Virus is an obligate intracellular parasite which infects human beings, lower animals, insects, plants, bacteria and fungus. Viruses of medical importance to humans comprise of seven families of DNA viruses and 14 families of RNA viruses. Laboratory diagnosis of viral infections is continuously being refined to accelerate the process of identification of viruses. Because of the expense & the delay involved in obtaining a definitive virological diagnosis, discrimination in their use has to be done

2. LABORATORY DIAGNOSIS
OF VIRAL INFECTIONS
PRESENTED BY
LAKSHMI S ANAND

3. Virus
• Obligate intracellular parasites infecting
humans,lower animals,insects, bacteria and fungi
• Word “ VIRUS ‘’ in latin – poison / other noxious
substances
• Attach to cell surfaces which act as receptor for their
entry to the cell

4. • Viruses of medical importance
• 7 family of DNA viruses
• 14 family of RNA viruses

5. Structure
• Consist of a nucleic acid either
DNA/RNA
• Surrounded by a protein coat
called capsid
• Capsid consist of capsomeres
• Capsid is antigenic
• Outer envelope is lipoprotein
in nature made of subunits
called peplomeres
• Peplomeres are antigenic

6. Classification
• Medically relevant DNA viruses

7. Medically relevant RNA virus

8. Rationale for performing laboratory virus
diagnosis
• Diseases in which management of patients as well as
prognosis depends on accurate diagnosis
• Epidemic diseases in which an early diagnosis helps
in immunization,quarantine and other forms of
control measures
• In epidemiological studies

9. • Confirm new viral pathogen or emerging viral
infection
• Vaccine study and novel test development for viral
diagnosis

10. Specimen collection
• Viral shedding is greatest during early stages of infection.
• So specimen should be collected as early as possible following the
onset of symptomatic disease
• Clinical sample should be taken from the right place at the right time
• Site:
• Depends on the clinical signs and symptoms together with
knowledge of the pathogenesis of the disease
• As a general rule , the epithelial surfaces that constitutes the portal
of entry and primary site of viral replication is usually the best site
for obtaining samples

12.  Vesicular fluid : HSV-1, HSV-2, Varicella-Zoster virus
 For slide : Use scalpel or gauge needle to unroof vesicle. Gently
scrape the base of the vesicle with a blunt end of a scalpel or
wooden applicator and smear on a slide.
 For electron microscope grid: an ultra-thin plastic covering can
be gently touched down (shiny side or plastic film-side) against
the lesion.
 Bronchial washes,nasal swab, rectal swab, stool specimens, urine,
bone marrow tissue are also collected for virus isolation.

13. • Specimens are to be collected as aseptically as possible.
1. Aspirated secretions – preferable
2. Swabs – easier to use for collection. These are made up
of cotton, dacron or nylon.
3. Tissue sample must be kept moist.
4. If serology is considered useful, serum is to be collected.
• Appropriate specimen selection dictates that the specimen
type and viruses suspected should be indicated on the
requisition.

14. Lifting a crust or ‘roof’ from the skin

15. Applying microscope slide to lesion

16. Applying EM grid to lesion

17. SPECIMEN TRANSPORT AND STORAGE
• Specimens intended for virus isolation must always be kept cold and
moist
• Immediately after collection the swab should be swirled around in a
small screw-capped bottle containing virus transport medium
• The swab stick is then broken off aseptically into the fluid, the cap is
tightly fastened and secured with adherent tape to prevent leakage,
and the bottle is labeled with the patient’s name, date of collection,
and nature of specimen.
• This is then dispatched immediately to the laboratory, accompanied by
a completed laboratory request form which must include an adequate
clinical history, a provisional diagnosis, request for a particular test, and
the date of onset of the illness

18. • If a transit time of more than an hour or so, the container should be
sent refrigerated (but not frozen), with cold packs (4°C) or ice in a
thermos flask or styrofoam box.
• International or interstate transport of specimens, particularly in
hot weather, generally requires that the container be packed in dry
ice (solid CO2) to maintain the virus in frozen state

19. • Blood for viral culture transported in a sterile tube
containing anticoagulant must be kept in refrigeration
temparature (4 degree C)
• Serum should be separated from the clot as soon as
possible. Serum can be stored for hours at 40C or for
weeks at -200C. Testing for virus specific IgM should be
done before freezing whenever possible because IgM
may form insoluble aggregates upon thawing giving a
false negative results.

