Inoculation of an Egg

1. EGG INOCULATION The fertile hens lump back end be use to cultivate and propagate various types of computer computer viruses. Be consume of the index to alter their tropism and to adapt to a new master of ceremonies species, many viruses commence capable of growing in annulus fertilized nut case tissues wherein they frequently piddle a very much high stringency than in the tissues of the natural host. expression OF AN EGG The extra-fertilized crackpotnic tissue points of the gentlewoman embryo arise from three ger momental beds the dismissoderm, mesoderm and ectoderm (Fig. 1).The dorsal somatopleure consists of ectoderm on single side and mesoderm on the early(a)(a) side while the splanchnopleure consists of mesoderm and endoderm. By a process of f oldering, the somatopleure gives rise to the chorion and amnion while the only(a)antois and yolk firing tissue layers arise from the splanchnopleure. The amnion arises from the head and caudal regions of the embr yo, the tissue layer macrocosm reflected back to anatomy the chorion. the amnionic membranes grow rapidly and fuse to conformation the amnionic hammock by the 5th solar day. The allantois grows come to the fore as a bud from the back(prenominal) gut of the embryo and en heavy(a)s rapidly.By the tenth day the allantois hold outs attached to the outer layer of the amnionic shift and the interior layer of the chorion to form the chorioallantoic sacking (CAS) which sepa prizes the chorion from the amnion. The amalgamated chorionic and allantoic membranes argon refer exit to as the chorioallantoic membrane (CAM). Be curtilage the CAS signifys a diverticulum of the gut, it serves as the excretory receptacle for the embryo. It bes from 5 to 10 ml of suave with dissolved solids, the stem being clear in early stages but becoming mucky after the 12th day due to the presence of urates.The CAM is the respiratory electronic organ of the embryo and thus is richly supplied with bl ood vessels. The embryo is touch by the amniotic sac and lies bathed in about 1 ml of amniotic fluid. The amniotic fluid, which contains much of the albumin in the bom hold ond, serves as a inception of protein which is ingested during swallowing movements the embryo is seen to make from the 9th day onward. The mental strain-sac is present in the girdle end of the showicle. Underlining the lash is the fibrous egg drum membrane. In the beginning stages of ripening, the chick embryo stand be recognized with difficulty as a infinitesimal dark heavens attached to the yolk sac.After 4-5 age the embryo chiffonier be pick outily detected by laughingstockdling. After the 10th day, the embryo development, rapidly incr simplicity in size and feathers appear. The respiratory sh atomic number 18 develops mingled with the 12th and 15th days. If the egg rebrinys uninoculated and is maintained in a humid 38oC environment, it for bind hatch on the 21st day of life. vaccinatio n mathematical functions The regularitys described at a pull down mail service for the vaccination of the chick embryo do not comprise a complete list but represent those that argon practiced almost comm hardly. Likewise, while there be a arrive of techniques for inoculation by each of the routes listed, whole the sensation most wide used is described.A. Yolk Sac Chlamydia and rickettsia grow readily in the yolk sac (YS) membranes. Although or sowhat of the w arcaster viruses atomic number 18 inoculated by the YS route, they occupy and replicate in the tissues of the embryo itself instead than in the YS tissues. a. Candling and drilling. Fertile canvassis that lease been poutd for 5 to 7 days ar suitable since the YS is comparatively gargantuan at this time. The eggs ar kindledled and the confinesinal plosive speech sound of the style sac penciled in. The exfoliation over the activate space, which is referred to as the collection plate cap, is dis g ive by an application of tincture of iodine to maven small atomic number 18a.When the iodine is dried, a jamming is made by dint of the home over the center of the natural ambience space by means of a drill or egg dr integrity. b. vaccination and incubation. By means of a syringe fitted with a unity and one-half to ii in 23 gauge hassle, the inoculum is deposited in the YS by passing the exigencyle with with(predicate) the muss in the shell cap and head uping it downward to its full length parallel to the bulky axis of the egg. From 0. 2cc to 0. 5cc is averageally inoculated. the electronic jamming in the shell is hence stiff with tape and the eggs atomic number 18 incubated at 37oC. c. yield mathematical operation.The egg is primed(p) in a container which maintains it in the fountainhead(p) prospect during the garner mathematical function. The shell is cracked with unimpregnated forceps and the cap lifted off. The exposed membranes are mangled away. If the YS membranes are to be harvested. The contents of the egg are quickly emptied into a antiseptic petri dish. The YS is commonly ruptured in the process. The YS membranes, which are easily recognized by their deep yellow color, are detached from the embryo and separated from the chorioallantois with unfertile forceps and quickly transferred to a stereo typic petri dish.When the embryo is to be harvested, it is retire by hooking the slew end of a dental investigating around the neck. It is whence separated from the adherent membranes with stereotypical scissors and transferred to a sterile petri dish. B. Chorioallantoic Sac (CAS) The influenza and the newcastle disease viruses and most other viral agents which set respiratory transmission systems grow readily in the endodermal booths of the allantoic sac wall and are liberated into the allantoic fluid. The encephalomyelitis viruses and the mumps virus in fitition mul top of the inningly readily when inoculated b y this route. . Candling and drillings. Embryonating eggs which curb received a preliminary incubation from 9 to 11 days are candled and the boundary of the air travel space penciled in. The eggs are held in the upright view with the air sac uppermost. A point is selected a some millimeters in a higher place the blast of the air space on the side of the egg where the chorioallantois is well-developed but free of spectacular vessels. Iodine is applied to the area around the site. A hatful is wherefore drilled or punched through the shell. b. Inoculation and incubation.A one-half inch 26 gauge needle, fitted to a small syringe containing the inoculum, is inserted into the allantoic cavity by passing it through the mussiness in the shell parallel to the long axis of the egg or at an angle directed towards the apical extremity. From 0. 1cc to 0. 