Diagnosis of viral infection

1. LABORATORY DIAGNOSIS
OF VIRAL INFECTION
- Dr. ANKUR KUMAR

2. TOPICS for discussion-
• PATHOGENESIS OF VIRUSES
• DIAGNOSTIC METHODS
• MICROSCOPY
• INCLUSION BODIES
• SEROLOGICAL TEST (CFT, HAI, Neutralisation)
• MOLECULAR METHODS
• CULTURE METHODS
• ANIMAL INOCULATION
• EMBRYONATED EGG INOCULATION
• TISSUE CULTURES
• CYTOPATHIC EFFECTS

3. PATHOGENESIS
• Attachment
• Penetration
• Uncoating
• Biosynthesis
• Assembly
• Maturation
• Release

4. Adsorption/Attachment
• The viruses have attachment sites on their
envelopes or capsid proteins that bind to the
complementary receptor sites present on the
host cell surface.
HIV: surface glycoprotein gp 120 binds to CD4
molecules on the host cells.
lnfluenza: Viral hemagglutinin (an envelope
protein) binds specifically to gp receptors present
on the surface of respiratory epithelium.

5. Penetration
1. Phagocytosis (or viropexis):
 It occurs through receptor mediated endocytosis resulting in the
uptake of virus particles within the endosomes of the host
cytoplasm.
2. Membrane fusion:
 Some enveloped viruses (HIV) enter by fusion of their envelope
proteins with the plasma membrane of the host cell so that only
the nucleocapsid enters into the cytoplasm, whereas the viral
envelope remains attached to the host cell membrane.
3. Injection of nucleic acid:
 Bacteriophages (viruses that infect bacteria) cannot penetrate
the rigid bacterial cell wall, hence only the nucleic acid is
injected; while the capsid remains attached to the cell wall.

6. • Uncoating
By the action of lysosomal enzymes of the host cells,
viral capsid gets separated and the nucleic acid is
released into the cytoplasm.
Absent in bacteriophages
• Biosynthesis
Components synthesized:
Nucleic acid
Capsid protein
Enzymes- required for various stages of viral replication.
Regulatory proteins- to shut down host cell metabolism.
In DNA viruses- DNA replication occurs in the nucleus
except in poxviruses
In RNA viruses- RNA replication occurs in cytoplasm
except in retroviruses and orthomyxoviruses

7. • Assembly
Viral nucleic acid and proteins are packaged
together to form progeny viruses (nucleocapsids)
Assembly take place in the host cell nucleus or
cytoplasm.
• Maturation
Maturation of daughter virions take place either in
the host cell nucleus or cytoplasm or membranes
(Golgi or Endoplasmic reticulum or Plasma
membrane)

8. Release
• Release of daughter virions occur either by:
1. Lysis of the host cells: Non enveloped viruses and
bacreriophages
2. Budding through host cell membrane:
• by enveloped viruses, during budding they
acquire a part of the host cell membrane to form
the lipid part of their envelopes.
• Envelope is acquired either from plasma
membrane (influenza virus) or from nuclear
membrane (e.g. herpesviruses).
• Viral glycoproreins are then inserted into the
envelopes.

10. Transmission and spread of viruses
Mode of transmission Produce local infection at
the portal of entry
Spread to distant sites
from the portal of entry
Respiratory route (most
common route)
Produce respiratory
infection
 Influenza virus
 Parainfluenza virus
 Respiratory syncytial virus
(RSV)
 Rhinovirus
 Adenovirus
 Coronavirus
 Herpes simplex virus (HSV)
 Measles v irus
 Mumps virus
 Rubella virus
 Varicella·zoster virus
 Cytomegalovirus (CMV)
 Parvovirus
 Smallpox virus
Oral route Produce gastroenteritis
 Rotavirus
 Adenovirus 40,41
 Calicivirus
 Astrovirus
 Poliovirus
 Coxsackie virus
 Hepatitis virus - A and E
 Cytomegalovirus
 Epstein-Barr virus (EBV)
Cutaneous route Produce skin lesions
 Herpes simplex virus (HSV)
 Human papillomavirus (HPV)
 Molluscum contagiosum virus
Herpes simplex virus

11. • Depend up on the site of infection -
Throat swab, nasopharyngeal swab, bronchial lavage
Blood
Bone marrow
Rectal swab, stool
Urine
Sterile body fluid
Tissues
CSF
Serum
• Collection swab is made of Dacron
• Specimen transportion in VTM
Specimen

12. • Viral diagnosis
Direct Detection Detection of Molecular Isolation
Demonstration of viral Specific Methods of Virus
of Virus antigens Antibodies
Electron microscopy ELISA ELISA Nucleic acid probe Animal inoculation
lmmunoelectron microscopy direct IF, ICT PCR Embryonated egg inoculation
Fluorescent microscopy ICT Flow through assays Tissue cultures:
Light microscopy Flow through assays HA/HAI, Organ culture
CFT Explant culture
Histopathological staining Neutralization test Cell line culture
To demonstrate inclusion bodies Primary
lmmunoperoxidase staining Secondary and
Continuous cell lines

