AP415A

AP415A - Recombinant tospovirus DNA constructs and plants comprising such constructs.

Info

Publication number: AP415A
Application number: APAP/P/1993/000496A
Authority: AP
Inventors: Grinsven Martinus Quirinus Joseph Marie Van; Haan Petrus Theodorus De; Johannes Jacobus Ludgerus Gielen; Dirk Peters; Robert Willem Goldbach
Original assignee: Novartis Ag
Priority date: 1992-03-19
Filing date: 1993-03-18
Publication date: 1995-09-29
Also published as: AU664241B2; EP0566525A2; AU3523793A; GB9206016D0; MX9301522A; EP0566525A3; US5773700A; ZA931985B; IL105086A0; CA2091806A1; AP9300496A0; JPH067170A

Abstract

Recombinant Impatiens Necrotic Spot Virus (INSV) DNA constructs

Description

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The present invention relates to. plants having reduced susceptibility to infection from tospoviruses, genetic material capable of generating tolerance to tospoviruses , probes suitable for isolating and diagnosing , and processes for obtaining such plants and genetic material and probes .

Viral infections in plants are frequently responsible for detrimental effects in growth, undesirable morphological changes , decreased yield and the like. Such infections often result in a higher susceptibility to infection in infected plants to other plant pathogens and plant pests.

' Transmission of plant viruses generally occurs via insect or fungal carriers or may occur through mechanical means.

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Plant breeders continuously look to develop varieties of crop plant species tolerant to or resistant to specific virus strains. In the past , virus resistance conferring genes have been transferred from wild types related to commercial plants into commercial varieties through breeding. The transfer of an existing resistance in the wild from the wild type gene pool to a cultivar is a tedious process in which the resistance conferring gene(s) must first be identified in a source (donor) plant species and then combined into the gene pool of a commercial variety. Resistance or tolerance generated in this way is typically active only against one or at best a few strains of the virus in question . One disadvantage of breeding cultivars

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AP Ο Ο Ο 4 Ο 3 for resistance to a particular virus species is that there is often a lack of a gene source suitable for conferring disease resistance within the crop species.

Other approaches to limit the effect of virus induced disease on plants include the use of chemicals such as insecticides, fungicides and the like which act against virus carriers , and/or rely on the employment of preventative methods such as efficient phytosanitary working conditions . However, the use of chemicals to combat virus disease by killing the carrier is' subject to increasingly tougher governmental regulations which present growers with a decreasing scala of permitted chemical plant-protectants.

In an alternative , a system referred to as crossprotection may be employed . Cross-protection is a phenomenon in which infection of a plant with one strain of a virus protects that plant against superinfection with a second related virus strain. The cross-protection method preferentially involves the use of avirulent virus strains to infect plants , which act to inhibit a secondary infection with a virulent strain of the same virus. However, the use of a natural cross-protection system can have several disadvantages. The method is very labour intensive because it requires inoculation of every plant crop , and carries the risk that an avirulent strain may mutate to a virulent strain, thus becoming a causal agent for crop disease in itself. A further possible hazard is that an avirulent virus strain in one plant species can act as a virulent strain in another plant species.

Several studies have indicated that the viral coat protein of the protecting virus plays an important role in crossprotection and that protection occurs when the resident virus and the challenging virus have the same or closely related coat protein structures.

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Recent developments in gene manipulation and plant transformation techniques have given rise to new methods for generating virus resistance in plants. Genetically engineered cross-protection is a form of virus resistance which phenotypically resembles natural cross-protection , but is achieved through the expression of genetic ( information of a viral coat protein from the genome of a genetically manipulated plant . Generation of virus resistance via genetic engineering has been described in for instance , EP 223 452 and reported by Abel et al [(1986) Science 232:738-743] . It was shown that expression of the tobacco mosaic virus strain Ul (TMV-Ul) coat protein gene from the genome of a transgenic plant resulted in a delay of symptom development after infection with any TMV strain. Similar results with respect to coat protein-mediated protection have also been obtained for alfalfa mosaic virus (AMV), potato virus X (PVX) and cucumber mosaic virus (CMV).

Although TMV, CMV, AMV and PVX belong to different virus

C groups, they share a common architecture: in all such viruses the viral RNA is a positive strand RNA encapsidated ( by a viral coat consisting of many individual but identical viral coat proteins.

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However , tospoviruses are essentially different from the plant viruses mentioned above. The genus tospovirus belongs to the family Bunyaviridae. All tospoviruses are transmitted by thrips. The virus particles are spherical in shape (80120 nm in diameter) and contain internal nucleocapsids surrounded by a lipid envelope studded with glycoprotein surface projections. The multipartite genome consists of linear single stranded RNA molecules of negative or ambisense polarity. The terminal nucleotides of these RNA molecules are characterised by a consensus sequence as follows: 5' AGAGCAAUX....................GAUUGCUCU 3', wherein X is C or U . Members of the tospovirus group include tomato spotted wilt virus (TSWV), Impatiens necrotic

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spot virus (INSV), and tomato chlorotic spot virus (TCSV), also known as tomato mottled spot virus (TMSV) or TSWV-like isolate BR-O3. A general description of a tospovirus, using TSWV as a representative of the genus tospoviruses can be found in our co-pending application EP 426 195 herein incorporated by reference .

The tospovirus particle contains at least 4 distinct structural proteins: an internal nucleocapsid protein N of 29 kd and two membrane glycoproteins: Gl, approximately 78 kd, and G2 approximately 58 kd. In addition, minor amounts of a large protein, L, approximately 260 kd have been detected in virus particles. Tospoviral genomes consist of three linear single stranded RNA molecules of about 2900 nucleotides (nt) (S RNA) , about 5000 nt , (M RNA) and about 8900 nt (L RNA) , each tightly associated with nucleocapsid proteins and a few copies of the L> protein to form circular nucleocapsids. A schematic structure outlining most properties of an INSV is given in Figure 1. Based on the above and other properties , INSV (like TSWV) has been classified as a member of the tospovirus genus.

Circumstantial evidence has been presented which suggests that an M RNA encoded gene is directly or indirectly involved in the synthesis of the Gl membrane glycoprotein [Verkleij and Peters ,(1983) J. Gen. Virol. 64:677-686] .

As mentioned above, tospoviruses such as TSWV , INSV and the like are transmitted by certain species of thrips. These tospovirus carriers belong to the family Tripidae and include tobacco thrips (Frankliniella fusca (Hinds.),, western flower thrips (F. occidentalis (Pergande)), common blossom thrips (F. Schultzei (Trybom)), chilli thrips (Scirtothrips dorsalis (Hood)), Thrips setosus (Moulton), onion thrips (T. tabaci (Lindeman, ), F. intonsa and melon thrips (T. palmi (Karny)). The tospovirus is acquired by

AP Ο Ο Ο 4 Ο 3 the virus before they pupate but adults more commonly transmit the virus. Adult thrips can remain infective throughout their lives .

Tospoviruses are widespread in temperate, subtropical and tropical climate zones throughout the world . The current distribution of tospoviruses covers all continents and makes them one of the most widely distributed of groups of plant viruses. At least 370 plant species representing 50 plant * ^J families, both monocotyledons and dicotyledons , are

-—i naturally infected by tospoviruses of the Bunvaviridae.

Tospoviruses seriously affect the production of food and ornamental crops . Symptoms of tospovirus infection in plants include stunting, ringspots, dark purple-brown sunken spots, stem browning, flower breaking, necrotic and pigmental lesions and patterns, yellows and non-necrotic mottle, mosaic in greens or even total plant death. Most plant hosts display only a few of these symptoms, however, the wide range of symptoms produced by tospovirus infection has complicated diagnosis of the disease and has led to individual diseases being given several different names . A further complication is that tospovirus symptoms within the same plant species may vary depending on the age of the plant, time of infection during the life-cycle of the plant , nutritional levels , environmental conditions, such as temperature , and the like.

Although TSWV has been known for many years, is widely distributed, and is the causal agent of a disease which leads to significant loss in yield in crops and ornamentals, limited progress has been made in identifying sources of genes capable of conferring resistance to TSWV or other tospoviruses . A monogenic TSWV tolerance has been identified in Lycopersicon peruvianum, but this trait has not been transferred to cultivated tomatoes so far , nor has a resistance source been identified for other crop species . The use of natural cross-protection systems to

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AP Ο Ο Ο 4 0 J decrease the invasive effects by tospovirus strains capable of causing damage is not well documented. Limited positive results have been reported for tomato and lettuce.

The introduction of genetic information capable of conferring resistance or tolerance to tospoviruses into plant gene pools by means of genetic manipulation provides the breeder and grower alike with a new method for combatting tospovirus induced disease. In particular, it has been found that genetic manipulation techniques may be employed to confer resistance to INSV related disease in plants .

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Detailed Description

According to the present invention there is provided a recombinant INSV DNA construct comprising a DNA sequence coding for transcription into

a) an RNA sequence of an INSV or an RNA sequence homologous thereto ;

b) an RNA sequence of an INSV or an RNA sequence homologous thereto capable of encoding for an INSV protein or a part thereof , in which one or more codons have been replaced by synonyms , or an RNA sequence homologous thereto ; or

c) an RNA sequence complementary to an RNA sequence according to a) or b) , which INSV DNA is under expression control of a promoter capable of functioning in plants and includes a terminator

V ' capable of functioning in plants.

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The DNA sequences defined under a), b) and c) above, for the purposes of the present invention will be referred to as INSV Related DNA Sequences hereinafter. An INSV Related DNA Sequence according to the invention may be modified as appropriate to create mutants or modified sequences homologous to such INSV Related DNA Sequences from which they are derived, using methods known to those skilled in the art such as site-directed mutagenesis and the like. Such mutants or modified coding sequences are embraced within the spirit and scope of the invention.

The term RNA sequence of an INSV may refer to a sequence of the S, M or L RNA strand, preferably an S or M

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The term ^Η RNA sequence homologous to an RNA sequence of an INSV refers to an RNA sequence of an INSV wherein a number of nucleotides have been deleted and/or added but which is still capable of hybridization to a nucleotide sequence complementary to an RNA sequence of an INSV under appropriate hybridization conditions. For the purposes of the present invention appropriate hybridization conditions may include but are not limited to , for example , an incubation for about 16 hours at 42’C , in a buffer system comprising 5 x standard saline citrate (SSC) , 0.5% sodium dodecylsulphate (SDS) , 5 x Denhardt ’ s solution , 50% formamide and 100 gg/ml carrier DNA· (hereinafter the buffer

Sx- system) , followed by washing 3x in buffer comprising 1 x

SSC and 0.1% SDS at 65'C for approximately an hour each time .

Preferably , hybridization conditions employed in the present invention may involve incubation in a buffer system for about 16 hours at 49'C and washing 3x in a buffer comprising 0.1 x SSC and 0.1% SDS at 55^-C for about an hour each time . More preferably , hybridization conditions may involve incubation in a buffer system for about 16 hours at 55‘C and washing 3x in a buffer comprising 0.1 x SSC and 0.1% SDS at 65’C for approximately an hour each time .

The length of the INSV Related DNA Sequence will i.a. depend on the particular strategy to be followed , as will become apparent from the description hereinafter . In general , the INSV Related DNA Sequence may comprise at least 20 , and suitably 50 or more nucleotides .

The term promoter refers to the nucleotide sequence upstream from the transcriptional start site and which contains all the regulatory regions required for transcription, including the region coding for the leader sequence of mRNA (which leader sequence comprises the

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AP Ο Ο Ο 4 Ο 3 ribosomal binding site and initiates translation at the AUG start codon) .

