction
• Plaques are enumerated under a microscope to determine the plaque forming units per
• This allows comparison of different viruses in the same unit
Calculation of PFU/mL(Figure 2-14)
Plaque Counts are averaged over wells
The average is then divided by the dilution times the volume
Figure (2-12)Plaque forming units calculation
Section II - Virology By Dr. Kareem Lilo
• Long period (up to 4 weeks) required for result.
• Often very poor sensitivity, sensitivity depends on a large extent on the condition of the
• Susceptible to bacterial contamination.
• Susceptible to toxic substances which may be present in the specimen.
• Many viruses will not grow in cell culture e.g. Hepatitis B, Diarrhoeal viruses,
• Some viruses agglutinate RBCs Mumps, measles, influenza
• Haemagglutination Clumps RBCs (Figure 2-13)
Section II - Virology By Dr. Kareem Lilo
Section II - Virology By Dr. Kareem Lilo
Figure(2-14)Direct Virological examination (A)Electron microscope (B)Principle of
immunofluorescence assay (C) immunofluorescence microscope.
Section II - Virology By Dr. Kareem Lilo
• Extremely high sensitivity, may detect down to one viral genome ( Figure 2-15) per
• Extremely liable to contamination
• High degree of operator skill required
• Not easy to set up a quantitative assay.
Section II - Virology By Dr. Kareem Lilo
• A positive result may be difficult to interpret, especially with latent viruses such as
CMV, where any seropositive person will have virus present in their blood irrespective
whether they have disease or not.
These problems are being addressed by the arrival of commercial closed systems such as
the Roche Cobas Amplicor which requires minimum handling. The use of synthetic
internal competitive targets in these commercial assays has facilitated the accurate
quantification of results. However, these assays are very expensive.
PCR - Polymerase Chain Reaction
PCR is an in vitro technique for the amplification of a region of DNA
which lies between two regions of known sequence. (Figure 2-16)
PCR amplification is achieved by using oligonucleotide primers.
These are typically short, single stranded oligonucleotides which are complementary to the outer
Figure (2-15 ) Structure of DNA
Figure(2-16) Two amplification regions of the DNA
Section II - Virology By Dr. Kareem Lilo
The oligonucleotides serve as primers for DNA polymerase and the denatured strands of
the large DNA fragment serves as the template.
• This results in the synthesis of new DNA strands which are complementary to the parent
• These new strands have defined 5' ends (the 5' ends of the oligonucleotide primers),
whereas the 3' ends are potentially ambiguous in length.
1. Target DNA - contains the sequence to be amplified
2. Pair of Primers - oligonucleotides that define the sequence to be amplified.
3. dNTPs - deoxynucleotidetriphosphates: DNA building blocks.
4. Thermostable DNA Polymerase - enzyme that catalyzes the reaction
5. Mg++ ions - cofactor of the enzyme
6. Buffer solution – maintains pH and ionic strength of the reaction solution suitable for
Section II - Virology By Dr. Kareem Lilo
How Gel Electrophoresis of DNA Works
Figure(2-17)A-PCR equipment (B)Polymerase chain reaction steps
Figure(2-18)DNA gel electrophorese A-Equipment B-Principle assay
Section II - Virology By Dr. Kareem Lilo
Viral infection induce immune response the detection of rising titers of antibody between acute and
convalescent stages of infection, or detection of IgM in primary infection .(Figure 2-20)
Primary and secondary responses to viral infection
Figure(2-19)Primary and secondary antibody responses toward a viral pathogen
Section II - Virology By Dr. Kareem Lilo
How useful a serological result is depends on the individual virus.
• For example, 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 titres of IgG
in the serum of the patient would indicate active disease.
• However, many viruses often produce clinical disease before the appearance of
antibodies such as respiratory and diarrhoeal viruses. So in this case, any serological
diagnosis would be retrospective and therefore will not be that useful.
• 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.(Figure 2-21)
Figure (2-20)Principle of ELISA assay
Section II - Virology By Dr. Kareem Lilo
• Long period of time required for diagnosis for paired acute and convalescent sera.
