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LABORATORY MANUAL

DIAGNOSTIC VIROLOGY
For Veterinary Microbiology Courses

Prepared by Dr Mohd Azmi Mohd Lila En Kamarudin Awang Isa En Rahim Osman

Research & Diagnostic Virology Laboratory Faculty of Veterinary Medicine Universiti Putra Malaysia http:/www.vet.upm.edu.my/~virolab/

Beta Version

1 LABORATORY INSTRUCTION A. Several viruses to be used in the laboratory are potentially pathogenic. The following rules should be followed to prevent undesired consequences. 1. 2. 3. 4. 5. No smoking or eating is allowed in the laboratory. Any accident with a risk of biological contamination should be reported to the instructor at once. At the beginning and end of each laboratory practise wipe the bench with a clorox solution provided. At the end of each laboratory practise turn off all gas and water receptacles and properly dispose all used and contaminated glasswares. Wash your hands thoroughly before leaving the laboratory.

B. Disposal of used and contaminated equipments 1. 2. 3. 4. 5. Used and/or contaminated plastic-ware should be discarded into a specific containers only, as instructed.. Used and/or contaminated surgical instruments and non-disposable syringes and needles should be discarded into a metal bucket provided. Used and/or contaminated glassware (test tube, flasks, beakers etc.) should be discarded into the large decontamination buckets. Contaminated disposable items (paper items, pasteur pipettes, disposable syringes and needles, etc.) should be placed into specified decontamination buckets. If these items are not contaminated, it may be placed in the wastebasket. Contaminated liquids should be disposed into a foil-covered beaker. Non-contaminated liquids may be poured down the drain.

C. Other Procedures 1. 2. All the materials which are necessary for each exercise will be placed on the front desk. Take only the required amount of each item that is called for in the laboratory exercise protocol. Many of the exercises will require several days to be completed. Items involved in the these experiments (cell cultures, eggs, test tube, etc.) should be clearly labeled with relevant informations (Name, date, exercise no., cell type, virus, etc.) so that confusion will not occur at a later time.

Preparation of glassware for virus work It is important that ALL glassware, pipettes and syringes to be used for virus work especially tissue culture, should be specially cleaned. Ordinary detergents must be avoided as they are proven to be toxic to cells and viruses.

Procedure 1. Completely submerge glasswares in a bucket of 0.1 % sodium carbonate solution taking care that no air bubbles are trapped. Autoclave 15 minutes. Scrub and rinse each pieces throughly under running tape water. Rinse three times in distilled water. Allow to dry. Pipettes are cleaned in concentrated sulfuric acid, throughly rinsed in tap water followed by three rinses in distilled water. Rubber stoppers are boiled for 1 hour in 0.1% sodium carbonate, rinses under running tap water followed by three time rinses distilled water. All beakers, flasks, bottles and cylinders are capped with heavy aluminum foil, all tubes with aluminum caps or foil. Pipettes, petri dishes, syringes and stoppers are packaged in stainless steel or glass containers. Cotton or paper products cannot being used since they contain harmful volatile oils. All glassware is dry air sterilized. Rubber stoppers are autoclave. Placed tisssue culture glassware into buckets of water so that it is completely submerged and immediately autoclaved before cleaning for re used. Do not used disinfectant for tissue culture glassware.

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2 COLLECTING AND HANDLING SPECIMENS FOR VIROLOGY Collection of Specimens The success in isolation of viruses from clinical material is greatly dependent on the proper collection and handling of specimens. Ideally, materials for examination should be collected as early as possible following autopsy, or on the day of examination and if the patient is examined alive. Specimens should be delivered to the laboratory promptly and placed in a refrigerator. All samples should be properly labelled and accompanied by information as the patients breed, age, sex, the date of onset of illness and the working clinical diagnosis should be included, since the clinical diagnosis may influence the test system selected. Throat or nasopharyngeal swabs Place in a tube containing 2 ml PBS. (Phosphate Buffer Saline) Rectal Swabs Place in a tube containing 2ml PBS) Stool specimens. 5 to 10 gm are preferable for rectal swabs, since at any given time only a small amount of virus may present in fecal materials. Cerebrospinal fluid (CSF) Collect at least several milliliters of fluid in a sterile tube. Pleural or pericardial effusions Collect several milliliters (5 to, 10 ml; if possible) in a sterile tube. Urine 10 to 50 ml. Collect freshly voided urine in a sterile tube. Blood samples Should be obtained from all patients. Collect the first sample (10 to 15 ml of clotted blood ) on examination and a second specimen 2 to 3 weeks later.

Autopsy materials Should be obtained as soon as possible after death; collect aseptically and never add preservatives. Stool 1. Make a 20% suspension of the stool by adding 4 gm stool to 16 ml PBS 2. Shake vigorously for 30 minutes in a stoppered flask containing glass beads. 3. Centrifuge at 3,000 rpm for 10 minutes, preferably in a refrigerated centrifuge. 4. Remove and recentrifuge the supernatant at 3,000 rpm for 10 minutes. 5. Add antibiotics to final a concentration of: Penicillin, 500 unit per ml Streptomycin, 500 ug per ml Mycostatin, 20 units. 6. Inoculate into suitable culture system.

Autopsy materials. (Including spinal cord, lung etc) 1. 2. 3. Grind autopsy tissues in a mortar using sterile alundum. Add 1 to 2 ml PBS and grind until smooth. Add an additional 1 to 2 ml PBS, grind and gradually add PBS until a 10% suspension is obtain. Collect supernatant and add penicillin and streptomycin, 200 unit per ml.