Examples of promoters suitable for use in DNA constructs of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in plant cells. The promoter may express the DNA constitutively or differentially . Suitable examples of promoters differentially regulating DNA expression are promoters inducible by disease carriers, such as thrips, e.g. so-called wound-inducible promoters. It will be j-- appreciated that the promoter employed should give rise to the expression of an INSV Related DNA Sequence at a rate sufficient to produce the amount of RNA necessary to decrease INSV susceptibility in a transformed plant. The required amount of RNA to be transcribed may vary with the type of plant. Particularly preferred promoters include the cauliflower mosaic virus 35S (CaMV 35S) promoter, derivatives thereof, and a promoter inducible after wounding by a disease carrier such as thrips, e.g. a wound inducible promoter. Examples of further suitable promoters include nopaline synthase, octopine synthase and the like.

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The term terminator refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription . Terminators are DNA 3 ‘ -non-translated sequences that contain a polyadenylation signal, that causes the addition of polyadenylate sequences to the 3 ¹ end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants. Examples of terminators particularly suitable for use in the DNA constructs of the invention include the nopaline synthase terminator of A. tumefaciens, the 35S terminator of CaMV and the zein terminator from Zea mays.

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In accordance with the present invention , an RNA sequence is complementary to another RNA sequence if it is able to form a hydrogen-bonded complex therewith , according to rules of base pairing under appropriate hybridization conditions (as described hereinabove) .

The present invention also provides a vector capable of introducing the DNA construct of the invention into plants and methods of producing such vectors.

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The term vector as employed herein refers to a vehicle with which DNA constructs of INSV or fragments thereof may be incorporated into the cells of a host organism.

The term plants refers to differentiated plants as well as undifferentiated plant material such as protoplasts, plant cells, including cybrids and hybrids, seeds, plantlets and the like which under appropriate conditions can develop into mature plants , progeny thereof and parts thereof such as cuttings , fruits of such plants and the like.

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The invention further provides plants comprising in their C genome a DNA construct of the invention, and methods of producing such plants. Such methods include plant breeding, plantlets derived from protoplast fusion and the like.

The plants according to the invention have reduced susceptibility to diseases induced by INSV or diseases related to INSV infection and suffer from substantially fewer or none of the disadvantages and limitations of plants obtained by classical methods as mentioned hereinabove.

Many types of plants are susceptible to INSV infection however only in some types is INSV infection known to give

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Such types of plants include the ornamental or flowering plants. Examples of such plants include but are not limited to Ageratum , Amaranthus , Anthirrhinum , Aquilegia , 3egonia , Chrysanthemum , Cineraria , clover. Cosmos , cowpea , Cyclamen , Dahlia , Datura , Delphinium ,

Gerbera , Gladiolus , Gloxinia , Hippeastrum , Impatiens , Mesembryanthemum , petunia , Primula , Saint Paulia , Salpiglossis , Tagetes , Verbena , Viola , Vinca ,

Zinnia , Pelargonium and the like .

Other types of plants may be susceptible to INSV infection but these plants may not present disease symptoms directly associated with INSV infection, however such plants may present symptoms of a disease as a result of a secondary infection by a different organism made possible as a result of an initial infection by INSV. Such plants may therefore be viewed as being the subject of an INSV infection related disease and may include plants selected from a wider group of plant types. Further examples of this group of plant types may include vegetable and other crops . Such crop types include alfalfa , aubergine , beet , broad bean , broccoli , brussels sprouts , cabbage , cauliflower , celery , chicory , cow pea , cucumber , endive , gourd , groundnut , lettuce , melon , onion , papaya , pea , peanut , pepper , pineapple , potato , safflower , snap bean , soybean , spinach , squash , sugarbeet , sunflower , tobacco , tomato , water melon and the like .

The invention relates in particular to ornamental plants and preferably to those listed ornamental plants comprising in their plant genome a DNA construct of the invention.

The particular features of tospoviruses including those of INSV are illustrated hereinafter.

The S, M and L RNA are single stranded RNA molecules. The S RNA of INSV is about 3000 nucleotides long( SEQ. ID No.l ;

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SEQ ID No. 2 ) and comprises two genes, one (SEQ ID No.3) encoding a non-structural protein (NSs) in viral sense, the other one (SEQ ID No.11) encoding the nucleocapsid protein (N) in viral complementary sense. The intergenic region between the NSs- and N-gene can be folded into a secondary structure (Seq ID No. 7 and SEQ ID No.8) . The 5'- and 3‘terminal sequences of the S RNA are capable of hybridizing to each other such that the first nucleotide is opposite (and complementary) to the last nucleotide of said S RNA strand . For the purposes of the description the doublestranded structure obtained by hybridizing both RNA termini (' will be referred to as a pan-handle (SEQ ID No.5 and SEQ

ID NO. 6) hereinafter.

The M RNA strand of INSV comprises about 5000 nucleotides (SEQ ID No. 14) . It contains at least two open reading frames, one encoding a non-structural protein (NSm) in viral sense ( SEQ ID No.15), and another open reading frame (SEQ ID No.21) in viral complementary sense. This open reading frame is translated on polysomes located on the endoplasmic reticulum where the nascent polypeptide chain is cleaved co-translationally to form the spike proteins G1 and G2 respectively. As with S RNA, the termini of the M RNA strand are complementary to each other and may likewise

C hybridize to form a pan-handle ( SEQ ID No.13 and SEQ

ID No.19).

The L RNA strand of INSV comprises about 8900 nucleotides. It contains complementary 3' and 5' ends for a length of from about 50 to about 80 nucleotides. The RNA has a negative polarity, with one open reading frame (ORF) located as the viral complementary strand. This ORF corresponds to a primary translation product of about 2875 amino acids in length with an anticipated Mw of between about 300,000 to about 350,000. Comparison with the polymerase proteins of other negative strand viruses indicates that this protein probably represents a viral

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Ο polymerase. In some mutant strains, shortened L RNA molecules have been found in addition to the wild type, full length L RNA. These shortened L RNAs however are observed to possess the characteristic terminal nucleotide sequences and thus are capable of forming “pan handle* structures. They are also encapsidated with nucleocapsid protein and are included in virus particles. Their presence suppresses symptom development resulting in less severe detrimental effect . Thus , these shortened L RNA molecules

L- can be regarded as defective interfering (DI) RNAs . A ς defective interfering RNA is one which is capable of interfering in replication by competing with other genomic RNAs for polymerases and therefore is capable of being replicated , and by so doing inhibits the replication and/or expression of other genomic RNAs with which it is competing .Thus , a DI RNA may comprise any RNA sequence which is capable of being replicated and may be an L , S , or M RNA within the context of the present invention . Such DI RNA sequences may comprise RNA sequences which have had nucleotides either deleted from or added thereto provided that they are capable of competing for polymerases and of £ replicating .

( A preferred embodiment of the invention relates to DNA constructs of the invention coding for transcription into INSV RNA sequences of a pan-handle (SEQ ID No.5 , SEQ ID NO. 6 ; SEQ ID No. 18 , SEQ ID No. 19), or into INSV RNA sequences homologous thereto.

Another preferred embodiment of the invention relates to DNA constructs of the invention coding for transcription into INSV-RNA sequences of an open reading frame in viral complementary sense i.e. having negative polarity , or into corresponding RNA sequences in which one or more codons have been replaced by their synonyms, or into RNA sequences

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A further preferred embodiment of the invention relates to DNA constructs of the invention coding for transcription into INSV-RNA sequences of a hairpin ( SEQ ID No.7 , SEQ ID No. 8 ; SEQ ID No. 13 , SEQ ID No. 16 ) , or into RNA sequences homologous thereto.

Preferably, the INSV-RNA sequence referred to hereinabove has at least 20 nucleotides. Preferably, the INSV-RNA sequence has at least 50 nucleotides.

Examples of DNA constructs suitable for use according to the invention include INSV-Related DNA Sequences coding for transcription into (reference is made to the sequence listing );

i) the viral S RNA nucleotide sequence from 1 to 3017 (SEQ. ID NO.1) ii) the viral S RNA nucleotide sequence from position 25 to 3017 (SEQ. ID No.2);

iii) the viral S RNA nucleotide sequence from 87 to 1436 (SEQ. ID NO.3);

iv) the viral S RNA nucleotide sequence from 2030 to

2868 (SEQ. ID No.4);

v) the viral S RNA pan-handle ’ structure comprising;

a) a first nucleotide sequence of from about 30 to about 36 nucleotides in length from the 5' end of the viral S RNA

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b) a second nucleotide sequence of from about 30 to about 36 nucleotides in length from the 3' end of the viral S RNA

vi)	the viral 2079; (SEQ	S ID	RNA No.	nucleotide 7)	sequence	from	1437	to
vii)	the viral	s	RNA	nucleotide	sequence	from	1440	to
	2041; (SEQ	ID	No.	8)

Q viii) the viral complementary S RNA nucleotide sequence from 1 to about 3017; (SEQ ID No.9) ix) the viral complementary S RNA nucleotide sequence from 1 to 2993; (SEQ ID No.10)

x) the viral complementary S RNA nucleotide sequence from 150 to 933; (SEQ ID No.11) xi) the S RNA nucleotide sequence from 1581 to 2930 of the viral complementary S RNA strand; (SEQ ID No.12);

c xii) the viral complementary S RNA secondary structure having a nucleotide sequence of 642 nucleotides from 939 to 1580; (SEQ ID No.13) xiii) S RNA nucleotide sequence from 87 to 1436 in which one or more codons have been replaced by their synonyms;

xiv) S RNA nucleotide sequence from 2080 to 2868 in which one or more codons have been replaced by their synonyms;

xv) the M RNA nucleotide sequence from 1 to 4970 (SEQ ID No.14);

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AP Ο Ο Ο 4 Ο 3 xvi) the Μ RNA sequence from 86 to 997 (SEQ ID No.15);

xvii) the M RNA sequence of the intergenic region from 998 to 1470 (SEQ ID No.16);

	xviii)	the M RNA 17)	sequence from 1471 to 4884; (SEQ ID No.
	xix)	the M RNA pan-handle structure comprising ; a) a first nucleotide sequence of from about 30 to about

nucleotides in length from the 5' end of the viral M RNA and

b) a second nucleotide sequence of from about 30 to about 36 nucleotides in length from the 3' end of the viral M RNA xx) the complementary viral M RNA sequence from 1 to 4970; (SEQ ID No.20)

O xxi) the complementary viral M RNA sequence from position 87 to position 3500 of the complementary viral M RNA sequence; ( SEQ ID No.21) xxii) the complementary viral M RNA sequence from position 3974 to 4885 (SEQ ID No.22) xxiii) rna sequences homologous to the nucleotide sequences defined under i) to xii) and xv, to xxii) hereinabove.

xxiv) fragments of sequences defined under i) to xxii)

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Preferred INSV-Related DNA Sequences code for transcription into the RNA sequences according to sequences iv) to xii) and xv) to xxii) as defined above, or into RNA sequences homologous thereto, or into fragments thereof comprising at least 15 nucleotides, more preferably at least 20 nucleotides, and most preferably at least 50 nucleotides.

According to another preferred embodiment of the invention the DNA constructs of the invention comprise INSV Related θ DNA Sequences coding for transcription into a combination of the 5' and 3' terminal sequences (ie pan-handles) of

C viral S, M or L RNA respectively, more preferably of S or M

RNA, and most preferably of S RNA . Examples of S RNA and M RNA terminal sequences include

i) a first nucleotide sequence 36 nucleotides in length from the 5' end of the viral S RNA :

' AGAGCAATNN NNNNNNNNNN NNNNGAACAAC CCAAGC 3 ' (SEQ ID No.5 ie nucleotides from position 1 to 36 of SEQ ID No.l , where N stands for A,T,G,or

C C) c

and a second nucleotide sequence 36 nucleotides in length from the 3‘ end of the viral S RNA:

5' GATTATATG ATGTTATATT CGTGACACAA TTGCTCT 3' (SEQ ID No.6 ie nucleotides from position 2981 to 3017 of SEQ ID No.l) ii) a first nucleotide sequence of 36 nucleotides in length

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5¹ AGAGCAATCA GTGCATCAAA ATTATATCTA GCCGAA 3¹ (SEQ ID No.18 ie nucleotides from position 1 to 36 of SEQ ID NO.13) and

b) a second nucleotide sequence 36 nucleotides in length from the 3' end of the viral M RNA

5' TGTTGTATGT AGAGATTTTG TTTGCACTGA TTGCTC T 3' (SEQ ID No.19 ie nucleotides from position 4941 to 4970 Of SEQ ID No. 13)

In the case of the terminus at the 5' end of the S RNA it is not known whether or not there are sixteen or seventeen nucleotides in the unknown region demarked by a series of N s , however the exact number of nucleotides in this region is not considered to be critical to the formation of pan-handle structures so long as the 5' end of the S RNA is capable of complementing the 3‘ end of the S RNA thus enabling the formation of a pan-handle structure .

