virus isolation at by molicular technique

1. Virus Isolation
Daniel Gizaw
nebiyudan@gmail.com
May 2021
Sebeta, Ethiopia

3. Requirement of virus Isolation
There are four basic requirements for successful cell
culture.
The fist one is well-established and properly equipped
cell culture facility.
• The level of biocontainment required (Levels 1–4)
This type of cells cultured and the risk that these cells
might contain, and transmit, infectious agents.
• a certified biological safety cabinet that protects both
the cells in culture and the worker from biological
contaminants

4. Requirement of virus Isolation
The second requirement for successful cell culture is the
practice of sterile technique.
 The third necessity for successful cell culture is
appropriate, quality controlled reagents and supplies.
There are numerous suppliers of tissue culture media (both
basic and specialized) and supplements.

5. Requirement of virus Isolation
The fourth necessity for successful cell culture is
the knowledge and practice of the fundamental
techniques involved in the growth of the cell type
of interest.

6. Type of cell lines
• Primary cell lines:- Non-transformed or normal
cells-having definite life span and primary cultures
from healthy tissues, require defined quantities of
proteins, growth factors and hormones even in the
best media
• Have definite number of passages
• Primary cell cultures have a short lifespan.

7. Type of cell lines
Continues cell lines :Cells immortalized either
spontaneously or by transfection with viral
sequences, produce most of these factors
• These cell lines do not have a definite number
of passages
• they change in their morphological and
genetic characteristics, unlike in finite cell
lines.
• Cell lines have a longer lifespan.
• They are more convenient than primary cell
lines

8. Cell lines
• Cell cultures are developed from tissue samples and
then disaggregated by mechanical, chemical, and
enzymatic methods to extract cells suitable for
isolation of viruses.
• Embryonated eggs and laboratory animals are also
used for isolation of viruses.
• Cell cultures are more convenient and less expensive
than egg and animal use and convenient to examine
microscopically for evidence of viral proliferation.
• Virus isolation in cell cultures has long served as the
“gold standard” for virus detection, and it is the
method to which all others have been compared

9. Cell culture
Can be grown as
1. Mono layers: Anchorage dependent cells
2. Suspension culture : hematopoietic cell lines
exhibit spontaneous growth in suspension .
All anchorage dependent cells require substrate to
which they attach during culture
• Tissue culture plastic has a net negative surface
charge which is produced by plasma treatment of
the polystyrene.

10. Compared with other technique
• Molecular detection of viral DNAs and RNAs and molecular
amplification by PCR and other techniques are now becoming more
widely available in diagnostic laboratories.
• The appropriate selection, collection, transport, and processing of
clinical samples are important for successful virus isolation.
• Collection of samples that contain the highest titer of virus is most
desirable
• Virus isolation, in general, continues to provide more sensitive virus
detection than rapid antigen assays and remains less costly and
generally better suited for detecting a wider range of viruses.
• Today, virus isolation in cell culture remains a useful approach for
viral disease diagnosis.

11. Examination cell culture
• Cell monolayers are screened by microscopic examination daily to
maximize the detection of viral growth.
• Degenerative changes in monolayer cells provide evidence of viral
presence.
The characteristic CPEs include
 Change in cell morphology, such Swelling, rounding and
aggregation of cells, shrinking
 Clustering or syncytia formation, and
 inclusion bodies formation.
 complete destruction of the monolayer.
• These changes are collectively called the cytopathogenic or
cytopathic effect (CPE) of the virus.
• In most cases, the CPE appears after 5-10 days of incubation

12. Cell attachment to the plate

13. RIA=Radio Immunosorbent Assay

14. A , D , G, J Normal Cell, other cells infected by virus

15. Examination of cell culture
The experienced observer may be able to predict which virus is present
based on
• the characteristics of the CPE,
• the cell line involved
• the length of incubation
• the type of clinical specimen
but confirmatory testing is needed to make a definitive viral identification.
 Confirmatory testing of virus cultures positive by CPE has traditionally
been based on the reaction of antibodies of known specificity with viral
antigens expressed in the infected cells.
 Most of this testing is accomplished at present through
immunofluorescence (IF) techniques that use fluoresce in isothiocyanate
(FITC)-labeled monoclonal antibodies(MAbs).