• Mild local infections such as HSV genitals may not produce a detectable humoral
• 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
• 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
• Used mainly for the diagnosis of herpes simplex and VZV encephalitis
• CSF normally contain little or no antibodies
Figure(2-21)Microplate ELISA for HIV antibody: Coloured wells indicate reactivity
Section II - Virology By Dr. Kareem Lilo
• presence of antibodies suggest meningitis or
𝟏𝟎𝟎 𝒊𝒔 𝒊𝒏𝒅𝒊𝒄𝒂𝒕𝒊𝒗𝒆 𝒐𝒇 𝒎𝒆𝒏𝒊𝒏𝒈𝒊𝒕𝒊𝒔
• Diagnosis depends on the presence of an intact blood-brain barrier
Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can
be separated, and often follows the use of a gel electrophoresis. The Southern blot is used
for transferring DNA, the Northern blot for RNA and the western blot for
Professor Sir Edwin Southern, Professor of Biochemistry and Fellow of Trinity developed this
Southern won the Lasker Award for Clinical Medical Research prize for the method of finding
specific DNA sequences he developed this procedure at Edinburgh University more than 30
years ago. The technique is known as DNA transfer or 'Southern blotting'
Figure (2-22)The three types of blotting techniques
Section II - Virology By Dr. Kareem Lilo
Southern Steps(Figure 2-24)( Figure 2-25)
1. DNA to be analyzed is digested to completion with a restriction endonuclease.
2. Electrophoresis to maximally separate restriction fragments in the expected size range. A set
of standards of known size is run in one lane of the gel.
3. Blot fragments onto a nitrocellulose membrane.
4. Hybridize with the 32P probe.
Figure (2-23) Professor Sir Edwin Southern
Section II - Virology By Dr. Kareem Lilo
Figure (2-24)Steps of southern blot technique
Section II - Virology By Dr. Kareem Lilo
Figure(2-25)General scheme for southern blot
Section II - Virology By Dr. Kareem Lilo
• Enveloped double stranded DNA viruses.
• Genome consists of long and short fragments which may be orientated in either direction,
A. Alphaherpesviruses - HSV-1, HSV-2, VZV
B. Betaherpesviruses - CMV, HHV-6, HHV-7
C. Gammaherpesviruses - EBV, HHV-8
• Set up latent or persistent infection following primary infection
• Reactivation are more likely to take place during periods of immunosuppression
• Both primary infection and reactivation are likely to be more serious in
HSV-2 virus particle. Note that all herpesviruses have identical morphology and cannot be
distinguished from each other under electron microscopy.(Figure2-26)
Figure (2-26) Herpesvirus Particle
Section II - Virology By Dr. Kareem Lilo
• Belong to the alphaherpesvirus subfamily of herpesviruses
• Double stranded DNA enveloped virus with a genome of around 150 kb
• The genome of HSV-1 and HSV-2 share 50 - 70% homology.
• Man is the only natural host for HSV.
• HSV is spread by contact, as the virus is shed in saliva, tears, genital and other secretions.
• By far the most common form of infection results from a kiss given to a child or adult from a
• Primary infection is usually subclinical in most individuals. It is a disease mainly of very
young children ie. those below 5 years.
• There are 2 peaks of incidence, the first at 0 - 5 years and the second in the late teens, when
• About 10% of the population acquires HSV infection through the genital route and the risk is
concentrated in young adulthood.
• During the primary infection, HSV spreads locally and a short-lived viraemia occurs,
whereby the virus is disseminated in the body. Spread to the to craniospinal ganglia occurs.
• The virus then establishes latency in the craniospinal ganglia.
• The exact mechanism of latency is not known, it may be true latency where there is no viral
replication or viral persistence where there is a low level of viral replication.
• Reactivation - It is well known that many triggers can provoke a recurrence. These include
physical or psychological stress, infection; especially pneumococcal and meningococcal,
fever, irradiation; including sunlight, and menstruation.
HSV is involved in a variety of clinical manifestations which includes ;-
2. Herpes Labialis (cold sore)
Section II - Virology By Dr. Kareem Lilo
5. Other forms of cutaneous herpes
• Acute gingivostomatitis is the commonest manifestation of primary herpetic infection.
• The patient experiences pain and bleeding of the gums. 1 - 8 mm ulcers with necrotic bases
are present. Neck glands are commonly enlarged accompanied by fever.
• Usually a self-limiting disease which lasts around 13 days.