20. Transport medium:
• Transport media : Stuart’s medium, Amie’s medium, Leibovitz – Emory
medium, Hanks Balance Salt solution(HBSS) and Eagle’s tissue culture
medium
• They contain albumin or gelatin - to stabilize virus
antimicrobials - to inhibit bacteria and fungi.

21. DIAGNOSIS OF VIRAL INFECTION
1. Direct Examination – directly detects virus particles.
2. Indirect Examination (Virus Isolation) : specimen inoculated into
cell culture, eggs or animals in an attempt to grow the virus
3. Serology: depends on the detection of rising titres of antibody
between acute and convalescent stages of infection, or the
detection of IgM. In general, the majority of common viral
infections can be diagnosed by serology.

22. Serology
Direct
examination
Indirect
examination
Cell Culture
A. LIGHT MICROSCOPY
Cytology
Histology
B. ELECTRON MICROSCOPY
Morphology of virus particles
Immune electron microscopy
C. IMMUNODIAGNOSIS
Immunofluorescence
Immunoassays
ELVIS
D. MOLECULAR METHODS
Hybridization with specific nucleic
acid probes
Amplification reaction
.
Animals
Eggs
Classical Techniques
1. Complement fixation tests
2. Haemagglutination inhibition
tests
3. Immunofluorescence
techniques
4. Neutralization tests
5. Single Radial Haemolysis
Newer Techniques
1. Radioimmunoassay
2. Enzyme linked immunosorbent
assay
3. Particle agglutination
4.Western blot

23. 1. Light microscopy
• The traditional means of directly demonstrating virus by observing
cellular changes, koilocytes, viral inclusions etc
• PAP/Giemsa/H&E are used.
• Smears or tissues
In general –
intranuclear inclusions Eg: HSV,VZV,CMV,Adenovirus
intracytoplasmic inclusions eg: rabies,molluscum
contagiosum

24. Tzanck smear
• Cytology is commonly used to detect infections with
HSV-Tzanck test
HSV inclusion bodies

25. Negri body in Rabies
CMV Inclusions
basophilic in an enlarged cell
pathognomonic
halo around inclusion
owl's eye" appearance.

26. Henderson patterson bodies in
molluscum contagiosum

27. 2. Electronmicroscopy
• Most useful in detecting noncultivable viruses – virus causing gastroenteritis &
encephalitis.
• In newly recognized viral syndromes can be used to identify characteristic viral
morphology
• Negative staining
• Viruses in the specimen are identified by their morphology,
dimensions and presence or absence of envelope.
• In order to be detected by EM there must be at least 107 virus
particles per milliliter of sample
APPLICATION

28. The penetration of the negative stain
into the herpesvirus particle may reveal
the presence of the viral capsid within
the envelope.
Coronaviruses (family Coronaviridae) are
spherical, lipidcontaining, enveloped
particles with tear-drop-shaped surface
projections or peplomers

29. Immune Electron Microscopy(IEM)
Addition of specific antiserum causes virus particles to form Ab-bound
aggregates which are more easily detected than the single virus.
The sensitivity and specificity of EM may be enhanced by at least a 100
fold by IEM.
IEM allows visualization of virus particles when present in too small
numbers.
Ind : adenovirus and influenza viruses

30. 2 variants:
Classical Immune electron microscopy (IEM) - sample
treated with specific anti-sera before being put up for EM.
Viral particles present will be agglutinated.
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.

31. Antigen detection
• Immunofluorescence
• Immunoassays
• Enzyme linked virus inducible system(ELVIS)

32. Immunoflourescence
• Immunofluorescence or fluorescent antibody staining is widely used
for the rapid diagnosis of virus infections by detection of virus
antigen in clinical specimens, as well as the detection of virus-
specific IgG or IgM antibody. The technique makes use of a
fluorescein-labeled antibody to stain specimens containing specific
virus antigens, so that the stained cells fluoresce under UV
illumination .
• Antigen is detected through the binding to the sample matrix of
specially modified, agent-specific antibodies. The modification is
the “tagging” of the antibody with a fluorochrome that absorbs
ultraviolet light of a defined wavelength, but emits light at a higher
wavelength.

