MAY

2019

VIRUS PURIFICATION
RICKY FERNANDO
WASISTA HANUNG PUJANGGA
PRINCIPLE
DNA PURIFICATION
RNA PURIFICATION

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• After a virus has been propagated it is usually necessary to remove
host cell debris and other contaminants before the virus particles
can be used for laboratory studies, for incorporation into a vaccine,
or for some other purpose.
• Many virus purification procedures involve centrifugation

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 SOURCE
• For purification studies, the starting material is usually large
volumes of tissue culture medium, body fluids, or infected cells.

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 SOURCE
• Many viruses can be isolated as a result of
their ability to form discrete visible zones
(plaques) in layers of host cells.
• Each plaque is formed when infection
spreads radially from an infected cell to
surrounding cells.
• Plaques can be formed by many animal
viruses in monolayers if the cells are
overlaid with agarose gel to maintain the
progeny virus in a discrete zone
• Plaques can also be formed by phages in
• lawns of bacterial growth 6
 STEPS

• Precipitation: technique concentration of the virus

particles
1. Separation virus particle from host cell :
a) Differential centrifugation.
b) Density gradient centrifugation.
c) Column chromatography.
d) Electrophoresis.

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 PRECIPITATION
• The first step frequently involves concentration of the virus
particles by precipitation with:
• Ammonium sulfate
• ethanol
• polyethylene glycol (PEG)
• by ultrafiltration.
• Hemagglutination and elution can be used to concentrate
orthomyxoviruses

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 Precipitation PEG
PEG-it™ Virus Precipitation Solution
provides a simple and highly effective
means to concentrate lentiviral particles,
such as those produced with System
Biosciences´ pPACK Lentivector Packaging
System. PEG-it™Virus Precipitation
Solution is a formulation of polyethylene
glycol optimized for the precipitation of all
lentiviral-based particles. The solution is
mixed with virus-containing cell culture
supernatant, incubated at 4°C overnight,
and centrifuged at 1500 × g to pellet
precipitated viral particles. As a result, viral
particle concentrations are increased 10- to
100-fold.
 Differential centrifugation
• Partial purification
• Differential centrifugation involves
alternating cycles of:
• low-speed centrifugation, after which most
of the virus is still in the supernatant, and
• high-speed centrifugation, after which the
virus is in the pellet
 Density gradient centrifugation

• Involves centrifuging particles (such as virions) or

molecules (such as nucleicacids) in a solution of
increasing concentration, and therefore density.
• The solutes used have high solubility: sucrose is
commonly used. There are two major categories of
density gradient centrifugation:
1. Rate zonal sentrifugation.
2. Equilibrium sentrifugation.
 Rate zonal sentrifugation
• In rate zonal centrifugation a particle moves through the
gradient at a rate determined by its sedimentation
coefficient, a value that depends principally on its size.
• Homogeneous particles, such as identical virions, should
move as a sharp band that can be harvested after the
band has moved part way through the gradient
• Sample of concentrated virus is layered onto a
preformed linear density gradient of sucrose or glycerol
 Equilibrium sentrifugation
• A concentration of solute is selected to ensure that the density at
the bottom of the gradient is greater than that of the
particles/molecules to be purified.
• A particle/molecule suspended in the gradient moves to a point
where the gradient density is the same as its own density.
• This technique enables the determination of the buoyant
densities of nucleic acids and of virions.
• Buoyant densities of virions (determined in gradients of caesium
chloride) are used as criteria in the characterization of viruses.
 Column chromatography

• Virus is bound to a substance such as

diethylaminoethyl or phosphocellulose
and then eluted by changes in pH or salt
concentration .
 Electrophoresis

• Zone electrophoresis permits separation of virus particles

from contaminants on the basis of charge.
• Specific antisera also can be used to remove virus
particles from host materials.
POINTS TO KNOW
• It is very difficult to achieve complete purity of
viruses.
• Small amounts of cellular material tend to adsorb to
particles and copurify.
• The minimal criteria for purity are a homogeneous
appearance in electron micrographs and the failure
of additional purification procedures to remove
“contaminants” without reducing infectivity
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POINTS TO KNOW
• Icosahedral viruses are easier to purify than enveloped
viruses. Because enveloped viruses usually contain
variable amounts of envelope per particle, the viral
population is heterogeneous in both size and density.

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IDENTIFICATION OF A PARTICLE AS A VIRUS
1. The particle can be obtained only from infected cells or tissues.
2. Particles obtained from various sources are identical regardless of
the cellular origin in which the virus is grown.
3. Particles contain nucleic acid (DNA or RNA), the sequence of
which is not the same as the species of host cells from which the
particles were obtained.
4. The degree of infective activity of the preparation varies directly
with the number of particles present.
5. Destruction of the physical particle by chemical or physical means
is associated with a loss of viral activity
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IDENTIFICATION OF A PARTICLE AS A VIRUS
6. Certain properties of the particles and infectivity must be shown to be
identical (eg, their sedimentation behavior in the ultracentrifuge and their
pH stability curves).
7. Antisera prepared against the infectious virus should react with the
characteristic particle and vice versa. Direct observation of an unknown
virus can be accomplished by electron microscopic examination of
aggregate formation in a mixture of antisera and crude viral suspension.
8. The particles should be able to induce the characteristic disease in vivo
(if such experiments are feasible).
9. Passage of the particles in tissue culture should result in the
production of progeny with biologic and antigenic properties of the virus.
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 Penyimpanan Virus

Virus sangat mudah mengalami perubahan sifat sehingga menjadi strain baru yang
berbeda dengan aslinya. Hal ini menambah cepat tumbuh dan berkembangnya
biodiversitas tersebut.  Oleh karena itu, perlu melakukan koleksi, menyimpan, dan
memeli-hara mikroba dengan baik.  Dasar dilakukannya penyimpanan Virus.
Metode yang dipilih sangat tergantung pada sifat mikroba dan tujuan
preservasi.