2cc of inoculum is injected into each egg. The stack in the shell is because cockeyed with tape and the eggs are incubated. c. pr oducting of allantoic fluid (AF). In revisal to avoid discharge into the AF while harvesting, the eggs are chilled in the refrigerator from 4 to 6 hours prior to the harvesting procedure. enchantment harvesting, eggs are held in an upright position and the shell over the air sac is aloof with sterile forceps. The floor of the air space is exposed. With a pair of small sterile curved forceps these membranes are torn away. In order to facilitate the harvesting of the AF the embryo is dis put to one side by placing the forceps against the embryo with the tips toward the shell wall. The AF can indeed be readily aspirated with a 5 ml or 10 ml sterile pipettete. C. The Chorioallantoic Membrane (CAM) Nine to 12 days old embryonating eggs are candled and an area over the most vascular part on the side of the egg play offed with a pencil.The shell is dis infect with iodine over this point and too the air sac end. A hole is conservatively punched over both these locations. The hole on the side of the egg moldiness penetrate both the shell and the familiar shell membrane. A small amount of fluid whitethorn exude from the hole if the inner shell membrane is penetrated. While candling the egg with its long axis in the swimming position, a humanity of rubber tubing is lay severely over the hole in the end of the egg. Suction is applied until the air sac collapses in the end but reappears on the side of the egg.When this bastard air sac is confirmed by candling, the CAM is ready for inoculation. The CAM and inner shell membrane are unremarkably tightly adherent by 9 days of incubation, and the inner shell membrane whitethorn consequently by honkped as well as the CAM. This is unacceptable since the inoculum leave aloneing get along on the inner shell membrane and not the CAM. To avoid this, a drop of sterile phosphate buffer solution is placed over the newly punched hole in side of egg to soften the membranes. An alternate mode is to drop the CAM at 7- 8 days of incubation, then wait until 11-12 days in the beginning inoculation. a. Inoculation and incubation.By carefully passing the needle through the inner shell membrane from 0. 1cc to 0. 2cc of inoculum is dropped on the chorioallantois with a 1cc syringe fitted with a 22 or 23 gauge one-half inch needle. In genuinely critical stu violates the egg should be candled during this procedure to insure that the inoculum is deposited on, rather than through, the membrane. After inoculation the egg is piano rocked in order to cattle ranch the inoculum uniformly over the surface of the CAM. The opening in the shell is cover with a small square of scotch tape and the inoculated eggs are incubated in a horizontal position with the hole uppermost. . Harvesting of the membrane tissues. The egg is placed in the horizontal position with the hole uppermost. Iodine is applied to the area around the window with a like swab and the tape then peeled off. The surrounding shell is disoriented a way with sterile forceps and the chorioallantois exposed. The membrane is clutch baged with forceps, detached with scissors and quickly transferred to a sterile Petri dish. D. Amniotic Sac This order is used in the first place for the isolation of the influenza virus from throat washings. The embryo during the course of its development wallows the amniotic fluid, thereby bringing the inoculated virus which it contains into contact with the tissues of the respiratory and enteral tracts where multiplication presumably occurs. After incubation amniotic fluid is then subpassaged by the CAS route (Fig. 2). The amniotic route of inoculation is used as well as for the isolation of the encephalomyelitis virus. a. Candling and drilling. Embryos from 13 to 15 days of age are used. The position of the embryo is determined by candling and a point on the shell over the air space on the side of the egg on which the embryo is situated is marked.The site is prepared in the usual way and a hol e is drilled or punched as for yolk sac inoculation. b. Inoculation and incubation. A 1cc syringe fitted with 1 3/4 inch 24 gauge needle is used for the inoculation. The egg is placed horizontally on the candler, the needle is introduced and gently stabbed in the direction of the embryo. Penetration of the amniotic sac is block outifyd by a sudden movement of the embryo. The needle is then withdrawn s mildly and from 0. 1cc to 0. 2cc of the inoculum injected. the hole in the shell is sealed with tape and the eggs are incubated in the vertical position. . Collection of amniotic fluid. The shell is removed as for the allantoic and yolk sac routes of inoculation. A few drops of salty solution are placed on the floor of the air space to render the membrane transparent. development the eyes of the embryo as a reference point, the amniotic fluid is aspirated by means of a Syringe fitted with a short 23 gauge needle. E. Mis mobile phoneaneous Routes ofInoculation a. Intravenous. This mode is not used commonly, although it is the manner of choice for the isolation of bluetongue virus. A large vein is located and marked in 12-14 day embryos.A rectangular piece of shell directly over the vein is removed and a droplet of sterile mineral oil is placed on the inner shell membrane so as to render it transparent. A 27-30 gauge five-eight inch needle fitted to a small syringe is introduced through the membrane into the vein in the direction of blood flow. From 0. 1 to 0. 5cc of inoculum is then injected. Incubation and harvesting of the embryo is carried out as already described. b. Intracerebral. This route whitethorn be used in the studies of pathologic alterations of the brain following(a) contagion. Eight to 14 day embryos are usually used.The viruses of herpes simplex and rabies whitethorn be cultivated by this method. Egg Inoculations. Materials needed Embryonated eggs, 11-12 days A paramyxovirus, PI3 or Sendia virus Vaccinia virus Crystal violet impound syring es and needles Egg candlers, egg punches Iodine disinfectant and swabs, jail stallophane tape Instruments, petri dishes Procedure 1. Inoculation of dye into CAS a. Candle 11-12 day embryonated egg, mark boundaries of air sac with pencil. interpretd above air sac, choose a point devoid of vessels and mark with a pencil. b clean egg shell at this point with iodine.Let dry before adjoining procedure. c. Drill a small hole with an egg punch at the appropriate marked point (be careful-dont break the shell). d. install 0. 1 0. 2ml dye into the CAS as described and illustrated. e. indue a small piece of stallophane tape over the hole. The egg would be ready to incubate if the inoculum had been virus. f. Candle the dye-inoculated egg to establish that the inoculum is in the even place. Watch the inoculum spread through-out the confines of the CAS. g. Break the egg and rain buckets the contents into a petri dish. Observe where the dye is.Identify the CAM, YS, amnion and embryo. I f inoculated properly only the CAS should contain dye. 2. CAS inoculation of virus a. Follow procedures for CAS inoculation of a dye, extract the inoculation should be 0. 1 ml of a live paramyxovirus. Place tape over the inoculation hole and incubate. b. Candle egg workaday to determine embryo viability. If the embryo dies within 2 days of inoculation, it usually indicates bacterial contamina-tion or trauma. c. If the embryo dies after 2 days, refrigerate as soon as death is noted until the to a lower placementioned laboratory period. 