1. Experiment five
Isolation and Identification of influenza virus

2. Isolation and Identification of viruses in lab
Specimen
Isolation and identification of virus
Serological diagnosis of viral infection
Rapid diagnosis of virus infections

3. Isolation and Identification of viruses in lab
Isolation of virus
Embryonated Eggs
Laboratory Animals
Tissue Culture
Quantitation of virus
50% tissue culture infectious dose (TCID50)
Plaque forming assay

4. Isolation and Identification of viruses in lab
Serological diagnosis of viral infection
Neutralization test(NT)
Complement fixation test(CF)
Titration of complement

5. Isolation and Identification of viruses in lab
Rapid diagnosis of virus infections
Identification virus particle
Detection of viral antigens
Detection of viral nucleic acid
Diagnosis of virus infections by detecting IgM antibodies
Other methods

6. Isolation and Identification of influenza virus
Purpose：
To obtain information about microbiological diagnosis methods of influenza virus
Procedure
Acquiring and managing specimen
Isolation and Identification of influenza virus
serological diagnosis of influenza virus

7. Isolation and Identification of influenza virus
Acquiring and managing specimen
Throat washing or gargling are obtained within 3 days after infection
Test at once or stored frozen.
Centrifuge the washings 15 min at 3000 rpm, Harvest 1 ml of supernatant.
Add antibiotic to prevent bacterial contamination.

8. Embryonated egg inoculation
Patient’s serum
Throat washings or gargling
Treated with penicillin and streptomycin
Hemagglutination test
Embryonated egg inoculation
(amniotic or allantoic route)
Harvest amniotic and allantoic fluids
Hemagglutination test + -
Hemagglutination inhibition test
complement fixation test
Inoculate to fresh embryos after two such passages
the result is negative

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

10. Mark the location of embryo air sac under egg-checking box, sterilize the shell with tincture and 75% ethanol, drill a small hole near the marked shell , inoculate 0.2 ml of virus inoculum into the allantoic cavity by pushing the needle just below the surface of the shell, seal the hole with melton wax.
Incubate the inoculated embryo at 35C, check it daily. After incubating for 72 h,place the embryo at 4C overnight. If the embryo dead within 72 h, it should be discard.
Harvest allantoic fluid.Assessing HA titer refers to Experiment 25.

11. Hemagglutination test, HT
MATERIALS
Virus:the allantoic fluid that contains influenza virus;
physiological saline;
0.5% chicken RBC suspension.
20-well plastic disposable microtiter plates and glass pipettes.

12. Hemagglutination test, HT
Materials well No.
(ml)
Saline
Virus
Dilution :10 1:20 1:40 1:80 1:160 1:3201:6401:12801:2560 con
0.5%RBC
incubate the plate at room temperature for 30 to 60 min.
Check the agglutination.

13. Hemagglutination test, HT
Analysis of the results

14. Hemagglutination test, HT
1:640
1:320
1:160
1: 80
1: 40
1: 20
1: 10

15. Hemagglutination test (HT)
RESULT
Record the hemagglutination as the followings:
++++ : All the RBC had been agglutinated.
+++ : 75% of RBC had been agglutinated
++ : 50% of RBC had been agglutinated.
+ : 25% of RBC had been agglutinated.
: No RBC was agglutinated.

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

17. Materials well No.
(ml)
Saline
Serum
Dilution :10 1:20 1:40 1:80 1:160 1:320 1:
Virus
Mix evenly incubation at 37C for 1h
0.5%RBC
Mix evenly incubation at 20c for 1 h
Result

19. Hemagglutination inhibition test (HIT)
RESULT
Assess hemagglutination as above, the hemagglutination inhibition (HI) titer is the reciprocal of the highest dilution of the patient's serum which shows complete inhibition of agglutination.1:40 dilution in Table is the HI titer.

21. Isolation and Identification of influenza virus
Hemagglutination inhibition test (HI)
--- subtype
Complement fixation test (CF)
---type