T.M. River, 1937 Modified from Koch’s Postulates (proof of bacterial diseases) Isolate virus from diseased hosts. Cultivation of virus in host cells. Proof of filterability. Production of a comparable disease when the cultivated virus is used to infect experimental animals. Reisolation of the same virus from the infected experimental animal. Detection of a specific immune response to the virus. Much more expensive and difficult to study animal viruses than bacteriophages Cultivation in host cells Living animal Embryonated chicken eggs Cell or tissue culture (= in vitro) Over 60% of all infectious disease cases seen by a physician are due to viral infections. Quality of patient specimens and their transport to the laboratory is importantViral Diagnostics in the Clinical Laboratory Types of specimens:- Respiratory tract infections: Nasal and bronchial washings, throat and nasal swabs, sputum Eye infections: throat and Conjunctival swab/scraping Gastrointestinal tract infections: stool and rectal swabs Vesicular rash: vesicle fluid, skin scrapings Maculopapular rash: throat, stool, and rectal swabs CNS (encephalitis and meningitis cases): stool, tissue, saliva, brain biopsy, cerebrospinal fluid Genital infections: vesicle fluid or swab Urinary tract infections: urine Blood borne infections: blood Sterile, leak proof container Minimal interval Transport media Viral infusion broth (VIB) Sucrose-phosphate-glutamate (SPG) Storage temperature: 4 deg C for up to 96 hours Minus 70 deg C beyond 96 hours Repeated cycles of freezing and thawing to be avoided 106 virus particles per ml required for visualization, 50,000 - 60,000 magnification normally used.Specimens are negatively stained by Potassium phosphotungstate and scanned under EM Viruses may be detected in the following specimens. Virus particles are detected and identified on the basis of morphology. A) Shape  Rabiesvirus –bullet shaped Rotavirus –Cart wheel Coronavirus –petal shaped peplomers Adenovirus –space vehicle shaped Astrovirus ---Star shaped B) Direct detection from specimens For viruses that are difficult to cultivate ,EM can be used as primary tool for diagnosis Faeces Rotavirus, Adenovirus Norwalk like viruses Astrovirus, Calicivirus Vesicle Fluid HSV VZV Skin scrapings papillomavirus, orf molluscum contagiosum As an alternative to tissue culture As tissues culture is time consuming and technically demanding ,EM is used as an alternative :- 1) Vesicular rashes –HSV and VZV detection from vesicular fluid 2) Meningitis—Detection of enterovirus and mumps from CSF. Virus detection from tissue cultures EM can be used for detection of viral growth in tissue culture The 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 Direct immumofluroscence

1. LABORATORY DIAGNOSIS
OF VIRAL INFECTIONS
DR MAYURI

2. River’s Postulates
 T.M. River, 1937
 Modified from Koch’s Postulates (proof of bacterial
diseases)
1. Isolate virus from diseased hosts.
2. Cultivation of virus in host cells.
3. Proof of filterability.
4. Production of a comparable disease when the
cultivated virus is used to infect experimental
animals.
5. Reisolation of the same virus from the infected
experimental animal.
6. Detection of a specific immune response to the
virus.

3. Methods of Study
Much more expensive and
difficult to study animal
viruses than bacteriophages
 Cultivation in host cells
 Living animal
 Embryonated chicken eggs
 Cell or tissue culture (= in vitro)

4. Viral Diagnostics in the Clinical
Laboratory
 Over 60% of all infectious disease
cases seen by a physician are due
to viral infections.
 Quality of patient specimens and
their transport to the laboratory is
important.

5. Indications for lab diagnosis of
viral infection
5
• If rubella is diagnosed in the first trimester of pregnancy,
abortion is recommended
• If baby is diagnosed to have Hepatites B –Igs within 12 hrs of
birth
For proper
management of
certain diseases
• for which antiviral chemotherapy is available (herpes viruses)
Diagnosis of
diseases caused by
viruses
• For hepatitis B & HIV virus helps to prevent spread of these
viruses
Screening of blood
donors
• To initiate appropriate control measures
Early detection of
epidemics

6. Cont….
11/28/2022
Cristi Francis
6
 To start post exposure prophylaxis  of
antiretroviral drugs to health care workers
following needle stick injuries .
 Survillance purpose- To asses the disease
burden in the community by calculating
prevalance and incidence of viral infections