33. • The emitted light is detected optically with a special
microscope equipped with filters specific for the emission
wavelength of the fluorochrome. The fluorochrome can
be bound directly to the agent-specific antibody (direct
immunofluorescence) or it can be attached to an anti-
immunoglobulin molecule that recognizes the agent-
specific antibody (indirect immunofluorescence)
• Commonly used for
adenovirus,influenza,parainfluenza,herpes
simplex,VZV,CMV,rabies

34. Direct IF
• Indirect
IF

35. Direct
Immunofluorescence
More rapid & specific
Best suited – large
quantities of virus are
expected
Indirect immunofluorescence
More sensitive & Less specific
When lower quantities of
virus
Whenever possible – direct test should be done – significantly faster & avoids using
secondary Ab

36. • Immunofluor
escent
analysis of
Herpes
Simplex Virus

37. Immunoassays
• Enzyme Immunoassay – (EIA)
• Optical Immunoassay
• Latex Agglutination
• Lateral flow immunoassay
Detects viral antigen

38. Enzyme Immunoassay – (EIA)
EIAs – been used successfully for the detection of viral antigens for
over 20yrs
• The assays used in this method rely on antibodies directed against a
specific virus or viral antigen that are adsorbed or directly linked to
polystyrene wells in microtiter plates, plastic beads, or membrane-
bound material. When viral antigen is present in a specimen, it is
coated into the well and the antibody conjugated to an enzyme
such as horseradish peroxidase or alkaline phosphatase is added
and attaches to the antigen.A substrate specific for the enzyme is
added and a color reaction occurs that can be monitored by
spectrophotometry or by direct visualization.

40. ELISA
Enzyme-linked immunosorbent
assay (ELISA) is a labeled
immunoassay that is considered
the gold standard of
immunoassays

42. • Technique:
• Coat the microtiter plate with purified antigen by letting an antigen solution
sit in the wells for 30-60 minutes. Wash away unbound antigen with buffer.
• Add serum sample to be tested for specific antibody to plate and allow
specific antibody to bind to the antigen. Wash off unbound antibody.
• Add anti-Ig that will bind to Fc region of specific antibody .The Fc region of
the anti-Ig is covalently linked with enzyme. Wash off unbound antibody-
enzyme complex.
• Add chromogenic substrate: colorless substrate that the enzyme will convert
to a colored product. Incubate until color develops; measure color in a
spectrophotometer. The more color that is detected, the more specific
antibody is present in the unknown sample

44. Solid phase ELISA
RSV,rotavirus,HSV, enteric
virus
Membrane ELISA
RSV, rotavirus.

45. OPTICAL IMMUNOASSAY
• The OIA utilizes a virus-specific antibody coated onto a thin
molecular film on a silicon wafer surface. The clinical sample is
treated to extract and expose any viral antigens present and is then
placed on the surface of the chip. Viral antigen is captured and the
resulting antigen–antibody complex changes the optical thickness of
the film on the chip. The change in the surface thickness is
magnified through addition of a second virus-specific antibody
conjugated to horseradish peroxidase followed by addition of a
substrate such as tetramethylbenzidine (TMB). The presence of
virus antigen is then detected by a change in color in the reflected
light from gold to purple
• Used – detect influenza virus – resp specimens

47. Latex agglutination –
Small latex spheres coated with
Ab – agglutinated with the viral
Ag present in the specimen
Highly non specific reaction
Intended to use – At bed side or
at doctor’s office

48. Lateral flow immunoassay
• The lateral flow immunoassay, also called the immunochromatographic
assay, is an immunoassay that is performed on chromatographic paper along
a single axis .The clinical sample is applied to an absorbent pad and then is
drawn by capillary action through a conjugate pad. If viral antigen is present
in the clinical sample, it will interact with a virus-specific antibody
conjugated to a colored particle (often colloidal gold). The fluid in the sample
carries the antigen–antibody complex to a reaction membrane to which
another virus-specific antibody has been immobilized in a line perpendicular
to the capillary flow direction. The antigen– antibody–conjugate complex is
captured and can be observed as a colored line on the membrane. The
sample is carried further across the reaction membrane to a control line.
Antibody specific for the antibody–conjugate is immobilized along the
control line, and visualization of the control line indicates that the sample
migrated across the membrane and picked up the antibody–conjugate as
designed.