The invention further provides probes suitable for use as diagnostic tools for the diagnosis of disease in plants suspected of being infected with INSV tospoviruses. Such probes comprise a labeled oligonucleotide (RNA or DNA) sequence complementary to an RNA sequence of an INSV tospovirus. The desired length of the sequence and appropriate method for diagnostic use of probes are known by those skilled in the art. A suitable probe may comprise a nucleotide sequence of at least 12 to about 800 nucleotides, preferably at least 15, more preferably more than 30 nucleotides, and most preferably from about 400 to bad ORIGINAL

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600 nucleotides complementary to an RNA sequence of an INSV tospovirus .

Probes according to the invention are helpful in identifying INSV tospovirus RNA or parts thereof in infected plant material i.a. for diagnostic purposes prior to full presentation of disease symptoms in plants .

The invention accordingly also provides a diagnostic method of determining INSV tospovirus infection in plants which comprises detecting INSV tospovirus replicative forms employing the probes of the invention in dot-blot type assays .

Probes according to the invention are useful in the construction of and use of chimeric genes comprising a DNA sequence corresponding to an RNA sequence of an INSV tospovirus.

The DNA constructs of the invention may be obtained by insertion of an INSV Related DNA Sequence in an appropriate expression vector, such that the sequence is brought under expression control of a promoter capable of functioning in plants and its transcription is terminated by a terminator capable of functioning in plants.

The term “appropriate expression vector as used herein refers to a vector containing a promoter region and a terminator region which are capable of functioning in plant cells.

The insertion of an INSV Related DNA Sequence into an appropriate expression vector may be carried out in a manner known per se. Suitable procedures are illustrated in

Likewise the construction of an appropriate expression vector may be carried out in a manner known per se.

AP000403

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Plants according to the invention may be obtained by

a) inserting into the genome of a plant cell a DNA construct as hereinbefore defined;

b) obtaining transformed cells; and

c) regenerating from the transformed cells genetically transformed plants.

DNA vectors of the present invention may be inserted into the plant genome of plants susceptible to INSV infection. Such plant transformation may be carried out employing techniques known per se for the transformation of plants, such as plant transformation techniques involving Ti plasmids derived from Agrobacterium tumefaciens, A. rhizogenes or modifications thereof, naked DNA transformation or electroporation of isolated plant cells or organized plant structures, the use of micro-projectiles to deliver DNA, the use of laser systems, liposomes, or viruses or pollen as transformation vectors and the like.

Plants of the invention may be monitored for expression of an INSV-Related DNA Sequence by mechods known in the art, including Northern analysis, Southern analysis, PCR techniques and/or immunological techniques and the like. The plants of the invention show decreased susceptibility to INSV infection as demonstrated by tests whereby the plants are exposed to INSV preferentially at a concentration in the range at which the rate of disease symptoms correlates linearly with INSV concentration in the inoculum.

Methods suitable for INSV inoculation are known in the art and include mechanical inoculation, and in particular, the use of appropriate vectors.

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Plants of the invention may also be obtained by the crossing of a plant obtained according to the methods of the invention with another plant to produce plants having in their plant genome a DNA construct of the invention.

The invention is illustrated by the following non-limiting examples and accompanying figures.

Figure 1: Schematic representation of an INSV particle .

Figure 2; Sequence strategy for INSV viral S RNA.

Figure 3: Open reading frame analysis of the INSV S RNA, full bars represent translational stop codons (TAA, TAG, TGA), half size bars indicate start codons (ATG).

Figure 4: Schematic review of the construction of a suitable expression vector (pZU-B).

Figure 5 suitable sequence.	: Schematic review of the	construction of a V N protein-coding
plasmid	comprising	the	INS’
Figure 6	: Schematic review	of	the	construction of a
suitable	plasmid	comprising	the	INSV	NSs protein-coding

sequence.

Figure 7: Schematic review of the construction of a suitable plasmid comprising the INSV NSm protein-coding sequence.

Figure 8: Schematic review of the construction of a suitable plasmid comprising the INSV G1/G2 glycoprotein precursor-coding sequence.

Figure 9: Schematic review of the construction of a INSV N gene-containing plant transformation vector.

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Figure 10: Schematic review of the construction of a INSV NSs gene-containing plant transformation vector.

Figure 11: Schematic review of the construction of a INSV G1/G2 glycoprotein precursor gene-containing plant transformation vector.

Figure 12: Schematic review of the construction of a INSV NSm gene-containing plant transformation vector.

Figure 13: The secondary structure located at the intergenic region of INSV S RNA.

Suitable examples of preferred INSV Related DNA Sequences coding for transcription into a sequence of the secondary structure of the intergenic region of S RNA or of RNA sequences homologous thereto are sequences coding for the 1437 to 2079 nucleotide sequence of S RNA or for a sequence homologous to such sequences.

Other advantageous features of the present invention will

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MATERIAL AND METHODS

All INSV RNA-derived sequences presented here are depicted as DNA sequences for the sole purpose of uniformity. It will be appreciated that this is done for convenience.

Cultivars of Nicotiana t a ba cum and Petunia hybrida, used in plant transformation studies, are grown under standard greenhouse conditions. Axenic explant material is grown on θ standard MS media [Muras’nige and Skoog, (1962) Physiol Plant

15: 473-497] containing appropriate phytohormones and sucrose concentrations.

E. coli bacteria are grown on rotary shakers at 37°C in standard LB-medium. Agrobacterium tumefaciens strains are grown at 28°C in MinA medium supplemented with 0.1 % glucose [Ausubel et al., (1987) Current Protocols in Molecular Biology , Green Publishing Associates and Wiley Intersciences , New York , Chichester , Brisbane , Toronto , and Singapore] .

In all cloning procedures the Ξ. coli strain JM83, (F~, ,—

V. A(lac-pro), ara, rpsL, 080, dlacZMl5) is used as a recipient for recombinant plasmids.

(

Binary vectors are conjugated to Agrobacterium tumefaciens strain LBA 4404, a strain containing the Ti-plasmid vir region, (Hoekema et al. , (1983) Nature 303:179-180] in standard triparental matings using the E. coli HB101, containing the plasmid pRK2013 as a helper strain. [Figurski and Helinski, (1979) Proc. Natl. Acad. Sci.USA 76:1648-1652] Appropriate Agrobacterium tumefaciens recipients are selected on media containing rifampicin (50 gg/ml) and kanamycine (50 μg/ml) .

Cloning of fragments in the vectors pUCl9 [Yanish-Perron

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ΡΒΙΝ19 [Bevan et al.,(1984) Nucl Acids Res. 12:8711-8721] or derivatives, restriction enzyme analysis of DNA, transformation to E. coli recipient strains, isolation of plasmid DNA on small as well as large scale, nicktranslation, in vitro transcription, DNA sequencing, Southern blotting and DNA gel electrophoresis are performed according to standard procedures [Maniatis et al., (1982) Molecular Cloning , a Laboratory Manual . Cold Spring Harbor Laboratory , New York ; Ausubel et al.supra, (1987)].

DNA amplification using the polymerase chain reaction (PCR) were performed as recommended by the supplier of the Tag polymerase (Perkin Elmer Cetus ).

Amplifications of RNA by reverse transcription of the target RNA followed by standard DNA amplification were performed using the Gene Amp RNA PCR Kit as recommended by the supplier (Perkin Elmer Cetus).

Examples

Example 1; Isolation of INSV particles and genetic material therein

INSV isolate NL-07, an isolate from Impatiens, is maintained on Impatiens by grafting. Virus is purified from systemically infected Nicotiana rustica leaves, after mechanical inoculation essentially as described by Tas et al. [(1977) J. Gen. Virol. 36:81-91 ] . All material used in the isolation procedure should be maintained at a temperatue of 4 °C . Twelve days after inoculation 100 grams of infected leaves are harvested and ground for 5-10 seconds at a low speed setting in 5 volumes extraction buffer (0.1 M NaH2PO4, 0.01 M Na2SO3, pH 7) in a Waring blender. The suspension is filtered through cheesecloth and the filtrate is centrifuged for 10 minutes at 16,000 x g. The resulting

AP Ο Ο Ο 4 Ο 3 pellet is resuspended in three volumes resuspension buffer (0.01 M NaH2PO4, 0.01 M Na2SO3, pH 7). The pellet is dissolved by stirring carefully at 4°C. After centrifuging for 10 minutes at 12,500 x g the pellet is discarded and the supernatant centrifuged again for 20 minutes at 50,000 x g. The pellet is resuspended in 0.2 volume of resuspension buffer (0.01 M NaH2PO4, 0.01 M Na2SO3, pH 7) and kept on ice for 30 minutes. Anti-serum raised in rabbits against material from non-infected Nicociana rustica is added to the solution and carefully stirred for 1 hour . Non-viral complexes are pelleted after 10 minutes centrifuging at 16,000 x g. The cleared supernatant is loaded on a linear 5% - 40 % sucrose gradient in resuspension buffer(0.01 M

NaH2PO4, 0.01 M Na2SO3, pH 7) , and spun for 45 minutes at 95,000 x g. The opalescent band containing INSV particles is carefully collected with a syringe and diluted 4 times with resuspension buffer. Washed viruses are pelleted by centrifugation for 1.5 hours at 21,000 x g and resuspended in one volume of resuspension buffer. Generally, 100 grams of leaf material yields approximately 0,5 mg of INSV viruses. INSV RNA is recovered preferentially from purified ζ virus preparations by SDS-phenol extractions followed by ethanol precipitation . From 1 mg INSV , 1-5 μg of RNA is

C extracted. The isolated RNA molecules are analysed for intactness by electrophoresis on an agarose gel. Three distinct RNA molecules are identified with apparent sizes of about 3000 nucleotides (S RNA), about 4900 nucleotides (M RNA) and about 8900 nucleotides (L RNA) respectively.

Example 2: Sequence determination of the 3'-termini of the INSV viral RNAs

In order to perform direct RNA sequencing , INSV RNA is extracted from purified nucleocapsids essentially according to Verkleij et al. (1983 ) supra . Twelve days after inoculation 100 grams of infected leaves are harvested and

AP Ο Ο Ο 4 Ο 3 volumes of TAS-E buffer (O.O1 M EDTA, 0.01 M NajSCb, 0.1 % cysteine, 0.1 M TRIS pH 8.0) in a Waring blender. The suspension is filtered through cheesecloth and centrifuged for 10 minutes at 1,100 x g. Nucleocapsids are recovered from the supernatant after 30 minutes of centrifuging at 66,000 x g. The pellet is carefully resuspended in one volume of TAS-R buffer (1 % Nonidet NP-40, 0.01 M EDTA, 0.01 M Na₂SO3, 0.1 % cysteine, 0.01 M glycine, 0.01 M TRIS , pH 7.9). The pellet is dissolved by stirring carefully for 30 minutes at 4 °C. The supernatant is cleared by centrifuging for 10 minutes at 16,000 x g. Crude nucleocapsids are collected from the cleared supernatant by sedimentation through a 30 % sucrose cushion for 1 hour at 105,000 x g. The nucleocapsid pellet is resuspended in 400 μΐ 0.01 M Na-citrate pH 6.5, layered on a 20 - 40 % sucrose (in 0.01 M Na-citrate pH 6.5) and spun for 2 hours at 280,000 x g. The three different opalescent bands, respectively L, M and S nucleocapsid, are collected separately. INSV RNA is recovered preferentially from purified nucleocapsid preparations by SDS-phenol extractions followed by ethanol precipitation. Generally , 100 μg of RNA are obtained from 100 grams of infected leaves. The 3'-ends of the separate INSV RNAs are labeled using RNA ligase and 5'-[^3:P]pCp. The end-labeled RNA molecules are separated on a low gelling temperature agarose gel [Wieslander, (1979) Anal Biochem 98: 305-309] . The enzymatic approach described by Clerx-Van Haaster and Bishop [(1980) Virology 105:564-574] and Clerx-Van Haaster et al. [(1982) J Gen Virol 61:289-292] is used to determine the 30 terminal nucleotides of the 3'and 5'-ends of both S and M RNA.