7. Storage and Collection of Biological
Specimens for Viral Testing
Types of specimens:-
 Respiratory tract infections: Nasal and bronchial washings, throat and
nasal swabs, sputum
 Eye infections: throat and Conjunctival swab/scraping
 Gastrointestinal tract infections: stool and rectal swabs
 Vesicular rash: vesicle fluid, skin scrapings
 Maculopapular rash: throat, stool, and rectal swabs
 CNS (encephalitis and meningitis cases): stool, tissue, saliva, brain
biopsy, cerebrospinal fluid
 Genital infections: vesicle fluid or swab
 Urinary tract infections: urine
 Blood borne infections: blood

8. TRANSPORT AND STORAGE OF SPECIMENS
8
 Sterile, leak proof container
 Minimal interval
 Transport media
 Viral infusion broth (VIB)
 Sucrose-phosphate-glutamate (SPG)
 Storage temperature:
 4 deg C for up to 96 hours
 Minus 70 deg C beyond 96 hours
 Repeated cycles of freezing and thawing to be
avoided

9. General approaches for Laboratory diagnosis of Viral
infections
Direct demonstration of
virus ,viral Ags or viral
genome
Isolation of virus
Detection of specific viral
antibodies
11/28/2022 Cristi Francis 9

10. Direct Demonstration of virus
• Examine specimen for viruses
Electron Microscope
• Labeled antibody
Immuno-electron
microscopy
• Fluorescent tag bound to Fc region of Ab
Immunofluorescence
• Histological appearance of affected cells
(Immunoperoxidase staining)
• Inclusion bodies
Light microscope
11/28/2022 Cristi Francis 10

11. 1) Electron Microscopy
11
 106 virus particles per ml required for visualization,
 50,000 - 60,000 magnification normally used.Specimens are negatively
stained by Potassium phosphotungstate and scanned under EM
 Viruses may be detected in the following specimens.
 Virus particles are detected and identified on the basis of morphology.
A) Shape 
 Rabiesvirus –bullet shaped
 Rotavirus –Cart wheel
 Coronavirus –petal shaped peplomers
 Adenovirus –space vehicle shaped
 Astrovirus ---Star shaped

12. Cont…
12
 B) Direct detection from specimens
 For viruses that are difficult to cultivate ,EM can be used as primary tool
for diagnosis
Faeces Rotavirus, Adenovirus
Norwalk like viruses
Astrovirus, Calicivirus
 Vesicle Fluid HSV
VZV
 Skin scrapings papillomavirus, orf
molluscum contagiosum

13. Cont..
13
c)As an alternative to tissue culture
 As tissues culture is time consuming and technically
demanding ,EM is used as an alternative :-
 1) Vesicular rashes –HSV and VZV detection from
vesicular fluid
 2) Meningitis—Detection of enterovirus and mumps from
CSF.
 Virus detection from tissue cultures EM can be used for
detection of viral growth in tissue culture

14. Electronmicrographs
Adenovirus Rotavirus

15. 2)Immune Electron Microscopy
• The 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
11/28/2022 Cristi Francis 15
Expensive equipment
Expensive maintenance
Require experienced observer
Sensitivity often low
Problems with Electron Microscopy:

16. 3) Fluroscent Microscope
 Direct immumofluroscence technique is used to
detect viral particles in the clinical samples.
 Procedure:-Specimen is mounted on slide stained
with antiviral antibody tagged with fluroscent dye and
viewed under fluroscent microscope
Advantages:
Convenient for many hospital (diagnostic level) labs
Easy availability of good quality commercial reagents
Disadvantages:
Cannot be used for viruses with complicated antigenic structure
(eg. Rhinoviruses, enteroviruses

17. Cont…
17
 Clinical applications:-
 a)rabies virus Ag in skin biopsy , corneal smear of
infected patients
 b)Syndromic approach-Rapid diagnosis of respiratory
infections by influenza virus, adenovirus, Herpesvirus
can be carried out by adding specific antibodies to
each of these viruses.
 c)Detection of adenovirus from conjuctival smears

18. 4) Light Microscopy inclusion
bodies
• It is basically crystalline aggregates of virions or
collections of replicating virus particles either in the
nucleus or cytoplasm.
• It is not sensitive or specific
Most characteristic histological feature in virus infected
cells
Structures with distinct size, shape, location & staining
properties
crystalline aggregates of virions or
Made up of virus antigens present at the site of virus
synthesis or
Degenerative changes produced by viral infection
18