50. Other methods to detect viral antigen
• Radioimmunoassay
• Immunohistochemical staining

51. ENZYME LINKED VIRUS INDUCIBLE SYSTEM (ELVIS)
• It uses a BHK (baby hamster kidney) cell culture system with a cloned
betagalactosidase gene that is expressed only when cells are infected
with a virus.
• ELVIS HSV test system - genetically engineered BHK cells are sold in
multiwelled microtitre plates.
• Following inoculation and overnight incubation of specimen, growth of HSV
results in the production of Betagalactosidase enzyme by the BHK cells.
• Beta galactosidase serves as a reporter molecule.
• When cells are fixed and stained for betagalactosidase activity, positive
staining indicates the presence of HSV 1 or HSV 2. Wells not containing
HSV donot show staining.

52. 4. Molecular Methods
Methods based on the detection of viral genome are also
commonly known as molecular methods.
Hybridization techniques
gene amplification assays

53. HYBRIDISATION TECHNIQUE
• Hybridisation is a renaturation between target single
stranded DNA and probes with base complimentary
to target sequence.
• Hybrid molecules are recognised using a label linked
to the probe

54. Hybridization techniques
Detection of viral
genomic DNA/RNA in a
thin tissue section, in liquid
or on a membrane – new,
sensitive diagnostic tech
Ideal method for
probing of viruses –
HPV, HSV, EBV, HIV

55. Probes
Probe – prepared from an organism
of known identity
Nucleotide bases complementary to
target DNA

56. Labelling
• Radioactive labelling
use of radioactive isotopes such as 32P, 33P and 35S
conjugated to a deoxy nucleotide
• Non radioactive labelling
Nonradioactive labelling involves the introduction into the
probe of nucleotides tagged with a hapten or reporter
molecule.eg: Digoxigenin,Biotin. The labelled probes are
usually detected with a highly specific anti-hapten antibody
conjugated to either alkaline phosphatase or horse radish
peroxidase enzyme, followed by enzymatic detection, using
the relevant substrate

57. Steps
• Denaturation of the target nucleic acid and
immobilised in a membrane
• Hybridisation with labelled probe
• Washing
• Detection by generating a signal

59. 3 main types
1. Solid support hybridization /Filter hybridization
• Southern hybridization
• Sandwich hybridization
• 2. In-solution hybridization
• 3. In-situ hybridization

60. Southern hybridisation

61. b. Gene amplification method
1. Polymerase chain reaction
2. Nucleic acid sequence based amplification
techniques
3. Ligase chain reaction

62. Polymerase chain reaction
• Polymerase Chain Reaction (PCR) a nucleic acid amplification
technique, in which specific genetic material is amplified to
reach detectable levels. In PCR, a certain kind of reagent
(primers) is used to target a small but specific part of the
virus-genome (deoxyribo-nucleic acid (DNA) or ribonucleic
acid (RNA)) in question, and with the help of an enzyme, this
small genomic area is amplified over and over again if the
target is present. As soon as the small target is amplified, a
third component of the PCR reaction (the probe), emits a
signal that can be detected by the PCR instrumentation

65. Types
• Standard PCR and its varients
• RT PCR
• Real time PCR

66. RT- PCR

67. REAL TIME PCR

68. • ADVANTAGES : simple to understand and use,rapid
results,increased sensitivity, can quantify the
products
• Disadvantages : liable to contamination,high degree
of operater skill required, prior information about the
target sequence is necessary in order to generate the
primers

69. Indirect Examination
(viral isolation)
1.Cell Culture Cytopathic effect (CPE)
Hemadsorption
Plaque assay
Immunofluorescence
2. Eggs Pocks on CAM
3. Animals Disease or death

70. •Cell culture is mostly used for identification and cultivation of viruses.
• Cell culture is the process by which cells are grown under controlled conditions.
• Cells are grown in vitro on glass or a treated plastic surface in a suitable growth
medium.
• On incubation the cell divide and spread out on the glass surface to form a
confluent monolayer. A thin layer of cells, or monolayer, is then inoculated with
viruses
– Cell cultures are of 3 basic types –
1. Primary cell culture
2. Semi-continuous or diploid cell culture
3. Continuous or heteroploid cell culture
Cell culture -

