cultivation of viruses

Viruses are obligate intracellular parasites so they depend on host for their survival. They cannot be grown in non-living culture media or on agar plates alone, they must require living cells to support their replication.Cultivation of viruses can be discussed under following headings:

Animal Inoculation
Inoculation into embryonated egg
Cell Culture

1. VIRUSES
intracellular❖Virusesareobligate
parasites.
❖Theymultiplyonlyinsidetheliving
hostcells.Animals,plants,humans,
bacteria,fungus,protozoaandalgae
arethenaturalhostsofviruses.

2. ❖Virusesarehostspecificandgrowonlyin
selectivehosts.Virologistsuseonlyasuitable
hostsystemforcultivationofavirus.
❖Virusescannotgrowinartificialmedia.They
cannotgrowninnon-livingcultureoronagarplates
alone,theymustrequirelivingcellstosupporttheir
replication.
❖Thereisnouniversalcellthatwillsupportall
viruses.

3. Main purpose of viruscultivation
❖To isolate and identify viruses in
clinicalsamples.
❖Topreparevirusesforvaccine
production.
❖To do research on viralstructure
,replication,geneticsand
effects on hostcells.

4. ❖Reedandcolleagues (1900)usedhuman
volunteersfortheirpioneeringworkonyellowfever.Dueto
theseriousriskinvolved,humanvolunteersareusedonly
whennoothermethodisavailableandwhenvirusisrelatively
harmless.
❖Monkeyswereusedfortheisolationofthepoliovirusby
LandsteinerandPopper(1909).However,dueto
theircostandrisktohandlers,monkeysfindonlylimited
applicationsinvirology.
❖Theuseofwhitemice,pioneeredbyTheiler(1903)
extended the scope of animalinoculation greatly.

5. ❖GoodPasturein1931firstusedtheembryonated
hen’seggforthecultivationofvirusandthismethodis
furtherdevelopedbyBurnet.
❖Thefirstapplicationoftissuecultureinvirologywasby
Steinhardtandcolleagues(1913),whomaintained
thevacciniavirusinfragmentsinrabbitcornea.
❖Maitland(1928)usedchoppedtissueinnutrient
mediaforcultivationofvacciniaviruses.
❖Theturningpointwhichmadetissueculturethemost
importantmethodforcultivationofviruswasthe
demonstrationbyEnders,WellerandRobins(1949),
thatpoliovirustillthenconsideredastrictlyneurotropic
virus,couldbegrownintissuecultureofnon-neuralorigin.

6. METHODS FOR CULTIVATION OFVIRUSES
Inoculation of virus
intoanimals.
Inoculation of virus
into embryonatedeggs.
Tissueculture.

7. ❖Viruseswhicharenotcultivatedin
embryonatedeggandtissuecultureare
cultivatedinlaboratoryanimals.e.g:mice,
guineapig,hamester,rabbitsand
primatesareused.
❖Theselectedanimalsshouldbehealthy
andfreefromanycommunicablediseases.
❖Sucklingmice(lessthan48hoursold)are
mostcommonlyused.
1).InoculationofVirusin
Animals

8. Different ways of inoculation in miceare:
1)Intracerebral.
2)Subcutaneous.
3)Intraperitoneal.
4)Intranasal.

9. 1).Intracerebral
➢Itoccurring or
introduced
within
or
administeredintothe
cerebrum.Itmeans
whenadiseasedblood
vesselwithinthebrain
burstsallowingblood
toleakinsidethebrain.

10. 2).Subcutaneous
❖Asubcutaneous
injectionisan
injectioninwhicha
needleisinserted
justundertheskin.

11. 3).Intraperitoneal
❖Intraperitoniuminjectionisthe
intotheinjectionofasubstance
peritonium (bodycavity).

12. 4).Intranasal
✓It lying within or administered by way
of the nasalstructure.