19. Presumptive etiologic diagnosis
• Intranuclear inclusions
(mostly DNA viruses)
• Acidophilic(pink ) Cowdry
type A(HSV, varicella zoster
virus)-Torres bodies in
yellow fever
• Cowdry type B(Adeno and
polio)
• Basophilic(blue) Owl eye
appaerance in CMV , adeno
virus
• Intracytoplasmic inclusions
(mostly RNA viruses)
• Guarnieri bodies(small pox)
• Negri bodies(Rabies)
• Paschen bodies in (variola )
• Bollininger bodies in
fowlpox
• HP bodies in Molluscum
contagiosum
Cristi Francis 19

20. Negri body ( Intracytoplasmic)- Rabies
Owl eye (Intranuclear )- CMV
11/28/2022 Cristi Francis 20

21. Immunoperoxidase staining
• Tissue sections or cells coated with viral
antigens are stained using antibodies tagged
with Horse radish peroxidase following which
hydrogen peroxide and a colouring agent
(benzidine derivative) are added.
• The color insoluble precipitate complex
formed can be viewed under light microscope
.
11/28/2022 Cristi Francis 21

22. Cont…
22
Tzanck test:
Performed by staining material from vesicular
scrapings with Giemsa, H&E or Papanicolaou stain
Multinucleated giant cells seen with the nuclei having
a ground-glass and molded or multifaceted
appearance
Herpes : Multinucleated giant cell--

23. 5) Antigen detection
23
 Include immunofluorescence testing of
nasopharyngeal aspirates for respiratory viruses e.g..
RSV, flu A, flu B, and adenoviruses,
 Detection of rotavirus antigen in diarrheic stool
 p24 Ag detection for HIV infected patients serum
 NS1 Ag for dengue virus
 Detection of HSV and VZV in skin scrappings,
 Detection of HBsAg and HbeAg for Hepatites B vitrus
from serum
 Advantage : rapid, result available within a few hours

24. 6)Detection of viral antibodies
24
 Antibody detection from serum
 Conventional diagnostic techniques:--
1)Heterophile agglutionation test(Paul Bunnel
test for EBV )
2) Haemagglutination inhibition (HAI) for
influenza virus and arbovirus infection
3) Neutralization test for poliovirus and
arbovirus
4) Complement fixation test for
poliovirus,arbovirus and rabies virus infection

25. Cont…
25
 Newer diagnostic techniques :-
 1)Anti-Hbc,AntiHBs and AntiHBe antibodies for
Hepaties B infection.
 2)Anti-Hepatitis C antibodies
 3)Antibodies against HIV 1 and HIV 2 antigens
from serum
 4) Anti-Dengue IgM/IgG antibodies from serum

26. Molecular Methods
26
 Advantages :-
 More sensitive ,Specific, Quick results
 Methods:--
1)Nucleic acid probes
2)PCR
3)RT-PCR
4)Real time PCR

27. 1)Nucleic acid probes
27
 Enzyme or radiolabelled nucleic acid sequence
complemantary to a part of nuclei acid sequence of
target virus.
 When added to clinical sample , it hybridizes to the
corresponding part of VNA .Depending on type of
label attached probe can be detected by calorimetric
methods (Dot Blot hybridization or gamma counting)
 Without amplification it detectes viral genes in the
specimen
 Used in identification of - CMV
Papillomavirus
Epstein – Barr virus

28. 2)PCR
28
 DNA Amplification involves 3 steps :-
 a) Viral DNA extraction from the specimen
 b) Amplification of specific region of viral DNA
 c) Detection of amplified products by gel
electrophoresis
 Used to in diagnosis of : HIV – 1 & 2,
Measles. Rota viruses.
Rhinovirus,Enteroviruses.HPV, Coxackie.
Parvoviruses B 19 ,Echoviruses, HSV, HHV –
Hepatitis (B,C,D,E). Rubella virus, V-Z virus.
.

29. Cont …
29
 Advantages of PCR:
 Extremely high sensitivity, may detect down to one viral genome
per sample volume
 Easy to set up
 Fast turn-around time
 Disadvantages of PCR
 Extremely liable to contamination
 High degree of operator skill required .
 .

30. 3) RT-PCR
30
 For detection of RNA viruses.
 After RNA extraction, viral RNA is reverse
transcribed to DNA then subjected for
amplification .
 Both PCR and RT-PCR cannot quantify the viral
load in specimen .

