Introduction Lab diagnosis Sample collection & Transportation Sample processing Demonstration of virus antigen Detection of viral growth in cell culture Serological diagnosis Molecular diagnosis

1. LAB DIAGNOSIS OF VIRAL
INFECTIONS
Ankur vashishtha
Microbiologist
Subharti university , Meerut

2. Lesson plan
• Introduction
• Lab diagnosis
• Sample collection & Transportation
• Sample processing
• Demonstration of virus antigen
• Detection of viral growth in cell culture
• Serological diagnosis
• Molecular diagnosis

3. INTRODUCTION
• Obligate intracellular .
• Contain only one type of nucleic acid, either DNA or RNA, but
never both.
• Lack enzymes necessary for protein and nucleic acid synthesis and
are dependent for replication on the synthetic machinery of host
cells.

4. • They multiply by a complex process and not by binary fission.
• They are unaffected by antibacterial drugs.
• The extracellular infectious virus particle is called the virion.
• Viruses are too small to be seen under the light microscope and
can only be seen under the electron microscope.

5. Lab Diagnosis
1) Specimen collection:
Depending on the site of involvement
E.g. Throat swab, CSF, Blood, Conjunctival scraping.
- Swabs with cotton / calcium alginate tips are not recommended.
Synthetic swab material, such as rayon and Dacron, are
acceptable.
2) Sample Transport: Specimens for viral isolation should not be
allowed to sit at room or higher temperature.
-Specimens should be kept cool 4⁰C and immediately transported to
the laboratory.
- If the delay in transport is unavoidable, the specimen should be
refrigerated, not frozen, until processed.

6. • Once collected, specimens on swabs should be emulsified in
viral transport medium before transport to the laboratory,
especially if transport will occur at room temperature and
require longer than 1 hour.
• Storage for 6 days or longer should be at -20⁰C or preferably
at - 70⁰C.

7. • Viral Transport medium (VTM) contain
- protein {e.g. serum(fetal calf serum), albumin, or gelatin} to stabilize
the viral agents
-Antimicrobials to prevent overgrowth of bacteria and fungi. [Mixture is
composed of vancomycin, gentamicin and amphotericin
Eg Hanks balanced salt solution (HBSS).
Eagle’s minimum Essential Medium (MEM).
other- Stuart’s medium

8. Transportation
1. Hank’s balanced salt solution (HBSS)
Contents: -Salts like Chloride of Sodium, potassium and calcium;
Phosphates Sodium and Potassium, Sulphate of magnesium and
Sodium bi carbonate.
-Glucose
- Phenol red indicator
2. Eagle’s minimum Essential Medium (MEM).
a) Amino acids
b) Glucose
c) Vitamins like folic acid
d) Phenol red indictor

9. 3) Sample processing
Microscopy
• Electron microscopy : Detection of the virus by electron
microscopy is being used increasingly. Eg . Viral diarrhea.
• Immunoelectron microscopy: In this method, specific
antibody is added to react with viral antigens and then
visualized by electron microscopy. Ex . Negri bodies (Seller’s
stain) is a routine diagnostic method for rabies in dogs.
• Fluorescent microscopy : DFA and IFA techniques- used for
examination of material from lesions as well as for early
demonstration of viral antigen in tissue cultures.
Eg. used for the microscopic diagnosis of rabies

10. Demonstration of virus antigen
• It is the demonstration by serological methods such as
precipitation in gel or immunofluorescence offers a rapid
method of diagnosis.
Ex . radioimmunoassay and enzyme- linked immunosorbent
assay(ELISA).

11. Isolation of virus
A) Animal inoculation
B) Embryonated Egg inoculation
C) Tissue culture

12. A. Animal Inoculation

13. -Mice are the most widely used animals in virology.
Eg: Infant (suckling) mice-susceptible to coxsackie virus and
arboviruses.
-Mice can be inoculated by several routes:
• Intracerebral
• Subcutaneous
• Intraperitoneal
• Intranasal

14. -Other animals- guinea pigs, rabbits, ferrets
- Growth of virus in an inoculated animal is indicated by-
• Visible lesions
• Signs of disease
• Death

15. Advantages-
a. Primary isolation of certain viruses
a. To study pathogenesis and immune response of viral
diseases
a. To study viral oncogenesis

16. Disadvantages-
1) Immunity may interfere with viral growth
2) Animals often harbor latent viruses

17. B. Embryonated Egg Inoculation
Good pasture (1931)
Burnet
*CAM ( Chorioallantoic membrane )
*Allantoic cavity
*Amniotic sac
*Yolk sac

18. * Chorioallantoic membrane (CAM)
- Produces visible lesions called as pocks.
- Each infectious virus particle can form one pock.
-Pock counting, therefore, can be used for the assay of
pock forming viruses like variola or vaccinia

19. Normal & Viral Infected Chorioallantoic Chick Membrane

20. *Allantoic cavity
- Provides rich yield of influenza virus and paramyxoviruses
- For growing influenza virus for vaccine production
- Chick embryo vaccines- e.g.
Yellow fever (17 D strain)
Rabies ( Flury strain )

21. Duck’s egg preferred- Bigger
-have a long incubation period
-Provide better yield of rabies virus
- Preparation of the inactivated non-neural rabies vaccine

22. *Amniotic sac
Primary isolation of influenza virus
*Yolk sac
Viruses
Chlamydiae
Rickettsiae

24. C. Tissue culture : 3 Types
1. Organ culture
2. Explant culture
3. Cell culture

25. 1. Organ culture
-Small bits of organs maintained in-vitro for days and weeks.
-Used for the isolation of some viruses which are highly
specialized parasites of certain organs
Eg– Tracheal ring culture for the isolation of coronavirus ,a
respiratory pathogen