Synthetic oligonucleotides complementary to the 3'-termini are synthesized using a commercially available system (Applied Biosystems) and used for dideoxy-sequencing with reverse transcriptase.

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Example 3: cDNA cloning of INSV genetic material

Oligonucleotides complementary to the 3' -end of the S RNA are used for priming first strand cDNA synthesis. With these primers, double stranded DNA to INSV RNA is synthesized according to Gubler and Hoffman ((1983) Gene 25:263-269].

Two different approaches are used to generate cDNA clones to the INSV viral RNAs. A first series of clones is obtained by random priming of the INSV RNA using fragmented single stranded calf thymus DNA, followed by first and second strand cDNA synthesis. cDNA is made blunt-ended using T4-DNA polymerase and ligated with T4 ligase into the Smal site of pUC19.

A second series of INSV cDNA clones is obtained by priming first strand DNA synthesis with the oligonucleotides complementary to the 20 terminal nucleotides at the 3 *-ends of the INSV RNAs. Blunt ended cDNA fragments are cloned into the Sma I site of pUCl9.

cDNA clones from both series containing viral inserts are ί selected via colony hybridization, essentially according to the method of Grunstein and Hogness [(1975) Proc. Natl.

I Acad. Sci. USA 72:3961-3965] using [32]p-labeled, randomly primed first strand cDNA as a probe. Sets of overlapping cDNA clones are selected by Southern analysis followed by plasmid walking, in order to construct a restriction map, based on cDNA derived sequences of the S RNA (Figure 2J.

Example 4: Sequence determination of the INSV S RNA

In order to determine the sequence of the S RNA 5 selected cDNA clones are subcloned into pBluescript, resulting in the plasmids pINSV-S2, pINSV-S15, pINSV-S61, plNSV-S60 and pINSV-S39 , (Figure 2). The clones are sequenced in both directions using the protocol of Zhang et al. ((1988) Nucl.

AP Ο Ο Ο 4 Ο 3 of the S RNA is determined by primer extension of the synthetic oligonucleotide INSV-S60 (5' d (AGAGCAATTGTGTCAJ. which is complementary to the 15 nucleotides of the 3'terminus. Sequence data from the INSV S RNA (3017 nt) is summarized in the sequence listing (SEQ ID No.l to SEQ ID No.12).

Computer simulated translation of the 6 different reading frames on the viral strand and viral complementary strand reveals the presence of two putative open reading frames (Figure 3). On the viral strand an open reading frame is found starting at position 87 and terminating at a UAA stopcodon at position 1436 encoding a protein of 449 amino acids with a predicted molecular mass of about 51.2 kd. This protein is a non-structural protein, tentatively designated NSs (Figure 3/ SEQ ID No. 26). The other open reading frame is located on the viral complementary strand from position 2080 to 2868 (SEQ ID No. 11) , encoding a 262 amino acid long polypeptide with a predicted molecular mass of about 28.7 kd. This open reading frame encodes the viral nucleocapsid protein N (Figure 3/ SEQ ID No 25). Thus Figure 3 shows the coding capacities of the viral and the viral complementary strand of INSV S RNA, indicating the NSs and N protein genes are expressed from subgenomic mRNAs (SEQ ID No.3 , SEQ ID No.11 respectively) . Thus, the situation occurs that a plant virus RNA has an ambisense gene arrangement. Other important features of this S RNA sequence is the existence of complementary terminal repeats capable of forming so-called pan-handle structures. These structures play an important role in replication and transcription of viral RNA. Another putative regulatory element is the secondary structure in the intergenic region of the S RNA, which most likely contains the transcription termination signals for both subgenomic mRNAs, encoding respectively the N and NSs-protein._ bad original

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The nucleotide sequence of the INSV M and L RNA is elucidated employing similar strategies and methods as used to determine the nucleotide sequence of the S RNA.

Example 5: Construction of an expression vector pZU-B

The recombinant plasmid pZO347 is a derivative of pBluescript carrying a 496 bp BamHI-Smal fragment containing a 426 bp 35S promoter fragment (Hindi fragment) of CaMV strain Cabb-S, linked to a 67 bp fragment of the nontranslated leader region, the so-called Ω-region, of the f tobacco mosaic virus. This results in a chimeric promoter with a complete transcriptional fusion between the promoter of CaMV to the untranslated leader of TMV. By using in vitro mutagenesis the original position of the TMV ATG startcodon is mutated to a Sinai site.

The plasmid pZO008 carries the nopaline synthase (NOS) terminator as a 260 bp Pstl-Hindlll fragment. This PstlHindlll fragment is excised from pZO008 and ligated using T4 ligase into Pstl-Hindlll linearized pZO347. The resulting recombinant plasmid pZU-B is another plant expression vector. The sequence of this 35S-Q promoter as used in the plant expression vector pZU-B is shown as SEQ ID No.23 .The resulting recombinant plasmid pZU-3 contains the 35S HincIITMV Ω fusion (35S-Ω) , unique Smal and Pstl sites and the NOS terminator (Figure 4 ). This expression vector is preferentially used in constructing translational fusions of the gene for expression downstream of the chimaeric promoter 353-Ω.

Example 6; Subcloning of the INSV N protein gene

The INSV N protein coding sequence is obtained by fusion of the cDNA clones plNSV-S60 and plNSV-S39 ( Figure 5) . The cDNA clone pINSV-S60 is subjected to Spel digestion and the fragment containing the 3¹-end of the INSV N protein gene is

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AP Ο Ο Ο 4 Ο 3 separated electrcphoretically and purified from the gel using a DEAE membrane (NA-45, Schleicher and Schall) and cloned in the largest Spel fragment of pINSV-S39 linearized resulting in the recombinant plasmid pINSV-N. Primers are designed homologous to the translational start and stop codon. Primer INSV-066 d(GCAGATATCATGAACAAAGC) creates an EcoRV site just proximal to the start codon.

Primer INSV-070 d(GCAACCTGCAGCTCAAATCTCTT) creates a Pstl site just distal to the stop codon. These primers are used in standard PCR experiments in which pINSV-N is used as the template. The resulting PCR fragment is isolated from the gel using a DEAE membrane (NA-45, Schleicher and Schall) and cloned in the Smal linearized pBluescript to generate plasmid pINSV-N2 . The added restriction sites, EcoRV and Pstl, facilitate the construction of further plasmids. (Alternatively, one may choose to add the sites in different ways such as but not limited to site-directed mutagenesis or by ligation of other synthetic oligonucleotide linkers. Such methods are all known to a person skilled in the art.)

Example 7 : Subcloning of the INSV non-etructural protein genes (NSs gene) of INSV S RNA

The sequence of the gene corresponding to the non-structural protein NSs is isolated using RNA based PCR on isolated INSV S RNA. Two primers are designed which are homologous to regions spanning either the translational start codon or stop codon . The start codon primer contains an EcoRV site proximal to the ATG codon , the stop codon primer has a Pstl site just distal thereto . Purified INSV S RNA is subjected to the Gene AMP RNA PCR. The resulting PCR fragment is isolated from the gel and cloned into Smal linearized pBluescript yielding the recombinant plasmid plNSV-NSs (Figure 6) .

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Example 8: Subcloning of the INSV non-structural protein gene (NSm gene) of the INSV M RNA

The sequence of the gene corresponding to the non-structural protein NSm is isolated using RNA based PCR on isolated INSV M RNA. Two primers are designed which are homologous to regions spanning either the translational start codon or stop codon . The start codon primer contains an EcoRV site proximal to the ATG codon, the stop codon primer has a Pstl site just distal thereto . Purified INSV S RNA is subjected to the Gene AMP RNA PCR. The resulting PCR fragment is isolated from the gel and cloned into Smal linearized pBluescript yielding the recombinant plasmid pINSV-NSm (Figure 7).

Example 9: Subcloning of the INSV G1/G2 glycoprotein gene (G1/G2 gene) of the INSV M RNA

The sequence of the gene corresponding to the G1/G2 glycoprotein precursor is isolated using RNA based PCR on isolated INSV M RNA. Two primers are designed homologous to regions spanning either the translational start codon or stop codon . The start codon primer contains an EcoRV site proximal to the ATG codon, the stop codon primer has a Pstl site just distal thereto . Purified INSV M RNA is subjected to the Gene AMP RNA PCR. The resulting PCR fragment is isolated from the gel and cloned into Smal linearized pBluescript yielding the recombinant plasmid pINSV-Gl/G2 (Figure 8).

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Example 10 : Construction of plant transformation vectors containing INSV sequences

Example 10A: N protein constructions in pZU-B

In order to make a fusion in which the ATG start codon from the N protein gene is fused directly to the 3'-end of the TMV untranslated leader of the 35S-Q promoter the start codon of the N gene has to be mutated using the PCR approach as hereinbefore described. The N protein gene is excised from the plasmid pINSV-N2 via an EcoRV-Pstl digestion. The fragment is isolated and inserted into the Smal-Pstl linearised pZU-B, resulting in recombinant plasmid pINSV-NB. The chimeric cassette containing the 35S-Q promoter, the N gene and the NOS terminator is excised from the plasmid pINSV-NB via a BamHI/Xbal digestion. The isolated chimaeric gene cassette is then inserted into the BamHI/Xbal linearized pBINl9 to create the binary transformation vector pINSV-NBB. The resulting plasmid pINSV-NBB (Figure 9) is used in plant transformation experiments using methods well known to a person skilled in the art.

Example 10B: NSs protein gene constructions in pZU-B

In order to create a fusion in which the ATG start ccdon from the NSs protein is fused directly to the 3'-end of the TMV leader of the 35S-Q promoter the start codon of the NSs gene is mutated, using the PCR approach. The plasmid pINSVNs is digested with EcoRV and Pstl and the NSs containing fragment is isolated from the gel and inserted into Smal/ Pstl linearized pZU-B resulting in the recombinant plasmid pINSV-NSsB. The chimaeric cassette containing the 35S-Q promoter, the mutated NSs protein gene and the NOS terminator is excised from the plasmid pINSV-NSsB via a BamHI/Xbal digestion. The isolated chimeric gene cassette is then inserted into the BamHI/Xbal linearized pBINl9 to create the binary transformation vector pINSV-NSsBB. The

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AP Ο 00 4 Ο 3 resulting plasmid pINSV-NSsBB (Figure 10) is used in plant transformation experiments using methods well known to a person skilled in the art.