31. 4)Real time PCR
31
 Advantage of quantifying viral nucleic acid load in
specimen.So used to moniter the treatment .
 It takes less time than PCR

32. Animal Inoculation
32
 Earliest method for cultivation of viruses.
 Reed and colleagues (1900) used human
volunteers for work on Yellow fever.
 Landsteiner and Popper (1909)- Monkeys for
isolation of polioviruses.
 Theiler (1903)- Pioneered use of white mice.
 Infant (suckling mice)- coxsackie and arboviruses
 Other animals – Guinea pig, rabbit and ferret
 Inoculation – Intracerebral, subcutaneous,
intraperitoneal or intranasal
 Growth detected – Death, disease or visible lesion

33. Cont…
33
 Disadvantage – 1) Immunity may interfere with viral
growth.
2) Animal often harbour latent
viruses
 Uses (Research use):-To study viral pathogenesis
or viral oncogenesis
 (Diagnostic use):-Primary isolation of virus which
are difficult to cultivate like arbovirus and
coxsackie virus.

34. Embryonated Egg
Dr.T.V.Rao MD
34
 Goodpasture (1931) – First used hen’s egg for
cultivation of viruses and the method was further
developed later by Burnett
 8-11 days old eggs are used.
 Choreoallantoic membrane (CAM) – visible lesion
(Pocks).

35. Embryonated Egg inoculation
35

36. 36
Site Organism Age of Embryo
Chorio - allantois Pox and Herpes group
Rous sarcoma virus
10-12 days
Amniotic sac Orthomyxoviruses and
paramyxoviruses
7-12 days
Allantoic sac Influenza virus for vaccine
production ,Mumps virus,
Newcastle virus
10-12 days.
Yolk sac Some viruses, Chlamydia
and rickettsiae
6-7 days

37. Cont….
37
CAM-Each infectious virus particle form one pock.So
number of pocks present represent number of viruses in
inoculum . Different virus have different morphology
 Vaccinia pocks –more haemorrhagic and necrotic
than variola .
 Pocks of HSV-2 are larger than HSV-1
 Ceiling temperature:-It is the maximum temperature
above which pock formation is inhibited .
 Eg:- Variola -37 C
Vaccinia -41 C
Amniotic cavity—Primary isolation of influenza virus .
Viral growth measured by detection of haemagglutinin
antigens in amniotic fluid .

38. Cont….
Dr.T.V.Rao MD
38
Allantoic cavity –Larger cavity so used for better
yield of viral vaccines .
 Egg derived vaccines:- Influenza vaccine , Yellow
fever (17D) vaccine and rabies (Flury Strain )
vaccine .
 Duck eggs are bigger than hens egg hence
produce better yield of rabies virus for preparation
of inactivated non neural vaccines .
Yolk sac –Growth of encephalities virus may result
in death of embryo .

39. Tissue Culture
Dr.T.V.Rao MD
39
 Steinhardt and colleagues (1913)- maintained the
vaccinia virus in fragments of rabbit cornea.
 Maitland (1928)- used chopped tissue in nutrient
media.
 Enders Weller and Robbins (1949)- grown
Poliovirus in tissue of non neural origin.

40. Types of tissue cultures
40
1.Organ Culture:
 Small bits of organ maintained preserving original
architecture.
 Isolation of fastidious viruses which are having
affinity to specific organs.
 e.g. Tracheal ring organ culture for Coronavirus.

41. 2.Explant Culture
41
 Fragments of minced tissue can be grown as
‘explants’ embeded in plasma clots.
 Originally known as tissue culture.
 Useful for isolation of viruses in the Latent state.
 e.g. Adenoid tissue explant culture.
Seldom employed.

42. 3.Cell culture
42
This is the only isolation method which is used now.
Tissues are completely digested by the action of
proteolytic enzymes like trypsin or collagenase followed
by machanical shaking which is done so that
component cells are dissociated into individual .Cells are
washed counted and suspended in growth medium

43. MEDIUM FOR CELL CULTURE
Dr.T.V.Rao MD
43
 Eagle’s minimum essential medium (MEM)
 Base (Hank’s or Earle’s balanced salt solution)
 L-amino acids
 Vitamins
 Glucose
 Salts and 5-10% fetal calf serum
 Antibiotics
 Phenol red as indicator
 Buffer- bicarbonate –pH-7.2-7.4
 Such media will enable cell to multiply with division
time 24-48 hour