13. Advantages anddisadvantages
of animal inoculation:
Advantages:
❖Production of antibodies can beidentified.
❖Diagnosis,pathogenesisandclinical
symptoms aredetermined.
❖Primary isolation of certainviruses.
❖Mice provideareliablemodelfor
studying viralreplication.
❖Used forthestudyofimmune
responses,epidemology andoncogenesis.

14. Disadvantages :
❖Expensiveanddifficultiesinmaintaince
ofanimals.
❖Difficultyinchoosingofanimalsfor
particularvirus.
❖Somehumanvirusescannotbegrown
inanimalsorcanbegrownbutdonot
causediseases.
❖Micedonotprovidemodelsforvaccine
development.

15. 2). Inoculation of virus into
embryonatedegg
Theprocessofcultivationofvirusesin
embryonated eggsdependuponthe
typeofeggbeingused.
Eggprovideasuitablemeansfor:
i.The primary isolation and
identification ofviruses.
ii.The production ofvaccines.
iii.The maintaince of stockculture.

16. After incubation , the egg is broken and virusis
isolated from tissue ofegg.
For inoculation , eggs are first prepared for cultivation ,
the sheel surface are first prepared for cultivation , the
shell surface is first disinfected with iodine and
penetrated with a small steriledrill.
Viruses are inoculated into chick embryo of 7-12
daysold.

17. Inoculation of virus into
embryonated eggs

18. ▪Virusgrowthandmultiplicationin
theeggembryoisindicatedbythe
deathoftheembryo,byembryocell
damage ,orbytheformationof
typicalpocksorlesionsontheegg
membrane.
▪Viruses canbecultivatedin
variouspatsofegglike:
1). Chorioallantoic membrane (CAM)
2). Allantoiccavity
3)Amnioticsac
4)Yolksac Pock

19. 1).Chorioallantoic
membrane (CAM)
▪Inoculationis
growingpoxvirus.
▪Afterincubation
mainlyfor
andvisible
lesionscalledpocksare
observed,whichisgreywhite
areaintransparentCAM.
▪Herpessimplexvirusisalso
grown.
▪Singlevirusgivessinglepocks.
▪Thismethodissuitablefor
plaquestudies.

20. Plaque:Aclearareainalawnof
hostcellsthatresultsfromthelysisof
hostcellsbyviruses.

21. 2). Allantoiccavity:
❖Inoculationismainlydonefor
productionofvaccineof
influenzavirus,yellowfever,
rabies.
❖Mostofavaianvirusescanbe
isolatedusingthismethod.
❖Allantoicinoculationisaquick
andeasymethodthatyields
largeamounts(8-15ml)of
virus-infectedeggfluids.

22. 3). Amniotic sac:
▪Inoculation is mainly done
for primary isolation of
influenza virus and the
mumpsvirus.
▪Growth and replication of
virus in egg embryo can be
detected by
haemagglutinationassay.
▪The virus is introduced
directly into the amniotic fluid
that bathes the developing
embryo.

23. 4). Yolk sac inoculation:
❖Itis
method
alsoasimplest
forgrowthand
multiplication ofvirus.
cultivation
viruses
of
and
❖Itisinoculatedfor
some
some
bacteria(Chlamydia,
interference
Rickettsiae).
❖Immune
mechanism can be
detected in most of avian
viruses.

24. Advantages of inoculation into
embryonatedegg
❖Widely used method for the isolation of virus and
growth.
❖Cost effective and maintenance is mucheasier.
❖The embryonated eggs are readily available.
❖Theyarefreefromcontaminatingbacteriaand
many latentviruses.
❖Ideal substrate for the viral growth andreplication.
❖less labor isneeded.
❖Widelyusedmethodtogrowvirusforsome
vaccineproduction.
❖Defensemechanismsarenotinvolvedin
embryonatedeggs.

25. Disadvantage of inoculation into
embryonatedegg
•The site of inoculation for varieswith
different virus . That is , each virus
have different sites for growth and
replication.

