MEKELLE UNIVERSITY

COLLEGE OF VETERINARY MEDICINE

Department of Veterinary Medicine

MSc in
Veterinary clinical Laboratory Science (VCLS) Year-I

Module: Clinical Virology

Biruk Mekonnen (DVM, MSc)

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 Outline of the Lecture
Unit 5. Diagnostic virology
✓Laboratory diagnosis of viral infections
o Tissue culture methods
o Cultivation and purification of viruses
oLaboratory animal inoculation
oMicroscopic techniques
oSerology
oNucleic acid based diagnosis

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 Learning objectives

➢ At the end of this lecture, students should be able to:

✓ list sources of specimens for laboratory diagnosis of viral

infections

✓ describe the different methods used in clinical and/or

research laboratories for:
o isolation,
o detection
o identification,
o Purification and quantification of viral etiologies

✓ describe advantages and disadvantages of each method

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 Laboratory diagnosis of viral infections

➢ Viral diseases can be diagnosed clinically,

➢ However, there are several conditions under which

laboratory confirmation of the specific virus is essential

1. For some life threatening viral infections

➢ Some viral infections have common clinical signs

✓Calf diarrhoea (rotavirus, coronavirus, calcivirus)

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2. Certification of freedom from specific infections
✓For sale, export: free from diseases like FMD

3. Public health importance in zoonotic diseases

Eg Rift valley fever, Rabies

4. Test and Removal program

✓ Eg. Marek’s diseases

5. For research and control activities

✓ Eg. NCD diseases

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 Successful laboratory investigations:

✓ Advance planning

✓ Collection of adequate and appropriate specimens

✓ Sufficient documentation

✓ Biosafety and decontamination

✓ Correct packaging

✓ Rapid transport

✓ Choice of a laboratory that can accurately perform the tests

✓ Timely communication of results

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 Viral specimen Sampling
(Selection, Collection and Submission)

➢ Diagnosis of viral disease greatly depends on appropriate

sampling which includes
➢ Selection:
✓ it refers the site from which the specimen is collected

➢ Collection: should be @ the right time

✓ usually during the acute stage of disease when the animal is
febrile (for isolation)
✓before infection: to determine the prevalence of NCD
(serology, serum)
➢ Submission:
✓ properly labeled and rapidly with transport media 7
 Appropriate sampling

Syndrome Specimen
Respiratory (eg NCD) - Nasal or throat swabs
Enteric syndrome - Feces
Genital - Genital swab
Eye - Conjunctival swab
Skin - Vesicle swab or skin scraping,
biopsy of solid lesion
CNS - Cerebrospinal fluid (CSF),
Generalized - Nasal swab, feces, blood
Postmortem - Autopsy

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 Specimen transportation

➢ Viral transport media (VTM)

✓ allows organisms (pathogens and contaminants) to survive

✓ non-nutritive :- does not allow organisms to proliferate

o Hanks balanced salt solution with antibiotics

✓ also known as Universal Transport Media

✓ needed for the transport of:

o Lesions, Mucous membranes and throats to the laboratory

o NCD infection: lesions/tissue like gizzard
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 VTM
• Salt solution – ensures proper ionic concentrations
• Buffer - maintains pH
• Protein - for virus stability
• Antibiotics or antifungals – to prevent contamination

➢ VTM specimens filtered (45nm filter) to eliminate bacteria in

specimen prior to being placed on cell monolayer

➢ For blood, tissue, & respiratory samples

• For short term transport storage is at 4˚C – 8 ˚C if up to 48 hours

• For long term transport (>72hours) storage should be at -70˚C

• Avoid any storage at -20ºC: greater loss in infectivity

• Non-enveloped viruses (nacked) (adenovirus, enteroviruses) more stable than

enveloped (e.g. RSV, VZV, CMV Herpesviridae).