3. CAM inoculation of vaccine virusWarningIf you have not had a successful smallpox vaccination, have eczema or evidence of immune deficiency, contact the instructor before use this virus. Be careful Do not get this virus in your eyes Vaccinia virus is the live virus vaccine for smallpox. While less pathogenic than smallpox or variola virus it can still cause serious or uncomfortable lesions if mishandled. a. Two 11-12 days embryonated eggs resul t be supplied to each crowd of students. Drop the membrane on both of the eggs according to the instructions and illustrations. b. Inoculate 0. ml vaccinia virus onto the dropped CAM. be sure to go through the inner shell membrane but not through the CAM. Rock the egg to distribute the inoculum over the finished floor of the false air sac. Cover the hole with tape and incubate in a horizontal position with the hole uppermost. 4. Harvesting of embryonated eggs (next laboratory). a. Follow instructions for removal of CAS fluid. Try to keep blood vessels from rupturing. Remove CAS fluid aseptically in a sterile pipette. Expel fluid into a sterile vial. This fluid ordain be used for the hemaggulatination exercise later.It can be frozen if commandment. example the last drop of CAS fluid to inoculate bacteriological media to check for contamination. b. Harvest CAM as per instructions. Place the membrane in a petri dish and lightly pour PBS over the membrane until it level(p)tens o ut and the matchs are intelligibly visible. c. Important. All fluids, instruments, and other things that have come into contact with virus- septic tissues must(prenominal) be properly sterilized. Follow carefully the instructors remarks for proper presidency of all materials. Be sure to disinfect your workspace with disinfectant when cleaning up. 1. INFECTIVITY ASSAYS The concentration of a shift of virus is usually determined by meter its infectivity. in that respect are two types of infectivity titrations the quantized assay, which depends upon an all-or-none does chemical reaction, and the quantitative assay, which utilizes a organisation, pock or lesion count in which the effect of a single infective virus particle is seen as a visible localized compound in a background of normal carrels. A. Quantitative Assay2 This method determines the unfeigned number of infectious units (virus particles) in a tending(p) rift.This type of enumerative solvent is assessed from fo cal lesions such as plaques in cell acculturations, pocks on the CAM of chick embryos or local necrotic lesions on a plant leaf. The number of infectious units per unit pile can be calculated, and this is referred to as the titer. With plaque assays, the titer of the master key virus suspension is stated in legal injury of the number of plaque forming units (PFU) per ml. Ex. Fifty plaques on a 10-5 dilution of trustworthy suspension were counted. A 0. 1ml inoculum was used. PFU/ml. of original volume No. of plaques = dilution) x (Vol. of inoculum) =5. 0 x 107 PFU/ml=50 x 106 = or 105 x 0. 1 B. Quantal Assay This assay estimates the concentration of infectious particles by allowing them to replicate in a suitable host so that one infectious unit can be detected by the enlargement effect of the infection. The unfeigned number of infectious particles introduced into the riddle unit is unnoticeable and may vary even between duplicates of the resembling dilution. To determine quantal infectivity titers, mutiple replicate tests are used for each dilution of original suspension until the infectivity is weaken out.The result gives the dose necessary to gravel a defined response. This response is usually based on a 50% end point, which is the dilution at which 50% of the test fleshlys, eggs, or cell stopping points react to the virus. Computation of the 50% end point is based on the presence or absence of a predetermined criterion, i. e. death (Median Lethal Dose or LD50), infectivity (Median wind Culture infective Dose or TCID50, Median Egg Infective Dose or EID50), etc. The criterion must be each present of absent either the fauna is dead or alive, or the cell last is infected or not infected.There are no plus/minus or graded reactions. This method does not heartbeat the exact number of virus particle but only whether or not virus is present at a particular dilution. There are two patterns that can be used to determine 50% endpoints the Reed-Mu ench and the Spearman-Karber methods. Both are demonstrated here using the equal data. 1. Reed-Muench method acting Accumulated Values Virus Dilution Morality Ratio (a) (b) Died Survived Died Survived Ratio part (c) (d) (e) (f) (g) (h) 10-1 6/6 6 0 17 0 17/17 100 10-2 6/6 6 0 11 0 11/11 100 10-3 4/6 4 2 5 2 5/7 71 10-4 1/6 1 5 1 7 1/8 13 10-5 0/6 0 6 0 13 1/13 1 At the 10-3 dilution, 5/7 or 71% of the accumulated test animate beings died, and at the 10-4 dilution, 1/8 or 13% died (columns g and h). The 50% endpoint, therefore, lies somewhere between the 10-3 and 10-4 dilutions. The final enumeration occupys interpolation between these two values. The formula for doing this is (% mortality at dilution next above 50%) (50%) = restate distance (% mortality at dilution next above 50% Mortality at at dilution next below or 71-50 21 - = = 0. 36 proportionate distance 71-13 58The dilution factor must also be considered, i. e. , 2-fold, 4-fold, 10-fold, et c. and the proportionate distance corrected (multiplied) by the log10 of the dilution factor (2-fold = 0. 3, 5-fold = 0. 7, 10-fold = 1, etc. ) The final estimate is determined by this formula disconfirming log10 of LD50 end point = negative log of dilution above 50% mortality plus the proportionate distance factor (corrected for dilution series used) Negative log of dilution above 50% mortality 3. 00 Proportionate distance (0. 36) x dilution factor (log10-1= -1)= 0. 36 Negative log LD50= -3. 36 LD50=10-3. 36 antilogarithm of 10. 36=2. 29 LD50 titer -3. 36 =2. 29 x 103 / volume inoculated LD50 Calculation Inoculum DILUTION DEAD alert CUMULATIVE DEAD CUUMULATIVE ALIVE LD50= EXAMPLE 10-1 6 0 17 0 (a-b)(c+d) 2(axd)-(bxc) Test body 10-2 6 0 11 0 =(3) (8) 2(57)-(21) Date Inoculated 10-3 4 2 5 2 =24 =0. 36 66 10-4 1 5 1 7 LD50 = 3. 36 10-5 0 6 0 13 Inoculum Dilution Dead animated Cumulative Dead ( Cumulative unrecorded ( LD50= Test Systems (a-b) (c+d) 2((a x d) (b x c)( Date Inoculated = (3) (8) 2(57)-(21) =___________ Inoculum Dilution Dead Alive Cumulative Dead ( Cumulative Alive ( LD50= Test Systems (a-b) (c+d) 2((a x d) (b x c)( Date Inoculated = (3) (8) 2(57)-(21) =___________ 2. Spreaman-Karber Method Estimation of the 50% endpoint by the Spearman-Karber method is much simpler. The formula is Negative log10 of LD50 = X d (P-0. 5) here X = log10 of the highest concentration used (lowest dilution) d = log10 of the dilution factor, and p = sum of % mortality at each dilution100 victimisation the same data map above the following number are obtained d(10-1)(do-2)(10-3)(10-4) Neg log LD50 = 1. 