26. 3). Tissueculture
❖Cultivationofbitsoftissuesandorgansin
vitrohadbeenusedbyphysiologistsand
surgeonsforthestudyofmorphogenesis
andwoundhealing.
❖Beforetheadventofcellculture,animalviruses
couldbepropagatedonlyonwholeanimalsor
embryonatedchickeneggs.

27. ✓Cellcultureshavereplaced
embryonatedeggsaspreferred
typeofgrowthmediumfor
manyviruses.
✓Cellcultureconsistsofcells
growninculturemediainthe
laboratory.

28. There are three types of tissueculture:
1)Organculture.
2)Explantculture.
3)Cellculture.

29. 1). Organculture
Example: Tracheal ring organ culture is employed for the isolationof
coronavirus, a respiratorypathogen.
Organ culture is useful for the isolation of some viruses whichappear
to be highly specialized parasites of certainorgans.
Small bits of organs can be maintained in vitro for days and weeks,
preserving their original architecture and function. Formalin is usedfor
thepreservation.

31. 2). Explant culture
The explants taken from laboratory animals such as mice , rabbit , guineapigs
, hamester and man can be grown in petridishes.
An explant is aseptically transferred into a sterile petri dish by using afine
tipped forceps and then a coverslip is placed over thatexplant.
Enough volume of medium is poured into the petri dish , which isthen
incubatedat37 ⁰C untill cellgrowth.
Example : Adenoid tissue explant culture were used for the isolationof
adenoviruses.
A small portion of tissue excised from animal’s body isexplant.

32. Explantculture

33. 3). Cellculture
Theessientialconstitutents of the growth medium are physiologic amountsof
essiential amino acids and vitamins , salts , glucose and a buffering system
generally consisting of bicarbonate in equilibrium with atmosphere containing
about 5% carbondioxide.
Tissues are dissociated into the components of cells by the
action of proteolytic enzymes such as trypsin and
mechanical shaking.
This is the type of culture routinely employed for growing
viruses.

34. Antibiotics are added to prevent thebacterial
contaminants and phenol red asindicator.
Such media will enable most cell types to multiplywith
a division time of 24-48hours.
The cell suspension is dispensed in bottles , tubesor
petridishes.
The cell adhere to the glass surface and on incubation,
divide to form a confluent monolayer sheet of cells
covering the surface within about aweek.
This is supplemented with up to 5% calf or fetal calf
serum.

35. Based on their origin , chromosomal
characters and the number of generations
through which they can be maintained , cell
culture are classified into three types:
Some fastidious virus grow only in such roller
cultures.
Cell culture tubes may be incubated in a
sloped horizontal position , eitheras
‘stationary culture’ or may be in special‘roller
drums’ to provide betteraeration.

36. Types of cellculture
1). Primary cellculture.
2). Continuouscell
lines.

38. 1). Primary cellculture
Examples : Kidney cells of monkey and man , embryo
cells , alveolar cells , macrophages and amniotic cells
are usually grown in primary cellcultures.
It is capable of only limited growth and hence it can
be subcultured once ortwice.
The cell culture established directly from cells taken
from animal’s tissue is called primaryculture.

39. Primary cell cultures are widely used for the isolation
of animal viruses and cultivation of viruses for vaccine
production.
The culture vessel is incubatedat37⁰Cfor a few daysto
get a primaryculture.
A small volume of cell suspension is aseptically
transferred to a culture flask or petri dishcontaining
nutrient medium , with the help ofpipette.

40. Secondary cell cultures are used for the isolation of wide group of animal
viruses and growing fastidious viruses. Some secondary cultures are usedfor
vaccineproduction.
Examples : Human embryonic kidney cells and skin fibroblastcells.
As secondary cell cultures can be maintained and subcultured for 20-50times
, they are called semi-continuouscells.
The cell culture established from primary cell culture are calledsecondary
culture orsub-culture.

41. The fragments of monoculture grow into large
monolayers. These are called secondaryculture.
It is then cut into small fragments. 2 or 3 fragments are
inoculated into a roller drum containing nutrientmedium
and the roller drum is incubated at 37⁰C for fewdays.
Monolayer produced as a result of primary cell cultureis
detached from the bottom of the culture flask by adding
trypsin orEDTA.