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 ➢ For sera specimen: store specimen at
– 4 °C to 8 °C for short periods of time
– -20 °C to - 40 °C for long term storage
• Avoid freeze-thaw cycles

Packing Specimens for Transportation

Goal: protect specimens during transportation

(arrival in good condition for analysis)

✓protection:
o the environment,
o the carrier and
o the sample

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➢ to pack specimens for transportation from the field to the
laboratory, three packaging layers are used:

➢ Three layers of protection are needed:

✓ Primary receptacle
✓ Secondary packaging
✓ Outer packaging

➢ If triple packaging not available

✓ Prepare according to international dangerous goods transportation rules

(see IATA guidelines)

➢ If transporting specimens a long distance, send on dry ice.

➢ If transporting a short distance, ice is acceptable

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1. Primary receptacle
✓ Leak-proof specimen container
✓ Packaged with sufficient absorbent material
❖ to absorb the entire content of the primary receptacle in case of breakage

2. Secondary receptacle/packaging
✓ Leak-proof secondary container
✓ Encloses and protects the primary receptacle(s)
✓ Sufficient additional absorbent material to absorb all fluid in case of breakage

3. Outer packaging

✓ Secondary packaging(s) are placed in outer shipping packaging with

suitable cushioning material

✓ Outer packaging protects contents from outside influences, physical

damage, while in transit
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 Category A “602 package”
Labels: UN 2814 UN 2900 Biohazard 14
There are different approaches for the diagnosis of viral infections:

1. Isolation (cell culture, chicken embryo and animal inoculation )

2. Direct microscopic identification in the specimen

3. Serologic procedures to detect a rise in antibody titer

4. Detection of viral antigens in blood or body fluids (serology)

5. Detection of viral nucleic acids in blood or the patient's cells

(molecular methods)

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 Isolation and Identification of Viruses

Isolation of Viruses

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 Isolation of viruses for identification

➢ It takes longer time, at least for a week and is expensive

➢ The primary purposes of viral cultivation are:

1. to isolate and identify viruses in clinical specimens
2. to prepare viruses for vaccines
3. to do detailed research on:
✓viral structure,
✓multiplication cycles,
✓genetics, and
✓ effects on host cells.

➢ Viruses can be isolated by cultivating them on a living cell. using:-

✓ Cell or tissue culture
✓ Chicken embryo inoculation
✓ Laboratory animal inoculation 17
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 1. Cell or tissue Culture
➢ Cell culture:

✓ is the most common method for primary isolation of viruses.

✓ this is where tissues are removed from an organism and are

grown “in vitro”, usually in flasks.

✓ the growth of viruses requires cell cultures, because viruses

replicate only in living cells.

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➢ Routinely used cell cultures for growing viruses classified into
3 types:
o Primary cell culture
o Diploid cell strains/secondary cell culture
o Continuous cell lines

1. Primary cell culture:

✓ normal differentiated cells freshly taken from body directly cultured,

✓ they have a limited life span (5 to 20 cell divisions)

✓ Most will grow attached to the flask as a monolayer of cells, one cell thick

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E.g.
1. Rhesus monkey kidney
2. Chick embryo fibroblast
3. Human amnion cell culture

Making a primary cell line

The cells may be:

• Separate the cell directly from the parent tissue (kidney, heart, liver
etc) and disaggregated by enzymatic means before cultivation, or

• they may be derived from a cell line or cell strain that has already
been established

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➢ to prepare the cell culture:
✓ tissues removed from live animals or recently killed animals
are dispersed in to single cells by treatment with proteolytic
enzymes

✓ then the cells are suspended in culture media (maintaining the

growth of the cell) using plastic flasks or plates and

✓as the cells divide, they cover the plastic surface and form a
monolayer

Culture medium
❖ Liquid or gel designed to maintain the growth of the cell
❖ It varies from different type of cells
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2. Secondary cell culture:

✓ It is also called a diploid cell strains (semi-continous cell lines)

