Practice 4: The Isolation, Cultivation and Identification of Viruses and serological diagnosis morphological laboratory centre
Outline Introduction of laboratory diagnosis of viral infection Virus isolation in tissue culture Virus isolation in embryonated hen s eggs Serological diagnosis_ Haemagglutination inhibition test (HI)
Part Ⅰ Introduction of laboratory diagnosis of viral infection
What is a virus? It is a segment of either RNA or DNA protected by a protein coat and in some families of viruses a host derived envelope with attached viral proteins
It lacks: - Protein synthesizing machinery - Energy producing system - No mitochondria - No stores of amino acids, nucleotides energy rich molecules It is a compulsory intra cellular parasite
It depends on three main principles: Direct detection of: Isolation on: Serology using: Virus particles Tissue culture IF Viral antigen Chick embryo HI Viral nucleic acid Cytopathology Laboratory animals NT ELISA
1.Direct detection Direct detection could be done by one of the following: Particle Electron microscopy. Antigen detection Fluorescent antibody test. ELISA Immunodiffusion. Nucleic acid
EM detection of corona virus
EM picture of rabies virus
Detection of virus by Immunofluorescent Technique
IF staining of rabies infected brain cells
Nucleic acid techniques PCR Probe Hybridization
2. Isolation and identification (gold standard) Isolation and identification of the virus from clinical specimens: three main systems are used for viral isolation: Tissue culture. Chick embryo. Laboratory animals
VIRUS CULTIVATION SYSTEMS 1. Laboratory animals 2. EMBRYONATED EGGS 3. TISSUE CULTURE SYSTEM
IN-VIVO / IN-VITRO? 1. Laboratory animals a) Natural host b) Experimental animals In - vivo c) Transgenic animals 2. Embryonated Eggs In - vivo & In - vitro 3. Tissue Culture System In - Vitro
ANIMAL INOCULATION- DISADVANTAGES Cost Maintenance Interference of immune system Individual variations Difficulty in choosing of animals for particular virus
EMBRYONATED EGGS ADVANTAGES Isolation and cultivation of many avian and few mammalian viruses Ideal receptacle for virus to grow Sterile & wide range of tissues and fluids
Cost- much less Maintenance-easier Less labour Readily available Free from bacteria and many latent viruses. Free from specific and non specific factors of defence. Sensitive to viruses which do not produce infection in adult birds.
TISSUE CULTURE SYSTEM a lot cheaper and easier to work with (contamination can be a problem however). Primary cell lines have a short lifespan in culture a few generations before reaching senescence. Diploid cell lines are derived from embryos and can grow for up to 100 population doublings before senescence. Continuous cell lines are derived from transformed cells and grow indefinitely in culture.
TISSUE CULTURE SYSTEM Hela cells 1st continuous cell line, derived from Helen Lane (fictional name - actually named Henrietta Lacks), a cervical cancer patient who died in 1951. This is the oldest continuous cell line and was first used to culture and identify polio virus.
Transformed Cells in Culture
Viral growth can cause cytopathic effects in the cell culture. Cytopathic effects can be so characteristic of individual viruses that they can often be used to identify viruses. (a) Uninfected cells in culture form a monolayer (b) Cells infected with vesicular stomatitis virus round up and pile up on top of each other
Non-cytocidal effects include acidophilic or basophilic inclusion bodies in the nucleus, cytoplasm, or both; cell fusion, and transformation. (a) Cytoplasmic inclusion body caused by rabies virus in brain tissue. (b) Syncytium formed by cell fusion due to infection by measles virus.
3. Serological demonstration of the antibodies by: Immunofluorescence (IIF). Enzyme immunosorbant assay (ELISA). Haemagglutination inhibition test (HI). Neutralization test (NT). Complement fixation
Part Ⅱ Virus isolation in tissue culture
1. Primary cell cultures When cells are taken freshly from animal tissue and placed in culture, the cultures consist of a wide variety of cell types, most of which are capable of very limited growth in vitro, usually fewer than ten divisions. These cells retain their diploid karyotype, i.e., they have the chromosome number and morphology of their tissues of origin. They also retain some of the differentiated characteristics that they possessed in vivo. Because of this, these cells support the replication of a wide range of viruses. Primary cultures derived from monkey kidneys, mouse fetuses, and chick embryos are commonly used for diagnostic purposes and laboratory experiments.
