Experiment five Isolation and Identification of influenza virus

Experiment fiveIsolation and Identification Isolation and Identification

of influenza virusof influenza virus

Isolation and Identification of viruses in lab

SpecimenSpecimen Isolation and identification of virusIsolation and identification of virus Serological diagnosis of viral infectionSerological diagnosis of viral infection Rapid diagnosis of virus infectionsRapid diagnosis of virus infections


Isolation and identification of virusIsolation and identification of virus Isolation of virusIsolation of virus

Embryonated EggsEmbryonated Eggs

Laboratory Animals Laboratory Animals

Tissue CultureTissue Culture

Quantitation of virusQuantitation of virus

50% tissue culture infectious dose (TCID50)50% tissue culture infectious dose (TCID50)

Plaque forming assay Plaque forming assay


Serological diagnosis of viral infectionSerological diagnosis of viral infectionNeutralization test(NT)Neutralization test(NT)Complement fixation test(CF)Complement fixation test(CF)Titration of complementTitration of complement


Rapid diagnosis of virus infectionsRapid diagnosis of virus infections Identification virus particleIdentification virus particleDetection of viral antigensDetection of viral antigensDetection of viral nucleic acidDetection of viral nucleic acidDiagnosis of virus infections by deteDiagnosis of virus infections by dete

cting IgM antibodiescting IgM antibodiesOther methodsOther methods

Isolation and Identification of influenza virus

PurposePurpose ：：To obtain information about microbiological To obtain information about microbiological

diagnosis methods of influenza virusdiagnosis methods of influenza virus

ProcedureProcedureAcquiring and managing specimenAcquiring and managing specimenIsolation and Identification of influenza virusIsolation and Identification of influenza virus serological diagnosis of influenza virusserological diagnosis of influenza virus


Acquiring and managing specimenAcquiring and managing specimenThroat washing or gargling are obtained within 3 Throat washing or gargling are obtained within 3

days after infectiondays after infectionTest at once or stored frozen. Test at once or stored frozen. Centrifuge the washings 15 min at 3000 rpm, Centrifuge the washings 15 min at 3000 rpm,

Harvest 1 ml of supernatant. Harvest 1 ml of supernatant. Add antibiotic to prevent bacterial contamination. Add antibiotic to prevent bacterial contamination.

Hemagglutination inhibition testHemagglutination inhibition testcomplement fixation testcomplement fixation test

Inoculate to fresh embryos Inoculate to fresh embryos after two such passagesafter two such passages

Treated with penicillin Treated with penicillin and streptomycinand streptomycin

Embryonated egg inoculationEmbryonated egg inoculation (amniotic or allantoic route)(amniotic or allantoic route)

Harvest amniotic and allantoic fluidsHarvest amniotic and allantoic fluids

Hemagglutination testHemagglutination test

the result is negativethe result is negative

++ --

Throat washings or garglingThroat washings or garglingPatient’s serumPatient’s serum

Hemagglutination testHemagglutination test


Isolation of influenza virusIsolation of influenza virus MATERIALS MATERIALS

1.Throat washings or garglings1.Throat washings or garglings 2.Embryos (within 9 to 10-day-old) 2.Embryos (within 9 to 10-day-old) 3.0.5% chicken RBC suspension, physiological 3.0.5% chicken RBC suspension, physiological saline saline 4. Scissors, forceps, drill, syringe, tubes, egg-4. Scissors, forceps, drill, syringe, tubes, egg- checking box, 75% ethanol, tincture of iodinechecking box, 75% ethanol, tincture of iodine

Mark the location of embryo air sac under Mark the location of embryo air sac under egg-checking box, sterilize the shell with tegg-checking box, sterilize the shell with tincture and 75% ethanol, drill a small holincture and 75% ethanol, drill a small hole near the marked shell , inoculate 0.2 ml e near the marked shell , inoculate 0.2 ml of virus inoculum into the allantoic cavity of virus inoculum into the allantoic cavity by pushing the needle just below the surfby pushing the needle just below the surface of the shell, seal the hole with melton ace of the shell, seal the hole with melton wax.wax.

