Diagnosis of Viral

Infection(Part 2)

Laboratory Safety

Monolayer culture

• Requires substrate or solid surface

for attachment and growth

: Anchorage dependent of growth

• Contact inhibition of movement: Monolayer

Primary cell culture

+ enzymes

time

Subculture

enzymes

time

Transformationloss of contact inhibition

Stationary cultivation

Culture flask

Culture Plate

Medium Constituents

* Balance salt solution : Phosphate buffer, Mg2+, Ca2+

* Inorganic ions and trace elements

: for membrane potential and osmotic pressure

: buffer

* Energy source : glucose, glutamine

* Amino acid : metabolism and biological synthesis

Role of Serum

• Buffer, Chelator, Carrier proteins

• Bind to toxin

• Protease inhibitor

• Promotes attachment of cell to substratum

• Source of Intermediate metabolites,

hormone and growth factor

Primary cell culture and Establishment cell line

• Preparation of cell suspension from intact tissue

1. Single cell preparation

: use mechanical, Chemical, and/or enzymatic method

2. Disaggregate or dissociate cell

: cutting, homogenizing, rotary shaker, vortex,

pipette

• Preparation of cell suspension from intact tissue (cont.)

** Enzymes **

• Trypsin (crude)

: from cattle and pig’s pancrease

• Preparation of cell suspension from intact tissue (cont.)

1. Removal of tissue and place in Isotonic

(or growth medium with antibiotic)

2. Trim off unwanted parts and cut into smaller pieces

3. Dissociate cells using enzyme

4. Remove large debris and wash cells

• Preparation of cell suspension from intact tissue (cont.)

5. Suspend cells in growth medium accordingly

6. Pipette into culture vessel and incubate

Common Cell line (cont.)

• HeLa : 1952

cancer tissue:cervical carcinoma

: harbors HPV type 18 genome

• Vero : 1962

: from African green monkey kidney

: preparation of Poliovirus vaccine

Work in Safety cabinet Class-II

UV

ethanol for decontamination

Culture medium and Reagent

1.PBS

2. Growth medium

: 5%FCS-DMEM

3. Trypsin-Versene

Subpassage

Remove growth medium

Wash with PBS

Detach cell with Trypsin-Versene

Remove TV

Refresh with growth medium

Mix cell by pipette

Count and calculate number of cell

: Hemacytometer

Detection of virus in cell culture:•Cytopathic effect -CPE:changed monolayer architecture, the entire cell/ nucleus thickening and shrinking (pycnosis), inclusion bodies formation, giant cell (syncytia)formation, nucleolar displacement,rearangement and margination of the nuclear chromatin,vacuolization,the type of CPE depends on virus specifity and the kind of cells, polio virus, adeno virus, HSV, CMV;•Viral hemagglutination–direct reaction with red cells, influenza;•Hemadsorption –adsorption of erythrocytes to infected cells;parainfluenza, influenza•Plaques effect –clear areas appear in culture (PFU)due to cell lysis;

Titer = 32 HA units/ml

Hemagglutination test: method

1:8

1:2 1:21:21:21:2

8 16 32 64 128 256

virus

serial dilution

mix with red blood cells

side view

top view

Hemagglutination assay. Seven different samples of influenza virus, numbered 1 through 7 at the left, were serially diluted as indicated at the top, mixed with chicken red blood cells (RBC), and incubated on ice for 1 to 2 hours. Wells in the bottom row contain no virus. Agglutinated RBCs coat wells evenly, in contrast to nonagglutinated cells, which form a distinct button at the bottom of the well. The HA titer, shown at the right, is the last dilution that shows complete hemagglutination activity. (From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.9)

Hemagglutination assay: influenza virus

Plaque Assays

   Quantitation of viruses

a. TCID50(50% Tissue culture infected dose ): Infections dose of 50% CPE of tissue cultures caused by viral minimum infected dose is called TCID50

b. PFU : under controlled condition, single plague can arise from a single infection virus particle, termed a plague-forming unit.

1:100

1:10 1:101:101:101:10

10-2 10-3 10-4 10-5 10-6 10-7

virus

serial dilution

plate 1 ml

plaques

100 10 1(1000)(100,000) (10,000)

Titer = 1 x 107 pfu/ml

Plaque assay: method

Virus cultivation 2. Inoculation of embryonated chick eggs

USE OF FERTILE EGGS

• Largely replaced by use of TC

• Still useful in cultivation and detection of some viruses, flu

• Three parts of the egg are of use – A   The amniotic cavity:

• Surrounds the embryo & lined by a single layer of epithelial cells. • The amniotic fluid bathes the external surface of the embryo and comes into contact

with the respiratory and alimentary tracts.

– B   The allantoic cavity: • Comprises an outgrowth of the hind-gut of the embryo and is lined with endoderm.

• Both are useful for the cultivation of viruses, particularly • orthomyxoviruses (e.g., influenza) and paramyxoviruses (e.g., mumps).

Membranes major source of cells in which virus growth occurs, but the embryo may also become infected.

• The allantoic cavity is routinely used because it is technically much simpler to inoculate. • However, some viruses (e.g., human influenza isolates) may need to be egg-adapted by growth

in the amniotic cavity before they will grow efficiently in the allantoic cavity; the reason for this is not known.

C   The chorio-allantoic membrane : The membrane consists of an outer layer of stratified epithelium which constitutes the respiratory surface of the egg, and an inner layer of endoderm (the lining of the allantoic cavity). The membrane may be used as a cell sheet provided it is first dropped away from the shell membrane. Dermatropic viruses (poxviruses and some herpes viruses) will grow on this membrane, and at low concentrations, will give discrete foci of infection which consist of centres of cell proliferation and necrosis (pocks). The membrane may therefore be used to assay these viruses. In addition, different viruses cause pocks of different colour and morphology, and this is of diagnostic value for distinguishing between different poxviruses.

Growth of virus on embryonated eggs

Davis, Duylbecco, Eisen, Ginsberg “Microbiology” 4th ed, J.B. Lippincott 1990, Fig. 48-1

•production of pocks or plaques on chorioallantoic membrane(herpes, smallpox, vaccinia);

•development of hemagglutinins in embryonic fluid(influenza);•death of embryo -encephalitis viruses;

Convenient, inexpensive, the viral suspension injected into the fluid of the egg, viral growth is detected by death of the embryo, or lesion on the membrane of the egg.

It is the most common method for viral growth and isolation , and used for preparation of viral vaccine.