Example 10C: Gl/62 glycoprotein gene constructions in pZU-B

In order to create a fusion in which the ATG start codon from the G1/G2 glycoproteinprecursor is fused directly to the 3'-end of the TMV leader of the 35S-Q promoter the start codon of the G1/G2 gene is mutated, using the PCR approach. The plasmid pINSV-Gl/G2 is digested with EcoRV and Pstl and the G1/G2 containing fragment is isolated from the gel and inserted into Smal/Pstl linearized pZU-B resulting in the recombinant plasmid pINSV-Gl/G2B. The chimeric cassette containing the 35S-Q promoter, the mutated G1/G2 glycoprotein gene and the NOS terminator is excised from the plasmid pINSV-Gl/G2B via a BamHI/Xbal digestion. The isolated chimeric gene cassette is then inserted into the BamHI/Xbal linearized pBlNl9 to create the binary f transformation vector pINSV-Gl/G2BB. The resulting plasmid pINSV-Gl/G2BB (Figure 11) is used in plant transformation experiments using methods well known to a person skilled in the art.

Example 10D: NSm protein gene construct iona in pZU-B

In order to create a fusion in which the ATG start codon from the NSm protein is fused directly to the 3'-end of the TMV leader of the 35S-Q promoter the startcodon of the NSm gene is mutated, using the PCR approach. The plasmid plNSVNSm is digested with EcoRV and Pstl and the NSm-containing fragment is isolated from the gel and inserted into Smal. Pstl linearized pZU-B resulting in the recombinant plasmid pINSV-NSmB. The chimeric cassette containing the 35S-Q promoter, the mutated NSm protein gene and the NOS terminator is excised from the plasmid pINSV-NSmB via a

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BamHi/Xbal digestion. The isolated chimeric gene cassette is then inserted into the BamHI/Xbal linearized pBINl9 to create the binary transformation vector pINSV-NSmBB. The resulting plasmid pINSV-NSmBB (Figure 12) is used in plant transformation experiments using methods well known to a person skilled in the art.

Example 10E: 5'- and 3'-termini pan-handle f constructions in pZU-B

A DNA analysis programme is used to. locate the pan-handle element of the loop in the viral INSV S RNA. The strongest pan-handle structure that is detected includes about the first 24-25 nucleotides at the 5‘-end (1 to 24 or 25) of the viral S RNA and about the last 36 nucleotides at the 3¹-end

of the viral S RNA length of the pan-ha nucleotides long.	(SEQ .ndle	ID Nos 5 element	and 6 of the	respectively).The loop is about 36
These regions are	synthesized	on a	commercial	DNA

v synthesizer and appropriate linker sequences are added.

Construction of the pan-handle vectors of S and M RNA

Z results in respectively: pINSV-termS and pINSV-termM. Using appropriate restriction enzyme combination these fragments are inserted between the 35S-Q promoter and the NOS terminator of pZU-B yielding the chimeric cassettes: pINSVtermSA , pINSV-termMA , pINSV-termSB and pINSV-termMB. These cassettes are then transferred into the binary transformation vector pBINl9 using appropriate enzyme combinations yielding the following plasmids: pINSV-termSAB, plNSV-termMAB, pINSV-termSBB and pINSV-termMBB. Alternatively, it is possible to design “pan-handle constructs including the 3'- and 5'-end termini that are larger than indicated above, or separated by any other DNA sequence in order to enhance the stability of the transcripts produced from these recombinant genes in plants.

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All pan-handle constructs resemble shortened tospovirus RNA molecules , specifically INSV RNA molecules and therefore can be regarded as defective interfering RNAs.

Example_1 OF-·._Construction_containing INSV 5 RNA g_eoondarY_structure region in dzu-b.

A DNA.analysis programme is used to locate a secondary structure in the viral INSV S RNA. The strongest secondary structure detectable starts at nucleotide 1440 and ends at nucleotide 2041 of SEQ ID No.l , (SEQ id No 8).

The DNA fragment carrying the secondary structure region is isolated from plNSV-S61 using a PCR approach similar to that described earlier. The two primers used contain the sequences 1440-1460 and 2021-2041 of SEQ ID No.l. The PCR fragment is excised from an agarose gel and subsequently treated with T4 polymerase to create blunt ends and is subsequently cloned into the Smal site of the expression vector pZU-B, resulting in the recombinant plasmid pINSVHpS3. The plasmid plNSV-HpSB is digested with Hindlll and the fragment containing the chimeric gene is excised from an agarose gel and ligated into Xbal linearized pBIN19, resulting in the transformation vector pINSV-HpSBB.

(It is clear to a person skilled in the art that other fragments can be isolated from the cDNA clones of the INSV S RNA containing the hairpin region as described above without interference to function. Also, a fragment containing the hairpin region may be synthesized using a DNA-synthesizer.)

Example 1 1 ; Transformation of binary vectors to tobacco plant material

Methods to transfer binary vectors to plant material are well established and known to a person skilled in the art.

Variations in procedures exist due to for instance differences in used Agrobacterium strains , different

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AP Ο Ο Ο 4 Ο 3 sources of explant material , differences in regeneration systems depending on as well the cultivar as the plant species used .

The binary plant transformation vectors as described above are used in plant transformation experiments according to the following procedures. The constructed binary vector is transferred by tri-parental mating to an acceptor Agrobacterium tumefaciens strain, followed by southern analysis of the ex-conjugants for verification of proper transfer of the construct to the acceptor strain, inoculation and cocultivation of axenic explant material with the Agrobacterium tumefaciens strain of choice, selective killing of the Agrobacterium tumefaciens strain used with appropriate antibiotics, selection of transformed cells by growing on selective media containing kanamycine, transfer of tissue to shoot - inducing media, transfer of selected shoots to root inducing media, transfer of plantlets to soil, assaying for intactness of the construct by southern analyses of isolated total DNA from the transgenic plant, assaying for proper function of the inserted chimeric gene by northern analysis and/or enzyme assays and western blot analysis of proteins.

Example 12 ; Expression of INSV 3 RNA sequences in tobacco plant cells

RNA is extracted from leaves of regenerated plants using the following protocol. Grind 200 mg leaf material to a fine powder in liquid nitrogen. Add 800 μΐ RNA extraction buffer (100 mM Tris-HCl (pH 8,0), 500 mM NaCl, 2 mM EDTA, 200 mM SMercapto-ethanol, 0,4% SDS) and extract the homogenate with phenol, collect the nucleic acids by alcohol precipitation. Resuspend the nucleic acids in 0,5 ml 10 mM Tris-HCl (pH 8,0), 1 mM EDTA, add LiCl to a final concentration of 2 M, leave on ice for maximal 4 hours and collect the RNA by centrifugation. Resuspend in 400 μΐ 10 mM Tris-HCl (pH 8,0),_ bad ORIGINAL

APO 00 4 Ο 3 mM EDTA and precipitate with alcohol, finally resuspend in 50 μΐ 10 mM Tris-HCl (pH 8,0), 1 mM EDTA. RNAs are separated on glyoxal/agarose gels and blotted to Genescreen as described by van Grinsven et al. [(1986) Theor Appl Gen 73:94-101]. INSV S RNA sequences are detected using DNA or RNA probes labeled with [³²P] , [³⁵S] or by using nonradioactive labeling techniques. Based on northern analysis, it is determined to what extent the regenerated plants express chimaeric INSV S RNA sequences.

Plants transformed with chimaeric constructs containing an INSV N protein-encoding sequence are also subjected to western blot analysis. Proteins are extracted from leaves of transformed plants by grinding in sample buffer according to the method of Laemmli [(1970) Nature 244: 29-30] . A 50 gg portion of protein is subjected to electrophoresis in a 12,5 % SDS-polyacrylamide gel essentially as described by Laemmli (1970) supra . Separated proteins are transferred to nitrocellulose electrop’noretically as described by Towbin et al. [(1979) Proc. Natl. Acad. Sci. USA 76:4350-4354]. Transferred proteins are reacted with antiserum raised against purified INSV structural or non-structural proteins (Towbin et al.(1979) supra. Based on the results of the western analysis, it is determined that transformed plants do contain INSV N proteins encoded by the inserted chimaeric

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Example 13: Resistance of plants against INSV infection

Transformed plants are grown in the greenhouse under standard quarantine conditions in order to prevent any infections by pathogens. The transformants are selfpollinated and the seeds harvested. Progeny plants are analyzed for segregation of the inserted gene and subsequently infected with INSV by mechanical inoculation. Tissue from plants systemically infected with INSV is ground in 5 volumes of ice-cold inoculation buffer (10 mM phosphate buffer supplemented with 1% Na2SO3) and rubbed in the presence of carborundum powder on the first two fully extended leafs of approximately 5 weeks old seedlings. Inoculated plants are monitored for symptom development during 3 weeks after inoculation.

Plants containing INSV Related DNA Sequences show reduced susceptibility to INSV infection as exemplified by a delay in symptom development, whereas untransformed control plants show severe systemic INSV symptoms within 7 days after inoculat ion.

Example 14: Use of synthetic oligonucleotides for diagnostic purposes

RNA is extracted from leaves of suspected plants using the following protocol: grind 1 gram of leaf material, preferentially showing disease symptoms, in 3 ml 100 mM Tris-HCl, 50 mM EDTA, 1.5 M NaCl and 2% CTAB (pH 8.0). After grinding, 1 ml of the homogenate is subjected to chloroform extraction and incubated at 65 °C for 10 minutes. The inorganic phase is then collected and extracted with phenol/chloroform (1:1), followed by a last extraction with chloroform. The ribonucleic acids are isolated from the inorganic phase, containing the total nucleic acids, by adding LiCl to a final concentration of 2 M. The preparation is incubated at 4°C for 1 hour, after which the ribonucleic

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APO 00 4 0 3 acids are collected by centrifugation. The ribonucleic acid pellet is resuspended in 25 μΐ 10 mM Tris-HCl, 1 mM EDTA (pH 8.0). The ribonucleic acids are recovered by standard alcohol precipitation. The ribonucleic acid pellet is resuspended in 25 μΐ 10 mM Tris-HCl, 1 mM EDTA (pH 8.0).

μΐ of the purified ribonucleic acids is spotted on a nylon blotting membrane (e.g. Hybond-N, Amersham UK). The presence of INSV in the plant is detected by standard hybridization, using any part or parts of the sequence isolated from virions or preferentially by designing synthetic oligomers on the basis of disclosed sequence information as a probe. (Alternatively, in vitro transcripts of regions of the INSV genome are used to detect INSV Related RNA Sequences in diseased plants.) A diseased plant is diagnosed by the occurrence of hybridization at the dot containing RNA material from the diseased plant.