2. Diploid cell strains Some primary cells can be passed through secondary and several subsequent subcultures while retaining their original morphological characteristics and karyotype. Subcultures will have fewer cell types than primary cultures. After 20 to 50 passages in vitro, these diploid cell strains usually undergo a crisis in which their growth rate slows and they eventually die out. Diploid strains of fibroblasts derived from human fetal tissue are widely used in diagnostic virology and vaccine production.
3. Continuous cell lines Continuous cell lines such as KB and HeLa, both derived from human carcinomas, support the growth of a number of viruses. These lines and others derived from monkey kidneys (e.g., Vero), mouse fetuses (L929), and hamster kidneys (BHK) are widely used in diagnostic and experimental virology. Continuous cell lines have been established from many types of vertebrate and invertebrate animal tissues and are available from the American Type Culture Collection.
4. PROTOCOL Culture of Primary Chick Embryo Fibroblasts (CEF) Materials 10- to 12-day-old embryonated eggs Forceps and scissors Sterile petri dishes Sterile 125-ml Erlenmeyer flask with magnetic stir bar Sterile 25-cm2 flasks containing MEM plus 10% fetal calf serum Sterile 0.5% trypsin in Saline A Sterile 15-ml centrifuge tubes containing 0.5 ml of serum Hemacytometers 1-ml and 10-ml pipettes Sterile Saline A
Procedure 1. Disinfect the surface of the egg over the air sac. With scissors or the blunt-end of a forceps, break the shell over the air sac. Sterilize forceps by dipping in alcohol and flaming. Cool forceps, then peel away the shell over the air sac, sterilize forceps again, and pull back the shell membrane and chorioallantoic membrane to expose the embryo. 2. Resterilize the forceps, grasp the embryo loosely around the neck, and remove the entire embryo from the egg to a sterile petri dish. 3. Using two forceps or a scissors plus a forceps, decapitate and eviscerate the embryo. Mince the embryo carcass into very small fragments with a scissors. 4. Add about 10 ml of sterile Saline A to tissue fragments in the petri dish, swirl gently for 1 to 2 minutes to resuspend and wash fragments, and carefully pour entire contents into a 125-ml Erlenmeyer flask. Tilt flask, allow fragments to settle, and gently decant saline. Discard saline.
Procedure continued 5. Add 10 ml of sterile warm trypsin solution to fragments in flask, cover, and stirslowly with magnetic bar for 5 to 10 minutes. Tilt flask, allow fragments to settle, and pour the trypsin-cell suspension into a 15-ml centrifuge tube containing 0.5 ml of serum. The serum contains a trypsin inhibitor that will prevent further damage to cell membranes by the enzyme. 6. Add 10 ml of sterile warm trypsin to fragments and repeat step 5. At the end of this second treatment, the size of tissue fragments will be greatly reduced and a large number of single cells should be suspended in trypsin. (Note: it is preferable to treat the tissue with multiple short applications of trypsin; however, if time is a limitation, for example in a lab class, this method will work).
Procedure continued 7. Visually balance volume in centrifuge tubes (transfer liquid if necessary) and centrifuge at 1,500 rpm for 10 minutes. Carefully decant and discard supernatant, resuspend pooled cell pellets in a total of 5 ml of MEM. (Resuspend the pellet in one tube, then transfer the suspension to the second tube and resuspend that pellet.) Mix well for counting in a hemacytometer. Be sure to keep your cell suspension sterile. 8. In most hemacytometers, each heavily etched square (surrounded by double or triple lines and containing either 16 or 25 smaller squares) is 1 mm on each side and 0.1 mm deep. Therefore, the area is 1 mm2 and the volume is 0.1 mm3 (Fig. 1). After centrifugation, the cells should be packed in a tight pellet at the bottom of each tube.
Procedure continued 9. Place the cover slip on top of the hemacytometer, bridged on the two glass arms beside the etched pattern. Add one drop of evenly-suspended cell suspension to the groove in the hemacytometer stage and allow it to fill the chamber under the cover slip. Examine with the 10X objective. If there are too many cells to count (>200), make a 1:10 dilution of the cell suspension in MEM (0.1 ml of cell suspension plus 0.9 ml of MEM) for counting.
Procedure continued The following example shows how to convert your cell count to the concentration of cells in your original suspension. Assume you made a 1:10 dilution, then counted a total of 168 cells in one 16-square grid: 168 x 10 4 x 10 = 1.68 x 10 7 cells/cm3 168 is the number of cells in the grid x 10 4 converts to cells per ml (10 4 is the number of 0.1 mm3 in 1 cm3 (1 ml)) x 10 accounts for the dilution 1.68 x 10 7 cells/ml(cm3) is the number of cells in your original suspension
FIG. 1. Hemacytometer (improved Neubauer counting chamber).