3 4. Inoculate into appropriate culture system.

Storage and Transportation of specimens Speed in delivering specimens to the laboratory is of great importance, and refrigeration is necessary if delays are unavoidable. If possible, specimens should be processed and inoculated immediately upon arrival at the laboratory; if there is a short delay, for example a few hours, refrigerate at 4oC. For longer periods of storage, freeze samples at 70oC. If mail shipment is made, specimens should be packed in sealed tubes or bottles and placed in an insulated container with dry ice. (-70oC)

Processing of specimens. Blood specimens. Centrifuge soon after the clot has formed to obtain the serum. In certain instances, heparinized or citrated whole blood may be used for virus isolation attempts.

Body fluids Including cerebrospinal fluid, pleural, vesicle fluid, etc., should be inoculated directly into test cultures.

Swabs Samples collected in this way are commonly trachea, eye, nose, or rectal swabbing. Use a sterile swab dampened (but not wetted) in a sterile diluent such as Hank's balanced salt solution. Insert the swab into the test area and rotate it gently. b) Immediately deposit it into a sterile tube containing diluent and snap off the extra length of swab stick before replacing the cap. c) At the laboratory, remove the cap with sterile technique grasp the swab with sterile forceps, agitate the swab in the diluent, press out the fluid against the wall of the tube, and remove the swab. Use the swab to make a sterility check. d) Centrifuge the fluid in the cold at 3,000 rpm for 10 minutes. Remove the supernatant add antibiotics, place in sterile vials, seal, label, and freeze. a)

4 CULTIVATION OF VIRUSES As viruses are capable of growing only in living cells, it is essential that the first step in experimental studies of a virus requires cultivation of virus in a convenient laboratory host. There are basic laboratory host for this purpose: 1) Embryonated eggs. 2) Tissue culture 3) Susceptible species or animals (laboratory animals).

Animals The ideal animal host for study of a virus is, of course, the natural host of the virus. This however, not always possible or practical, and quite often it is necessary to use any animal that is susceptible to infection. Among the laboratory animals commonly employed for this purpose are: mice, rats, rabbits, guinea pigs, hamster, chickens, and monkey. The choice of an animal is obviously dictated by the nature of the virus under investigation, since to all animals are equally susceptible to all viruses; moreover, not all viruses have been successfully cultivated in the experimental animals. One of the most useful laboratory animals is the mouse which may be infected with a variety of viruses by various routes. Its immune system is well studied. The particular route to be used is determined by the ability of tissues to support virus multiplication (e.g. viruses such as influenza viruses are usually introduced intranasally; viruses such as arboviruses, on the other hand, are generally introduced intra cerebrally).

Embryonated eggs Although not all viruses can be grown in embryonated eggs, many viruses are cultivable in this host. The embryonated eggs can be infected by various routes (e.g. amniotic, allantoic, chorioallantoic and via the yolk sac). Since embryonating eggs having many cells of various origins (ie. ectodermal, endodermal, and mesodermal), various tissues exhibit varying susceptibility to viral infection, and the choice of a particular route is again dependent upon the ability of tissues to support virus replication. The chorioallantoic membrane (CAM) is a useful tissue for those viruses which produce lesions i.e. formation of pocks by poxviruses, and the development of these pocks affords a readily demonstrable effect. The CAM, in essence provides an equivalent solid sheet of cells on which virus can grow. Since each infective viral particle is capable of giving rise to pock, the CAM may be used to assay many viruses, much as a nutrient agar plate may used for determination of bacterial cell numbers. The most convenient method of infection of eggs is via the allantoic cavity which is lined by cells of endodermal origin. Viruses which are capable of multiplication in these cells are eventually released from the cells and accumulate in the allantoic fluid and bath the allantoic cavity. The virus may, therefore, can be obtained from allantoic fluid.

Tissue culture In recent year, a great deal of emphasis has been placed on the use of tissue cultures for investigations of many different viral problems. The basis of tissue culture is the cell can be obtained alive from animal and it does not necessarily result in death of the cell (i.e. in other environment). With adequate supports and nourishment, the isolated cell can be maintained and made to proliferate in vitro. The ability of these cells or tissue cultures to support the growth of many viruses has made them highly useful host systems. Several basic types of tissue cultures are available (eg.suspension, monolayers, or surfaces on glass beads tissue culture) and the use of any particular will depend in part on the objectives of the investigator. In recent years, the technique of preparing monolayers of cells on glass or some other solid substrate has been used extensively. This technique affords a continuous single layer of cells on which viruses can be grown an studied. Many viruses produce localized and well-define lesions (plaques)when growing in a cell monolayer and are therefore easily assayed in this manner (analogous to pock method of assay).

5 The application of tissue culture methods in virology has also been facilitated by the development of pure lines of mammalian cell strains since they constitute easily cultivable and fairly homogenous cells of known susceptibility to different viruses. In any tissue culture used for growth of virus, it is necessary to establish as to whether the virus is replicating. This may be achieved in a number of ways: 1. Determination of pH of medium, for example, growth of poliomyelitis virus results in death of cells and ceasation of cell metabolism and a consequent failure to change the pH of environment as contrasted with uninfected and actively metabolizing cells. Direct observation of culture for evidence of cell destruction (cytopathic change). Determination of viral concentration by measuring viral infectivity or of some other viral activity (eg. hemagglutination).

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The three basic methods described above are useful not only for cultivation of viruses but also for various other purposes (eg. titration of virus, assay of antibody, studies of viral genetics, production of vaccines, studies of viral replication).