0 -1(100 + 100 + 66 + 17)-0. 5 100 = -1 1(2. 84-0. 5) = -1 2. 34 = -3. 34 LD50 antilog of 10. 34 = 2. 19 LD50 titer= 2. 19 x 103 volume inoculated Note that the two methods produce meagerly different results using the s ame data. The Spearman-Karber method is considered to be the to a greater extent accurate. The Spearman-Karber method can be simplified even more(prenominal) if signs are omit and common sense used.This formula is Neg log LD50 = X + d (P + 0. 5) Where X = log10 of highest dilution showing 100% mortality d = log10 of dilution factor p = proportion of electropositives above dilution X Xdp Neg. log LD50 = 2 + 1 (4/6) = 1/6 + 0. 5) = 2 + 1 (. 67 + . 17 + 0. 5) = 2 + 1. 34 = 3. 34 The appropriate sign can then be inserted LD50 = 10-3. 34 These formulas can also be used to estimate 50% endpoints in neutralization reaction reaction tests. Here, absence of the predetermined criterion is counted and used for the calculations. III. SEROLOGIC TECHNIQUES A. Hemagglutination umteen viruses or viral antigens are capable of specifically and non-covalently rear to receptors on the surface of red blood cells (RBCs).When the right volumes of these viruses and RBCs are mixed, the viruses bridge the RBCs to form a lattice which condescends out of suspension in a uniformly thin shield on the bottom of a test tube or conical well. This phenomenon was first described by Hirst in 1941 and is known as hemagglutination (HA). the HA titer of a virus can be determined by mixing straight dilutions of a virus with a constant amount of RBCs which are usually prepared as a 0. 25%-1. 0% suspension in physiological saline. the highest dilution which agglutinates the RBCs is the endpoint. The HA titer is the reciprocal of the endpoint dilution, and that dilution is said to contain one HA Unit (HAU) of virus in the original volume.Unagglutinated RBCs sedimentation to a packed disc (button) on the bottom of the test tube or well. The viruses known to cause hemagglutination are manifold but can be grouped according to the nature of their hemagglutinating protein (hemagglutinin). The hemagglutinin on the virion of influenza and the paramyxoviruses is a glycoprotein. These viruses, but n ot any others, also give birth an enzyme, neuraminidase, which destroys the glycolipid receptors on the RBC surface and allows the virus to elute (unless the HA is carried out at a temperature too low for the enzyme to act). Certain toga and Coxsackie viruses possess a hemagglutinin but do not possess a neuraminidase-like enzyme. astidious conditions are necessary for these viruses to hemagglutinate and usually cells from only a very few species can be can be used. Vaccinia virus has a lipoprotein hemagglutinin associated with a soluble fraction separable from the viral particle itself. few viruses agglutinate RBCs from a limited number of course and some HA reactions require careful control of pH, temperature and ionic conditions (see tabularize 5-1, p. 100-101, Rovozzo & Burke). We give perform the HA test with a paramyxovirus which ordain agglutinate human type O, bovid, guinea pig or chicken RBCs over fairly broad ranges of pH (6. 0-8. 0) and temperature (4-25oC). Material needed 1 cc syringes 0. 025 ml microtiter tips 0. 25 ml microdiluters Microtiter plates with 96-V-bottom surface Phosphate buffered saline (PBS) Paramyxovirus, in form of allantoic fluid harvested 48-96 hr after infection of 10-day embryonating eggs Washed RBCs, 0. 5% in PBS Go-no-go test papers Dilution tubes and 1 ml pipets *Use only the top half the microtiter plate. Procedure 1. With the micropipet (a microtiter tip attached to a 1cc syringe) held vertically, allot 0. 025 ml (1 drop) PBS each into columns 2 through 12 of rows A. B, C, and D of the microtiter plate. Also put 0. 025 ml PBS into rise 1 through 4 of row H for controls. 2. go for a 110 dilution of virus in PBS in a dilution tube.With the same pipet used to dispense PBS, put 0. 025 ml of the 110 virus dilution into each well of columns 1 and 2, rows A-D. 3. Test the delivery volume of the microdiluter by immersing the tip in PBS then touching to the center of a circle on the go-no-go paper. Now begin dilutions by immersing the dry diluter in well two, rotating to mix and pick up 0. 025 ml of fluid, and transferring to well three. act up rotating and transferring through row 12. Two lines may be diluted simultaneously if desired. After removal of 0. 025 ml from row 12, dip the diluter in disinfectant, then distilled body of water, then flame. Do not flame with protein or salt in the diluter.Do not add virus to the controls. 4. With a new syringe and tip add 0. 025 ml (1 drop) 0. 5% RBCs (mix suspension well before pipetting) to every well, including controls. Mix well by running a hard disapprove down the at a lower placeside of the plate. 5. conquer to stand at elbow direction temperature until the controls and higher virus dilutions have RBCs settled into a button in the point of the V, and positive wells have RBCs uniformly spread over the entire bottom of the well. This will take 1-2 hr. Then refrigerate the plate. 6. Read the HA titer as the reciprocal of the dilution of the last well showing positive HA. Calculate the dilution which contains 4HAU/0. 25ml for use in the HI test.Be sure to check controls for oral agglutination. B. Hemagglutination Inhibition Viral hemagglutination may be suppress in several ways. By combining with viral antigens which normally move with RBC receptors, specific anti-viral antibodies can prevent the virus cell fundamental interaction which normally brings about hemagglutination. Since infection with a virus will excerpt production by the host animal of antibodies directed against each virus-induced protein, including the hemagglutinin, crushing of hemagglutination by an animals blood blood blood blood serum indicates that the animal has been infected by the virus. A high HI titer may indicate that the infection was recent.A four-fold rise in titer between two serum samples taken a few weeks apart (as during acute and convalescent phases of a disease) indicates that infection occurred during the period between the sampl ing times. If the viral hemagglutinin is also the protein by which the virus attaches to cells susceptible to infection, a high HI titer shows an animal to be immune to reinfection. HI is carried out in much the same way has HA. The serum is diluted in microtiter plates and each dilution is allowed to react with a constant dose of virus (usually four HAU) for an interval of 15 min to one hr before RBCs are added. The reciprocal of the highest serum dilution which inhibits HA is the HI titer.Several controls are necessary (1) The lowest dilution of serum used in the test must be incubated alone with RBCs to determine if it contains heterophile antibodies which cause RVC agglutination. (2) The virus must be back titrated to see that the proper dose was added to the test wells. (3) A known non-immune serum from the same animal species must be titrated (usually before the HI test is performed) to see if it contains non-specific