Sifat mikroba tercermin dalam (1) ciri-ciri morfologi mikroba yang beragam
(virus), (2) ciri-ciri fisiologi dan biokimia mikroba, dan (3) kemampuan
mikroba bertahan hidup baik dalam lingkungan alaminya maupun
lingkungan buatan.
Tujuan Penyimpanan Virus

Tujuan koleksi dan preservasi meliputi tujuan jangka pendek

dan jangka panjang. Preservasi jangka pendek dilakukan
untuk keperluan rutin penelitian yang disesuaikan dengan
kegiatan program atau proyek tertentu. Preservasi jangka
panjang dilakukan dalam kaitannya dengan koleksi dan
konservasi plasma nutfah mikroba, sehingga apabila suatu
saat diperlukan, dapat diperoleh kembali atau dalam
keadaan tersedia.
Beberapa cara yang dapat digunakan supaya kualitas partikel virus tidak
berubah adalah :

1. Temperatur
2. Bahan kimia
3. Proses kering beku
Temperatur

• Kebanyakan virus tahan hidup selama beberapa hari dalam tempratur 4oC.
• Keuntungan penyimpanan virus dalam suhu ini ialah dapat menghindari proses
pembekuan dan pencairan(freeze-thawing) suspensi virus yang dapat merusak partikel
virus.
• Untuk penyimpanan virus dalam waktu lama (berbulan-bulan atau sampai bertahun-
tahun ) digunakan tempratur -70oC (dalam freezer) atau -196oC (dalam tabung berisi
nitrogen cair.
• Bagi virus-virus yang berada dalam sel (Cell associated) perlu ditambahkan serum atau
gliserol sampai 10% untuk mengawetkan sel-sel tersebut sehingga virus tetap hidup.
 Bahan Kimia

• Jika virus disimpan pada tempratur -70oC, bahan kimia

yang dapat dipakai untuk mengurangi kerusakan virus
adalah DMSO dengan konsentrasi 10%.

• Bila virus tersebut Cell associated, disamping DMSO 10%,

pada media penyimpanan virus ditambahkan pula serum
sampai 10%untuk menjaga keutuhan sel.

• Gliserol sebagai alcohol polihidrat dapat menstabilkan

dinding sel dan partikel virus. Pada konsentrasi 50%,
gliserol digunakan untuk mengawetkan virus pox dan sel
epitel yang mengandung virus PMK.
Proses Kering Beku (Freeze-Drying)/liofilisasi .

• Fungsi preservatif adalah menstabilkan protein,

mencegah kerusakan akibat pembekuan, dan
melindungi dari kekeringan yang berlebihan.

• Cara ini juga disebut liofilisasi dan merupakan

yang terbaik dalam mengawetkan virus,
terutama bila sebelumnya suspensi virus
tersebut mengandung 10% serum anak sapi.

• Virus yang sudah kering beku dapat disimpan

dalam tempratur 4oC selama berbulan-bulan.
Metode ini digunakan dalam penyimpanan vaksi
aktif
Media/ larutan yang digunakan untuk penyimpanan kering beku:

• Mist dessicants (Sly, 1983) yang merupakan cairan dengan komposisi pepton
Difco 12 g dan glukosa 30 g dalam 100 ml akuades.

• Larutan pepton 1%, larutan susu skim 1%, larutan Naglutamat 1%.

• Larutan campuran serum kuda dengan pepton 10% (Sly, 1983).

Penyimpanan secara Kriogenik / ultra-low temperatures

Liquid Nitrogen Freezing

Metode ini memerlukan alat khusus untuk mengontrol tingkat pembekuan sebelum disimpan dalam
waktu lama dalam nitrogen cair. Media yang digunakan adalah 10 % (vol/vol) gliserol atau 5% (vol/vol)
DMSO. Suhu penyimpanan pada metode ini adalah 77 K atau -1960C yang merupakan titik didih dari
nitogen cair.

Pada suhu rendah ini beberapa aktivitas biologis termasuk reaksi biokimia yang menyebabkan
kematian pada sel dapat diperlambat (Suriawiria, 2005). Penyimpannan dengan menggunakan
metode ini dapat berlangsung hingga 100 tahun.
• Untuk virus penyebab infeksi saluran nafas : sukrosa-fosfat-glutamat yang
mengandung 1% bovine albumin (SPGA) dan hipertonik sukrosa.

• Selain isolat murni spesimen tinja yang mengandung patogen enterik virus dapat
dibekukan pada suhu −70 hingga −85 ° C selama 6 hingga 10 tahun dengan
mempertahankan karakteristik morfologis astrovirus,, adenovirus enterik, rotavirus
dan calicivirus

• Isolasi HIV  Untuk penyimpanan limfosit darah tepi yang terinfeksi HIV yang
dicampur kedalam 10% serum janin sapi dan 10% DMSO dan menyimpannya pada
suhu −60 ° C
REFERENCES
• Carter JB., Saunders VA. Virology Principles and Applications.
Chichester : Wiley, 2007.
• Brooks GF., et al. Jawetz, Melnick, & Adelberg’s Medical
Microbiology. 26th ed. New York : McGraw-Hill, 2013.
• Dongyou Liu. Handbook of Nucleic Acid Purification. 2009.

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THANK YOU!
Alexander Martensson

Phone
678-555-0143
Email