6 A. Cultivation of virus in Embryonated Eggs. Inoculation of eggs by the allantoic route Materials: 10 or 11-day embryonating eggs 1 ml tuberculin syringe Newcastle Disease virus (NDV) swabs 70% alcohol paraffin Vaseline mixture Phosphate buffer saline Egg candle, iodine

Procedure: 1. 2. 3. 4. 5. 6. 7. Candle eggs and mark (a) air sac and (b) a non-vascular spot near embryo. Label eggs with your name, type of virus and date of inoculation Sterile the shell and marked spot over embryo by painting shell with iodine. Drill hole (a) At spot marked over embryo and air sac of egg. Paint holes with a fairly dry iodine swab. Fill a syringe with virus. (or suspected materials). Hold syringe at a slight angle from horizontal and insert needle into hole over Embryo to a depth of 3 mm. Inoculate 0.1 ml of virus and withdraw needle. Repeat until all the eggs have been injected. Discard syringe into a pan of water For control use PBS as inoculum. 8. After inoculation of eggs, paint the holes with iodine (use a fairly dry swab). 9. Seal both holes in each egg with a paraffin-Vaseline mixture . 10. Incubate eggs at 37oC for 2-5 days., 11. Candle eggs everyday and discard all eggs that die on 1st day of incubation.

Harvesting Allantoic Fluids Materials: Pasteur pipettes, rubber bulb, blunt and curved forceps, 1 thioglycolate medium, 1 blood agar plate, bijou bottle, blocks for eggs, towels, clorox, 70% alcohol, iodine, cotton swabs. Procedure: 1. 2. At the end of the incubation period chill embryos in ice box or fridge for 1 hour. Arrange eggs in the wooden blocks or egg holder over a piece of cloth or paper towel which has been soaked in clorox (1:1000). Disinfect eggs by painting shells with iodine. Allow iodine to dry and wash off iodin with 70% alcohol. When the egg shell is fairly dry, crack air sac of each egg with a pair of blunt forceps. With a fresh pair of blunt forceps, remove shell above the pencil line. Use 1 forceps for all of the eggs. 4. Pull shell membrane and underlying CAM with a pair or curved forceps. (Allantoic fluids should be clear. Turbidity may (or may not) indicate bacterial contamination. If there is any doubt on sterility, discard egg and use a fresh pair of forceps for remaining eggs. Push embryo and membrane to one side with a sterile pair of curved forceps and withdraw allantoic fluid with a pasteur pipette equipped with a rubber bulb. Place fluids in a bijou bottle and label properly. Test for bacterial contamination by adding one drop of fluid on agar plate. Incubate for 2 days at 37oC. Store virus in refrigerator until results of sterility test are available.

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7 When all the allantoic fluids have been collected the presence of virus can be determined by hemagglutination (HA) and hemagglutination inhibition (HI) test. Place a drop of 1% chicken RBC on an enamel plate and mix a drop of the allantoic fluid to be tested with the cell suspension. Rotate the plate gently. If hemagglutinins are present, the red cells will clump in a minute. To identify the virus, mix a loopful of known Newcastle antiserum and loopful of allantoic fluid on an enamel plate and then add a drop of washed RBC's. Mix and rotate as above. Agglutination inhibition is presumptive evidence that NDV is present. As control allantoic fluid of control chick embryo should be used. If hemagglutinins are present and fluids are sterile, store in refrigerator for future use. Label and store at ultra-low temperature.

Inoculation of Egg by Chorioallantoic Membrane (CAM) Route. a. b. c. d. e. f. g. Candle eggs and mark a one inch line on the long axis of the egg and not over major blood vessels. Make a cross on this line at an approximate midpoint of the egg. Mark the position of the air space. Puncture a hole over the air space with a needle. Carefully puncture a second hole at the "x" deep enough to make a depression in the shell without puncturing the Chorioallantoic membrane. While candling the egg, apply gently suction to the hole over the original air space. As the air is removed, the CAM will separate from the shell, creating an artificial air space. Inoculate 0.1ml of virus suspension with a 1/4 inch, 27 gauge needle onto the dropped membrane. Gently rotate the egg to insure an even distribution of the inoculum.

Harvest the chorioallantoic membrane of the two eggs which were inoculated with virus suspension.

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Clean the shell with 70% alcohol. With sterile scissors, cut a window over the dropped CAM to reveal the CAM. Aseptically remove the CAM. Store in sterile bijou bottle at -70oC

Inoculation of Eggs by Yolk Sac Route a. b. c. d. e. Candle eggs and mark the position of the air sac. Puncture the shell over the center of the air cac. Use a 1ml syringe fitted with a 1 inch, 22 gauge needle and inoculate the egg through the puncture in the shell Point the needle straight down for a depth of about 1 Harvest the yolk sac of the eggs which were inoculated with virus suspension.

Wash the yolk sac by transferring to a series of petri dishes containing PBS. Most of the yolk material adhering. To the membrane will washed off. Blot the yolk sac on sterile cotton gauze to remove the excess yolk material.

8 Isolation and Cultivation Of Infectious Laryngotracheitis and Virus Fowl Pox Virus Materials ILT Virus Embryonated Eggs. Tuberculin syringes. Procedure 1. 2. 3. Inoculate 0.1ml of each virus suspension (ILTV and FPV) into 2 eggs by the CAM method. One egg will serve as control. Half of the eggs are opened after 3 days. If negative, the remainder are examined after a further 2 days of incubation. The growth of the viruses is indicated by the production of pocks on the CAM.