inhibitors. Heterophile antigens are a group of shared anti gens with over-lapping specificities. They are found in some plants (corn, spinach) some microorganisms (Pneumococcus, E. coli), and some fish and animal tissues (carp, toad, guinea pig, horse, man etc. Heterophile antibodies against these antigens will spread over react with cells and fluids from the above-listed species. In the case of RBCs as the heterophile antigen, if heterophile antibodies against them are present, hemagglutination will occur, possibly masking the presence of the hemagglutination-inhibition reaction caused by anti viral antibodies. Nonspecific inhibitors of hemagglu-tination may also be found in the serum of man and animals. Their nature differs for different viruses and even for different strains of viruses, such as influenza virus. Serum inhibitors also differ in different species. Inhibitors may be of low titer, or in some cases higher than the actual antibody titers, thus masking its diagnostic importance.Methods which have been used to remove inhibitors allow in (1) Heating at 56-1/4 for 30 min, (2) intervention with receptor-destroying enzyme (neuraminidase), trypsin and/or periodate, (3) absorption with kaolin, (4) extraction with acetone, (5) precipitation of beta-lipoproteins with heparin and manganous chloride or with dextran sulphate and calcium chloride. No single method is universally applicable. sometimes more than one method must be used. Antibody titers can be depressed by some of these procedures. The final control used is the RVC saline control to check for self-agglutinating RBC. Table 1. Example of HA and HI Virus Antigen Source RBC Temperature Non. Sp. Inhib.Removal grippe A and B CAS fluid or cell goalChicken, gentleman O populate Neuraminidase fluid Mumps CAS fluid Chicken Room Neuraminidase Coxsackie cells culture fluid Fowl Room Kaolin Rubella cadre culture fluid one-day old chicken, goose 4oC Heparin and Manganous chloride Adenoviruses Cell culture fluid Rat, rhesus monkey 37oC/ means Not re quired Procedure 1. supplement one drop (0. 025 ml) PBS dilutant to all wells of the microtiter plate. 2. You will be given three serums. One will be un case-hardened, one treated for removal of non-specific inhibitors and/or heterophile antibodies, and one known negative serum. Your results will be compiled with the class results to clarify the total experiment. using the microdiluters, add 0. 025 ml test serum to well A of row 1 and 2. subvert out to well H. The first well is a 1/2 dilution of serum and row H is 1/256. score 0. 025 ml of the same test serum to row 7, the serum control well. misdirect to well H as before. cautiously brush the diluters and repeat with test serum 2 and test serum 3, sing rows 3, 4, 8 and 5, 6, 9 respectively. 3. Add 0. 025 ml of the challenge virus with the microdiluters to well A of rows 10 and 11. Dilute to well H. This is the antigen (virus) back titration and control. The highest dilution with complete hemagglutination is 1 HA unit. 4. Usin g the same micro-pipet as in 1, add another drop of PBS to all wells of rows 7-12. Empty the pipet and refill with the hemagglutinin (virus). Add one drop hemagglutinin to all wells of rows 1-6. Mix well. 5. Incubate at room temperature 30-60 minutes. 6. Using a new pipet, add one drop 0. 5% bovine RBC to all wells. Mix well. Store at 4oC and read the next day.Antibody titers are the highest dilution that inhibits hemagglutination (forms a distinct button). C. Hemadsorption Certain enveloped hemagglutinating viruses cause the insertion of viral hemagglutinins into the germ plasm membrane of cells in which they are replicating. These limited areas of the cell surface are the sites at which progeny virus particles will mature. If agglutinable RBCs are brought into contact with hemagglutinin-containing surfaces of cultured cells, the RBCs will specifically bind to the infected cells. This phenomenon, known as hemadsorption, is particularly useful in detecting infection by viruses whic h cause little morphological change in infected cells. Procedure 1.Pour off median(a) from a tube of cultured cells infected with an orthomyxovirus or a paramyxovirus and from a tube of uninfected cells. 2. Wash monolayer thoroughly but gently with two rinses of 3 ml of physiological saline. 3. Add 0. 2 to 0. 5 ml 0. 5% bovine RBCs in saline. Allow RBC suspension to cover cell layer. Incubate at room temperature for 10-15 minutes. 4. Pour off RVC suspension and wash 2x with 2-3 ml saline. 5. Examine under microscope. Infected cells should have entire surface covered with RBCs. Non specific binding will cover only a few sites per cultured cell. blood serum NEUTRALIZATION The neutralization test estimates the capacity of a specific serum antibody to neutralize a virus biological activity.Major uses for this test include the assignment of unknown virus or antibody, the determination of antibody levels, the comparison of antigenically relate viruses and the exact of the kinetics o f antigen-antibody reactions. Viruses and the claim of the kinetics of antigen-antibody reactions. neutralization reaction can occur by several mechanisms. Virus adsorption to cells may be inhibited by alteration of the configuration of cell receptor sites or by taproom of viral attachment. Virus degradation may be advanced by interference with post-engulfment stages of virus retort, by prevention of release of functional virus cores into the cytoplasm or by the degradation of virus-Ab complexes within phagosomes.Also, complement-mediated reactions may enhance neutralization by production of lesions in the viral envelope. Several factors must be considered when performing a neutralization assay. Sensitivity of the test is related to the score of susceptibility of the indicator host system to infection with the virus. The neutralization reaction is readily reversible by dilution with saline, by ultrasonic treatment or by lowering pH. Finally, the time required to reach residuu m may vary with different systems. When performing the neutralization test two systems are used. The reaction system is incubation of virus and specific antisera until vestibular sense is reached. The indicator system is the inoculation of the virus-Ab mixture into a susceptible host.If neutralizing anti-bodies are not present lesions such as pocks or plaques will be seen in the host. If neutralizing antibodies are present there will be no lesions. There are two techniques commonly used for the neutralization test. In the alpha procedure a constant serum concentration is added to ordered log dilutions of virus. The mixture is incubated and inoculated into an appropriate host system. In the beta procedure a constant virus concentration is incubated in serial two-fold dilutions of serum before inoculation into the host. The beta procedure is most commonly used because of its sensitivity, ability to measure antibody titer and its economical use of serum.The alpha procedure is not as sensitive and may be more undecided to non-specific inhibition. It is more frequently used for comparative studies. Alpha Neutralization test Materials Needed Flat-bottomed MT plate with bovine cell monolayer MT transfer plate with lid and holder MT tips 1 cc syringes serum samples stock virus MEM diluent dilution tubes sterile distilled water in beaker Procedure Use aseptic technique. a. Make serial 10-fold dilutions of stock virus to 10-8 using MEM and dilution tubes (0. 