Perbanyakan Massal Virus sebagai Agens

Antagonis

Oleh Irda Safni

Kuliah ke-11

Virus sebagai agens antagonis

patogen tumbuhan

Ada beberapa cara virus dapat digunakan untuk mengendalikan patogen tumbuhan, yaitu :

1. Virus dapat membunuh atau mengurangi patogenisitas bakteri patogen dan jamur patogen tumbuhan.

2. Virus dapat membunuh vektor invertebrata virus pada tanaman tingkat tinggi sehingga mencegah penyebarannya.

3. Strain yang agak lemah yang menyebabkan sedikit atau tidak menimbulkan dapat melindungi tanaman dari infeksi laten oleh strain yang lebih parah.

• Stanway (1985) mendeteksi infeksi virus sebanyak 126 dari 157 isolat

jamur take-all pada tanaman gandum (Gaeumannomyces graminis

var. tritici).

• Kultivar gandum yang terinfeksi Helminthosporium victoriae (penyakit

hawar) adalah penyakit penting gandum di Amerika pada 1947

dan1948.

• Lindberg (1959) menemukan beberapa koloni jamur H. victoriae

menjadi memendek, ditandai dengan daerah pada pinggir koloni,

miselium udara menjadi lisis dan menghambat perkembangan koloni.

• Penyakit ini juga ditransmisikan ke kultur yang sehat oleh anastomosis

hifa dan mungkin disebabkan oleh 1 atau 2 dsRNA virus yang biasa

dijumpai pada H. victoriae (Ghabrial 1986).

• Tanaman gandum yang dijumpai isolat jamur tidak menyebabkan

kehilangan hasil yang besar, kemungkinan disebabkan penyebaran

virus pada populasi H. victoriae, karena isolat menghasilkan sedikit

toksin victorin dan menjadi kurang patogenik dibanding isolat yang

normal (Lindberg 1960).

• Isolat kultur Rhizoctonia solani mengandung 3 segmen virus

dsRNA, sedangkan tidak dijumpai virus dsRNA pada ujung hifa

yang sehat dari isolat yang mengifeksi.

Virus sebagai entomopatogen

Penyakit yang disebabkan virus entomopatogen mulai

diketahui sejak abad ke-16.

Penyakit yang disebut Jaundice o graserrie, sekarang

diidentifikasi sebagai nucleopolyhedrosis, ditemukan pada

ulat sutra (Bombyx mori) oleh Vida pada tahun 1524 dan

kemudian juga diisolasi dari lebah madu (Apis mellifera).

Pada tahun 1856, dua orang ahli Italia (Maestri dan

Cornalia) menjelaskan occlusion bodies (OBs) ulat sutra

nucleopolyhedrosis.

Pada tahun 1926 Paillot mendeskripsikan Granulovirus

(GVs) pertama sekali.

Pada tahun 1934 Ishimori menjelaskan jenis baru

polyhedrosis di dalam ulat sutra OBs dibentuk didalam

sitoplasma sel yang diinfeksi (bukan pada asam nukleat)

sekarang dikenal dengan Cypovirus.

Sejak tahun 1950 s/d 1970, Steinhaus dan koleganya menguji

baculovirus sebagai agens hayati di lapangan dengan

mengaplikasi nucleopolyhedrovirus (NPV) untuk mengendalikan

ulat alfalfa (Colias eurytheme Boisduval; Lepidoptera: Pieridae).

Bioinsektisida komersil berbahan aktif virus pertama

dikembangkan pertama sekali pada tahun 1975 oleh

Perusahaan Sandoz (dengan nama dagang Elcar) untuk

mengendalikan Heliothis/Helicoverpa Lepidoptera: Noctuidae).

Selama tahun 1979 s/d 1980, penemuan penting pada

genetika virus entomopatogen, khususnya baculovirus.

Hingga saat ini studi genetika virus entomopatogen difokuskan

pada studi genom lengkap telah ada 29 sekuensing genom

lengkap virus entomopatogen.

Keuntungan dan Kerugian Penggunaan Virus untuk

mengendalikan hama

Keuntungan

1. Selektif dan efektif terhadap hama sasaran.

2. Aman bagi serangga dan organisme bukan sasaran serta tidak menyebabkan

resistensi.