Isolation and Cultivation of Newcastle Disease Virus (NDV) and Infectious Bronchitis Virus (IBV) Materials ND Virus. IB Virus. Embryonated eggs. Tuberculin syringes Procedure 1. 2. 3. 4. Inoculate 0.1 ml of each virus suspension (NDV and IBV) into 2 eggs by the allantoic cavity route. One egg will serve as control. Candle daily. Evidence of growth for NDV is indicated by the embryo mortality (48 - 72 hours) and by presence of hemagglutinin in allantoic fluid. The growth of infectious bronchitis virus is indicated by the dwarfing and curling effect of embryo with slower mortality (5 - 7 days) and failure of allantoic fluid to hemagglutinate RBC.

Preparation of Monolayer Cultures Of Chick Fibroblast Cells. 10 Days old chick embryo, PBS, 0.25% trypsin in PBS, centrifuge tube, sterile petri dish, 50 ml flasks (3 oz bottles), forceps, nutrient medium, lamb serum, tissue culture glassware.

Procedure: 1. Remove chick embryo from shell using sterile techniques, and place in sterile petri dish. Discard wings, feet, head and viscera if possible and minced the embryo. Collect minced embryo and placed in a 50 ml Erlenmeyer flask or 3 oz. bottle . Add approximately 10 mls. Phosphate buffered Saline (PBS) and wash the embryo fragments by swirling the flask. Let the suspension settle for 5 minutes, then decent and discard the supernatant. Add 10 mls 0.25% trypsin in PBS. Place sterile plastic covered magnet bar is suspension and agitate for 20 minutes on magnetic stirrer at 37oC for 5-10 minutes. If magnetic stirrer is not available, digestion may be carried out by intermittently Shaking the bottle and putting the bottle back into the incubator. Care must be taken to avoid contamination during this procedure. Permit any undigested fragments to settle to the bottom of the flask, then pour supernate into a sterile 15 ml. tube. Centrifuge tube containing 0.1-0.2ml. of lamb serum to stop the action of the trypsin. Over-trypsinization will result in a stringy mass which will interfere with sedimentation of the tissue cells in the centrifuge. Sediment cells by centrifugation (800-1000rpm for 5 minutes) and pour off supernatant trypsin and cellular debris. Determine packed cell volume and resuspend to 20% concentration in a nutrient medium (MEM with 5% calf serum). Prepare 20 tube cultures by further diluting the suspension in nutrient medium to a final dilution of 1-200 and distributing 1ml. of the diluted suspension into sterile culture tubes, stoppered with

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9 rubber stoppers. (Calculation for dilution of packed cells to yield final 1-200 dilution: 1 ml packed cells plus 4mls nutrient medium = 20% suspension (1-5 dilution). 0.5ml of 20% suspension plus 19.5ml. nutrient medium = 1-40 dilution, yielding a final dilution of (1-200). Incubate at 36-37oC and observe for cell growth. When growth of cells is apparent, the cell cultures may be seeded with virus and used for various purposes (study of morphological changes, titration of virus, assay of antibody, etc).

Preparation of continuous cell culture. Materials 1. 2. 3. 5. Monolayer of MDBK cell line, Vero cell, PK-15 cells in 3 oz.bottle. Trypsin-Versene solution. Minimum Essential medium 4. Fetal calf/bovine serum Sterile centrifuge tube, pipettes, roller tubes, rubber stopper, test tube rack and 3 oz. bottle.

Procedure 1. 2. 3. Observe the morphology of the monolayer under the microscope. Decant the medium from stock cell culture completely and drain the bottle. Add 10ml of serum free memdium and wash the cell monolayer free of any serum. Add enough trypsin versene solution to cover the monolayer (about 10ml) and trypsinise in the incubator at 37oC and rock the bottle at frequent interval. Usually the cells will be off the glass in 2-3 minutes. Do not trypsinise longer than necessary. When trypsinization is complete, pour of the cell into a centrifuge tube containing 0.5ml serum (to inactivate or stop the action of trypsin). Disperse the cell well by repeated mixing with pasteur pipette. Centrifuge the cell suspension at 1000 rpm for 5 minutes and discard the supernatant. Dilute the cell suspension with enough MEM containing 5% fetal calf serum to give a final cell concentration of 105 cell per ml. (initially a cell count is necessary, however through experience a rough estimate can be made). Dispense cell suspension in 3 oz. bottle or roller tube. Incubate culture at 37oC. Observe the growth of cells daily until the cell sheets are confluent.

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Cultivation (isolation) of Pseudorabies virus, Newcastle disease virus in Tissue Culture Materials Suspension of pseudorabies Virus (PsR), PBS, MEM, 3 oz. bottle of PK-15 cells or Vero. Procedure 1. 2. 3. Pour off the culture medium from the tissue culture bottle and replace with 5ml of PBS. Rotate the bottle gently and remove the PBS. Remove the PBS from the cell culture as completely as possible and inoculate 0.1ml of virus suspension. Inoculate control with 0.1ml of PBS. Incubate at 37oC for 1 hour to allow adsorption of virus to cells. At 20 minutes interval during the adsorption period redistribute the inoculum by tilting the cultures. At the end of the 1 hour period, add 5 ml of media (1-2% serum) and incubate again. Examine for changes (compared with controls) daily for a total of 7 days and record any changes. When half of the cell population is found to be affected and the virus are to be harvested, place the bottle in the freezer for 5-6 hours and then thaw them. During the process thawing the cells will be loosen from the bottles. Dispense the virus suspension in sterile bottles, label and freeze it. Sometimes the process of freezing and thawing may repeated 2-3 times before virus is harvested.