✓ When primary cell is sub-cultured then it becomes/known as a

Diploid cell line or subclone

o Cells of single type (fibroblast cells) that can be sub-cultivated

for limited number of times, mostly 50 -100 times
o E.g.
1. WI-38: human embryonic lung cell
2. HL-8: Rhesus embryo cell

➢ Sub culture:
✓ transfer of cell from one culture vessel to another
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 ✓ Method:
❖ remove growth medium;
❖detach adherent cell (enzymatic method)

➢ Why sub culture is required:

✓ to provide fresh nutrient

✓ to provide more space

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3. Continuous cell lines – malignant cells:
✓ Immortalized cells

✓ are cells that have a mutation or mutations that allow the cells to be
passed many times

❖ i.e. they don’t have a limited life span (possible for

indefinite sub-cultivtion).
✓ Most were originally derived from a tumor.

✓ Most grow as mono-layers, though a few grow in suspension

✓ E.g.
1. HeLa: Human Ca of cervix cell line
2. HEP-2: Human epithelioma of larynx
3. Vero: Vervet monkey kidney
4. McCoy, Detroit-6, BHK-21, Kb
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.
Viral isolation in cell culture
1. primary cell
2. secondary cell lines
3. continuous cell lines

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 Summary of cell/tissue culture preparation for viral infection
The process to culture cells
Harvest cells
Normal cell from Cancer cell from
human or animal human or animal

Isolate cells with

appropriate enzymes Primary cell
culture

Apply the isolated cells on to growth

media in culture dish subculture

Culture cells by placing the

culture dish in a cell incubator
Cell lines

Subculture cells to
Verify the cultured cells are of obtain a pure culture
the cell type of interest or to bypass some
problems Immortalization Continuous cell lines
(such as senescence) via virus or
chemicals

Cells are ready to be manipulated

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 Infection with virus and detection of Viral growth

➢ The cell lines suspended in suitable media are infected with any
desired virus which replicates inside the multiplying cells.

✓ If the virus is virulent, they cause lysis of cells and virus particles
are released in the surrounding medium.

✓ These newly produced virus particles (virions) infect the adjacent cells

✓ As a result, localized areas of cellular destruction and lysis (called

plaques) often are formed

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 Viral detection
I. Presumptive identification
1. Cytopathic effect (CPE)
➢ Virus growth in cell culture frequently produces a characteristic CPE
that can provide a presumptive identification

✓ CPE is a change in the appearance (morphological change) of

the virus-infected cells.

✓ CPE is usually a manifestation of virus-infected cells that are

dying or dead due to:
• Multiplying viruses,
• Inhibition of DNA, RNA or protein synthesis,
• Effects on permeability of membrane.

✓ CPEs of infected cells can be observed with inverted light

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microscopes
➢ Morphological change of the virus-infected cells includes:

❖ Cell rounding, disorientation, swelling or shrinking, loss of

adherence (detachment from the surface)
Eg: - Enterovirus infection

❖ Syncytial Formation: fusion of cells to form multinucleated

giant cells
Eg:- Herpesvirus, and Paramyxovirus

2. Inclusion body
✓ Inclusion bodies are abnormal structures within cells that
are the result of virus infection.

✓ Inclusion bodies may be of considerable diagnostic aid 31

✓ Intra-cytoplasmic/ intra-nuclear virus specific structures formed
during viral multiplication.

✓ In general DNA viruses lead to nuclear inclusion bodies while RNA

viruses cause cytoplasmic inclusion bodies

✓ Examples:
❖Herpesvirus & adenovirus (in nucleus),
❖ Poxvirus (in cytoplasm),
❖ Measles virus (both)

✓ For many families of viruses the staining characteristics and

appearance of inclusion bodies are characteristic and can be
used as a way of identifying the virus.

✓ For example, the intracytoplasmic inclusion bodies in nerve cells-the

Negri body- is pathognomonic for rabies. 32
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II. Definitive identification

➢ A definitive identification of the virus grown in cell culture is

made by using known antibody in one of several tests.