44. 44
Tissues are dissociated into component
cells.
Washed, counted and suspended in
growth medium.
Antibiotics and phenol red are added
Dispensed in bottles, tubes or Petri
dishes
Cells adhere to glass surface
Confluent monolayer sheet of cells

45. 1. Primary cells cultures
45
 Normal cells freshly taken from organs and
cultured.
 Capable of only limited growth in cultures.(5-10
divisions)
 They maintain a diploid karyosome .
 e.g. Monkey kidney, Human amnion and Chick
embryo cell cultures.
 Use – Primary Isolation and cultivation for vaccine
production.
Ex-Rhesus monkey kidney cell culture.
Human amnion cell, Chick embryo cell line

46. 2.Secondary or diploid cell strains
46
 They can divide maximum upto 10-50 divisions
before they undergo death .
 They are derived from normal host cells and
maintain diploid karyosome .
 Derived from human fibroblasts so useful for
isolation of fastidious viruses and viral vaccine
preparation .
Ex- WI-38 Human embryonic lung cell strain.
HL-8 Rhesus embryo cell strain
Human fibroblast cell line
MRC-5

47. 3.Continuous/Established cell
lines
47
 Derived from cancerous cells ,hence are capable of
indefinite growth .
 They possess altered haploid chromosome.
 They are easy to maintain in the laboratories by serial
subculturing so most widely used cell lines .
Ex- HeLa-Human carcinoma of cervix cell line
HEP-2-Human epithelioma of larynx cell line
Vero-Vervet monkey kidney cell line
BHK 21– Baby Hamster Kidney cell line
McCoy –Human synovial carcinoma cell line
KB –Human carcinoma of nasopharynx cell line

48. Detection of virus growth in
cell culture
48
 1 Cytopathic effect
 2 Metabolic inhibition
 3 Haemadsorption
 4 Interference
 5 Transformation
 6 Immunoflurescence
 7 Detection of viral specific Nucleic acid
 8 Detection of enzymes
 9 Electron microscopy

49. 1 Cytopathic effect
49
 Roundening of cells- Picornavirus (Viral replication
leads to neclear pyknosis , rounding, refratility,
degeneration)
 Cell necrosis and lysis –Enterovirus
 Syncytium formation- measles, RSV, HSV
(neighbouring cell fused together to form
multinucleated giant cell).
 Discreate focal degeneration –Herpes virus
 Rounding and aggregatation –Adenovirus (large
granular clumps like bunches of grapes )
 Cytoplasmic vacuolation-SV-40

50. Dr.T.V.Rao MD
50

51. 2.Metabolic inhibition
Dr.T.V.Rao MD
51
 The medium turns acid due to cellular metabolism
in normal cell cultures .
 When viruses grow in cell cultures ,Cell
metabolism is inhibited and no acid production
 Indicated by change in color of indicator (phenol
red) of medium

52. 3.Haemadsorption
Dr.T.V.Rao MD
52
 When haemagglutinating viruses grow in cell
culture , their prescence is indicated by addition
of guinea pig erthrocytes to cultures .They will
absorb onto surface of cells if viruses are
multiplying in culture ….K/a Haemadsorption

53. 4. Interference .
Dr.T.V.Rao MD
53
 The multiplication of one virus in cell usually
inhibits multiplication of second virus called the
challenge of virus , when added to culture
 The growth of a non cytopathogenic virus in cell
culture can be tested by subsequent challenge
with a known cytopathogenic virus .
 The growth of first will inhibit infection by the
second virus by interference .

54. 5.Tranformation
54
 Tumor forming (oncogenic viruses ) induce cell
tranformation and loss of contact inhibition so
growth appears as piled up fashion forming
microtumors
 Ex-Herpes virus , adenovirus ,hepadnavirus ,
papovavirus , retrovirus

55. 6 .Immunofluroscence and
others
55
Cell from virus infected cultures can be stained by
flurescent conjugated antiserum and examined
under UV microscope for presence of virus antigen
.
 7.Detection of viral specific nucleic acid –PCR
 8.Detection of enzymes –Reverse transcriptase in
retrovirus
 9.Electron microscopy –viruses have distinctive
appearance and can be detected by EM of
ultrathin sections of infected cells