3. Persisten dan tidak meninggalkan residu beracun di alam.

4. Tidak menyebabkan pencemaran lingkungan.

5. Efektif menginfeksi ulat yang telah terkena insektisida kimia.

6. Tidak menyebabkan peningkatan populasi hama sekunder.

7. Dapat ditularkan oleh parasitoid dan predator ke inang yang sehat.

8. Dapat mengendalikan ulat instar V-VI.

9. Tidak menyebabkan penyakit virus pada tanaman.

10.Kompatibel dengan teknik pengendalian yang lain, termasuk insektisida kimia.

11.Mudah diproduksi dengan teknik sederhana (menggunakan alat semprot

standar).

12.Berpotensi sebagai pengendali hama jangka panjang.

13.Dapat beradaptasi dengan teknologi modifikasi secara genetik (bioteknologi).

Kerugian

1. Hanya spesifik terhadap hama sasaran.

2. Kemungkinan berbahaya bagi serangga bukan sasaran.

3. Waktu aplikasi harus tepat untuk memaksimalkan efektivitas.

4. Memerlukan pendistribusian secara merata pada kanopi tanaman

untuk meningkatkan kontak dengan hama sasaran.

5. Daya bunuh lambat.

6. Rentan terhadap pengaruh lingkungan.

7. Kehilangan virulensi dan patogenitas jika diperbanyak secara terus

menerus (jika tidak dilakukan penggantian inang baru).

8. Infektivitasnya di lapangan singkat dan membutuhkan penanganan

tertentu.

9. Kekhawatiran masyarakat terhadap kemungkinan

patogenik/menyebabkan alergi.

10. Menurunkan dosis perlakuan melalui bioteknologi menyebabkan

resurgensi hama atau serangga bukan sasaran.

Saran untuk aplikasi virus entomopatogen

Virus tidak dapat diaplikasikan sendiri, tetapi kerkonjugasi dengan teknik

pengendalian yang lain.

Virus entomopatogen bersifat spesifik, sehingga serangga target farus

diidentifikasi secara benar.

Lahan dicangkul terlebih dahulu sebelum aplikasi dilakukan, dan virus

diaplikasikan pada serangga target sewaktu masih mudah tetapi aktif makan.

Virus yang Menginfeksi Invertebrata

Virus DNA

Double stranded DNA

- Poxviridae

- Iridoviridae

- Baculoviridae: NPV & GV

- Polydnaviridae

Single stranded DNA

- Parvoviridae

Virus RNA

Double stranded RNA

- Rheoviridae: Cypovirus

Single stranded RNA (-)- Rhabdoviridae

- Bunyaviridae

Single stranded RNA (+)

- Picornaviridae, Togaviridae,

Tetraviridae, Flaviridiae,

Nodaviridae

FAMILY NUCLEIC

ACID

NUCLEOCAPSID

SIMETRY

OCCLUSION

BODY

Baculoviridae dsDNA Baciliform +

Reoviridae dsRNA Isometric +

Poxviridae dsDNA Ovoid +

Iridoviridae dsDNA Icosahedral -

Parvoviridae ssDNA Isometric -

Picornaviridae ssRNA Spherical -

Ascoviridae dsDNA Allantoid -

Polydnaviridae dsDNA Ovoid -

Rhabdoviridae ssRNA Baciliform -

Nodaviridae ssRNA Icosahedral -

Rhabdoviridae ssRNA Baciliform -

NON-CLASSIFIED RNA VIRUSESs

Divided genome ssRNA Isometric -

Nodaurelia ssRNA Isometric -

Kelply group ssRNA Isometric -

5-virus group ssRNA Isometric -

Minivirus ssRNA Isometric -

Ovoid virases ssRNA Ovoid -

Drosophila X Virus dsRNA Isometric -

Tabel 1. Kelompok Virus Entomopatogen

ds= double-stranded, ss= single-stranded.

6

2 microns

Baculoviruses

Spodoptera littoralis

From Hunter-Fujita et al

7

Baculoviruses -

Mode of action

From Hunter-Fujita et al

8

Found only in invertebrates

No member of the family is known to infect plant or

vertebrate

Most have narrow host insect range, and infectivity is

restricted to the original host genus or family

Baculoviruses

Susceptibility of Alternative Hosts

9

Baculoviruses

Toxicity studies - mammals

Toxicity test results from 1970s/80s of 29 NPVs

indicated no toxicity or pathogenicity. Doses were

generally 10 – 100 x the “per acre” (1 acre = 0.45ha)

field rate equated to a 70kg person.