71. Primary cells –
•These are normal cells
derived from animal or
human cells.
•They are able to grow
only for limited time and
cannot be maintained in
serial culture.
•They are used for the
primary isolation of
viruses and production of
vaccine.
•Examples: Monkey kidney
cell culture, Human
amnion cell culture
Semi-continuous or diploid
cell culture
•They are diploid and
contain the same number of
chromosomes as the parent
cells.
•They can be sub-cultured up
to 50 times by serial transfer
following senescence and
the cell strain is lost.
•They are used for the isolation
of some fastidious viruses and
production of viral vaccines.
•Examples: Human embryonic
lung strain, Rhesus embryo cell
strain
Continuous or heteroploid
cell culture
•They are derived from
cancer cells.
•They can be serially
cultured indefinitely so
named as continuous
cell lines
•They can be maintained
either by serial subculture or
by storing in deep freeze at -
70°c.
•Due to derivation from cancer
cells they are not useful for
vaccine production.
•Examples: HeLa
(Human Carcinoma of
cervix cell line), HEP-2
(Human Epithelioma of
larynx cell line) etc.

72. • Viruses readily isolated by
cell culture
• Herpes Simplex
• Cytomegalovirus
• Adenoviruses
• Polioviruses
• Coxsackie B viruses
• Echoviruses
• Influenza
• Parainfluenza
• Mumps
• Respiratory Syncytial Virus

73. CYTOPATHIC EFFECT
• Detection of growth of virus is observed by the changes in the cell
culture monolayer
• – Cytopathic Effect (CPE)
• CPE is characterized by:
• Rounding
• Detachment (plaques)
• Clumping
• Ballooning (Giant cell)
• Fusion (syncytium formation)
• Inclusion body formation

74. Bovine fetal spleen cells 2 days postinfection
with HERPES VIRUS

75. • Haemadsorption: Cells acquire the ability to stick
to mammalian red blood cells. Haemadsorption is
mainly used for the detection of influenza and
parainfluenza viruses.
Haemadsorption of influenza virus
HAEMADSORPTION

76. PLAQUE ASSAY
•Plaque-based assays are the standard method used to determine virus concentration
in terms of infectious dose.
•Viral plaque assays determine the number of plaque forming units(pfu) in a virus
sample, which is one measure of virus quantity.
•This assay is based on a microbiological method conducted in petri dishes or multi-
well plates. A series of 10-fold dilutions of a viral suspension is inoculated onto
monolayers of cultured cells for an hour or so to allow the virions to adsorb to the
cells. The infected cells are then overlaid with medium in an agar or methylcellulose
gel, to ensure that the spread of viral progeny is restricted to the immediate vicinity of
the originally infected cell.

77. •A viral plaque is formed when a virus infects a cell within the
fixed cell monolayer.
• The virus infected cell will lyse and spread the infection to
adjacent cells where the infection-to-lysis cycle is repeated.
•The infected cell area will create a plaque (an area of infection
surrounded by uninfected cells) which can be seen with an optical
microscope or visually by adding a crystal violet solution for 15
minutes until it has colored the cytoplasm, gently removing the
excess with water will show uncolored the location of dead cells

79. • Shell vial culture
Shell vial culture is a modification of the conventional cell culture technique for rapid
detection of viruses in vitro. The technique involves inoculation of the clinical specimen on to
cell monolayer grown on a cover slip in a shell vial culture tube, followed by low speed
centrifugation and incubation. This system works on the principle that the low speed
centrifugation enhances viral infectivity to the susceptible cells. It is thought that the minor
trauma to the cell surface produced as a result of low speed centrifugation mechanical force
enhances the viral entry in to the cells, which in turn reduces the total time taken for the virus
to produce infection of cells. Virus may be detected by direct fluorescent antibody (DFA) or
indirect fluorescent antibody (IFA) staining within hours or days of inoculation.

80. Figure 5.17a: Tissue culture cells are grown on
coverslips on the bottom of shell vials.
Modified from J. H. Shelhamer, et. al., Ann. Intern. Med. 124 (1996): 585-599.
Figure 5.17b: Detection of Herpes Virus
Simplex 1 using the shell vial technique and
immunofluorescence.
Reproduced from Athmanathan, S., S. R. Bandlapally, and G. N. Rao, BMC Clin. Pathol. 2 (2002):
1-5.

81. Problems with cell culture
• Long period (up to 4 weeks) required for result.
• Often very poor sensitivity, sensitivity depends on a large extent on the
condition of the specimen.
• 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 virus,
parvovirus, papillomavirus.