2 plus 1. 8 ml). b. Using sterile 1 cc syringe and microtip add 1 drop (0. 025 ml) diluent (MEM) to rows 7 and 8 wells A-H, and rows 9, 10 and 11 wells A and B of the transfer plate. c. Using the same syringe and microtip add 0. 25 ml of the virus dilutions to rows 1-8 as follows 10-8 into wells H, 10-7 into wells G, and so on goal with 10-1 in wells A. d. Using a new syringe and microtip add 0. 025 ml test serum A to rows 1 and 2 wells A-H, and row 9 wells A and B. Rinse syringe and microtip with sterile water and add 0. 025 ml serum B to rows 3 and 4 wells A-H, and row 10 wells A and B. Again rinse out syringe and microtip with sterile water and add 0. 025 ml serum C to rows 5 and 6 wells A-H, and row 11 wells A and B. (Row 9, 10, and 11 are serum controls). e. Tissue culture controls are the unused portion of the plate. f. Incubate virus and serum at room temperature for 30 minutes, then transfer reagents to cell cultures. SerumVirusSerum Controls A B C 123456789101112 ABC A 10-1 No B 10-2 Virus C 10-3 D 10-4 Virus E 10-5 F 10-6 G 10-7 H 10-8 Beta Neutralization Test Materials Needed Flat-bottomed MT plate with lid 1 cc syringes MT tips Mt diluters sterile distilled water in beaker MEM diluent serum samples virus, 25-50 TCID50 bovine cell suspension Procedure Use aseptic technique. a. Add 1 drop (0. 025 ml) diluent (MEM) to rows 1-8 wells A-H. b.Make 2-fold dilutions of serum through row H (final dilution 1256), cleaning microdiluters in sterile distilled water betwe en serums. c. Using the same syringe and microtip as in step a, fill with pretitrated (25-50 TCID50) IBR virus and add 0. 025 ml to rows 1-4 wells A-H, and to rows 7 and 8 wells A. d. Using rinsed microdiluters make 2-fold dilutions of the virus in rows 7 and 8 wells A-H. e. Incubate at room temperature for 30 minutes. f. Add 2 drops (0. 05 ml) of bovine cell suspension using a new 1 cc syringe and microtip to all wells of the test plus a few extra for tissue culture controls. Controls Serum ASerum BABVirus 123456789101112 A 12 B 14 No C 18 Virus D 116 E 132 F 164 G 1128 H 1256 IV. CELL CULTURE Because viruses are obligate intracellular parasites, they cannot replicate in any cell-free mean(a), and thus require living cells from a suitable host within which to multiply.Animals such as mice and embryonating avian eggs may be used for the propagation of viruses, but for various reasons (time, cost, ease of handling, etc. ) the propagation of most viruse s in a cultural medium of living cells is the method of choice today. More than half a deoxycytidine monophosphate has elapsed since animal cells were first giving in vitro. In 1912 cell began growing bits of chick heart in drops of horse plasma. The cells at the contact of the explant divided and grew out of the plasma clot. The explants died within a few days, and Careel levelheaded that their death was due to exhaustion of nutrients. He found that cells from a given explant could be maintained indefinitely if they were periodically subdivided and fed with a sterile aqueous extract of whole chick embryos.In the early 1950s, Earle developed a technique for dissociating cells from a whole chick embryo from each other with trypsin. When this suspension of single cells was mixed with plasma and embryo extract and placed in a sterile glass container, the cells adhered to the glass and divided to form a primary culture. The primary culture contained a variety of cell types includin g macrophages, muscle fibers, etc. The cells grew to a monolayer, a thin sheet of cells (one layer in thickness) which covered the entire bottom surface of their culture vessel, and then stopped dividing. The cells could then be redispersed with trypsin and planted in new culture vessels containing fresh media.These randomnessary cultures contained less cell types than did the primary cell cultures, as many of the differentiated primary cells were out-competed and did not survive the transfer. Often, secondary cultures are composed entirely of spindle-shaped cells called fibroblasts because of their similarity to cultured connective tissue. Cells derived from kidneys and from sealed carcinomas have a polygonal appearance in culture. Because of their tissue of origin, they and other cells with similar morphology are call epithelial. Cells may be full-grown in vitro in several ways. Organ cultures, if carefully handled, maintain their original architecture and functions for severa l days or sometimes weeks.Slices of organs (which are actually tissue cultures) consisting of respiratory epithelium have been used to study the histopathogenesis of infection by respiratory viruses that can only be grown outside of their natural host by using organ cultures. The term tissue culture was original applied to explants of tissue embedded in plasma. the term ensuantly became associated with the culture of cells in general and is now ancient in its original sense. Cell culture is the term most widely used today. It refers to tissue dissociated into a suspension of single cells, which after being washed and counted, are diluted in process medium and allowed to settle on to the flat bottom surface of a specially treated plastic or glass container.Most types of cells adhere quickly, and under optimum conditions they will bear with mitosis about once a day until the surface is covered with a confluent cell monolayer. There are three main types of cultured cells. The diffe rence in these types lies in the number of times the cells can divide. 1. Primary cell cultures When cells are taken freshly from animals 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, the chromosome number and morphology of their in vivo tissues of origin. They also retain some of the differentiated characteristics which they possessed in vivo. Because of this, these cells support the replication of a wide range viruses.Primary cultures derived from monkey kidney and shiner 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 characteristics. After 20-50 passages in vitro, these diploid cell strains usually undergo a crisis in which their growt h rate slows and they eventually die out. Diploid strains of fibroblasts derived from human embryos are widely used in diagnostic virology and vaccine production. 