Heliothis zea NPV most extensively tested for toxicity in

humans and led to registration of “Elcar” by Sandoz in

USA.

10

Baculoviruses

Toxicity studies – mammals cont.

No effects of HzNPV found in:

Acute toxicity-pathogenicity tests in mouse, rat, guinea pig, rabbit,

monkey and man at 6x109 – 3 x 1012 OB / kg.

Skin irritation sensitivity tests in guinea pigs, rabbits and man at 106

and 107 OB / mm2skin.

Eye irritation tests in rabbits with 105 and 2x106 OB / eye

Subacute toxicity-pathogenicity tests and subcutaneous injection into

mice, rats, dogs and rhesus monkeys.

Teratogenicity and carcenogenicity studies in rats and mice at 109 –

3.5x1012 OB / kg.

Similar but less extensive results for many other NPVs from the

1970s/80s

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BaculovirusesToxicity studies – wildlife

Birds

Able to pass NPV through the alimentary tract unaffected

No deleterious effects

Aquatic organismsNo adverse effects

Beneficial insects

No direct effect on parasitoids, predators and pollinators

Indirect effects on parasitoids resulting from host death

12

Toxicity tests designed for testing effects of chemicals on

vertebrates are insufficient

Results reported in Gröner (1986) indicate no virus induced

antibody production in test mammals and chicken.

No cytogenetic effects of baculoviruses in mammalian cells either

in vivo or in vitro.

BaculovirusesPathology studies

13

AcNPV inoculated into vertebrate cells can be taken-

up and the degree of up-take depends on cell type,

temperature, time and viral phenotype.

BUT, none of the human and nonhuman vertebrate

lines tested showed evidence of viral replication.

NPVs unable to activate retroviruses in mammalian

cell lines

BaculovirusesVirus-cell interactions in vitro

15

Baculoviruses

a list of the baculoviruses regulated as pesticideactive ingredients by the US EPA Office of PesticidePrograms as of May 2005

Anagrapha falcifera NPV

Cydia pomonella GV

Douglas fir tussock moth NPV

Gypsy moth NPV

Helicoverpa zea NPV

Indian meal moth GV

Mamestra configurata NPV (pending)

Spodoptera exigua NPV

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Baculoviruses – US EPA fact sheet

III. ASSESSING RISKS TO HUMAN HEALTH

These viruses infect only the target insect larvae and closely related

species. Toxicity tests show that the viruses pose no risk to the public.

Workers wear protective clothing to prevent possible irritation from

handling and applying the product.

IV. ASSESSING RISKS TO THE ENVIRONMENT

Tests show that the GV and NPVs that EPA has registered as pesticide

active ingredients specifically infect only certain species of moth larvae.

The viruses do not harm other organisms, including plants, beneficial

insects, other wildlife, or the environment. These viruses occur naturally

in their insect hosts.

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Cypoviruses: Mode of action

Polyhedra ingested and dissolved in larval midgut

Virions released and attach to midgut columnar cells

Viral core enters cell cytoplasm

RNA transcription and replication

RNA occluded in capsules

Virus capsules occluded by virogenic stroma to form occlusion

bodies

18

Cypoviruses (Rheoviridae)

No CPV has been found infecting vertebrates or plants

(Belloncik, 1989)

Dendrolimus spectabilis CPV registered in Japan in

1974. Safety test results generally negative.

– Katagiri, K. (1981) Pest control by cytoplasmic polyhedrosos viruses. In:

Microbial control of pests and plant diseases 1970-1980. (Ed Burges, H.D.)

Academic Press.

Poxviridae:Entomopoxvirus

Member of the family of Poxviridae has a wide host,

including vertebrates and invertebrates.

Chicken pox and Small pox virus belong to this family.

The show allantoid – to brick-shaped virions, occluded within

ovoid OBs called Spheroids.

Entomopoxvirus has been isolated from 27 orthopterans,

lepidopterans, dipterans and coleopterans.

