Isolasi dan Identifikasi Virusvlm.ub.ac.id/pluginfile.php/44399/mod_resource/content/1... · 2020. 4. 8. · Isolasi dan Identifikasi Virus drh. Sruti Listra Adrenalin, M.Sc. 8 April

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Isolasi danIdentifikasi Virusdrh. Srut i Listra Adrenal in, M.Sc .

8 A p r i l 2 0 2 0

Click to edit Master title styleVirus Isolation

• Unlike bacteria, viruses require a living host cell for replication parasite obligat intraseluler.

• Virus isolation remains the "gold standard" against which newer methods must be compared.

• There is an excitement in isolating viruses.

Click to edit Master title style… Virus Isolation

• Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate).

• Animal virus cultivation is important for:

1. identification and diagnosis of pathogenic viruses in clinical specimens.

2. production of vaccines.

3. basic research studies.


• Virions in the liquid medium can be separated from the host cells by centrifugation or filtration.


Advantages Disadvantages

‘Catch-all’ (as long as viable within

the chosen cell line(s)).

Only detects ‘viable’ virus.

Sensitive. Slow (conventional cell culture).

Generates isolate for further study, for

example phenotyping.

Multiple cell lines required.

Can be adapted for a more rapid result. Labour intensive and requires skilled

Staff.

Safety concerns, laboratory security.


Stadium/ fase Virus dalam spesimen Deteksi antibodi

Inkubasi Ya (jarang) Tidak

awal kejadian Ya Ya (jarang)

Fase akut Ya Ya

Penyembuhan Ya (jarang) Ya

Mati Ya Tidak


Host Asli Hewan Coba TAB Cell Culture

Advantage Paling baik Lebih praktis Penanganan

praktis

Lebih luas (dapat

dipilih jenis sel

yang sesuai)

Disadvantage Pemeliharaan

sulit, mahal

Terbatas Tidak semua virus

dapat tumbuh

Mahal

… Virus Isolation

Click to edit Master title styleSpecimens for Virus Diagnosis

Click to edit Master title styleVirus Isolation and Identification

Method Specimens/Findings Characteristics

Virus isolation in cultured

cells

Tissues, cells, secretions,

excretions/ Specimens inoculated

into suitable cell cultures and

presence of virus detected by

various methods, usually

immunological methods

Relatively slow, expensive, and

technically demanding. However, this

is the only method that provides a

virus isolate for further testing

(eg, strain typing) and is therefore

widely used in reference centers

Virus isolation in animals Tissues, cells, secretions,

excretions/ Specimens inoculated

into animals, usually newborn or

3-week-old mice, usually by the

intracerebral or intraperitoneal

routes, with sickness or death as

indication of viral growth.

Identification of virus by various

methods, usually immunological

methods

Even slower, more expensive, and

technically demanding than virus

isolation in cell culture.

However, for viruses that do not grow

well in cell culture, this is the only

method that provides a virus isolate

for further testing (eg, strain typing)

and is therefore still used in `reference

centers in special circumstances

Click to edit Master title styleDetection and Identification of Viral Antigens

Method Specimens/Findings Characteristics

Enzyme immunoassay methods (eg,

antigen-capture enzyme

immunoassay).

Tissues, cells, secretions, excretions/

Reaction of viral antigen with antibody of

known specificity.

Rapid, sensitive and specific. Most common

methods in use today.

Immunochromatography,

immunogold-binding assays

(the equivalent of the home

pregnancy test).

Blood, secretions, excretions/Viral

antigen identified by reaction with

antibody of known specificity.

Rapid, sensitive, specific, suitable for testing

of individual specimens in the clinical setting.

Immunofluorescence. Tissues and cells/Viral antigen identified

in situ by reaction with


Rapid, sensitive and specific. Localization of

antigen in specific cells adds to confidence in

diagnosis; technically demanding.

Immunohistochemistry

(immunoperoxidase

staining).

Tissues and cells/Viral antigen

identified in situ by reaction with


Slow, but sensitive and specific. Localization

of antigen in specific cells adds to confidence

in diagnosis; technical expertise involved is

more like an extension of histopathology.

Immunoelectron microscopy. Tissues, cells, secretions, excretions/

Character and aggregation of virus by

specific antibody of known specificity.

Extension of diagnostic electron microscopy.

Rapid, sensitive and specific. Expensive and

technically demanding; expertise unavailable

in many settings

Click to edit Master title styleCell Culture

• A single viable virion present in a specimen can be grown in cultured cells, thus expanding it many million fold to produce enough material to be characterized antigenically.