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10 Viruses which are known or suspected to be sensitive to freezing and thawing may be collected by gently scraping the cell sheet loose with a sterile rubber policemen. When trying to isolate virus from the clinical specimen, culture which does not show CPE should be repassanged 3 times in that cell type before the specimen is considered negative. If a virus isolated comes from a suspected case of pseudorabies, the virus can be identified by neutralization test or immunofluorescence technique.

Hemagglutination (HA) and Hemagglutination-inhibition (HI) test Many viruses are capable of agglutinating red blood cells of certain avian and mammalian species. These viruses can be grouped according to the nature of their hemagglutinating property or hemagglutinin. 1. For the myxovirus group, the hemagglutinin is the viral particle itself and processes an enzyme which destroys cell receptors as the hemagglutinin elutes from the cell. This phenomenon has been used as a model to study cell receptors and viral absorption. The virus particle is the hemagglutinin but there is no enzyme associated with the agglutination reaction. Some of the Enteroviruses and Arboviruses belong to this group. Condition of the test, such as pH, temperature and source of red blood cells, are critical for obtaining hemagglutination with these viruses. The hemagglutinin of several poxviruses is a lipoprotein that is separable from the viral particle (i.e. a soluble hemagglutinin).

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Since hemagglutinins are antigenic, they stimulate the production of specific antibodies which have the capacity to inhibit agglutination of red blood cells by hemagglutinin. The hemagglutination inhibition test can be employed as an aid in the laboratory diagnosis of viral diseases or to identify an unknown virus. This test can also be applied on a quantitative basis by using fixed concentrations of hemagglutinin and red blood cells and varying the deletions of immune sera. In this way antibody concentration in sera can be obtained and the titer expressed as the reciprocal of the highest dilution of serum that completely inhibits hemagglutination.

Materials Microtiter plates, 0.025 ml, microtiter pipette droppers. 0.05ml micropipette droppers, 0.05ml microtiter loops, microtiter blotting paper, 1 and 10ml pipettes, 15 ml graduated centrifuge tubes, Mc Cartney bottles, phosphate buffer saline, (PBS pH 7.2- 7.3) chicken RBC, Newcastle disease virus and its antiserum.

Procedure 0.25% erythrocyte suspension: Filter cells through a piece of cheesecloth into centrifuge tube, Wash cells with cold saline, centrifuge at 1500 rpm for 5-10 minutes, pour off supernatant fluid, resuspend cells in saline and repeat process until cells have been washed 3 times. Suspend cells in fresh saline and centrifuge cells for 10 minutes (time it) at 1500 rpm. Take reading of packed cells and make up to 0.25% suspension of cells with saline.

Antigen Titration: The antigen is reconstituted or thawed and the appropriate dilution is determined by titration as follows: 1. 2. 3. Volumes of 0.05ml of PBS are added into rows 2 of 12 wells (duplicate titration) starting from second well. Place 0.1ml of antigen in well no 1 of each row. Using the 0.05 ml diluting loops carry out serial two fold dilutions (1-2 to 1-2048) of the antigen (the loop should be rotated at least 180 degrees 4 times in each well) leaving the last well without antigen as a cell control. Place 0.05 ml of 0.25% RBC suspension in each well. The plates are covered and kept at room temperature (20-25oC) until a distinct button has been formed by the cells settling out in the cell control well. The end-point of the titration is the highest dilution of the antigen with 100% hemagglutination.

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11 The HA units in 0.05 ml of the undiluted antigen is equal to the dilution factor of the endpoint, eg. if the endpoint dilution is 256 then there are 256 HA units in 0.05 ml of antigen. To determine the antigen dilution that contain 8 HA units per 0.05 ml or 4HA units per 0.025 ml, Divide the endpoint titer by 8, eg. 256/8 = 32; therefore, antigen with a titer of 256 units is diluted 1:32 for used in the hemagglutination- inhibition (HI) test.

Serum Titration: 1. Serial twofold dilutions of each serum are prepared so that the serum is diluted out through 12 wells. the no. 1 well in each row receives no antigen and serves as a control of nonspecific agglutination. a. Place 0.025 ml of PBS in all 12 wells in each row. b. Place 0.025 ml of the serum in well no. 1 c. Prepare serial dilutions with the loop calibrated to deliver 0.025 ml 2. 3. 4. 5. 7.

from well no. 1 through well no. 11.

Place 0.025 ml of the appropriate antigen dilution containing 4 HA units as determined above under "Antigen Titration" in wells no. 2 through no. 11. Mix by placing the plate on the jogger for about 10 seconds and maintain at room temperature (20-25oC) for 0 minutes or shake by hand. Place 0.05 ml of 0.025% RBC suspension in each well and maintain at room temperature. Read the test as soon as the RBC have settled out (approximately 30 minutes). Serum titers are expressed as the reciprocal of the highest dilution having complete inhibition of the hemagglutination of the antigen.

Control of the test On each series of test the following controls are required: The antigen dilution used in the test is titrated to make certain it contains at least 4 HA units. a. b. c. d. e. Place 0.05 ml PBS in row of wells except first well. Place 0.1 ml of standard antigen dilution in well no. 1. Using an 0.05 diluter loop make serial twofold dilutions of the antigen up 11 the well. Place 0.05 ml of standard RBC suspension in each well. If the antigen contains 4 HA units per 0.025 ml the hemagglutination should be complete through well no.4

Results Antigen titration - HA test. Tube 1 2 3 Virus dilution HA * * * *

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HA titer = 128 HA units. Standardized antigen dilution = 128/8 = 18 Serum titration-HI test Tube 1 Anti-serum dilution

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12 HI * * * * * * * * * HI titer = 256.