1. Complement fixation

✓ If the antigen (the unknown virus in the culture fluid) and the known
antibody are homologous, complement will be fixed to the
antigen–antibody complex.

✓ This makes it unavailable to lyse the "indicator" system, which is

composed of sensitized red blood cells

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 Positive test (CFT)

Ag (the unknown virus in the culture fluid)

+
Serum with known Ab against Ag
+
Complement fixation on
antigen–antibody complex
+
Sheep RBC = no hemolysis

❖ All available complement is fixed by Ag+Ab reaction; no hemolysis occur, so

the test is positive for the presence of antibodies

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 Negative test (CFT)

Ag (the unknown virus in the culture fluid)

+
Serum without Ab against Ag
+
No Complement fixation
(b/c no antigen–antibody complex)
+
Sheep RBC = hemolysis

❖ No antigen-antibody reaction occurs. The complement remains, and the RBC

are lysed , so the test is negative.

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2. Neutralization test

➢ Neutralization of the CPE are the most frequently used tests.

✓ If the virus and antibody are homologous, the antibody bound
to the surface of the virus blocks its entry into the cell.

✓ This neutralizes viral infectivity, because it prevents viral

replication and subsequent CPE formation or animal infection

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 3. Hemagglutination inhibition test

➢ Is an indirect test for antibody against specific viruses that can

agglutinate RBCs.
✓Only viruses that agglutinate red blood cells can be identified by
this method. Eg. NCD virus

✓ Mix serial dilutions of patient’s sera (for direct detection of virus

from patient samples) and then add RBC’s.

✓If the patient has antibodies specific to the virus, they will bind
to the virus and prevent the virus from agglutinating the RBCs.

➢ If the virus and antibody are homologous, the virus is blocked from
attaching to the erythrocytes and no hemagglutination occurs.
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 4. Other confirmatory tests
1. Immunofluorescence assay (fluorescent antibody)

➢ If the virus-infected cells and the fluorescein-tagged antibody

are homologous, the typical apple-green color of fluorescein is
seen in the cells by ultraviolet (UV) microscopy
➢ picture

2. Enzyme-linked immunosorbent assay (ELISA)

3. Immunoelectron microscopy are also used in special instances.

➢ If the antibody is homologous to the virus, aggregates of

virus–antibody complexes are seen in the electron microscope
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 Problems with cell culture
1. Long period (up to 4 weeks) required for result

2. Often very poor sensitivity,

✓sensitivity depends on a large extent on the condition of the
specimen.

3. Susceptible to bacterial contamination.

4. Susceptible to toxic substances which may be present in the

specimen.

5. Many viruses may not grow in cell culture

✓e.g. Hepatitis B, Diarrhoeal viruses, parvovirus,
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 2. Chick Embryo Inoculation

➢ The process of cultivation of some viruses in embryonated eggs

depend on the type of egg which is used:

✓ Embryonated chicken, duck or turkey for inoculation of viral

suspension

✓ Only embryonated egg suitable for viral growth

➢ The egg used for cultivation must be sterile and the shell should be
intact and healthy.

➢ 7 -10 days old embryonated eggs are used.

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➢ Before inoculation of the viral suspension:
✓the egg must be cleaned,
✓the shell decontaminated with a disinfectant and
✓checked in ovoscope if it is alive

❖ Ovoscope is the equipment used for candling

Procedure
1. Candling
2. Drill the Hole
3. Inject the Suspension with Syringe
4. Hole Sealed With Paraffin Wax
5. Eggs Incubate at 36 0c for 2-3 Days
6. Harvesting of Embryos picture 45
PROCEDURE

1. CANDLING 2.DRILL THE HOLE

3.Inject The Suspension With Syringe

4 Hole Sealed With Paraffin Wax

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5. Eggs Incubate At 360c For 2-3 Days 6. HARVESTING OF EMBRYOS

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 Detection of viral growth in the embryo
➢ The signs of viral growth include:

i) Death of the embryo (mortality of the embryo):

ii) Defects/abnormalities in embryonic development

✓Deformities,
✓hemorrhages, and
✓edema of the developing embryo.

iii) Localized areas of damage in the membranes, resulting in

discrete, opaque spots called pocks

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iv) The embryonic fluid and tissue can be prepared for examination
with an electron microscope.

v) Some can also be detected by their ability to agglutinate red

blood cells if it is present.