13. DIRECT DEMONSTRATION OF VIRUS
• Electron Microscopy - Specimens are negatively
stained by potassium phosphotungstate and scanned
under EM.
Rabies virus- Bullet shaped
Rotavirus- Wheel shaped
Coronavirus- Petal shaped peplomers
Adenovirus- Space vehicle shaped
Astrovirus- Star shaped peplomers
• Drawbacks: EM is highly expensive, has low
sensitivity with a detection threshold of 107
virions/mL. Specificity is also low

14. DIRECT DEMONSTRATION OF VIRUS
• lmmuno-electron Microscopy-
sensitivity and specificity of EM can be improved by
adding specific antiviral antibody to the specimen to
aggregate the virus particles which can be
centrifuged.
The sediment is negatively stained and viewed under
EM.
• Fluorescent Microscopy
Procedure: Specimen is mounted on slide, stained
with specific antiviral antibody tagged with
fluorescent dye and viewed under fluorescent
microscope
Diagnosis of rabies virus antigen in skin biopsies,
adenovirus from corneal smear of infected patients.
Syndromic approach: Rapid diagnosis of respiratory
infections (caused by influenza virus, rhinoviruses, respiratory syncytial
virus, adenoviruses and herpesviruses).

15. Light Microscopy
• Inclusion bodies:
Histopathogical staining of tissue sections used for
detection of inclusion bodies.
e.g. Negri bodies detection in brain biopsies of
patients or animals died of rabies
• lmmunoperoxidase staining:
Tissue sections/ cells coated with viral antigens
are stained using antibodies tagged with horse
radish peroxidase following which
Hydrogen peroxide and a coloring agent
(benzidine derivative) are added
Color complex formed can be viewed under under
microscope.

16. INCLUSION BODY
• They are the aggregates of virions or viral proteins and
other products of viral replication that confer altered
staining property to the host cell.
• They have distinct size, shape, location and staining
properties by which they can be demonstrated in virus
infected cells under the LM.
• Characteristic of specific viral infections.
• lntracytoplasmic IB:
 Generally acidophilic
 Seen as pink structures when stained with Giemsa or eosin
methylene blue stains
 e.g. most poxviruses and rabies
• lntranuclear IB:
 Basophilic in nature.
 Cowdry (1934) had classified them into
• Cowdry type A inclusions- variable in size and have granular
appearance.
• Cowdry type B inclusions- more circumscribed and multiple

17. INCLUSION BODY

18. DETECTION OF VIRAL ANTIGENS
• Test used for detection of viral antigens in
serum and other samples are ELISA, ICT, flow
through assays etc.
HBsAg and HBeAg antigen detection- for hepatitis
B virus infection from serum
NS1 antigen detection- for dengue virus infection
from serum.
p24 antigen detection- for HIV infected patients
from serum.
Rotavirus antigen detection- from diarrheic stool.
CMV specific pp65 antigen detection- from serum

19. DETECTION OF VIRAL ANTIBODIES
• Most commonly used method in diagnostic virology
• Conventional Diagnostic Techniques- less commonly
used now a day. Examples
Heterophile agglutination test (e.g Paul-Bunnell rest for
Epstein-Barr virus).
Hemagglutination inhibition (HAI) test for influenza virus
and arbovirus infection.
Neutralization test- for poliovirus and arbovirus infections
CFT- for poliovirus, arbovirus and rabies virus infections
• Newer Diagnostic Formats -ELISA, ICT, flow through
assays example:
Anti-HBc, Anti-HBs and Anti-HBe antibodies for Hepatitis B
infection.
Anti-Hepatitis C antibodies
Antibodies against HIV-1 and HIV-2 antigens
Anti-Dengue IgM/IgG antibodies

20. MOLECULAR METHODS
• More sensitive, specific and yield quicker results
than culture.
• Nucleic Acid Probe-
An enzyme or radio-labelled nucleic acid sequence
complementry to a part of nucleic acid sequence of
the target virus.
Added to the clinical specimen- hybridizes to the
corresponding part of viral nucleic acid.
Both DNA and RNA probes are commercially available
• Polymerase Chain Reaction
• Reverse Transcriptase PCR (RT-PCR)
• Real Time PCR

21. ISOLATION OF VIRUS
• Viruses cannot be grown on artificial culture
media.
• They are cultivated by
Animal inoculation-
• Infant (suckling) mice are used.
• Specimens are inoculated by intracerebral or
intraperitoneal routes.
• Eg- intracerebral inoculation of Coxsackie virus into
suckling mice-
• Coxsackie-A virus produces flaccid paralysis
• Coxsackie-B virus produces spastic paralysis
Embryonated egg inoculation or
Tissue cultures.