16. Media and reagent for cell culture
A media comprises an appropriate source of energy for the cells
which they can easily utilize and compounds which regulate the cell
multiplication.
• DMEM+ growth medium: high-glucose DMEM supplemented with
10% FBS, 4 mM glutamine, 100 IU penicillin, and 100 μg/mL
streptomycin. Store at 4°C.
• Fetal bovine serum (FBS). Sera should be aliquoted and stored at –
20°C.
• Penicillin–streptomycin solution. 100X stock solution. Aliquot and
store at –20°C
• L-Glutamine, 200 mM stock solution. Aliquot and store at –20°C.
• Trypan blue stain (0.4% w/v trypan blue in phosphate-buffered
saline [PBS] filtered to remove particulate matter) or eosin stain
(0.14% w/v in PBS; filtered) for determination of cell viability.

17. • Tissue-culture-grade dimethyl sulfoxide (DMSO) (i.e.,
Sigma) stored at room temperature.
• Freezing medium, freshly prepared and chilled on ice,
consisting of 90% FBS and 10% DMSO
• Trypsin is an enzyme that is active at 37°C. it is advisable
to thaw the solution overnight at 4°C and then aliquot into
convenient sizes (i.e., 40 mL/tube) for storage at –20°C
• CO2 concentration is a complex factor need to be regulated in cell
culture as it influence the pH of the media and HCO¯3 ion
concentration of the culture.

18. Some equipment in the cell culture lab

19. Culture Media
• The nutrient media used for the culture of animal cells and tissues,
must be able to support their survival as well as growth, in other
words , they must provide nutritional, hormonal and stromal
factors.
• Tissue culture media are broadly grouped into two categories-
Natural media and artificial media.
• The choice of the medium depends mainly on the type of cells to
be cultured-normal, immortalized or transformed-and the
objective of the culture-growth, survival, differentiation or
production of desired proteins.

20. Natural media
These are the natural source of nutrient sufficient for
growth and proliferation of animal cells and tissues. These
are of 3 types.
1. Clots available on market or prepared from blood
2. Biological fluids It is obtained in the form of serum from
human adult blood, placental, cord blood, horse blood,
calf blood,
3. Tissue extracts Extract from tissues such as embryo,
liver, spleen, leukocyte, tumor, bone marrow

21. Artificial media
Artificial media are used for cell cultures. The
various artificial media developed for cell cultures
may be grouped into the following classes.
• Serum-containing media
• Serum-free media
• Chemically defined media
• Protein free media

22. Cell viability
 Every cell line has an optimal concentration for maintaining growth
and viability. Until sufficient experience is gained with a new cell
line, it is recommended to check cell densities and viability every
day or two to ensure that optimal health of the cultures is
maintained.
 Maintenance of healthy, viable cells requires routine medium
exchanges or passage of the cells to ensure that the nutrients in the
medium do not become depleted and/or that the pH of the medium
does not become acidic (i.e., turn yellow) as a result of the presence
of large amounts of cellular waste by Sub culture.

23. Cont.
• Enzyme such as trypsin, dipase, collagenase in
combination with EDTA breaks the cellular glue that
attached the cells to the surface
• Subculture It’s the passage of cells when they reach to
80-90% confluency in flask/dishes/plates

26. Cryopreservation of cell culture
• Cells for cryopreservation should be in log growth phase
with greater than 90% viability.
• The only insurance against loss of the cell line is to ensure
that adequate numbers of vials (i.e., at least 10) are
cryopreserved for future use.
• For newly acquired cell lines, cryopreservation of stock
(master cell bank) vials should be done as soon as
possible after the cell line has been confirmed to be free
of mycoplasma

27. Storage for future use

28. Cryopreservation of cell culture
• Most cell culture media contain phenol red as a pH
indicator. Repeated entry into the medium bottle can result
in a shift in the pH and, thus, a change in the color from red
to a more purple color.
• Note the information recorded on the vial and then rinse
the outside of the vial with 70% ethanol or isopropanol to
decontaminate it prior to proceeding with the thawing
procedures.

29. Cryopreservation of cell culture
• It is critical to record the passage number of all
primary cells and to ensure that aliquots are
frozen for future use as soon as possible if future
experiments are anticipated.

31. Cell count
A haemocytometer has an exact volume under the coverslip,
enabling determination of the concentration (cells/ml) of live
and dead cells in the chamber.
The cell concentration of the original cell suspension will be
the same as that of the chamber except for any dilutions
made.
Dead cells take up the trypan blue, and appear blue under
the microscope. Living cells exclude trypan blue, and appear
white.