Procedure continued 10. Calculate what volume of the original cell suspension you will need to add to the growth medium in the flask to give between 2 x 105 and 8 x 105 cells/ml (~5 x 105 cells/ml). (Hint: if you have 5 ml of growth medium in the flask, you need a total of 5 ml x 5 x 105 cells/ml = 2.5 x 106 cells.) Add the appropriate volume of original cell suspension (not the 1:10 dilution, if you made one) to the medium in your flask. 11. Be sure to examine cell cultures both macroscopically and microscopically each day. Actively growing cells produce acidic metabolic by-products and their medium becomes yellow, and thus the ph of the medium may need to be adjusted by the addition of a drop or two of 7.5% NaHCO3. If floating (dead) cells and debris are present or the color of the medium indicates a basic ph, the medium should be changed.
Virus isolation in tissue culture cell line
Viral identification: This is achieved by: (a) The effect on cell culture: i.e. cytopathic effect, (b) Neutralization test. This is based on the neutralization of the virus infectivity by mixing it with specific antibody before inoculation into cultures.
Part Ⅲ Virus isolation in embryonated hen s eggs
METHODS OF CULTIVATION Various routes of inoculation a)yolk sac b) Allantoic sac c) Chorioallantoic membrane d. Amniotic cavity e. Intravenous
EMBRYONATED EGG
BLOOD VESSELS
1.YOLK SAC ROUTE
YOLK SAC ROUTE Advantages Simplest method Mostly mammalian viruses Immune interference for most of avian viruses Disadvantages Not suited for avian viruses
2.ALLANTOIC ROUTE INOCULATION SITE DETERMINATION
ALLANTOIC ROUTE Most popular Most of avian viruses High titered virus Simple technique
3.CHORIOALLANTOIC SAC ROUTE OF INOCULATION
4.CHORIOALLANTOIC MEMBRANE ROUTE Pox and Herpes viruses. Pock Lesions Suitable for plaque studies
A VIRUS INOCULATION BEING DROPPED ONTO THE CHORIOALLANTOIC MEMBRANE OF THIRTEEN DAY OLD CHICK EMBRYO.
CAM ROUTE OF INOCULATION
Herpes virus lesion on the chorioallantoic membrane
5.AMNIOTIC ROUTE Primary isolation of influenza and mumps viruses Growth of virus detected by haemagglutination Influenza Virus Hemorrhagic lesions in the proventriculus, seen at necropsy in fowl with avian influenza Mumps Virus
Inoculation into the amniotic cavity of the chick embryo.
6.INTRAVENOUS ROUTE Blue tongue virus Cherry red embryo Highly cumbersome Most sophisticated procedure
EGG INOCULATION ROUTES - COMBINED
Equipment Needed for Egg Candling and Inoculation 4 eggs/student Egg flat Drill 70% Ethanol spray bottle Marker Glue 23 guage 1 needle Egg labels Sharps container
CANDLING BOX
Candle 10-11 day-old embryos and check for embryo vitality mark the air cell line
Check for embryo location and mark the side opposite the embryo midway along the long axis where the vein structure is well developed
Disinfect egg shell surface on both the air cell end and side
Drill a small hole through the shell and eggshell membrane in the air cell end of the embryo
HARVEST OF ALLANTOIC FLUID
HARVEST OF ALLANTOIC FLUID 1.Disinfect egg shell surface 2. Only open eggs from a single specimen at one time in the BSC 3. Open egg from air cell end with forceps 4. Break allantoic sac with sterile forceps 5. Hold membranes and embryo away from pipette tip with forceps 6. Harvest AAF
Haemagglutination & Haemagglutination inhibition HAI HA
Part Ⅳ Serological diagnosis Hemagglutination (HA) Test Hemagglutination-Inhibition (HI) Test
HA/HI Tests Supplies Needed U- or V-bottom microtiter plates (non-sterile) Pipettors with tips (sterile ART, nonsterile) Single channel (calibrated) 25-200ul Multichannel (calibrated) 25-200ul Phosphate buffered saline (0.01M, ph 7.2) Washed chicken erythrocytes (0.5%) Virus:the allantoic fluid that contains influenza virus;
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) 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.
Hemagglutination inhibition test (HIT) 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 - - 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 37C for 1h 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.