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SEQUENCE LISTING

SEQ. ID No. 1

Sequence type: Nucleotide Sequence length: 3017 nucleotides Strandedness: single stranded

Molecule type: Viral S RNA nucleotide sequence from position 1 to position 3017

Original Source Organism:	Impat iens	Necrotic Spot Virus
AGAGCAATNN	NNNNNNNNNN	NNNNGAACAA	CCAAGCTACA	ACAAATCTTA	CAATATTGTC	60
AATTACATTA	CTACTTCCAT	TTTAACATGT	CTAGTGCAAT	GTATGAAACA	ATTATCAAAT	120
CGAAGTCCTC	AATCTGGGGA	ACAACATCTT	CGGGTAAAGC	AGTAGTAGAT	AGTTATTGGA	180
TTCATGATCA	ATCTTCCGGA	AAGAAGTTGG	TCGAAGCTCA	ACTCTATTCT	GACTCCAGGA	240
GCAAGACCAG	TTTCTGTTAC	ACTGGTAAAG	TTGGCTTTCT	CCCAACAGAA	GAAAAAGAAA	300
TTATAGTGAG	ATGTTTTGTG	CCTATTTTTG	ATGACATTGA	TCTGAATTTC	TCCTTTTCAG	360
GGAATGTTGT	CGAAATTCTG	GTCAGATCTA	ACACAACAAA	CACAAACGGT	GTTAAACATC	420
AAGGTCATCT	CAAAGTGTTA	TCCTCTCAGT	TGCTCAGAAT	GCTTGAAGAG	CAAATAGCAG	480
TGCCTGAAAT	TACTTCAAGA	TTCGGTCTGA	AAGAATCTGA	CATCTTCCCT	CCAAATAATT	540
TCATTGAAGC	TGCAAATAAA	GGATCATTGT	CTTGTGTCAA	AGAAGTCCTT	TTTGATGTCA	600
AGTATTCAAA	CAACCAATCC	ATGGGCAAAG	TCAGTGTTCT	TTCTCCTACC	AGAAGTGTTC	660
ATGAATGGCT	GTACACACTT	AAGCCTGTTT	TTAACCAATC	CCAGACCAAC	AACAGGACAG	720
TAAACACTTT	GGCTGTAAAA	TCACTGGCAA	TGTCTGCAAC	TTCTGATTTA	ATGTCAGATA	780
CTCATTCGTT	TGTCAGGCTC	AATAATAACA	AGCCTTTTAA	AATCAGCCTT	TGGATGCGCA	340
TCCCTAAAAT	AATGAAATCA	AACACATACA	GCCGGTTCTT	CACCCTGTCT	GATGAATCTT	900
CTCCTAAAGA	GTATTATATA	AGCATTCAAT	GTCTTCCGAA	TCACAACAAT	GTTGAAACAG	960
TCATTGAATA	TAACTTTGAT	CAGTCAAACC	TCTTCTTGAA	TCAACTCCTT	CTAGCAGTGA	1020
TTCATAAAAT	TGAGATGAAT	TTTTCTGATC	TAAAAGAACC	TTACAATGTT	ATCCATGATA	1CS0
TGTCGTATCC	TCAAAGAATT	gttcattcac	TTCTTGAAAT	CCACACAGAA	CTTGCTCAAA	1140
CTGTCTGTGA	CAGTGTTCAG	CAAGACATGA	TTGTCTTCAC	TATAAATGAG	CCAGATCTAA	1200
AGCCAAAAAA	GTTTGAGCTA	GGGAAAAAGA	CTTTAAATTA	TTCAGAAGAT	GGTTATGGGA	1260
GAAAATATTT	CCTTTCTCAG	ACCTTGAAAA	GTCTTCCGAG	AAACTCACAA	ACAATGTCTT	1320
ATTTGGATAG	CATCCAGATG	CCCGATTGGA	AATTTGACTA	TGCTGCAGGT	GAAATAAAAA	1380

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TTTCTCCTAG	ATCAGAGGAT	GTTTTGAAAG	CTATTTCTAA	ATTAGATTTA	AATTAACCTT	1440
GGTTAAACTT	GTCCCTAAGT	AAAGTTTGTT	TACATGCATT	TAGATCAGAT	TAAACAAATC	1500
TAATAACAGA	TAAACCAAAA	ACAATCATAT	GAAATAAATA	AATAAACATA	AAATATATAA	1560
AAAATACAAA	AAAAATCATA	AAATAAATAA	AAACCAAAAA	AGGATGGCCT	TCGGGCACAA	1620
TTTGGTTGCT	TTAATAATGC	TTTAAAATGA	ATGTATTAGT	AAATTATAAA	CTTTAAATCC	1680
AATCTACTCA	CAAATTGGCC	AAAAATTTGT	ATTTGTTTTT	GTTTTTGTTT	TTTGTTTTTT	1740
GTTTTTGTTT	TGTTTTATTT	GTTTTTTATT	TTGTTTTTTG	TTTTTTGTTT	TTTATTTTAT	1800
TTATATATAT	ATATATATAT	ATTTTGTAGT	ggtttttatt	gtttttatta	TTTTTTGTAG	1860
CTTTTTTACT	TGTTTATTTC	ACACGCAAAC	ACACTTTCAA	GTTTATATAT	TAAAACACAC	1920 *
ATTAAACTTA	TTTCAAATAA	TTTATAAAAG	CACACTTAAT	ACACTCAAAC	AATAATTAAT	1980
TATTTTATTT	TTTATTTTAT	TTTTTATTTT	TATTATTTTT	ATTTTTATTT	ATTTAAATGC	2040
ATTTAACACA	ACACAAAGCA	AACCAAGCTC	AAATCTCTTT	TAAATAGAAT	catttttccc	2100
AAAATCAATA	GTAGCATTAA	ACATGCTGTA	AATGGATGTA	AGCCCTTCTT	TGTAGTGGTC	2160
CATTGCAGCA	AGTCCTTTAG	CTTTCGGACT	ACAAGCCTTT	AGTATATCTG	CATATTGTTT	2220
AGCCTTGCCA	ATTTCAACAG	AGTTCATGCT	ATATCCTTTG	CTTTTTAGAA	CTGTGCACAC	2230
TTTCCCAACT	gcctctttag	TGCTAAACTT	AGACATGTCA	ATTCCAAGCT	CAACATGTTT	2340
AGCATCTTGA	TAAATAGCCG	GAACTAGTGC	AGCTATTTCA	AAATTCAGTA	CAGATGCTAT	2400
CAGAGGAAGA	CTTCCTCCTA	AGAGAACACC	CAAGACACAG	GATTTCAAAT	CTGTGGTTGC	2460
AAGACCATAT	GAGGCAATCA	GAGGGTGACT	TGGAAGGCTA	TTTATAGCTT	CAGTCAGAGC	2520
AGATCCATTG	TCCTTTATCA	TTCCAACAAG	ATGAACTCTC	ACCATTGCAT	CAAGTCTTCG	2530
GAAAGTCATA	TCATTGACCC	CAACTCTTTC	TGAATTGTTT	CTAGTTTTCT	TAATTGTGAC	2640
TGATCCAAAA	GTGAAGTCAG	CACTCTTAAT	GACTCTCATT	ATAGATTGCC	TATTCTTGAG	2700
GAAGGATAGG	CAGGATGCAG	TAGTCATGTT	CTGAATCTTT	TCACGGTTGT	TGGTAAAGAA	2760
GTCAGTGAAA	TTGAAAGACC	CTTCATTTTG	AGTTTCCTCA	AATTCTAAGG	AATCAGATTG	2820
AGTCAAAAGC	TTGACTATGT	TCTCCTTGGT	AATCTTTGCT	TTGTTCATCT	TGATCTGCTG	2880
ACTTTACTAA	CTTTAAAGCT	TAAAGTGTTC	AAATTACTAA	ATAGTACTTG	CGGTTAAAGT	2940
AGTATTTGGT	AAAATTTGTA	ATTTTTCAGT	TTCTAGCTTT	GGATTATATG	ATGTTATATT	3000
CGTGACACAA	TTGCTCT					3 017

BAD ORIGINAL

AP Ο Ο Ο 4 Ο 3

SEQ ID NO: 2

Sequence type: nucleotides

Sequence length: 2993 nucleotides

Strandedness: single stranded

Molecule type: Viral S RNA nucleotide sequence from position 25 to position 3017 of SEQ ID NO.l

Original Source Organism: Impatiens Necrotic Spot Virus

GAACAACCAA	GCTACAACAA	ATCTTACAAT	ATTGTCAATT	ACATTACTAC	TTCCATTTTA	60
ACATGTCTAG	TGCAATGTAT	GAAACAATTA	TCAAATCGAA	GTCCTCAATC	TGGGGAACAA	120
CATCTTCGGG	TAAAGCAGTA	GTAGATAGTT	ATTGGATTCA	TGATCAATCT	TCCGGAAAGA	180
AGTTGGTCGA	AGCTCAACTC	TATTCTGACT	CCAGGAGCAA	GACCAGTTTC	TGTTACACTG	240
GTAAAGTTGG	CTTTCTCCCA	ACAGAAGAAA	AAGAAATfAT	AGTGAGATGT	TTTGTGCCTA	300
TTTTTGATGA	CATTGATCTG	AATTTCTCCT	TTTCAGGGAA	TGTTGTCGAA	ATTCTGGTCA	360
GATCTAACAC	AACAAACACA	AACGGTGTTA	AACATCAAGG	TCATCTCAAA	GTGTTATCCT	420
CTGAGTTGCT	CAGAATGCTT	GAAGAGCAAA	TAGCAGTGCC	TGAAATTACT	TCAAGATTCG	480
GTCTGAAAGA	ATCTGACATC	TTCCCTCCAA	ATAATTTCAT	TGAAGCTGCA	AATAAAGGAT	540
CATTGTCTTG	TGTCAAAGAA	GTCCTTTTTG	ATGTCAAGTA	TTCAAACAAC	CAATCCATGG	600
GCAAAGTCAG	TGTTCTTTCT	CCTACCAGAA	GTGTTCATGA	ATGGCTGTAC	ACACTTAAGC	660
ctgtttttaa	CCAATCCCAG	ACCAACAACA	GGACAGTAAA	CACTTTGGCT	GTAAAATCAC	720
TGGCAATGTC	TGCAACTTCT	GATTTAATGT	CAGATACTCA	TTCGTTTGTC	AGGCTCAATA	780
ATAACAAGCC	TTTTAAAATC	AGCCTTTGGA	TGCGCATCCC	TAAAATAATG	AAATCAAACA	840
CATACAGCCG	GTTCTTCACC	CTGTCTGATG	AATCTTCTCC	TAAAGAGTAT	TATATAAGCA	900
TTCAATGTCT	TCCGAATCAC	AACAATGTTG	AAACAGTCAT	TGAATATAAC	TTTGATCAGT	960
CAAACCTCTT	CTTGAATCAA	CTCCTTCTAG	CAGTGATTCA	TAAAATTGAG	ATGAATTTTT	1020
CTGATCTAAA	AGAACCTTAC	AATGTTATCC	ATGATATGTC	GTATCCTCAA	AGAATTGTTC	1080
ATTCACTTCT	TGAAATCCAC	ACAGAACTTG	CTCAAACTGT	CTGTGACAGT	GTTCAGCAAG	1140
ACATGATTGT	CTTCACTATA	AATGAGCCAG	ATCTAAAGCC	AAAAAAGTTT	GAGCTAGGGA	1200
AAAAGACTTT	AAATTATTCA	GAAGATGGTT	ATGGGAGAAA	ATATTTCCTT	TCTCAGACCT	1260
TGAAAAGTCT	TCCGAGAAAC	TCACAAACAA	TGTCTTATTT	GGATAGCATC	CAGATGCCCG	1320
ATTGGAAATT	TGACTATGCT	GCAGGTGAAA	TAAAAATTTC	TCCTAGATCA	GAGGATGTTT	1380

BAD ORIGINAL

AP Ο Ο Ο 4 Ο 3

TGAAAGCTAT TTCTAAATTA GATTTAAATT AACCTTGGTT AAACTTGTCC CTAAGTAAAG 1440

TTTGTTTACA TGCATTTAGA TCAGATTAAA CAAATCTAAT AACAGATAAA CCAAAAACAA 1500

TCATATGAAA TAAATAAATA AACATAAAAT ATATAAAAAA TACAAAAAAA ATCATAAAAT 1560

AAATAAAAAC CAAAAAAGGA TGGCCTTCGG GCACAATTTG GTTGCTTTAA TAATGCTTTA 1620

AAATGAATGT ATTAGTAAAT TATAAACTTT AAATCCAATC TACTCACAAA TTGGCCAAAA 1680

ATTTGTATTT GTTTTTGTTT TTGTTTTTTG TTTTTTGTTT TTGTTTTGTT TTATTTGTTT 1740

TTTATTTTGT TTTTTGTTTT TTGTTTTTTA ΤΤΤΤΑΤΤΤΑΤ ΑΤΑΤΑΤΑΤΑΤ ΑΤΑΤΑΤΑΤΤΤ 1800

TGTAGTGGTT TTTATTGTTT ΤΤΑΤΤΑΤΤΤΤ TTGTAGCTTT TTTACTTGTT TATTTCACAC 1860 ί'’', ' GCAAACACAC TTTCAAGTTT ATATATTAAA ACACACATTA AACTTATTTC ΑΑΑΤΑΑΤΤΤΑ 1920 ( TAAAAGCACA CTTAATACAC TCAAACAATA ΑΤΤΑΑΤΤΑΤΤ ΤΤΑΤΤΤΤΤΤΑ ΤΤΤΤΑΤΤΤΤΤ 1980