• Culture is the only method of producing a supply of live virus for further examination, such as antigenic variation.

• To produce diagnostic antigens and monoclonal antibodies for distribution to other laboratories.

• Primary cells derived from fetal tissues of the same species usually provide the most sensitive cell culture substrates for virus isolation.

• Continuous cell lines derived from the homologous species (Cell lines offer some advantages and are available for most domestic mammals).

Click to edit Master title style… Cell Culture

Cell culture:

• Primary cells culture

• Secondary cells culture

• Continuous cells line

Detection virus growth:

• CPE (cytopathic effects): morphologic changes of culture cells

• Hemaglutination

• Hemadsorbtion

• Phenomena of interference (non-CPE virus)

• Transformation of cell (oncogenic viruses)

Click to edit Master title styleMonolayer Culture

Sel-sel yang tumbuh monolayer, melekat di dasar tabung perbenihan.

Perbenihan sel primer (Primary Cell Culture)

• Menggunakan organ segar (jaringan embrio, organ hewan/ manusia, tumor) Tidak dapat dibuat subkultur.

Continous Cell Lines

• Biakan sel ganas/tumor sehingga mudah untuk dibuat subkultur berseri takterhingga.

• Contoh: sel Hela (dari jaringan Ca-cervix uteri); Sel Vero (ginjal kera); BHK-21 (ginjal anak hamster).

• Tidak untuk pertumbuhan virus dalam pembuatan vaksin )karena akanmemindahkan faktor karsinogenik pada resipien vaksin).


Derivation of continuous cell lines ofhuman and animal cells


Cells for culture are prepared by separating them from their tissue matrix

Click to edit Master title style… Cell Culture

• Special types of cell and organ culture are utilized for particular viruses beta and gammaherpesviruses may be recovered from monolayer cultures propagated directly from tissues taken from the diseased animal.

• Coronaviruses and Rhinoviruses growth may occur in explant cultures (e.g., small cubes of tissue with intact epithelium taken from the trachea or gut).

• Cocultivation of explant tissues with cell monolayers latency viruses.

• Arthropod cell Arboviruses.

Click to edit Master title styleVirus Isolation in Animals

• Many viruses will grow satisfactorily in chick embryos or newborn mice but neither is now commonly used because cell culture is generally the simpler option.

• Mice arboviruses and rabies virus <24 hours old are injected intracerebrally and/or intraperitoneally observed for up to 2 weeks (pathognomonic signs) euthanizing (histopathology, immunofluorescence, immunohistochemistry, or serology).

• Natural host animals pathogenesis and immunity research.

Click to edit Master title styleVirus Isolation in Chick Embryos

• For many avian viruses.

• There are several important pathogens that replicate much better in eggs than in cell cultures derived from chick embryo tissues.

• Many viruses have a tissue tropism, and must therefore be introduced into a specific site for growth.

• Inoculated in the amniotic cavity, the allantoic cavity, the yolk sac, or on the chorioallantoic membrane.

• Viral infection may damage tissue membranes, producing lesions called pox; disrupt embryonic development; or cause the death of the embryo.



Click to edit Master title stylePox Virus

6 hari setelah inokulas

terdapat bercak putih di

membrane chorioallantois

Lesi pox di seluruh

tubuh

Click to edit Master title styleInfectious Bronchitis

• Embrio kerdil

• Amnion & allantois

menebal

• Yolk sac tidak terserap

Click to edit Master title styleNewcastle Disease

Kongesti dan hemoragi

jaringan SC.

Click to edit Master title style… Virus Isolation in Chick Embryos

• Pertumbuhan virus di TAB tanpa perubahan/ dengan perubahanembrio sifat keganasan virus pada embrio (kematian, perdarahan, cacat, lesi).

Click to edit Master title styleDetection of a Virus

• CPE: distinct observable cell abnormalities due to viral infection The type or severity of the CPE depends on the type of virus involved:

1. loss of adherence to the surface of the container.

2. changes in cell shape from flat to round.

3. shrinkage of the nucleus.

4. vacuoles in the cytoplasmfusion of cytoplasmic membranes and the formation of multinucleated syncytia, inclusion bodies in the nucleus or cytoplasm, and complete cell lysis.