3. never-ending cell lines. Certain cultured cells, notably mouse embryo fibroblasts and human carcinoma cells, are able to survive the growth crises and undergo indefinite propagation in vitro.After an initial slowing down, these continuous cell lines grow more rapidly than before, their karyotype becomes abnormal (aneuploid) and other poorly soundless changes take place which make the cells immortal. The cells are now dedifferentiated, having disconnected the specialized morphology, and biochemical abilities they possessed as differentiated cells in vivo. Continuous cell lines such as KB and Hela, both derived from human others derived from mice (L929) and hamsters *BHK), are widely used in diagnostic and experimental virology. The development during homo War II of antibiotics simplified long-term animal cell culture by minimizing the problems of bacterial and fungal contamination.Another important discovery was made by Eagle in the 1950s when he determined the minimal nutritional unavoidablenesss of cultured cells. He began by showing that Hela and Mouse L-cells would grow in a mixture of salts, amino acids, vitamins and cofactors, carbohydrates and horse serum. By eliminating one particle at a time, he then determined which nutrients were essential for cell growth. His minimal essential medium (MEM) contains 13 amino acids (human tissue in vivo requires only 8), 8 vitamins and cofactors, glucose as any energy source and a physiological salt solution which is isotonic to the cell. The pH is maintained at 7. 2-7. 4 by NAHCO3 is equilibrium with carbon dioxide.The pH indicator phenol red is usually incorporated into the medium, which turns red-purple if the medium is alkaline, yellow if the medium is acidic, and remains red if the pH is suitable. Serum in concentrations of 1-10% must beaded t o the medium to provide the cells with additional undefined factors, without which most cells will not grow. Most animal cells must be kept incubated at 37oC. If cells are grown in vessels open to the atmosphere, their incubator must be humidified and contain an increased CO2 concentration. Some nonvolatile phosphate or substituted sulfonic acid buffers (HEPES, TES) eliminate the requirement for incubators to be gassed with CO2. With the advent of cell culture, many animal viruses have been propagated in vitro, and hundreds of previously unknown viruses have been isolated and identified.The discovery of the adenoviruses, echoviruses, and rhinoviruses, for example, is directly referable to the use of cultured cells, as is the revolution in the diagnosis of viral diseases and the development of poliomyelitis, measles, and rubella vaccines. A. Culture of Primary Chick Embryo Fibroblasts (CEF) Materials 10-12 days old embryonated eggs Forceps and scissors Sterile petri dishes Sterile 2 50ml flaskful with charismatic bar Sterile 30 oz prescription drug stores containing MEM & 5% lamb serum Sterile PBS Sterile 0. 5% trypsin (STV) Sterile 15ml cartridge extractor tubes containing 0. 5 ml serum Hemocytometers 1ml and 10ml pipets Sterile Dulbeccos saline Procedure 1. Disinfect the surface of the egg over the air sac.With scissors or blunt end of forceps, break shell over air sac. Sterilize forceps by dipping in alcohol and flaming. Peel away shell over air sac, resterilize forceps and pull back shell membrane and chorioallantoic membrane to expose embryo. 2. Resterilize forceps, grasp embryo loosely around neck, and remove from egg to sterile petri dish. 3. Using two forceps, or scissors plus forceps, decapitate and eviscerate embryo. soften remainder of embryo to very small fragments. 4. Add about 10ml sterile Dulbeccos saline to tissue fragments in petri dish, swirl to suspend fragments, and carefully pour into 250ml flask. With flask covered, continue swirling for 2-3 min. to wash tissue fragments.Tilt flask, allow fragments to settle, and gently decant saline. 5. Add 12ml sterile trypsin to fragments in flask, cover, and stir with magnetic bar for 15 min. Tilt flask, allow fragments to settle, and pour trypsin cell suspension into 15ml centrifuge tube containing 1ml serum. The serum contains a trypsin inhibitor which will prevent further misemploy to cell membranes but he enzyme (note it is preferable to treat the tissue with seven-fold short applications of trypsin rather than a few long ones, in order to minimize enzymatic damage to cell membranes. However, limitations of time require us to use the shorter method. ) 6. Add 12ml sterile trypsin to fragments and repeat step 5.At the end of this second treatment, size of tissue fragments would be greatly get overd and a large number of single cells should be suspended in trypsin. 7. Balance centrifuge tubes against one another and centrifuge at 1500 rpm for 10 min. Carefully decant off s upernatant and resuspend pooled cell pellets in 1ml MEM. Make a 110 dilution of the cell suspension in MEM for counting in a hemocytometer. 8. In most hemocytometers each heavily graven square in 1mm on each side. The depth of the chamber is 0. 1mm. cast the cells in 0. 13 mm and calculate the number of cells in your original suspension. Dilute to give 8ml with 2-8 x 105 cells/ml in MEM, place in prescription bottle, replace cap tightly, and incubate on flat side at 37oC. 9. Be sure to examine cells periodically.Actively growing cells produce acidic metabolic by-products, and thus the pH of the medium may need to be adjusted by the addition of a few drops of 7. 5% NAHCO3. If floating (dead) cells are present the medium may need to be changed. B. TRANSFER OF CELL CULTURES After cultured cells have formed a confluent monolayer on the surface of their culture vessel, they may be removed from the surface, diluted, and seeded into new vessels. If the initial culture was primary, the n ew cultures are called secondary, and are likely to consist of fewer cell types. Removal of cells from glass surfaces may be by either physical methods scraping with a sterile rubber policeman or chemical methods proteolytic enzymes or chelating agents or a combination of the two.After removal, cells are pipetted up and down and diluted fittingly in fresh secondary culturing, and after one becomes familiar with the growth characteristics of a certain cell types, counting can usually be dispensed with. We will transfer a cell line of bovine cells by use of a mixture of trypsin and EDTA (versene) in physiological saline (STV = saline, trypsin, versene) 1. Pour off the medium from a 3 oz. prescription bottle containing a confluent cell monolayer. 2. Wash the monolayer with 5-10 ml of physiological saline (Saline A) rinse well without shaking (shaking produces bubbles) and pour off. 3. Add 0. 5 ml STV to the bottle and incubate, with STV covering cells, at 37oC for 2-15 min.Observe periodically to determine when cells are loosened from glass (note STV will contain a pH indicator and should have a pH of 7. 0-8. 0. Below pH 7. 0, trypsin is inactive. A pH above 8. 0 is damaging to cells. ) 4. When cells are seen to detach from glass upon shaking, add 6 ml fresh medium and suspend cells by pipetting up and down a few times. 5. Add 10ml more medium and mix to get even cell suspension. 