MEASUREMENT OF VIRAL INFECTIVITY It is often necessary to determine the amount of infective virus which is present in a given sample of virus-containing material (eg. in following the course of purification, in assaying the results of immunization). There are several methods available for quantitative estimation of viruses: 1. Dilution End-point: This method is most commonly used and involved determination of dose of virus needed to effect 50% of host (Animal, Eggs, Tissue Culture). This dose (commonly designated ID50 or LD50 defending on weather the end result is infection or death) is established by making serial dilutions of virus, inoculating a suitable host with the dilutions and observing the numbers of positive and negative responses. From these results, it is possible to calculate the 50% endpoint by the method of Reed and Muench. The particular response or endpoint used varies with the virus being tested (eg. if virus usually causes death of infected host, death may be used as the endpoint: if virus is nonlethal, it will be necessary to establish presence of infection by other criteria such as production of visible lesions, demonstration of virus in tissues by staining, hemagglutination or other suitable procedures). 2. Plaque Counts: Titration of virus in tissue cultures is usually made by dilution of virus, infection of tissue cultures with these (localized lesions on solid sheet of cells) or cytopathogenic effect (death of cells without plaque formation). The plaque method is particularly useful since there is a definite relationship between numbers of viral particles and numbers of plaques (one infective viral particle gives rise to one plaque); the number of plaques when multiplied by the dilution factor will yield the total number of infective viral particles in any given viral suspension.

3. Pock Counts: The basis of pock counting as well as the procedure itself is similar to that of plaque counting except that the host system is the chorioallantoic membrane of the embryonated egg.

A. Titration of virus infectivity by EID50 Materials Phosphate buffer saline PBS MEM, Embryonated eggs, dilution tubes, 1 ml pipettes, Newcastle disease virus. Procedure 1. Prepare the embryonated eggs as in the cultivation of virus. 2. Prepare 10 fold dilution of the given virus starting with 10-1 to 10-9 . 3. Label 20 eggs with virus dilution 10-6 through 10-9 (5 eggs per dilution). Label 5 as uninfected control. With sterile technique, inject 0.1 ml of each virus dilution into the eggs beginning at the most dilute (10-9) and working toward the most concentrated level of virus. The same syringe can be used to inoculate several dilutions. 4. Incubate eggs at 37oC for 2-6 days. 5. Candle eggs everyday and discard all eggs that die on 1 st day of incubation. 6. Record any death after the 2nd day and test allantoic fluid for HA. 7. Harvest the allantoic fluid of all eggs that do not die after the 6 th day and test allantoic fluid for HA and record.

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Example of EID50 Calculation (Reed muench Method): Egg HA Data Virus dilution 10-7 10-8 10-9 10-10

Death and HA+ 4 4 2 0

Alive HA0 1 3 5

HA+ Ratio 4/4 4/5 2/5 0/5

Arrows indicate direction of addition for accumulated value Accumulated Value Result Virus HA+ dilution 10-7 10-8 10-9 10-10 10 6 2 0

HIRatio 0 1 4 9 10/10 6/7 2/6 0/9

HA+ Percent 100 86 33 0

The next step is to find the proportionate distance between 2 dilutions wherein the 50% end-point lies (between 10-8 and 10-9)

PD = % HA+ above 50% - 50% % HA+ above 50% - % HA+ below 50% = 86 - 50 86 - 33 = 36 = 0.7 53 The dilution at which the 50% endpoint lies 10-8.7 The titre + 109.7 EID50 / ml.

14 Titration of Pseudorabies Virus By Plaque-Forming units. Materials Six prescription bottle cultures of chick fibroblast (CF) , or pig kidney cells, phosphate buffer saline (PBS) virus suspension 3% agar in 3 oz bottles, nutrient overlay medium (2x concentrated), neutral red solution (1- 2000 concentration), water bath (46oC), pipettes, Dilution tube. Procedure: 1. 2. 3. Pour off the culture medium from the normal chick fibroblast cultured and replace with 10-12 mls PBS. After 15 minutes, remove the first PBS. Wash by pouring and replace with a second 10-12 mls volume. Prepare 10-fold dilution of (PrV in PBS. Remove the second wash fluid from cell cultures as completely as possible; inoculate cultures with 0.1/ml volumes of 10-4 10-5 and 10-6 dilutions of virus, starting with the 10-6 dilution. Infect 2 cultures/dilution. Distribute the inoculum throughly over the cell sheet by tilting the cultures in various directions; incubate horizontally at 37oC for 1 hour to allow adsorption of virus by cells. At 10 minutes intervals during the adsorption period, redistribute the inoculum by tilting the cultures. During the 1 hour adsorption, melt some 3% agar in boiling water and equilibrate at 46oC. Warm the concentrated (2x) nutrient overlay medium to 37oC. When the adsorption period is completed, remove cultures from the incubator. Add 1.5 ml neutral red solution and 30 mls. nutrient overlay medium to the melted agar. Mix well by inverting the bottle (after replacing the plastic cap) and immediately add 10-12 mls of the nutrient agar to the cell cultures by pouring along the inside of the infected cultures. Place cultures horizontally until agar is completely gelled. Incubate at 37oCin an inverted horizontal position for 48 hours. Count the plaques which have formed. These plaques will appear as clear, circular areas against a pink stained background formed by the living cells which retain the vital dye, neutral red. Examine an infected culture with the low-power objective of the microscope and note the difference in appearance between plaque areas and living cells. Calculate the plaque-forming units (pfu) in the undiluted inoculum. pfu/ml = average number of plaques x 10x dilution factors.