32. Cont.
Calculate the number of cells by using the following equation:
× 10,000
=
Viability:
% Viability = × 100%

34. Thawing, reseeding of frozen cell
Cell are frozen or kept at -70 C, so it at dormant state so it
should come to 37 oC
Thaw cell sample vials in a 370C water bath.
 Transfer the thawed cells dropwise into the centrifuge
tube containing the desired amount of pre-warmed
complete growth medium appropriate cell line
Centrifuge the cell suspension at approximately 1500rpm
for 10 minutes. Decant the supernatant
Gently reconstitute the cells in complete growth
medium, and transfer them into labeled culture flask and
incubate a t 370c with 5% CO2

35. Inoculation of Embryonated Chicken
Eggs for the Isolation of Viruses
Several avian viruses grow much better in embrocated eggs
than in primary or continuous cell cultures.
 Depending on the virus, various routes of egg inoculation
are used: allantoic, amniotic, chorioallantoic, or yolk sac).
 The chorioallantoic route is the most widely used for
common viruses such as influenza, Newcastle disease
and avian bronchitis.
Chicken eggs from a Specific Pathogen Free (SPF) source. SPF =
Breeders that have not been exposed to common avian pathogens

36. 37°C incubator with source
of humidity
Intense light source used to
check viability
 Dark area to candle eggs

37. Chorioallantoic membrane (CAM) In 9‐11 day
of embryonation embryos (doe)
 Amniotic cavity: in 10‐11 doe
 Yolk sac: in 5‐6 doe
 Chorioallantoic sac (CAS): in 9‐11 doe

38. Rout of egg inoculation

39. Chorioallantoic Sac (CAS) Route

40. Chorioallantoic Sac (CAS) Route
Preferred route of egg inoculation for
primary isolation of:
– Avian influenza [AIV]
– Newcastle disease virus [NDV]
– Infectious bronchitis virus [IBV]
Read out:
– Test allantoic fluid for hemagglutination@ 48‐72
hours post‐inoculation
– Evaluate embryos for lesions @ 7 days

41. Candling Eggs to Check for Viability
Is egg viable or dad ?

42. Step for inoculation
 Spray Eggs with Disinfectant and Allow to Dry
 Punch Hole in Egg at Marked Area
Label Inoculate 0.1 ml per Egg
Seal Inoculation Site and Return to the
Incubator

43. Daily fellow up inoculated eggs
Candle eggs for viability daily
– Record mortality on worksheet
– Mortality at 24 hrs is likely due to trauma.
At 48 hrs post inoculation
– Aseptically harvest allantoic fluid from embryo to test
for hemagglutination with chicken red blood cells (CRBC)
• Reseal embryos with glue and return to incubator
• At 7 days post inoculation, open embryos and look for
lesions

44. Tissue culture infective dose (TCID50)
 TCID 50 is the tissue culture infectious dose defined as
that dilution of virus required to infect 50% of the cell
monolayers.
 Cells are cultured on a well plate titer, and then varying
dilutions of the testing viral fluid are added to the wells.
 After incubation, the percentage of infected wells is
observed for each dilution, and the results are used to
calculate the TCID50 value
 Not all virus types cause CPE in tissue culture, and the
cell line and virus must be carefully matched in order to
see a cytopathic effect.

45. TCID50 Assay
• The procedure is performed to determine the infectious
titer of any virus which can cause cytopathic effects (CPE)
in tissue culture over a reasonable period of 5 to 20 days
while cells in culture remainviable.
• This procedure is performed to quantify how much
infectious virus is in a preparation.
• The TCID50 is determined in replicate cultures of serial
dilutions of the virus sample.
• The titer of the virus stock is expressed as the TCID50
which can be calculated by hand or statistical Excel
program and is more accurate than a negative end-point.

46. Cell synchronization
Is a process by which cells in a culture at different
stages of the cell cycle are brought to the same
phase.
• Cell synchrony is a vital process in the study of
cells progressing through the cell cycle as it allows
population-wide data to be collected rather than
relying solely on single-cell experiments.
• The types of synchronization are broadly
categorized into two groups;
physical fractionization and chemical blockade.

47. Physical fractionation
is a process by which continuously dividing cells are
separated into phase-enriched populations based
on characteristics such as the following:
– Cell density
– Cell size
– The presence of cell surface epitopes marked
by antibodies
– Light scatter
– Fluorescent emission by labeled cells.