82. 2. Embryonated Eggs
Embryonated hen’s egg – first used for cultivation of
viruses
Now for vaccine production – esp influenza
Inoculation produce visible lesions – POCKS

83. Inoculation of Virus
•Chicken, duck, and turkey eggs are the most common
choicesfor inoculation
•The egg used for cultivation must be sterile and the shell
should be intact and healthy
•Rigorous sterile techniques must be used to prevent
contamination by bacteria and fungi from the air and the outer
surface of theshell
•The egg must be injected through the shell, usually by drilling
a hole or making a small window
•The viral suspension or suspected virus- containing fluid is
injected into the fluid of the egg
•The exact tissue that is inoculated is guided by the type of
virus being cultivated and the goals of the experiment

84. Routes of Viral Inoculation
• An embryonated egg offers
various sites for the
cultivation of viruses.
• The different sites of viral
inoculation in
embryonated eggs are:
1.Chorioallantoic membrane
(CAM)
2.Amniotic Cavity
3.Allantoic Cavity
4.Yolk sac

86. The signs of viral growth include:
•Death of the embryo
•Defects in embryonic development and
localized areas of damage in the
membranes, resulting in discrete opaque
spots called pocks

87. smallpox virus pocks on the chorioallantoic membrane of a
developing embryonic chick.

88. 3. Animal Inoculation -
Earliest method for isolation of viruses
– Susceptible experimental animals like Mice, Monkey, Rabbits, Guinea Pigs
etc. are used for the cultivation of viruses. Virus sample to be cultivated
should injected into the experimental animal. It is important to select
specific host animal for particular viruses. Route of inoculation of viral
sample in the host cell also play important role in cultivation of viruses

89. Laboratory animals used

90. Routes of inoculation
•Intracerebral
•Subcutaneous
•Intraperitoneal
•Intranasal

91. • The animal is :
• observed for visible lesions
• observed for signs of
disease
• or it is killed to examine
the infected tissue for virus

92. Disadvantages –
Immunity of the animal may interfere with viral growth
Animals may harbor latent virus
Animal inoculation – used to study
• Pathogenesis of the disease
• Immune response in the host
• Oncogenesis

93. Serological Assay
• The detection of newly developed, virus-specific antibody or the detection
of an increase in titer of preexisting antibody is important in viral diagnosis
and is one of the most commonly used methods in epidemiologic studies of
viruses. Most primary infections or reinfections result in the production of
specific antibodies.
• Viral serology – now used primarily to
detect immune status
make a diagnosis in which virus cannot be cultivated or detected by
immunoassay

94. Humoral immune response –
IgM – produced by the host –
first interaction with the virus –
relatively short period of time
Later cells producing IgM switch
to produce IgG
A second encounter – produce
only IgG - B lymphocytes retain
the memory of Virus and respond
quickly with large no of Abs than
at the initial interaction –
Anamnestic response

95. • Serological diagnosis is usually based on either
the demonstration of the presence of specific
IgM antibodies or a significant increase in the
levels of specific IgG antibodies between two
consecutive samples taken 7–10 days apart.

97. Complement fixation
test
• The complement fixation
test (CFT) is a classical
laboratory diagnostic test
• The test mainly measures
IgG antibodies.

98. Hemagglutination inhibition test
• Many viruses bind to
hemagglutininmolecules found at the
surface of red blood cells of various animal
species and this can cause aggregation of
red cells in suitable conditions.
• Prevention of this aggregation, called
hemagglutination inhibition, by specific
antiviral antibodies in the patient’s serum
has been widely used for diagnostic
purposes.
• Both IgM and IgG antibodies are able to
inhibit hemagglutination.

100. Neutralization test
Antibodies that decrease the infectious capacity of
the virus are called neutralizing antibodies
Both IgM and IgG antibodies participate in
the neutralization.
In the assay, known amounts of infectious virus are
mixed with the serum sample and incubated for a
short period after which the residual infectivity is
measured using cell cultures or test animals. This
infectivity is then compared with the infectivity of
the original virus and the neutralizing capacity is
calculated from this result.