4.

5.

Titration of Virus By Tissue Culture Infective Dose 50 (TCID50) The rate of cellular changes and patterns of CPE induced by different viruses varies greatly depending upon: a. The type of culture system used. b. The concentration of virus in the specimen. c. The properties of each virus strain. Once CPE is obtained, virus infectivity titers can be estimated by the Reed and Muench method for determination of the 50% end point (16). Materials Virus, tissue culture cells (roller tubes, 3 oz bottle or macro titer plate), sterile pipettes medium and PBS. 1. 2. 3. Pour off medium form tissue culture tubes and rinse with 2ml PBS. Prepare 10 folds dilution of the given virus starting with 10-1 to 10-9 Label 16 tissue culture tubes with virus dilution 10-3 through 10-6 ( 4 tubes per dilution).label 4 as uninfected control. With sterile technique, inoculate 0.1 ml of each virus dilution into the tissue culture tube beginning at the most dilute (10-6) and working toward the most concentrated level of virus. The same pipette can be used to inoculate several dilutions. Incubate at 37oC for 2-6 days. Check all tubes for CPE. Record any CPE after the 2 nd day up to the 6th day.

4. 5. 6.

15

Dilution of virus

No. of Cultures Showing CPE/No. Inoculated

Cumulati ve No. Infected

Cumulative No. Not Infected

Calculated Infectivity

Ratio

Percent

4/4 10-3 3/4 10-4 2/4 10-5 0/4 10-6

9 5 2 0

0 1 3 7

9/9 5/6 2/5 0/7

100 83 40 0

In this example the proportionate distance between the two dilutions (10-4 and 10-5) where the 50% end point lies is equal to: Infectivity above 50% - 50% Infectivity above 50% - Infectivity below 50% = 83-50 = 0.7 83-40

Therefore, virus titer, represented by 104.7 per ml, is the TCID50 (tissue culture infectious dose, infecting 50% of the cultures inoculated), and a dilution of 102.7 of this virus suspension contains 100 TCID50 in a volume of 0.1 ml.

16 VIRUS IDENTIFICATION Neutralization Test (Inhibition of CPE) 1. 2. Determine virus titer as illustrated above Add equal volume of a constant virus dilution containing 100 TCID50 per 0.1 ml to a known type-specific antiserum at a concentration of 20 units and shake well. (Note: The highest serum dilution that neutralizes 100 TCID50 of virus represents 1 unit. Allow the virus-serum mixture and the virus control of serial 10-fold dilutions to remain at room temperature for 1 hour. Inoculate 0.2ml of the virus-serum mixture each into 2-4 culture tubes. For the control, inoculate 0.1ml of each virus dilution into a set of cultures, 2-4 tubes per dilution. Check all tubes for CPE at 3, 5 and 7 days post inoculation.

3. 4. 5. 6.

Neutralization test (Plaque Reduction) 1. 2. 3. 4. 5. 6. 7. 8. 9. Determine virus titers by inoculating serial 10-fold dilutions of the virus suspension into tissue cultures; overlay with nutrient agar and determine plaque counts. Add equal volumes of a constant virus dilution containing approximately 50-100 PFU (Plaque Forming Units) per 0.1 ml to a known antiserum of 20 units and shake well. Allow the virus-serum mixture(s) and virus serial 10-fold dilutions to remain at room temperature for 1 hour. Inoculate 0.2 ml of each mixture and 0.1 ml of each test virus dilution into a bottle culture. Incubate culture bottles at 37oC for 1 hour to allow virus adsorption. Remove bottles from incubator and overlay with nutrient medium containing agar (avoid exposure to light during and after overlay). Invert bottles after the agar has solidified, and incubate at 37oC. Observe the bottle cultures daily for the appearance of plaques, and count the number as they appear. An 80% or greater reduction of plaques is considered a positive serum neutralization test, and confirms the identity of the virus.

Immunofluorescence Technique Immunofluorescence technique is employed in both diagnostic and research work for detecting antibody antigen reactions by the use of fluorescent antibodies. The principle of immunofluorescent staining can be applied either for demonstrating viral antigen present in cells (smear, section, monolayers) or viral antibodies contained in sera. In each case the sites or specific antigen antibody reactions are made fluorescent by tagging immunoglobulin with suitable fluorochromic dye. The most commonly use fluorochrome dye is fluorescein isothiocyanate (FITC) which is coupled to the immunoglobulin of antisera. The specific immunoglobulin which have been tagged with a FITC dye fluoresces on exposure to ultraviolet light. There are several variants of the technique but the most common ones are the direct and the indirect immunofluorescence.

Direct Staining Method 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Remove coverslip containing infected cells from Lighten culture tubes. As a control, uninfected cultures should be treated in the same manner. Wash coverslip with PBS 2-3 times. Dry at room temperature. Fix with acetone for 10 minutes. Dry at room temperature (at this step the fixed cells on coverslip can be stored at -20oC in stoppered-tubes). Overlay infected cells on coverslip with fluorescein-labeled antiserum. Let stand 30 minutes in petri dish containing moist gauze. Wash in PBS to remove excess labeled antiserum (Three changes). Dry coverslip at room temperature. Mount with buffered glycerin (1 ml PBS + 9 ml glycerin that has been prepared within a 3-week period). Cover with a 22 x 50 mm (00 thickness) coverslip and seal with colorless nail polish to prevent evaporation. Examine the preparations under a microscope with a dark-field condenser and UV light source.