22. Embryonated Egg Inoculation
• Embryonated hen’s eggs are used
• Specimens inoculated into embryonated 7 to
12 days old hen's eggs
• Incubated for 2-9 days.
• Routes of inoculation
Yolk Sac Inoculation
Amniotic Sac
Allantoic Sac
Chorioallantoic Membrane

23. Embryonated egg

24. Embryonated Egg Inoculation
• Yolk Sac Inoculation
Arboviruses- e.g. Japanese B encephalitis virus
Saint Louis encephalitis virus
West Nile virus and
Some bacteria such as Rickettsia, Chlamydia and
Haemophilus ducreyi
• Amniotic Sac
Primary isolation of the influenza virus
Viral growth measured by detection of
hemagglutinin antigens in amniotic fluid

25. Embryonated Egg Inoculation
• Allantoic Sac
Used for yield of viral vaccines like- influenza
vaccine, yellow fever (17D) vaccine and Rabies
(Flury strain) vaccine.
• Chorioallantoic Membrane (CAM)
Poxviruses, HSV and other viruses
Produce visible lesions called as pocks on CAM
Each pock derived from a single virion

26. Tissue Cultures
• Tissue culture - 3 types
• Organ culture:
For certain fastidious viruses that have affinity to
specific organs.
e.g. tracheal ring culture for isolation of corona virus
• Explant culture:
Obsolete now
Fragments of minced tissue can be grown as ‘explants’
e.g. Adenoid explants used for adenoviruses.
• Cell line culture:
Currently used method
Types - Primary cell lines, Secondary or diploid cell
lines, Continuous cell lines

27. Preparation of the Cell Lines
• Tissues are completely dissociated into individual cells
and dispensed in tissue culture flasks containing viral
growth medium
• Digested by- treatment with proteolytic enzymes
(trypsin or collagenase) followed by mechanical
shaking
• Viral growth medium: Contains balanced salt solution,
essential amino acids, vitamins, salts and glucose
supplemented by 5- 10% of fetal calf serum, antibiotics
and phenol red. pH of 7.2 to 7.4
• lncubation:- Tissue culture flasks are incubated
horizontally in presence of CO2 either as a stationery
culture or as a roller drum culture.
• Monolayer sheet formation: On incubation, the cells
adhere to glass surfaces of the flask and divide to form
a confluent monolayer sheet of cells within a week.

28. Cell lines
1. Primary cell lines
 Derived from normal cells freshly taken from the organs and cultured.
 Capable of very limited growth in culture, maximum up to 5-10
divisions.
 Maintain a diploid karyosome.
 examples –
• Monkey kidney cell line- useful for isolation of myxoviruses,
enteroviruses and adenoviruses
• Human amnion cell line
• Chick embryo cell line
2. Secondary or diploid cell lines:
 Derived from the normal host cells and they maintain the diploid
karyosome
 Divide maximum up to 10-50 divisions
 Common examples:
• Human fibroblast cell line: CMV
• MRC-5 and WI-38 (human embryonic lung cell strain):- HSV, VZV,
CMV, adenoviruses, and picornaviruses also for vaccine for rabies,
chickenpox, hepatitis-A and MMR vaccines

29. Cell lines
3. Continuous Cell Lines
Derived from cancerous cell lines, hence are immortal &
capable of indefinite growth.
Possess altered haploid chromosome.
Easy to maintain in the laboratories by serial sub culturing
for indefinite divisions.
Examples
• HeLa cell line (Human carcinoma of cervix cell line).
• Hep-2 cell line (Human epithelioma of larynx cell line)-
widely used for RSV, adenovairuses and HSV.
• KB cell line (Human carcinoma of nasopharynx cell line).
• McCoy cell line (Human synovial carcinoma cell line)- useful
for isolation of viruses as well as Chlamydia .
• Vero cell line (Vervet monkey kidney cell line)-used for rabies
vaccine production.
• BHK cell line (Baby hamster kidney cell line)

30. DETECTION OF VIRAL GROWTH IN THE CELL CULTURES
• Methods used are
Cytopathic Effect (CPE)
Viral Interference
Hemadsorption
Direct immunofluorescence Assay
lmmunoperoxidase Staining
Electron Microscopy
Viral Genes Detection

31. Cytopathic Effect
• Morphological change produced by the virus
in the cell Iine, detected by light microscope.
• The type of CPE is unique for each virus and
that helps for their presumptive identification

32. Viral Interference
• The growth of a non-CPE virus in cell culture
can be detected by the subsequent challenge
of the cell line with a known CPE virus.
• Viral interference- The growth of the first virus
would inhibit infection by the second virus.
• For example, rubella is a non-CPE virus but
prevents the replication of enteroviruses.

33. • Hemadsorption-
The process of adsorption of erythrocytes to the
surfaces of infected cell lines cells is known as
hemadsorption.
Hemagglutinating viruses (e.g; influenza virus)
when grown in cell lines, they produce
hemagglutinin antigens which are coated on the
surface of the cell lines and detected by adding
guinea pig erythrocytes to the cultures.
• Direct immunofluorescence Assay
Virus infected cells are mounted on a slide and
stained with specific antibodies tagged with
fluorescent dye
Viewed under fluorescent microscope for the
presence of viral antigens on the surface of
infected cells.

34. • lmmunoperoxidase Staining
Cells coated with viral antigens are stained by
immunoperoxidase tagged specific antibodies and
viewed under Light microscope.
• Electron Microscopy
Viruses can also be demonstrated in infected cell
lines by EM
• Viral Genes Detection
By using PCR or nucleic acid probes

35. THANK
YOU