Paramyxovirus

Syncytium and

faint basophilic

cytoplasmic

inclusion bodies

Poxvirus

Pink

eosinophilic

cytoplasmic

inclusion

bodies (arrows)

and cell

swelling

Herpevirus

Cytoplasmic

stranding

(arrows) and

nuclear

inclusion

bodies (dashed

arrow)

Adenovirus

Cell

enlargement,

rounding, and

distinctive

grape-like

clusters

Click to edit Master title stylePerhitungan Jumlah Virus

• Titer virus: Jumlah virus infeksius pe satuan volume

• Unit infeksius: jumlah terkecil virus yang menyebabkanterdeteksinya efek pada hospes.

1. Quantal Assay

Tidak menghitung jumlah partikel virus infeksius dalam inokulum(suspensi virus), tetapi menentukan titer virus.

2. Quantitative Assay

Menghitung jumlah partikel virus infeksius pada inoculum.

Click to edit Master title styleQuantal Assay

Dapat dilakukan di kultur sel, TAB, hewan model.

Endpoint: efek virus pada host Rumus Reed and Muench.

LD50: titer virus yang mampu menyebabkan kematian 50% hospesyang diinokulasi

ID50: titer virus yang mampu menginfeksi 50% hospes yang diinokulasi

• TCID50: titer virus yang mampu menimbulkan terbentuknya CPE 50% pada kultur sel

• EID50: titer virus yang mampu menginfeksi 50% embrio TAB yang diinfeksi

Click to edit Master title styleReed & Muench

• 50% endpoint terletak antara pengenceran 10-6 dan 10-7

• Titer ditentukan berdasarkan angka kebalikan dari pengenceranvirus pada end point

Log pengencer-

an virus

Unit tes

yang

diinfeksi

Kumulatif

terinfeksi (A)

Kumulatif yang

tidak terinfeksi (B)

Rasio

A/(A+B)

Presentasi

terinfeksi

-5 5/5 9 0 9/9 100

-6 3/5 4 2 4/6 66,7

-7 1/5 1 6 1/7 14,3

-8 0/5 0 11 0/11 0

Click to edit Master title style… Reed & Muench

• 50% endpoint terletak antara pengenceran 10-6 dan 10-7

• Jarak sebanding= % positif di atas 50% -50%

% pos di atas 50% - % pos. di bawah 50%

= 66,7% - 50,0%

66,7% - 14,3%

= 0,3

• Log pengenceran di atas 50% = -6

• Jarak sebanding = 0,3

• Faktor pengenceran = -1 (pengenceran serial 10x)

• Log ID50 = (log pengenceran di atas 50%) + (jarak

sebanding x log faktor pengenceran)

= (-6) + (0,3 x -1)

• ID50= 6,3

Click to edit Master title styleTCID50

10-3 A

10-4 B

10-5 C

10-6 D

10-7 E

10-8 F

10-9 G

Tanpa H

virus

v v v v vvv

v v v

v v v v

v v vv

v

v

v

v

v

v

v

v

v

v

v

v

v

v

v

v

1 2 3 4 5 Pengenceran

virus Skoring

1: tidak ada CPE

2: CPE <50%

3: CPE 50%

4: CPE ± 75%

5: Monolayer cell rusaksecara total

Reed & Muench

Click to edit Master title styleQuantitative Assay

• Pada bakteri adalah bacterial colony count:

Bakteri ditumbuhkan pada medium koloni diestimasikansebagai jumlah sel bakteri yang hidup.

• Virus: menghitung jumlah plak yang terbentuk pada kultur selmonolayer yang diinfeksi virus Plaque Assay

• Contoh lain quantitative assay: pock assay, infectious center assay, transformation assay, electron mikroskop.

Click to edit Master title stylePlaque Assay

• CPE viruses.

• Plaque assay menggunakan kultur sel monolayer yang konfluen, dan diinfeksi dengan berbagai pengenceran virus, kemudian ditutupdengan media semisolid atau padat.

• Media pada dipergunakan untuk melokalisir virus hanya di sekelilingsel.

• Visualisasi terbentuknya plak: neutral red, tetrazolium

• Plak terbentuk apabila sel yang diinfeksi mengalami lisis zona bersih (tidak lisis terwarnai).

Click to edit Master title style… Plaque Assay



• PFU (plaque forming unit) titer infektivitas virus (yang dihitung 10-100 plak/pengenceran).