17

Indirect Staining Method Steps 1 to 5 are the same as for the direct method. 6. Overlay infected cells on coverslip with unlabeled antiserum (eg. produced in rabbits) and let stand for 30 minutes in a petri dish containing a piece of moist gauze (as controls, normal rabbit serum can be used instead of antiserum). Wash off excess antiserum with PBS (three changes). Dry coverslip by letting stand at room temperature. Add fluorescein-labeled antiglobulin (in this instance, anti-rabbit globulin produced in goats) and let stand to conjugate with the rabbit antiserum (added in step 6) for 30 minutes under petri dish cover with moist gauze. Wash off excess labeled antiglobulin with PBS (three changes). Dry coverslip at room temperature. Mount with buffered glycerin and cover with a coverslip, then examine under a dark-field microscope with UV light source.

7. 8. 9. 10. 11. 12.

Agar Gel Precipitation Test This method permits the comparison of antigen-antibody reactions between an antigenic solution and various antiserum and various antigenic solutions on the same plate. Through the agar-gel medium contained in a petri-dish, antigen and antibody diffuse towards each other from separate wells. One well is centrally located and the others are peripheral to it at equal distance. The antigen solution may be placed in the central reservoir and the various antisera in the others, or vice versa. Patterns or lines of precipitate form between the central and one or more of the peripheral wells containing serologically related materials. The number of precipitate lines indicates the minimum number of antigen-antibody systems present.

Gel preparation 1. 2. Buffer is prepared by mixing 0.15 M Na2HPO4 and 0.15 M KH2PO4 until a pH of 7.4 is obtained. Methiolate 0.001% or Sodium Azide (NaN3) 0.01% is added as a preservative. Difco Noble agar (washed with distilled water and acetone if necessary) is added to the buffer to a 1% concentration and heated in a boiling water-bath until dissolved. NaCl is added to the hot agar to a final 8% concentration. Dispense in 15 ml lots and store at 4oC.

3.

Slides 1. 2. 3. 4. 5. Glass microscope slides are washed with dichromosulphuric acid and store under ethanol until used. Before use they are dried, coated with a thin layer of the agar to be dilute 1: 5 in hot distilled water. When it dries, slides are placed on a horizontal surface and flooded with approximately 3.5 ml agar per slide. Place 3 slides in each side of a gelman slide tray and flood at 12ml/3 slides. These slides are usually left overnight at 4oC prior to use. Well of 3 mm diameter are cut in a pattern of 6 wells around a central well with 6 mm between centers of wells. Agar plugs are aspirated just before used.

Test 1. 2. 3. 4. Antigen is added to the central well and antiserum added to peripheral wells. A positive control serum is always included. Volume of reagents per well are 10 microliter. Slides are incubated at room temperature in a humidified box and daily observed. Lines are only considered to be positive if they form a line of identity with the positive control system.

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Hemadsorption Some hemagglutination viruses, e.g myxoviruses can be multiply in cell culture without producing CPE but the infected cells contain hemagglutinin and therefore adsorb erythrocytes. This phenomenon is called hemadsorption and can be utilised for virus identification and for the titration of these viruses in cell culture.

Identification of Myxovirus by Hemadsorption Materials Bovine kidney cell culture monolayer, Influenza virus/paramyxovirus. Earle's medium with 2% fetal calf serum, 0.5% fresh guinea pig erythrocytes, Phosphate buffered saline (PBS). Procedure 1. 2. Remove culture medium from monolayers and wash with PBS. Inoculate cultures with 0.1-0.3 ml of virus dilution. Add Earle's Medium Incubate at 37oC Test for hemadsorption after 1 day incubation. 3. 4. 5. 6. 7. Wash fresh guinea pig erythrocytes three times in cold PBS and prepare a 0.5% suspension in cold PBS. Add 0.2 ml of 0.5% red cell suspension to each culture and spread over surface. Place culture in horizontal position in the refrigerator for 20 minutes. Remove unadsorbed RBC by washing cultures with cold PBS. Read cultures microscopically for hemadsorption. The virus can further be identified if a known antiserum against the virus is available. An addition of appropriate dilution of the serum to the cell culture before adding the erythrocytes inhibit the adsorption of the erythrocytes to the infected cells.

Electron Microscopy Electron Microscopy has been shown to be a rapid and reliable tools in identifying viruses commonly encountered in the veterinary diagnostic laboratory. The negative staining technique or the negative contrast electron microscopy with the use of electron dense salts of phosphotungstic acid (PTA) has permitted the visualization of the shape and size of viruses. Morphologic identification of the viruses together with the clinical history is sufficient to make a diagnosis of infection. There are three basic applications of the negative staining technique in the veterinary diagnostic laboratory: 1. 2. 3. Morphologic identification of cytopathogenic virus in the cell culture or embryonating eggs. The morphologic identification of viruses in clinical specimen . The serologic identification of viruses in both clinical materials and virus cultures.

Negative staining A drop of purified virus suspension is placed on a 300-mesh collodion-covered carbon-coated copper grid for several minutes. Excess fluids is gently blotted away and the grid immersed face down in a solution of sodium phosphotungstate (1% PTA mixed with NaOH until it reaches pH 7.0) for 1 minute. After excess fluid is blotted away, the grid is air-dried and examined immediately in an electron microscope.