The subfamily Poxvirinae includes three genera, i.e.

Entomopoxvirus A, Entomopoxvirus B, and Entomopoxvirus C

Entomopoxvirus A infects only coleopteran species;

Entomopoxvirus B infects lepidopteran and coleopteran

species; Entomopoxvirus C infects only dipteran species.

The fourth group, group D, has been proposed by ICTV

which only attacks hymenopterans.

Poxviridae:Entomopoxvirus

Anomala cuprea (Coleoptera) larvae infected with an

entomopoxvirus show the symptoms of the infection

such as a whitish appearance and underdevelopment

(left, infected larva; right, healthy one).

Ascoviridae: Ascovirus

Members of the family of Ascoviridae are double strande

DNA (dsDNA) viruses that infect lepidopteran insects and

cause the unique pathology of forming virion containing

vesicles in the hemolymph of infected hosts.

The presence of the vesicles gives the hemolymph

a milky white appearance, which is a major characteristic

of the disease.

A few species of Ascovirus has been isolated only from

insects, specifically from Lepidopterans (Noctuidae).

Enveloped virions of ascoviruses are bacilliform, ovoid or

allantoid in shape, and occluded within vesicle-like OBs.

An ascovirus-infected caterpillar

Ascovirus symptoms

In cases where a Microplitis wasp has both parasitised a caterpillar and

infected it with ascovirus, the symptoms seen are those of the disease rather

than of the parasitoid. When ascovirus kills the caterpillar, it also kills the

developing Microplitis larva.

Caterpillars infected with ascovirus will generally stop eating within two

days. They stop growing, but can live for weeks in a lethargic state before

they die.

The blood of an ascovirus-infected caterpillar is white and creamy,

whereas the blood of a healthy caterpillar is clear. Blood colour gives the

best diagnosis in the laboratory and can be tested by splitting or pricking the

caterpillar.

Ascoviridae: Ascovirus

Iridoviridae: Iridovirus

Invertebrate Iridescent Viruses (IIVs) (family Iridoviridae)

are known to infect a number of agricultural pests,

medically important insect vectors, and terrestrial isopods that

live in damp or aquatic habitats.

The major characteristic of this family is the presence of

iridescent blue, green, orange, or purple coloration in heavily

infected individuals.

The small iridovirus tend to display colors from violet to

turquoise.

The viral structure is non-enveloped, non-occluded,

isocahedral viral particles.

Iridoviridaeviridae: Iridovirus

Although some iridoviruses infect frogs and fishes, those

infecting insects belong to two genera: Iridovirus, whose viral

particles fluctuate between 120 to 130 nm in size.

They mostly infect arthropods, particularly insects, in damp

or aquatic habitats worldwide (see complete list of

invertebrate hosts.

They are highly infectious by injection but have low

infectivity by ingestion.

Horizontal transmission can occur by cannibalism or

predation of patently infected individuals, or the virus may

even be vectored by nematodes and parasitoid wasps that

introduce viral particles into the host insect during the act of

penetration or oviposition.

Iridoviridaeviridae: Iridovirus

Iridovirus infected (blue) larva of Aedes aegypti

next to a healthy larva.

Polydnaviridae

Polydnaviridae only infects endoparasitic Hymenoptera.

Member of this family show non-occluded, ovoid virions,

containing multipartite dsDNA

ICTV recognizes two genera within this family, including

Ichneovirus and Bracovirus.

Life cycle of of parasitoid wasps and Polydnaviruses (PDVs) parasitizing a

lepidopteran larval host

Perbanyakan Virus

A. Perbanyakan in vivo

Walaupun produksi skala besar virus di dalam serangga hidup

membutuhkan tenaga kerja yang banyak, perbanyakan secara in vivo

masih layak digunakan.

Bagi laboratorium skala kecil, memberi makan virus diikuti dengan

memanen serangga terinfeksi adalah standar produksi stok virus.

Perbanyakan virus di dalam kultur sel akan beresiko kehilangan

kekhasan genetik pada sistem in vitro .

1. Propagation of Cydia pomonella granulovirus

(CpGV) in C. pomonella larvae

1.Rear neonate codling moth larvae for 10 days on virus-free artificial diet.