ΤΑΤΤΤΤΤΑΤΤ ΑΤΤΤΤΤΑΤΤΤ ΤΤΑΤΤΤΑΤΤΤ AAATGCATTT AACACAACAC AAAGCAAACC 2040

AAGCTCAAAT CTCTTTTAAA TAGAATCATT TTTCCCAAAA TCAATAGTAG CATTAAACAT 2100

GCTGTAAATG GATGTAAGCC CTTCTTTGTA GTGGTCCATT GCAGCAAGTC CTTTAGCTTT 2160

CGGACTACAA GCCTTTAGTA TATCTGCATA TTGTTTAGCC TTGCCAATTT CAACAGAGTT 2220

CATGCTATAT CCTTTGCTTT TTAGAACTGT GCACACTTTC CCAACTGCCT CTTTAGTGCT 2280

AAACTTAGAC ATGTCAATTC CAAGCTCAAC ATGTTTAGCA TCTTGATAAA TAGCCGGAAC 2340

TAGTGCAGCT ATTTCAAAAT TCAGTACAGA TGCTATCAGA GGAAGACTTC CTCCTAAGAG 2400

AACACCCAAG ACACAGGATT TCAAATCTGT GGTTGCAAGA CCATATGAGG CAATCAGAGG 2460 θ GTGACTTGGA AGGCTATTTA TAGCTTCAGT CAGAGCAGAT CCATTGTCCT TTATCATTCC 2520 ζ- . AACAAGATGA ACTCTCACCA TTGCATCAAG TCTTCGGAAA GTCATATCAT TGACCCCAAC 2580

TCTTTCTGAA TTGTTTCTAG TTTTCTTAAT TGTGACTGAT CCAAAAGTGA AGTCAGCACT 2640

CTTAATGACT CTCATTATAG ATTGCCTATT CTTGAGGAAG GATAGGCAGG ATGCAGTAGT 2700

CATGTTCTGA ATCTTTTCAC GGTTGTTGGT AAAGAAGTCA GTGAAATTGA AAGACCCTTC 2760

ATTTTGAGTT TCCTCAAATT CTAAGGAATC AGATTGAGTC AAAAGCTTGA CTATGTTCTC 2320

CTTGGTAATC TTTGCTTTGT TCATCTTGAT CTGCTGACTT TACTAACTTT AAAGCTTAAA 2830

GTGTTCAAAT TACTAAATAG TACTTGCGGT TAAAGTAGTA TTTGGTAAAA TTTGTAATTT 2940

AP Ο Ο Ο 4 Ο 3

SZQ ID NO. 3

Sequence type: Nucleotide

Sequence length: 1350 nucleotides

Strandedness: Single stranded

Molecule type: Viral S RNA nucleotide sequence coding for the non-structural (NSs) protein from position 87 to position 1435 of SEQ ID NO :1

Original source organism: Impatiens Necrotic Spot Virus

ATGTCTAGTG	CAATGTATGA	AACAATTATC	AAATCGAAGT	CCTCAATCTG	GGGAACAACA	60
TCTTCGGGTA	AAGCAGTAGT	AGATAGTTAT	TGGATTCATG	ATCAATCTTC	CGGAAAGAAG	120
TTGGTCGAAG	CTCAACTCTA	TTCTGACTCC	AGGAGCAAGA	CCAGTTTCTG	TTACACTGGT	180
AAAGTTGGCT	TTCTCCCAAC	AGAAGAAAAA	GAAATTATAG	TGAGATGTTT	TGTGCCTATT	240
TTTGATGACA	TTGATCTGAA	TTTCTCCTTT	TCAGGGAATG	TTGTCGAAAT	TCTGGTCAGA	300
TCTAACACAA	CAAACACAAA	CGGTGTTAAA	CATCAAGGTC	ATCTCAAAGT	GTTATCCTCT	350
CAGTTGCTCA	GAATGCTTGA	AGAGCAAATA	GCAGTGCCTG	AAATTACTTC	AAGATTCGGT	420
CTGAAAGAAT	CTGACATCTT	CCCTCCAAAT	AATTTCATTG	AAGCTGCAAA	TAAAGGATCA	480
TTGTCTTGTG	TCAAAGAAGT	CCTTTTTGAT	GTCAAGTATT	CAAACAACCA	ATCCATGGGC	540
AAAGTCAGTG	TTCTTTCTCC	TACCAGAAGT	GTTCATGAAT	GGCTGTACAC	ACTTAAGCCT	500
GTTTTTAACC	AATCCCAGAC	CAACAACAGG	ACAGTAAACA	CTTTGGCTGT	AAAATCACTG	550
GCAATGTCTG	CAACTTCTGA	TTTAATGTCA	GATACTCATT	CGTTTGTCAG	GCTCAATAAT	720
AAC zirtGC C TT	TTAAAATCAG	CCTTTGGATG	CGCATCCCTA	AAATAATGAA	ATCAAACACA	730
TACAGCCGGT	TCTTCACCCT	GTCTGATGAA	TCTTCTCCTA	AAGAGTATTA	TATAAGCATT	840
CAATGTCTTC	CGAATCACAA	CAATGTTGAA	ACAGTCATTG	AATATAACTT	TGATCAGTCA	900
aacctcttct	TGAATCAACT	CCTTCTAGCA	GTGATTCATA	AAATTGAGAT	GAATTTTTCT	950
GATCTAAAAG	AACCTTACAA	TGTTATCCAT	GATATGTCGT	ATCCTCAAAG	AATTGTTCAT	1020
TCACTTCTTG	AAATCCACAC	AGAACTTGCT	CAAACTGTCT	GTGACAGTGT	TCAGCAAGAC	1080
ATGATTGTCT	TCACTATAAA	TGAGCCAGAT	CTAAAGCCAA	AAAAGTTTGA	GCTAGGGAAA	1140
AAGACTTTAA	ATTATTCAGA	AGATGGTTAT	GGGAGAAAAT	ATTTCCTTTC	TCAGACCTTG	1200
AAAAGTCTTC	CGAGAAACTC	ACAAACAATG	TCTTATTTGG	ATAGCATCCA	GATGCCCGAT	1260
TGGAAATTTG	ACTATGCTGC	AGGTGAAATA	AAAATTTCTC	CTAGATCAGA	GGATGTTTTG	1320
AAAGCTATTT	CTAAATTAGA	TTTAAATTAA				1350

bad original

AP Ο Ο Ο 4 Ο 3

SEQ ID NO: 4

Sequence type: Nucleotide

Sequence length: 789 nucleotides

Strandedness: Single stranded

Molecule type: Viral S RNA nucleotide sequence from 2080 to 2868 of SEQ ID NO: 1

Original Source Organism: Impatiens Necrotic Spot Virus < TTAAATAGAA TCATTTTTCC CAAAATCAAT AGTAGCATTA AACATGCTGT AAATGGATGT 60

AAGCCCTTCT TTGTAGTGGT CCATTGCAGC AAGTCCTTTA GCTTTCGGAC TACAAGCCTT 120

TAGTATATCT GCATATTGTT TAGCCTTGCC AATTTCAACA GAGTTCATGC TATATCCTTT 180

GCTTTTTAGA ACTGTGCACA CTTTCCCAAC TGCCTCTTTA GTGCTAAACT TAGACATGTC 240

AATTCCAAGC TCAACATGTT TAGCATCTTG ATAAATAGCC GGAACTAGTG CAGCTATTTC 300

AAAATTCAGT ACAGATGCTA TCAGAGGAAG ACTTCCTCCT AAGAGAACAC CCAAGACACA 360

GGATTTCAAA TCTGTGGTTG CAAGACCATA TGAGGCAATC AGAGGGTGAC TTGGAAGGCT 420

ATTTATAGCT TCAGTCAGAG CAGATCCATT GTCCTTTATC ATTCCAACAA GATGAACTCT 480

CACCATTGCA TCAAGTCTTC GGAAAGTCAT ATCATTGACC CCAACTCTTT CTGAATTGTT 540 θ TCTAGTTTTC TTAATTGTGA CTGATCCAAA AGTGAAGTCA GCACTCTTAA TGACTCTCAT 600

TATAGATTGC CTATTCTTGA GGAAGGATAG GCAGGATGCA GTAGTCATGT TCTGAATCTT 660

TTCACGGTTG TTGGTAAAGA AGTCAGTGAA ATTGAAAGAC CCTTCATTTT GAGTTTCCTC 720

AAATTCTAAG GAATCAGATT GAGTCAAAAG CTTGACTATG TTCTCCTTGG TAATCTTTGC 780

APO00403

SEQ ID KO i 5

Sequence Type: Nucleotide

Sequence length: 36 nucleotides

Strandedness: Single stranded

Molecule type: Viral S RNA nucleotide sequence from the 5' end of the viral S RNA sequence

Original Source Organism: Impatiens Necrotic Spot Virus

AGAGCAATNN NNNNNNNNNN NNNNGAACAAC CCAAGC

SEQ ID NO :6

Sequence Type: Nucleotide

Sequence length: 36 nucleotides

Strandedness: Single stranded

Molecule type: Viral S RNA nucleotide sequence from the 3' end of the viral S RNA sequence

Original Source Organism: Impatiens Necrotic Spot Virus

GATTATATGA TGTTATATTC GTGACACAAT TGCTCT

SEQ ID Ho. 7

Sequence type: Nucleotide

Sequence length:643 nucleotides

Strandedness: Single stranded

Molecule type: Viral S RNA

Original Source Organism: Impatiens Necrotic Spot Virus

CCTTGGTTAA	acttgtccct	AAGTAAAGTT	tgtttacatg	CATTTAGATC	AGATTAAACA
AATCTAATAA	CAGATAAACC	AAAAACAATC	atatgaaata	AATAAATAAA	CATAAAATAT
ATAAAAAATA	CAAAAAAAAT	CATAAAATAA	ataaaaacca	AAAAAGGATG	GCCTTCGGGC
ACAATTTGGT	TGCTTTAATA	ATGCTTTAAA	ATGAATGTAT	TAGTAAATTA	TAAACT7TAA
ATCCAATCTA	CTCACAAATT	GGCCAAAAAT	TTGTATTTGT	TTTTGTTTTT	GTTTTTTGTT
T-tttgtTTTT	GTTTTGTTTT	atttgttttt	TATTTTGTTT	TTTGTTTTTT	GTTTTTTATT

BAD ORIGINAL

AP Ο Ο Ο 4 0 j

ΤΤΑΤΤΤΑΤΑΤ ATATΑΤΑΤΑΤ ATATATTTTG TAGTGGTTTT TATTGTTTTT ΑΤΤΑΤΤΤΤΤΤ 420

GTAGCTTTTT TACTTGTTTA TTTCACACGC AAACACACTT TCAAGTTTAT ATATTAAAAC 480

ACACATTAAA CTTATTTCAA ATAATTTATA AAAGCACACT TAATACACTC AAACAATAAT 540

ΤΑΑΤΤΑΤΤΤΤ ΑΤΤΊΤΊΤΑΤΤ ΤΤΑΤΤΤΤΤΤΑ ΊΤΤΤΤΑΤΤΑΤ ΤΤΤΤΑΤΤΤΤΤ ΑΤΤΤΑΤΤΤΑΑ 600

ATGCATTTAA CACAACACAA AGCAAACCAA GCTCAAATCT CTT 643

SSQ ID No. 9

Sequence type: Nucleotide

Sequence length: 602 nucleotides

Strandedness: Single stranded

Molecule type: Viral S RNA from 1440 to 2041 of SEQ ID No.l Original Source Organism: Impatiens Necrotic Spot Virus