48. Chemical blockade
• The addition of exogenous substrates used to block cells in
certain phases of the cell cycle and frequently target cell cycle
checkpoints.
• The most common type of chemical blockade is arrest-and-
release, which involves treatment of a culture with a chemical
block and subsequent release by washing or addition of a
neutralizing agent for the block.
• Chemical blockade is typically more effective and precise than
physical separation, some methods can be imperfect for various
reasons
• the proportion of synchronized cells is insufficient
• chemical manipulations may disrupt cellular function and/or kill a
portion of cells
• the treatment is toxic and not applicable in vivo (only relevant if
considering clinical application)

49. Quality assurance and contamination
Quality is important in all aspects of tissue culture.
The main areas of quality control that are of concern for tissue
culture are:
– The quality of the reagents and materials
– The cell line integrity and identification
– The avoidance of microbial contamination

50. Reagents and Materials
Bovine serum - bovine viral diarrhea virus (BVDV).
Porcine trypsin is also a potential source of Mycoplasma.
Use mycoplasma and BVD screening test
• Equipment used needs to sterile plasticware (flasks,
centrifuge tubes, pipettes)
Keep certificate of analysis of reagent and materials

51. Cell Line Integrity and Identification
The sourcing of cell lines can have an important
effect on quality;
So obtain cell lines from a recognized that are:
• Contaminant free
• Certified
• With known species of origin
• Supplied with a detailed data sheet

52. Microbial Contamination
The three main types of microbial contaminants
in tissue culture are:
• Bacteria and Fungi
• Mycoplasma
• Viruses

54. Aseptic Technique and Contamination Control
• Personnel hygiene
• Working within the Microbiological Safety
Cabinet
• Pipetting and Prevention of Aerosols Disposable
plastic pipettes (1ml, 2ml, 5ml, 10ml and 25ml)
are the easiest forms to use for cell culture.
• Adherence to GLP

55. Viral interference tests,
• Viral interference/ superinfection resistance, is the inhibition
of viral reproduction caused by previous exposure of cells to
another virus.
• Factors that have been implicated are the generation
of interferons by infected cells, and the occupation
or downmodulation of cellular receptors.
• Viral interference appears to be independent of any antigenic
similarities between the viruses.
• The interval between infections and the sequential combination of
viruses were important determinants of viral interference.
• state of temporary immunity from infection, is proposed to be
induced by viral infection

56. Infectivity assays
• Electron microscopy: EM is useful to count virus
particles directly in a negatively stained viral
suspension.
• The virus suspension is mixed with a known
concentration of latex particles, and the number
of virus particles in the suspension is estimated
by a ratio between the virus and latex particles
demonstrated by EM.

57. Infectivity assays
• Hemagglutination: Quantitation of
hemagglutinating viruses is carried out by
determination of hemagglutination titers.
• Not a sensitive method, but convienent
• approximately, 107 influenza virions are essential to
produce microscopic agglutination in cultured
cells.

58. Plaque assay, Pock assays
• Quantitative assay of infectivity: Quantitative
assay is usedto estimate the presence of
actual number of viable infectious viral
particles in the inoculum.
• Two methods are available for the purpose,
which include plaque assay in monolayer cell
culture and pock assay on chick embryo CAM.

59. Plaque assay: This is based on the principle that each
infectious viral particle gives rise to a localized focus of
infected cells that can be visualized by the naked eye.
• Such foci are called plaques, and each plaque indicates
an infectious virus.
• The test is performed by adding a viral inoculum to a
monolayer of culture cells in a bottle or Petri dish.
• This allows adsorption of viruses.
• The liquid medium is removed and replaced with a solid
agar gel to ensure that the spread of progeny virus is
confined to the immediate vicinity of infected cells.

60. Plaque assay
Living cells were stained with crystal violet. The viral
plaques, each from one virion, remained transparent

61. Pock assays
• Pock assay: Viruses that form pocks on CAM can be assayed
by counting the number of pocks formed on the inoculated
CAM. Each pock on CAM arises from a single virus particle.
This is known as pock assay. Vaccinia and variola viruses can
be assayed by pock assay.
• P ractically abandoned and replaced by the plaque assay
• diluted viral sample is inoculated into the epithelial layer of
the chrioallantoic membrane (CAM) of a chick embryo in an
embryonated chicken egg