Kuliah 11_Perbanyakan virus sebagai agens antagonis_2018

Perbanyakan Massal

Perbanyakan Massal

Virus sebagai Agens

Virus sebagai Agens

Antagonis

Antagonis

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

• 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).



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)





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

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.

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.

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

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,

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

6

2 microns

Baculoviruses

Spodoptera littoralis

7

Baculoviruses

-Mode

of

action

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

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.

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.

11

Baculoviruses

Toxicity

studies

–

wildlife

Birds

Able to pass NPV through the alimentary tract unaffected No deleterious effects

Aquatic organisms

No adverse effects

Beneficial insects

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.

Baculoviruses

13

AcNPV inoculated into vertebrate cells can be

taken-up and the degree of taken-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

Baculoviruses

15

Baculoviruses

a list of the baculoviruses regulated as pesticide

active ingredients by the US EPA Office of Pesticide

Programs 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)

16

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

17

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

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.

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

Poxviridae:Entomopoxvirus

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

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.

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,

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

Iridoviridaeviridae: Iridovirus

Iridovirus infected (blue) larva of Aedes aegypti

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

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.

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.

 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.

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

B. Isolasi virus dari serangga

 Isolasi virus dari inang yang terinfeksi biasanya tahapan lanjut setelah perbanyakan virus.

 Sangat diperlukan suspensi virus yang sangat murni.

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.

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}