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Route of inoculation

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Candling

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 Advantages of culturing in embryonated eggs

✓ Isolation and cultivation of many avian and few mammalian viruses

✓ Cost- much less
✓ Maintenance-easier
✓ Less labour
✓ Readily available

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 3. Laboratory Animal Inoculation/
Viral Growth in Whole Animals

➢ Involves using live animal eg. mice, rats, rabbits, guinea pigs,
hamster, and monkey.

➢ We can easily cultivate viruses and diagnose viral diseases by

inoculating suspected specimen into susceptible animals.

➢ viruses which are not cultivated in embryonated egg and

tissue culture are cultivated in laboratory animals

➢ The animal should be susceptible and free from antibodies

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➢ the animal is exposed to the virus by injection of a viral
preparation or specimen into:
✓ the brain,
✓ blood,
✓ muscle,
✓ body cavity,
✓ skin, or footpads.

➢ The route of inoculation depends on

✓the nature of the virus,
✓the possible tissue affinity,
✓the age and
✓specie of the experimental animals.

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 Detection of viral growth

➢ The signs of viral growth include:

i) death of the animal

ii) defects in animal development.

iii) the infected animal tissue can be prepared for

examination with an electron microscope

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 Virus separation and purification from clinical samples and cell/tissue/animal
cultures
• Isolation, separation and purification refer to:
• techniques used to
• isolate,
• concentrate or
• purify
• cells,

• viruses,

• cell fractions,

• organelles or

• biological macromolecules for subsequent analysis

• e.g. proteins,

• protein complexes,

• chromatin,

• nucleic acids,

• carbohydrates or lipids

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 Isolation/purification of virions: achieved via centrifugation and filtration
• Centrifugation
1. Differential centrifugation - high vs low speed to separate cells from viruses

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2. Gradient centrifugation - separate by size or density

Equilibrium density gradient centrifugation

4/23/2015 35

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3. Rate zonal centrifugation separates viruses from cells and cellular components based
on their size and density.
Rate-zonal centrifugation – similar to density gradient centrifugation, but uses a
preformed gradient rather than generating a gradient during the centrifugation process

4/23/2015 36

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•2. Filtration

•Size by filtration- molecular sieve chromatography.

– Uses a column filled with beads containing holes.
•Large molecules are excluded from the holes and come off the column
first.
•Small molecules enter the holes in the beads and therefore move slower
down the column, coming off the column after large molecules.

•Purification of specific viruses can be achieved by:

❖ affinity chromatography using antibodies directed to the virus
of interest.

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 . Identification of Viruses

A. Direct Identification of Viruses, Viral Antigens or Nucleic Acids

➢ We can directly demonstrate the virus or it’s remainant in the

specimen.
❖Electron Microscopy- for the virion and its ultrastructure

❖Immunofluorescence- for viral antigens of any specimen

❖Immunohistochemistry- for viral antigens in tissues

❖Polymerase Chain Reaction (PCR)- for viral nucleic acids

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 B. Indirect Identification of viruses by detection of Antiviral
Antibodies (Serological Diagnosis)

➢ Some of the common serological tests includes:

1. ELISA ..widely used

2. Other common serological assays includes:-

❖ Serum Neutralization Assay

❖ Western Blotting

❖ Heamaggltunation test (HA)Indirect

Complement Fixation Test (CFT)
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