• Following primary infection, 45% of orally infected individuals will experience reactivation.
The actual frequency of recurrences varies widely between individuals.
• Herpes labialis (cold sore) is a recurrence of oral HSV.
• A prodrome of tingling, warmth or itching at the site usually heralds the recurrence. About
12 hours later, red-ness appears followed by papules and then vesicles.
Figure (2-27)Gingivostomatitis
Section II - Virology By Dr. Kareem Lilo
HSV causes a broad spectrum of ocular disease, ranging from mild superficial lesions
involving the external eye, to severe sight-threatening diseases of the inner eye. Diseases
caused include the following :
- Primary HSV keratitis – dendritic ulcers
• Genital lesions may be primary, recurrent or initial.
• Many sites can be involved which includes the penis, vagina, cervix, anus, vulva, bladder,
the sacral nerve routes, the spinal and the meninges. The lesions of genital herpes are
particularly prone to secondary bacterial infection eg. S.aureus, Streptococcus, Trichomonas
• Dysuria is a common complaint, in severe cases, there may be urinary retention.
• Local sensory nerves may be involved leading to the development of a radiculitis. A mild
• 60% of patients with genital herpes will experience recurrences. Recurrent lesions in the
perianal area tend to be more numerous and persists longer than their oral HSV-1
• Herpes Simplex encephalitis is one of the most serious complications of herpes simplex
• Neonatal – there is global involvement and the brain is almost liquefied. The mortality rate
• Focal disease – the temporal lobe is most commonly affected. This form of the disease
appears in children and adults. It is possible that many of these cases arise from reactivation
of virus. The mortality rate is high (70%) without treatment.
• It is of utmost importance to make a diagnosis of HSE early. It is general practice that IV
acyclovir is given in all cases of suspected HSE before laboratory results are available.
Section II - Virology By Dr. Kareem Lilo
• Disseminated herpes simplex are much more likely to occur in immunocompromised
individuals. The widespread vesicular resembles that of chickenpox. Many organs other than
the skin may be involved e.g. liver, spleen, lungs, and CNS.
• Other cutaneous manifestations include
- eczema herpeticum which is potentially a serious disease that occurs in patients with
- Herpetic whitlow which arise from implantation of the virus into the skin and typically affect
- “zosteriform herpes simplex". This is a rare presentation of herpes simplex where HSV
lesions appear in a dermatomal distribution similar to herpes zoster.
- Electron microscopy of vesicle fluid - rapid result but cannot distinguish between HSV and
- Immunofluorescence of skin scrappings - can distinguish between HSV and VZV
- PCR - now used routinely for the diagnosis of herpes simplex encephalitis
- HSV-1 and HSV-2 are among the easiest viruses to cultivate. It usually takes only 1 - 5 days
- Not that useful in the acute phase because it takes 1-2 weeks for before antibodies appear
after infection. Used to document to recent infection.
A-Cytopathic Effect of HSV in cell culture: Note the
ballooning of cells. (Linda Stannard, University of Cape
B-Positive immunofluorescence test for HSV antigen in epithelial
cell. (Virology Laboratory, New-Yale Haven Hospital(
Section II - Virology By Dr. Kareem Lilo
drugs being a main consideration. Generally, antiviral chemotherapy is indicated where the primary
infection is especially severe, where there is dissemination, where sight is threatened, and herpes
Acyclovir – this the drug of choice for most situations at present. It is available in a number of
- I.V. (HSV infection in normal and immunocompromised patients)
- Oral (treatment and long term suppression of mucocutaneous herpes and prophylaxis of
HSV in immunocompromised patients)
- Cream (HSV infection of the skin and mucous membranes)
Famciclovir and valacyclovir – oral only, more expensive than acyclovir.
Other older agents – e.g. idoxuridine, trifluorothymidine, Vidarabine (ara-A).
• These agents are highly toxic and is suitable for topical use for opthalmic infection only
• Belong to the alphaherpesvirus subfamily of herpesviruses
• Double stranded DNA enveloped virus
• Genome size 125 kbp, long and short fragments with a total of 4 isometric forms.
• One antigenic serotype only, although there is some cross reaction with HSV.
• Primary varicella is an endemic disease. Varicella is one of the classic diseases of childhood,
with the highest prevalence occurring in the 4 - 10 years old age group.
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