56. Viral assay
Dr.T.V.Rao MD
56
The virus content in specimen can be assayed in 2
ways :-
A). Total virus particle count
1 Electron Microscopy
2 Haemagglutination
B). Infectious virions assay
1 Quantal assay
2 Quantitative infective assay :--a) Plaque assay
b) Pock assay

57. A). Total virus particle count
Dr.T.V.Rao MD
57
1)Electron Microscopy:-
 By simple negative staining ,the virus particles in
a suspension can be accounted directly under
EM .
 The virus suspension can be mixed with a known
latex particles.
 The ratio between particles under EM gives an
indication of the virus count

58. Cont..
58
2)Haemagglutination :-
 Determination of haemagglutinating titres with
haemagglutinating viruses.
 Approx 107 influenza virions are required to
produce macroscopic agglutination of convenient
quantity of chicken erthrocytes (0.5 %
suspension )

59. B). Infectious virions assay
59
1) Quantal assay :-
 Introduced in animal virology by Dulbecco (1952)
 Indicate Presence or absence of infectious viruses.
 Assays can be carried out in animals , eggs or tissue
culture. Endpoints used for infectivity titration are
death of animals , haemagglutination production in
amniotic fluid or appearance of CPE in cell cultures .
 These assays determine the extent to which a virus
suspension can be diluted and still containes
infectious viruses
 The virus titre expressed as ID50/ml the highest
dilution of inoculum that would produce an effect in 50
% of animals , eggs or cell cultures

60. 2).Quantitative Assays
a) Plaque assays
 Lytic viruses only
 Steps
 Serial dilution of virion-
containing solution
 Create tissue culture plates
 Spread diluted virus
 Overlay with agar—
prevents diffusion
 Count number of plaques
 Each plaque represents 1
PFU (Plaque Forming Unit)
Figure : Plaque assays used to
quantitate a viral stock.
Courtesy of Teri Shors

61. Cont…
61
b) Pock assay:-
 Viruses like herpes and vaccinia form pocks when
inoculated on CAM on embryonated egg .
 Such viruses can be assayed by counting number
of pocks on CAM by viruses
Advantages:
• Specificity maximum
• Sensitivity usually adequate
• Isolate available for characterization
Disadvantages:
• Cell-culture facilities needed
• Slow (results in days to weeks)
• Doesn’t work for all viruses.

62. Serology
Dr.T.V.Rao MD
62
Detection of rising titers of antibody between acute and
convalescent stages of infection, or the detection of IgM in primary
infection.
Classical Techniques Newer Techniques
1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)
2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)
3. Immunofluorescence techniques (IF) 3. Particle agglutination
4. Neutralization tests 4. Western Blot (WB)
5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay

63. 63

64. Serology
Dr.T.V.Rao MD
64
 Since the isolation and identification of viruses
is not commonly done in the clinical
laboratory, the clinical picture and serology
plays a greater role in the diagnosis of viral
disease. The major types of antibodies that
are assayed for are neutralizing,
haemagglutination inhibiting and complement
fixing antibodies. Complement fixing
antibodies follow the kinetics of IgM and are
most useful in indicating a current or recent
infection

65. Serology
Dr.T.V.Rao MD
65
 The development of
antibodies to different
components of the
virus is used in
staging the disease.
For example in
hepatitis B and HIV
infections this
approach is used.

66. Viral Serology
 Indirect
 Primary and secondary responses to viral infections
 IgM (1st exposure)
 IgG (2nd exposure)
Figure 5.18: Primary (1 degree) and secondary (2 degree) antibody responses toward a viral pathogen.

67. Serological Diagnosis
Dr.T.V.Rao MD
67
 Detection of
Immunologlublins Ig G.
Ig M Ig A
 Raise of titers Ist
sample later sample
(convalescent sample)
tested after 10 – 14
days Raise of titer is
diagnostic

68. Viral Serology
Enzyme-Linked Immuno-
Sorbant Assays (ELISAs)
Enzyme reacts with substrate to
produce colored product
Very sensitive
 HIV test
 If positive twice, Western Blotting is performed
next

69. ELISA for HIV antibody
Micro plate ELISA for HIV antibody: colored wells indicate reactivity
Dr.T.V.Rao MD
69

70. © Hank Morgan/Science Photo Library/Photo Researchers, Inc.
HIV ELISA test.

71. ELISA
Procedures
Figure 5-19a
Modified from Specter, S. C., R. L. Hodinka and S. A. Young. Clinical Virology Manual, Third
Edition . ASM Press, 2000.
Figure 5-19b