42. ➢Animalcellscapableofindefinitegrowthare
calledcontinuouscelllinesorcelllines.
➢Thesearethecellsofasingletype,usually
derivedfromcancercells,thatarecapableof
continuousserialcultivationindefinitely.
➢Standardcelllinesderivedfromhuman
cancers,suchasHeLa,HEp–2andKBcell
lineshavebeenusedinlaboratories
throughouttheworldformanyyears.

43. ❖Thesecelllinesmaybemaintainedby
serialsubcultivationorstoredinthecold(
-70⁰C ) for use whennecessary.
❖Somecelllinesarenowpermittedtobe
used for vaccine manufacture, forexample
: Vero cells for rabiesvaccine.

44. Advantages of cellculture
❖Relativeease,broadspectrum,cheaper
andsensitivity
Disadvantage of cellculture
❖Theprocessrequirestrainedtechnicians
withexperienceinworkingonafulltime
basis.
❖Statehealthlaboratories
laboratoriesdonotisolate
andhospital
andidentify
viruses in clinicalwork.
❖Tissueorserumforanalysisissentto
central laboratories to identifyvirus.

45. Detection of virusgrowth
•The following methods are available to
detect the virus growth in the cell or tissue
cultures.
a).Cytopathiceffect.
b).Haemadsorption.
c).Interference.
d)Transformation.
e)Immunofluorescence.
f).Metabolicinhibition.

46. ❖Manyvirusescausemorphologicalchanges
inculturedcellsinwhichtheygrow.These
changescanbereadilyobservedby
microscopicexaminationofthecultures.
❖Thesechangesareknownas‘cytopathic
effects’(CPE)andthevirusescausingCPE
arecalled‘cytopathogenicviruses’.
❖TheCPEproducedbydifferentgroupsof
virusesarecharacteristicandhelpin
presumptiveidentificationofvirusisolates.

47. •For example, enteroviruses produce
rapid CPE with crenation of cells and
degeneration entire cell sheet ;measles
virus produce syncytium formation;
herpes virus causes discrete focal
degeneration; adenovirus produce large
granular clumps ofgrapes.

49. ❖Whenhemagglutinatingviruses(suchas
influenzaandparainfluenzaviruses)grow
incellcultures,theirpresencecanbe
indicatedbytheadditionofguineapig
erythrocytestothecultures.
❖Ifthevirusesaremultiplyinginthecultures,
theerythrocyteswilladsorbontothesurface
ofcells.Thisisknown as
‘hemadsorption’.

51. 3).Interference
•Thegrowthoffirstviruswillinhibitsecond
virusinfectiondue
effect.Thisproperty
tosomeinhibitory
ofcellculturesis
celledinterference.itisusefultodetect
thegrowthofnon-cytopathicvirusesincell
cultures

52. 4).Transformation
•Ifoncogenicvirusesareinoculatedinto
cellcultures,theinfectedcellgrowfast
andproducemicrotumoursintheculture.
Thisiscalledtransformation.Itindicated
thepresenceofoncogenicvirusesinthe
culture.

53. 5). Immunofluorescence test
•Somecellfromthecellculturearestained
withafluorescentdyeconjugatedantiserum
andviewedunderanUVmicroscope.
•Viralantigenpresentonthecellsurfacebind
withtheantiserum.
•Fluorescencefromthecellisthepositive
indicationforpresenceofvirusinthecell.
Thisisawidelyusedmethodindiagnostic
virology.

54. ImmunofluorescenceAssay

55. 6). Metabolicinhibition
❖Innormalcellcultures,themedium
turnsacidduetothecellular
metabolism.
❖Whenvirusesgrowincellcultures,the
cellmetabolismisinhibitedandthereis
noacidproduction.
❖Thiscanbemadeoutbytheindicator
(phenolred)incorporatedinthe
medium.

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