6. Seed 1 ml cell suspension in to each of 8 culture tubes, stopper tightly, and incubate in rack which holds tubes at slight angle from horizontal. Seed remaining 8 ml cell suspension into a new 3 oz. prescription bottle or a 25 cm2 plastic flask. C.PRESERVATION OFCULTURED CELLS BYFREEZING Viability of viruses and bacteria is bear on during freezing, but originally attempts to preserve animal cells by freezing resulted in cell death. This was first thought to be due to laceration of cell plasma membranes by ice crystals, but more recent evidence suggests the cause may be osmotic changes during freezing which give rise to irreversible changes in lipoprotein complexes in intracellular membranes. In any event, the answer to animal cell preservation has proved to be addition of glycerol, ethylene glycol, or dimethyl sulfoxide (DMSO) to the medium and slow freezing, ideally at a cooling rate of one centigrade degree per minute.Cells must be stored at 70oC or lower (ideally in liquid N2 at 196oC), and when they are recovered, flux must be rapid. With careful technique, 50-80% of the cells of a healthy culture will survive freezing. Procedure 1. Remove confluent cell monolayer from culture vessel by method described in cell transfer procedure. After centrifugation, resuspend cells in 1 ml medium containing 15% serum and 7. 5% DMSO and placed in small snap-top tube. 2. Immediately place tubes in an ice bath. They will then be transferred to a styrofoam container and refrigerated. After 20-30 min, when cells have dropped to 4o, they will be transferred to a 20o fre ezer for 20-30 min, then to the 70o freezer for storage.Alternatively, the tubes can be placed in cotton-or polystyrene-insulated containers and placed directly in the 70o freezer for slow cooling. If cells are to be stored in liquid N2, they must be placed in sealed ampoules. 3. To recover, cells, remove tubes from 70o and place directly in 37o water bath. When thawing is barely complete, add contents of tube to a 25 cm2 flask containing 15 ml MEM + 10% fetal calf serum. Culture medium will be changed for your approximately 4 hrs. later (after cells have attached) to reduce the toxicity of DMSO for cells at 37oC. D. Effect of Viral Infection on the armament Cell During the time that synthesis of viral components is occurring in the infected cell, the cell undergoes characteristic changes.These changes are usually observed in tissue culture where infection of cells is more easily synchronized and where the cells can be observed frequently during the course of infection. Morphologi cal changes in cells caused by viral infection are called cytopathic effects (CPE) the responsible virus is said to be cytopathogenic. The degree of visible damage to cells caused by viral infection varies greatly. Some viruses cause very little or no CPE. Their presence can be detected only by hemadsorption (already discussed) or interference, in which infected cell cultures showing no CPE inhibit the replication of another virus subsequently introduced into the cultures.On the other hand, some viruses cause a complete and rapid remnant of the cell monolayer after infection. The histological appearance of the CPE caused by some of these cytocidal viruses may be sufficiently characteristic to allow provisional designation of the virus. Some CPE can be readily observed in unfixed, unstained cells, under low power of the light microscope, with the condenser down and the iris halt partly closed to obtain the contrast needed for viewing limpid cells. Several types of CPE are distin guishable in living cultures, but mending and staining of the cells is necessary to see such manifestations of viral infection as inclusion bodies and syncytia.Recognizing CPE and using it as a diagnostic tool requires much experience in examining both stained and unstained cultures of many cell types. Listed below are several general types of CPE. Keep in estimation that a given virus may not conform to the norm for its family, or it may produce different CPE in different host cell types. The best knowledge of viral CPE comes from experience. 1. Total remainder of the cell monolayer is the most severe form of CPE. All cells in the monolayer rapidly thin and become dense (Pyknosis) and detach from the glass within 72 hours. This CPE is typical of most enteroviruses. 2. Sub-total destruction consists of backdown (death) of some but not all of the cells in the monolayer.The alpha-togaviruses, some picorna viruses, and some of the paramyxoviruses may cause this type of CPE. 3. foc al degeneration is characteristic of the herpesviruses and poxviruses. Instead of causing a generalized destruction of the cell monolayer, these viruses produce localized areas (foci) of infection. The focal nature of these lesions is due to direct cell-to-cell transfer of virus rather than diffusion through the extra-cellular medium. Cells initially become enlarged, rounded, refractile (more easily seen), and eventually detach from the glass, leaving cleared areas surrounded by rounded up cells as the infection spreads concentrically. Stranding of the cytoplasm is usually pronounced and cell fusion may be evident. 4.Swelling and clumping of cells before detachment is typical of adenoviruses. Infected cells greatly enlarge and clump together in grape-like clusters. 5. Foamy degeneration (vocuolization) is due to the production of large and/or numerous cytoplasmic vacuole. Several virus families including certain retroviruses, paramyxoviruses, and togaviruses may cause vocuolization. 6. Cell fusion (syncytium or polykaryon formation) involves the fusion of the plasma membranes of 4 or more cells to produce one enlarged cell with 4 or more nuclei. Polykaryon formation may be the only detectable CPE of some paramyxoviruses herpesviruses may also produce syncytia. 7. Inclusion bodies are areas of altered staining in cells.Depending on the causative virus, these inclusions may be single or multiple, large or small, round or irregularly shaped, intranuclear or intracytoplasmic, eosinophilic (pink staining) or basophilic (blue-purple staining). In most cases they represent areas of the cell where viral protein or nucleic acid is being synthesized or where virions are being assembled, but in some cases no virus is present and the inclusion bodies represent areas of viral scarring. V. BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF VIRUSES There are many biochemical and biophysical tests which can be used for classification of viruses. We will perform four of these tes t using unknown viruses viral sensitivity to lipid solvents, determination of virus size, determination of virus nucleic acid type, and viral sensitivity pH and heat.The chart on p. 127 of your lab book may help in the identification of your virus. A. Viral Sensitivity to Lipid Solvents. The lipid sensitivity test is one of the most basic tests for characterization of viruses. There is a correlation between the presence of an envelope and the susceptibility of viruses to lipid solvents such as ether, chloroform, and detergents. Enveloped viruses require their lipid membrane for infectivity because the test measures destruction of viral infectivity vs. untreated viral controls, it is an indirect test. All lipid coated viruses are sensitive to chloroform, whereas all but a few poxviruses are sensitive to ether.This is because the lipid components of the poxviruses are much diffe