Incubate the inoculated embryo at 35C, cIncubate the inoculated embryo at 35C, check it daily. After incubating for 72 h,plaheck it daily. After incubating for 72 h,place the embryo at 4C overnight. If the embce the embryo at 4C overnight. If the embryo dead within 72 h, it should be discard.ryo dead within 72 h, it should be discard.

Harvest allantoic fluid.Assessing HA titer Harvest allantoic fluid.Assessing HA titer refers to Experiment 25.refers to Experiment 25.

Hemagglutination test, HT

MATERIALSMATERIALSVirus:the allantoic fluid that contains influenVirus:the allantoic fluid that contains influen

za virus; za virus; physiological saline; physiological saline; 0.5% chicken RBC suspension.0.5% chicken RBC suspension.20-well plastic disposable microtiter plates 20-well plastic disposable microtiter plates

and glass pipettes.and glass pipettes.

Materials Materials well No.well No.

(ml)(ml) 1 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 10

Saline Saline 0.45 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.250.45 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

Virus Virus 0.05 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 -0.05 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 -

Dilution Dilution 1:10 1:20 1:40 1:80 1:160 1:3201:6401:12801:2560 con1:10 1:20 1:40 1:80 1:160 1:3201:6401:12801:2560 con

0.5%RBC 0.5%RBC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.20.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.255

incubate the plate at room temperature for 30 to 60 min. incubate the plate at room temperature for 30 to 60 min.

Check the agglutination.Check the agglutination.



Analysis of the resultsAnalysis of the results


1:6401:640

1:3201:320

1:1601:160

1: 801: 80

1: 401: 40

1: 201: 20

1: 101: 10

Hemagglutination test (HT)

RESULTRESULT

Record the hemagglutination as the followings:Record the hemagglutination as the followings:

++++ : All the RBC had been agglutinated. ++++ : All the RBC had been agglutinated.

+++ : 75% of RBC had been agglutinated+++ : 75% of RBC had been agglutinated

++ : 50% of RBC had been agglutinated.++ : 50% of RBC had been agglutinated.

+ : 25% of RBC had been agglutinated.+ : 25% of RBC had been agglutinated.

- : No RBC was agglutinated. - : No RBC was agglutinated.

Hemagglutination inhibition test (HIT)

MATERIALSMATERIALS

Patient‘s serum: deactivated 30 min at 56C;Patient‘s serum: deactivated 30 min at 56C; influenza virus: 4 HA units;influenza virus: 4 HA units;0.5% chicken RBC suspension;0.5% chicken RBC suspension;physiological saline physiological saline 20-well microtiter plates, pipettes and tubes. 20-well microtiter plates, pipettes and tubes.

Materials Materials well No.well No.

(ml)(ml) 1 2 3 4 5 6 7 8 9 101 2 3 4 5 6 7 8 9 10

Saline Saline 0.9 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.250.9 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

Serum Serum 0.1 0.25 0.25 0.25 0.25 0.25 0.25 - 0.25 -0.1 0.25 0.25 0.25 0.25 0.25 0.25 - 0.25 -

Dilution Dilution 1:10 1:20 1:40 1:80 1:160 1:320 1:640 - - -1:10 1:20 1:40 1:80 1:160 1:320 1:640 - - -

Virus Virus 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 - -0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 - -

Mix evenly incubation at 37C for 1hMix evenly incubation at 37C for 1h

0.5%RBC 0.5%RBC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 - 0.250.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 - 0.25

Mix evenly incubation at 20c for 1 hMix evenly incubation at 20c for 1 h

ResultResult - - - + ++ +++ ++++ ++++ - - - - - + ++ +++ ++++ ++++ - -

Hemagglutination inhibition test (HIT)

RESULTRESULT

Assess hemagglutination as above, the hAssess hemagglutination as above, the hemagglutination inhibition (HI) titer is the emagglutination inhibition (HI) titer is the reciprocal of the highest dilution of the preciprocal of the highest dilution of the patient's serum which shows complete inatient's serum which shows complete inhibition of agglutination.1:40 dilution in Thibition of agglutination.1:40 dilution in Table is the HI titer.able is the HI titer.


Identification of influenza virusIdentification of influenza virus

Hemagglutination inhibition test (HI)Hemagglutination inhibition test (HI) --- subtype --- subtype Complement fixation test (CF)Complement fixation test (CF) ---type ---type

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Experiment five Isolation and Identification of influenza virus