Experiment five Isolation and Identification of influenza virus

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

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

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

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

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

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.

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

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

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.

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.

Hemagglutination test, HT Materials well No. (ml) 1 2 3 4 5 6 7 8 9 10 Saline 0.45 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 Virus 0.05 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 - Dilution 1:10 1:20 1:40 1:80 1:160 1:3201:6401:12801:2560 con 0.5%RBC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 incubate the plate at room temperature for 30 to 60 min. Check the agglutination.

Hemagglutination test, HT Analysis of the results

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

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.

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.

Materials well No. (ml) 1 2 3 4 5 6 7 8 9 10 Saline 0.9 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 Serum 0.1 0.25 0.25 0.25 0.25 0.25 0.25 - 0.25 - Dilution 1:10 1:20 1:40 1:80 1:160 1:320 1:640 - - - Virus 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 - - Mix evenly incubation at 37C for 1h 0.5%RBC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 - 0.25 Mix evenly incubation at 20c for 1 h Result - - - + ++ +++ ++++ ++++ - -

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.

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