• Pfu/ml= jumlah plak x kebalikan pengenceran x kebalikan volume (ml)

• Contoh: kultur sel diinfeksi 0,1 ml virus dengan pengenceran 10-5, diperoleh rerata jumlah plak yang terbentuk 45

• Titer virus= 45 x 105 x 10

= 4,5.107 pfu/ml

Click to edit Master title styleDetection of Virus-Specific Ag-Ab

1. Hemaglutination (HA)

2. Hemaglutination Inhibition (HI)

3. Agar Gel Presipitation Test (AGPT)

4. Complement Fixation Test (CFT)

5. Virus Neutralization (VN)

6. Enzim Linked Immunosorbent Assay (ELISA)

7. Immunohistochemistry (IHC)

Click to edit Master title styleSample

• Serum, plasma, fluids.

• Antibodies in serum are very stable.

Click to edit Master title styleHemaglutination (HA)

• HA: viruses (ag) & RBC are mixed.

• Advantages: simple, inexpensive, available instruments, results within a few hours, well established in many laboratories around the world, allowing some measure of credibility, comparison, and standardization.

Click to edit Master title style… Hemaglutination (HA)

• Untuk mengetahui titer virus uji HI.

• Mengidentifikasi virus yang memiliki hemagglutinin.

Click to edit Master title styleHemaglutination Inhibition (HI)

• Titer serum HI: The highest dilution of serum (Ab) that prevents hemagglutination.


Click to edit Master title styleAgar Gel Presipitation Test (AGPT)

Prinsip :

1. Reaksi antara Ag dan Ab dapat berlangsung dalam lapisan agar.

2. Ag/Ab di dalam agar akan berdifusi ke segala arah & pd titikpertemuannya akan membentuk kompleks Ag – Ab yg mengendapsebagai garis presipitasi.

Click to edit Master title style… Agar Gel Presipitation Test (AGPT)

• Manfaat:

1. Menentukan adanya Ag atau Ab secara kualitatif atau semi kuantitatif.

2. Mengetahui adanya reaksi silang/ hubungan imnologik daribeberapa macam Ag.

Click to edit Master title styleVirus Neutralization (VN)

• Prinsip: Virus menjadi non infektif bila digabungkan dgn Ab spesifikthd virus tsb.

• Kegunaan:

1. Untuk identifikasi virus.

2. Untuk mendeteksi reaksi silang di antara bbrp strain virus.

3. Untuk membandingkan level Ab dlm serum pd stadium akut dankonvalesen.

Click to edit Master title style… Virus Neutralization (VN)

• Hasil berdasarkan:

1. Angka kesakitan / kematian hewan coba.

2. Angka kecacatan / kematian embrio.

3. Adanya kerusakan sel (CPE) pd kultur sel.

4. Hitung dgn Rumus Reed and Muench atau Spearman-Karber.

5. Indeks Netralisasi (IN) = log titer serum standard – log titer serum uji(Signifikans bila >log 1,5).

Click to edit Master title style… Virus Neutralization (VN)

Click to edit Master title styleComplement Fixation Test (CFT)

Click to edit Master title styleEnzim Linked Immunosorbent Assay (ELISA)

• Sbg detektor digunakanenzim untuk melabel Ab/ Ag.

• Reaksi dpt dibaca dgnpenambahan substratdan ntuk melihatnyadiperlukanspektrofotometer (ELISA reader)

Click to edit Master title styleImmunohistochemistry (IHC)

• Pemeriksaan untuk mengukur kadarab/ ag dalam sediaan jaringan.

• Tujuan: mengidentifikasi komponenjaringan yang memiliki ciri tertentudengan menggunakan interaksiantara antigen target dan antibodispesifik yang diberi label.


Thank You


Click to edit Master title styleReferences

• MacLachlan, N.J., Dubovi, E.J. 2016. Fenner’s Veterinary Virology 5th Edition. Academic Press is an Imprint of Elsevier.

• Murphy, F.A., Gibbs, E.P.J., Horzinek, M.C., Studdert, M.J. 1999. Veterinary Virology 3rd Edition. Academic Press.

• Tim Dosen Mikrobiologi dan Imunologi. Isolasi dan Identifikasi Virus (PPT). Fakultas Kedokteran Hewan Universitas Brawijaya, Malang.

• Zuckerman, A.J., Banatvala, J.E., Schoub, B.D., Griffiths, P.D., Mortimer, P. 2009. Principles & Practice of Clinical Virology 6th

Edition. Wiley-Blackwell.

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Isolasi dan Identifikasi Virusvlm.ub.ac.id/pluginfile.php/44399/mod_resource/content/1... · 2020. 4. 8. · Isolasi dan Identifikasi Virus drh. Sruti Listra Adrenalin, M.Sc. 8 April