101. ELISA

102. WESTERN BLOTTING
Serum antibodies

103. Some major drawbacks of serology are –
1. Serological assays measure host response rather than
detection of virus
2. Ab producing capabilities of human host varies widely
3. Ab level does not necessarily correlate with the activity
level of the infection

105. DNA VIRUSES
HSV
• Light microscopy- intranuclear inclusion bodies
• EM of vesicle cell smears - cannot be distinguished from VZV particles.
• Direct IF of vesicular cell smears
• PCR
• Immunoperoxidase technique
• Virus isolation – virus culture(Vero, Hep-2, human diploid fibroblasts),mice
inoculation,embryonated eggs
• Serology - primary infection may be diagnosed by rising antibody titre of
IgM.CFT,RIA,ELISA

106. VZV
• LM & EM – similar to HSV
• Virus isolation - human diploid (HEL, HEK), typical CPE. Virus is
difficult to isolate because it is quite labile and vesicle fluid must
be inoculated as soon as possible after collection
• Direct IF of vesicular cell smears
• PCR
• Serology - CFT can be used for diagnosis but not sensitive enough
for immunity screening. IF and ELISA may be used for immune
status screening

107. CMV
• LM – intranuclear and intracytoplasmic inclusion
bodies,large owl eye cells
• EM – morphology of viruses
• Virus isolation - HEL (human embryonic lung
fibroblasts)used, takes 2 to 4 weeks for a characteritic CPE to
appear.
• Immunohistochemistry
• Insitu Hybridisation
• PCR for CMV- there is a trend towards the use of
quantitative PCR assays.
• Serology - ELISA

108. Papillomaviruses
• Human papillomaviruses may be detected by EM, IF,
hybridization, and PCR techniques on swabs, scrapings or
biopsies of lesions.
• Serology has no role in the diagnosis of HPV infection
because the Ab response may require several months to
form and Ab levels are usually low.

109. RNA viruses
INFLUENZA VIRUS
• Virus isolation- MK,LLC-MK2,MDCK
• IF&EIA
• RT-PCR
• Serology- CFT,HAI

110. Parainfluenza virus
• Virus isolation-MK,LLC-MK2
• IF
• RT-PCR
• Serology- CFT,HAI,Neutralisation,EIA

111. MUMPS
• Virus culture- MK, LLC-MK2, Vero cell lines
• RT-PCR
• Serodiagnosis –Mumps specific IgM antibodies or four
fold rise of mumps specific IgG titers between acute
and convalescent phases

112. MEASELS
• Virus isolation - MK, HEK cells may be used. CPE takes a week or more to
appear. CPE consists of multinucleated giant cells. Definitive identification
requires neutralization. Measles may be isolated from throat swabs,
conjunctival swabs, and urine. Recovery of measles from SSPE specimens
require special processing and cocultivation techniques.
• RT-PCR
• Direct detection - typical multinucleated giant cells may be found by direct
examination of nasopharyngeal secretion or conjunctival swab. IF may be
used to stain buccal, nasopharyngeal, or urinary sediment smears.
• Serology – IgM antibody assay,CFT and HAI are widely used.

113. HIV
• Virus isolation
• Detection of viral antigen eg: p24
• Detection of viral nucleic acid by PCR
• Detection of specific antibody- ELISA,Western blot
Note: in children below 18 months,antibody detection
is not used,p24 antigen detection ,viral nucleic acid
detection by PCR is commonly used

114. CORONA VIRUS
• NUCLEIC ACID AMPLIFICATION TEST(NAAT)- RT-PCR,Real
time RT PCR
• Antigen Detection–ELISA
• Antibody Detection - ELISA
• Viral culture is not recommended for routine diagnosis.

115. Conclusion
• Advances made in diagnostic techniques over the past decade
have significantly improved the accuracy and timeliness of a
viral diagnosis. Molecular and serological techniques should be
used in a complimentary fashion for the diagnoses of virus
infections. Accurate diagnosis of viral infections enhances the
ability of the clinician to make decisions on appropriate
treatment of patients, evaluate disease progression and
prevent misuse of antibiotics. Knowledge of the pathogen
involved also allow implementation of infection control and
monitoring of success of antiviral treatments that may affect
the prognosis of patients.

116. • Epidemiological data collected through accurate
diagnostics play an important role in public health
through identification and control of outbreaks,
implementation of appropriate diagnostic tests,
vaccination programs and treatment but also to
recognize common and emerging pathogens in a
community.

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