2.After 10 days, larvae reach instar L4eL5.

3.Pipette 1 ml virus suspension containing 1000 OBs on the surface of a

small piece of diet (about 2 × 2 ×2 mm).

4.Keep larvae single in a well containing one piece of contaminated diet

and incubate them for 24 h at 26○C.

5.Transfer larvae, which have eaten the contaminated

diet completely, to virus-free diet.

6.Incubate larvae at 26○C until infection is visible (usually after 5e6 days)

and monitor daily.

7.Collect infected larvae before tissue rupture.

8.Collected larvae can be frozen at —20○C until virus purification

Virus propagation follows the same protocol for small- and large-scale

production. Infection of about 200 codling moth larvae following this

protocol will usually result in 5 ml purified virus suspension with a

concentra- tion of about 1011 OB/ml.

It is recommended to feed more larvae, because not every larva will

eat its diet plug completely or show symptoms of virus infection.

Quality control of in vivo virus propagation

Perbanyakan virus di dalam tubuh serangga hidup dapat

menyebabkan variasi produk dalam hal komposisi dan kemurnian

– mikroorganisme lain yang hadir di dalam serangga dapat

menyebabkan kontaminasi produk akhir.

Oleh karena itu ―quality control" yang baik sangat diperlukan.

Stok virus yang digunakan untuk perbanyakan harus

menggunakan strain virus yang sudah dikarakterisasi sebelumnya

dengan analisa DNA restriction analysis untuk menentukan

apakah isolat tunggal atau campuran genotip.

Purifikasi inokulum dengan meminimalisasi resiko kontaminasi

dengan protozoa atau spora bakteri yang dapat mempengaruhi

replikasi virus.

Setiap virus yang dihasilkan harus diestimasi dengan

penghitungan atau metode lain yang sesuai dan diuji aktivitas

biologinya dengan bioassay.

B. Isolasi virus dari serangga

Isolasi virus dari inang yang terinfeksi biasanya tahapan lanjut

setelah perbanyakan virus.

Sangat diperlukan suspensi virus yang sangat murni.

Untuk uji bioassay, suspensi virus harus bebas dari mikroorganisme

yang mempengaruhi proses infeksi.

1. Homogenization and filtration

Serangga mati di-homogenisasi di dalam deterjen anionic yang rendah

konsentrasi untuk memfasilitasi lepasnya jaringan tubuh serangga dan

melepaskan partikel.

Membekukan larva sebelum homogenisasi juga membantu untuk merusak

sel.

Suspensi homogenisasi masih mengandung residu makanan rering, kapsul

kepala, dan bagian besar integumen, sehingga perlu disaring dengan is

therefore filtered through saringan kain (cheese-cloth / gauze).

2. Centrifugation

Suspensi virus yang disaring masih mengandung lemak, bakteri dan

partikel halus non-virus lainnya.

Dengan proses sentrifugasi beberapa kali virus dapat terpisah dari

kontaminan ini.

3. Purifikasi

Figure 1. Example of a NPV [Agrotis segetum NPV Oxford strain (AgseNPV-

B)] under the light microscope using an improved Neubauer hemocytometer

(depth 0.1 mm). The edge of each small square is 0.05 mm × 40 (for further

information see text). B. A granulovirus [Cydia pomonella granulovirus

(CpGV)] as seen by dark-field illumination under the light microscope. For

observation a PetroffeHausser counting chamber (depth 0.02 mm) is used.

The edge of each small square is 0.05 mm × 40.

Figure 2.. Basic bioassay procedures for LD50 and LC50 determination. A. For the diet plug method, a known

dosage of virus suspension is pipetted on a small piece of diet and fed to one test larvae each until full

consumption. Larvae are then reared on virus-free diet. B. Droplet feeding: single droplets of virus suspension

containing a known dosage of virus mixed with food dye are fed to single larvae. Larvae which have ingested the

droplet are then reared on virus-free diet. C. For surface contamination, virus suspension is added to a known

unit of artificial diet to cover the surface. After a short time of feeding, larvae are transferred to virus-free diet.

Concentration is given per mm2. D. Diet incorporation: virus suspension is mixed directly to a measured volume

of artificial diet. Test larvae feed on the contaminated diet until the end of the bioassay.

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