26. 2. Explant culture
Minced tissue fragments can be grown as explants embedded
in plasma clots
eg– Adenoid tissue explant culture for the isolation of
adenoviruses

27. 3. Cell culture
Routinely employed for growing viruses
Tissues are dissociated into component cells – Trypsin
and mechanical shaking
Cells are washed ,counted & suspended in a growth medium
Cell suspension is dispensed in bottles, tubes or
Petri dishes
Cells adhere to the glass surface and divide to form a confluent sheet
of cells

28. Constituents of the growth medium : Physiologic amounts of
essential amino acids and vitamins, salts, glucose, and a buffering
system generally consisting of bicarbonate in equilibrium with
atmosphere containing about 5 % carbon dioxide.
• Supplemented with up 5% fetal calf serum.
• Antibiotics are added to prevent bacterial contaminants
• phenol red is used as indicator.

29. Cell culture tubes may be incubated in a sloped horizontal position –
‘stationary culture’
Rolled in special roller drums to provide better aeration – Fastidious
viruses

30. Based on their origin, chromosomal characters & the no. of
generations through which they can be maintained –
a. Primary cell cultures
a. Diploid cell strains
a. Continuous cell lines

31. a. Primary cell cultures
Normal cells
Freshly taken from the body & cultured
Limited growth
Cannot be maintained in serial cultures
Useful for isolation of viruses & their cultivation for
vaccine production
Ex – Monkey kidney, Human embryonic kidney, Human
amnion & Chick embryo cell culture

32. A monolayer of monkey kidney cells is a cell culture enabling
the propagating viruses
NORMAL CELLS VIRAL GROWTH WITH CPE

33. b. Diploid cell strains
These are Cells of single type that retain the original diploid
chromosome number and karyotype during serial sub cultivation for a
limited number of time.
After about fifty serial passages, they undergo ‘Senescence’.
Useful for isolation of fastidious pathogens.
For the production of viral vaccines.
E.g – Rabies vaccine production in WI-38( Human
embryonic lung cell strain)

34. c. Continuous cell lines
Serially cultivated indefinitely
From cancerous tissue
Haploid chromosomes
HeLa, HEp2, KB cell lines – in labs.
Maintained by serial subcultures or by cold storage at -70°C
Not used for manufacture of viral vaccines

35. Detection of viral growth in cell culture

36. 1. Cytopathic effect (CPE)
-Morphological changes in the cultured cells in which they
grow
-Cytopathogenic viruses
-CPE are characteristic
-Help in presumptive identification of virus isolates

37. Examples –
a) Enteroviruses produce rapid CPE with crenation of cells &
degeneration
b) Measles virus produces ‘Syncytium formation’
c) Herpes virus causes balloning of cells, discrete focal degeneration.
d) Adenoviruses produce large granular clumps
e) Picornaviruses cause rounding of cells

38. Inverted Microscope
• Examination of culture for cytopathic effect can be performed .
• inverted microscope the objective is located under the specimen
and the condenser above, in the upright microscope (the most
used microscope) the objective is located above the specimen
and the condenser below.

40. CRENATION OF CELLS
ENTEROVIRUSES

41. 2. Metabolic inhibition
In normal cell cultures – Medium turns acidic
When viruses grow in cell cultures ,cell metabolism is
inhibited- no acid production
Indicated by change in the colour of the indicator

42. 3. Haemadsorption
Haemagglutinating viruses
Presence detected by
adding guinea pig RBC’s
If viruses are multiplying
in the culture – RBC’s
adhere to the infected cells
E.g. Influenza virus

43. Hemagglutination

44. 4. Interference
Non-cytopathogenic virus tested with known cytopathogenic
virus
Growth of the first will inhibit the infection by the second
virus by interference
Eg. Rubella virus

45. 5. Transformation
Tumors forming viruses induce cell transformation
Growth in a ‘piled –up’ fashion producing microtumours
Ex. is some herpes ,adenoviruses and retroviruses

46. CELL TRANSFORMATION

47. 6. Immunofluorescence
Detected in infected cells by staining with fluorescent
conjugated antiserum
Fluorescent microscope – virus antigen
Positive results earlier- wide application in diagnostic virology

48. Shell vial cell culture
• Shell vial cell culture : rapid modification of conventional
cell culture.
• The infected cell monolayer is stained for viral antigen
produced soon after infection.
• Before the development of CPE.

49. • Method : A shell vial culture tube, a 15 x 45 mm vial, is
prepared by adding a round cover slip to the bottom of the
tube, covering this with growth medium, and adding
appropriate cells.
• During incubation, a cell monolayer forms on top of the cover
slip.
• Shell vials should be used 5 to 9 days after cells have been
inoculated.
• Shell vials can be purchased with the monolayer already
formed.

50. • Specimens are inoculated onto the shell vial cell monolayer by
low – speed centrifugation.
• This enhances viral infectivity for reasons that are not well
understood.
• Cover slip are stained using virus – specific
immunofluorescent conjugates.
• The presence and visualization of characteristic fluorescing
inclusions are used to confirm the presence of an infecting
virus.

51. • Serological diagnosis : demonstration of a rise in titer of
antibodies to a virus in paired sera 10 – 14 days apart.
• - neutralization , complement fixation test , latex agglutination
test, ELISA , hem agglutination inhibition tests

52. Molecular diagnosis:
Molecular diagnosis: nucleic acid amplification techniques
• PCR,
• Reverse transcriptase PCR
• Real time PCR.