	TGGTTAAACT	TGTCCCTAAG
	CTAATAACAG	ATAAACCAAA
	AAAAATACAA	AAAAAATCAT
	ATTTGGTTGC	TTTAATAATG
	CAATCTACTC	ACAAATTGGC
C-	TGTTTTTGTT	TTGTTTTATT
(	TTTATATATA	TATATATATA
V	GCTTTTTTAC	TTGTTTATTT
	CATTAAACTT	ATTTCAAATA
	TTATTTTATT	TTTTATTTTA
	CA

TAAAGTTTGT	TTACATGCAT	TTAGATCAGA	TTAAACAAAT	60
AACAATCATA	TGAAATAAAT	AAATAAACAT	AAAATATATA	120
AAAATAAATA	AAAACCAAAA	AAGGATGGCC	TTCGGGCACA	180
CTTTAAAATG	AATGTATTAG	TAAATTATAA	ACTTTAAATC	240
CAAAAATTTG	TATTTGTTTT	TGTTTTTGTT	TTTTGTTTTT	300
TGTTTTTTAT	TTTGTTTTTT	gttttttgtt	TTTTATTTTA	360
TATTTTGTAG	TGGTTTTTAT	tgtttttatt	ATTTTTTGTA	420
CACACGCAAA	CACACTTTCA	agtttatata	TTAAAACACA	480
ATTTATAAAA	GCACACTTAA	TACACTCAAA	CAATAATTAA	540
TTTTTTATTT	TTATTATTTT	ΤΑΤΤΤΤΤΑΤΤ	TATTTAAATG	600

602

BAD ORIGINAL

AP Ο Ο Ο 4 Ο 3

SEQ ID HO: 9

Sequence type: Nucleotide

Sequence length: 3017 nucleotides

Strandedness: single stranded

Molecule type: Complementary viral S RNA nucleotide sequence from 1 to about 3017

Original Source Organism: Impatiens Necrotic Spot Virus

AGAGCAATTG	TGTCACGAAT	ATAACATCAT	ATAATCCAAA	GCTAGAAACT	GAAAAATTAC	60
AAATTTTACC	AAATACTACT	TTAACCGCAA	GTACTATTTA	GTAATTTGAA	CACTTTAAGC	120
TTTAAAGTTA	GTAAAGTCAG	CAGATCAAGA	TGAACAAAGC	AAAGATTACC	AAGGAGAACA	180
TAGTCAAGCT	TTTGACTCAA	TCTGATTCCT	TAGAATTTGA	GGAAACTCAA	AATGAAGGGT	240
CTTTCAATTT	CACTGACTTC	TTTACCAACA	ACCGTGAAAA	GATTCAGAAC	ATGACTACTG	300
CATCCTGCCT	ATCCTTCCTC	AAGAATAGGC	AATCTATAAT	GAGAGTCATT	AAGAGTGCTG	360
ACTTCACTTT	TGGATCAGTC	ACAATTAAGA	AAACTAGAAA	CAATTCAGAA	AGAGTTGGGG	420
TCAATGATAT	GACTTTCCGA	AGACTTGATG	CAATGGTGAG	AGTTCATCTT	GTTGGAATGA	430
TAAAGGACAA	TGGATCTGCT	CTGACTGAAG	CTATAAATAG	CCTTCCAAGT	CACCCTCTGA	540
TTGCCTCATA	TGGTCTTGCA	ACCACAGATT	TGAAATCCTG	TGTCTTGGGT	GTTCTCTTAG	600
GAGGAAGTCT	TCCTCTGATA	GCATCTGTAC	tgaattttga	AATAGCTGCA	CTAGTTCCGG	660
CTATTTATCA	AGATGCTAAA	CATGTTGAGC	TTGGAATTGA	CATGTCTAAG	TTTAGCACTA	720
AAGAGGCAGT	TGGGAAAGTG	TGCACAGTTC	TAAAAAGCAA	AGGATATAGC	ATGAACTCTG	730
TTGAAATTGG	CAAGGCTAAA	CAATATGCAG	ATATACTAAA	GGCTTGTAGT	CCGAAAGCTA	840
AAGGACTTGC	TGCAATGGAC	CACTACAAAG	AAGGGCTTAC	ATCCATTTAC	AGCATGTTTA	9C0
ATGCTACTAT	TGATTTTGGG	AAAAATGATT	CTATTTAAAA	GAGATTTGAG	CTTGGTTTGC	960
TTTGTGTTGT	GTTAAATGCA	TTTAAATAAA	TAAAAATAAA	AATAATAAAA	ATAAAAAATA	1020
AAATAAAAAA	TAAAATAATT	AATTATTGTT	TGAGTGTATT	AAGTGTGCTT	TTATAAATTA	1080
TTTGAAATAA	GTTTAATGTG	TGTTTTAATA	TATAAACTTG	AAAGTGTGTT	TGCGTGTGAA	1140
ATAAACAAGT	AAAAAAGCTA	CAAAAAATAA	TAAAAACAAT	AAAAACCACT	ACAAAATATA	1200
TATATATATA	TATATAAATA	AAATAAAAAA	CAAAAAACAA	AAAACAAAAT	AAAAAACAAA	1260
TAAAACAAAA	CAAAAACAAA	AAACAAAAAA	CAAAAACAAA	AACAAATACA	AATTTTTGGC	1320
CAATTTGTGA	GTAGATTGGA	TTTAAAGTTT	ATAATTTACT	AATACATTCT	TTTAAAGCAT	1330
TATTAAAGCA	ACCAAATTGT	GCCCGAAGGC	CATCCTTTTT	TGGTTTTTAT	TTATTTTATG	1440
attttttttg	TATTTTTTAT	ATATTTTATG	TTTATTTATT	TATTTCATAT	GATTGTTTTT	1500
GGTTTATCTG	TTATTAGATT	TGTTTAATCT	GATCTAAATG	CATGTAAACA	AACTTTACTT	1560
AGGGACAAGT	TTAACCAAGG	TTAATTTAAA	TCTAATTTAG	AAATAGCTTT	CAAAACATCC	1620
TCTGATCTAG	GAGAAATTTT	TATTTCACCT	GCAGCATAGT	CAAATTTCCA	ATCGGGCATC	1630

BAD ORIGINAL

AP Ο Ο Ο 4 Ο 3

TGGATGCTAT	CCAAATAAGA	CATTGTTTGT	GAGTTTCTCG	GAAGACTTTT	CAAGGTCTGA	1740
GAAAGGAAAT	ATTTTCTCCC	ATAACCATCT	TCTGAATAAT	TTAAAGTCTT	TTTCCCTAGC	1800
TCAAACTTTT	TTGGCTTTAG	ATCTGGCTCA	TTTATAGTGA	AGACAATCAT	GTCTTGCTGA	1860
ACACTGTCAC	AGACAGTTTG	AGCAAGTTCT	GTGTGGATTT	CAAGAAGTGA	ATGAACAATT	1920
CTTTGAGGAT	ACGACATATC	ATGGATAACA	TTGTAAGGTT	CTTTTAGATC	AGAAAAATTC	1980
ATCTCAATTT	TATGAATCAC	TGCTAGAAGG	AGTTGATTCA	AGAAGAGGTT	TGACTGATCA	2040
AAGTTATATT	CAATGACTGT	TTCAACATTG	TTGTGATTCG	GAAGACATTG	AATGCTTATA	2100
TAATACTCTT	TAGGAGAAGA	TTCATCAGAC	AGGGTGAAGA	ACCGGCTGTA	TGTGTTTGAT	2160
TTCATTATTT	TAGGGATGCG	CATCCAAAGG	CTGATTTTAA	AAGGCTTGTT	ATTATTGAGC	2220
CTGACAAACG	AATGAGTATC	TGACATTAAA	TCAGAAGTTG	CAGACATTGC	CAGTGATTTT	2280
ACAGCCAAAG	TGTTTACTGT	CCTGTTGTTG	GTCTGGGATT	GGTTAAAAAC	AGGCTTAAGT	2340
GTGTACAGCC	ATTCATGAAC	ACTTCTGGTA	GGAGAAAGAA	CACTGACTTT	GCCCATGGAT	2400
TGGTTGTTTG	AATACTTGAC	ATCAAAAAGG	ACTTCTTTGA	CACAAGACAA	TGATCCTTTA	2460
TTTGCAGCTT	CAATGAAATT	ATTTGGAGGG	AAGATGTCAG	ATTCTTTCAG	ACCGAATCTT	2520
GAAGTAATTT	CAGGCACTGC	TATTTGCTCT	TCAAGCATTC	TGAGCAACTG	AGAGGATAAC	2580
ACTTTGAGAT	GACCTTGATG	TTTAACACCG	TTTGTGTTTG	TTGTGTTAGA	TCTGACCAGA	2640
ATTTCGACAA	CATTCCCTGA	AAAGGAGAAA	TTCAGATCAA	TGTCATCAAA	AATAGGCACA	2700
AAACATCTCA	CTATAATTTC	TTTTTCTTCT	GTTGGGAGAA	AGCCAACTTT	ACCAGTGTAA	2760
CAGAAACTGG	TCTTGCTCCT	GGAGTCAGAA	TAGAGTTGAG	CTTCGACCAA	CTTCTTTCCG	2320
GAAGATTGAT	CATGAATCCA	ATAACTATCT	ACTACTGCTT	TACCCGAAGA	TGTTGTTCCC	2330
CAGATTGAGG	ACTTCGATTT	GATAATTGTT	TCATACATTG	CACTAGACAT	GTTAAAATGG	2940
AAGTAGTAAT	GTAATTGACA	ATATTGTAAG	ATTTGTTGTA	GCTTGGTTGT	TCNNNNNNNN	3000
NNNNNNNNNA	TTGCTCT					3 017

SEQ ID NO: 10

Sequence Type: Nucleotide

Sequence length: 2993

Strandedness: Single stranded

Molecule type: Complementary S RNA nucleotide sequence from 1 to 2993 zt SEQ ID NO :9

Original Source Organism: Impatiens Necrotic Spot Virus

AGAGCAATTG TGTCACGAAT ATAACATCAT ATAATCCAAA GCTAGAAACT GAAAAATTAC 60

AAATTTTACC AAATACTACT TTAACCGCAA GTACTATTTA GTAATTTGAA CACTTTAAGC '.20

TTTAAAGTTA GTAAAGTCAG CAGATCAAGA TGAACAAAGC AAAGATTACC AAGGAGAACA '.30 bad original O

AP Ο Ο Ο 4 Ο 3

TAGTCAAGCT TTTGACTCAA TCTGATTCCT TAGAATTTGA GGAAACTCAA AATGAAGGGT 240 CTTTCAATTT CACTGACTTC TTTACCAACA ACCGTGAAAA GATTCAGAAC ATGACTACTG 300

CATCCTGCCT ATCCTTCCTC AAGAATAGGC AATCTATAAT GAGAGTCATT AAGAGTGCTG 360

ACTTCACTTT TGGATCAGTC ACAATTAAGA AAACTAGAAA CAATTCAGAA AGAGTTGGGG 420 TCAATGATAT GACTTTCCGA AGACTTGATG CAATGGTGAG AGTTCATCTT GTTGGAATGA 480 TAAAGGACAA TGGATCTGCT CTGACTGAAG CTATAAATAG CCTTCCAAGT CACCCTCTGA 540 TTGCCTCATA TGGTCTTGCA ACCACAGATT TGAAATCCTG TGTCTTGGGT GTTCTCTTAG 600 GAGGAAGTCT TCCTCTGATA GCATCTGTAC TGAATTTTGA AATAGCTGCA CTAGTTCCGG 660 CTATTTATCA AGATGCTAAA CATGTTGAGC TTGGAATTGA CATGTCTAAG TTTAGCACTA 720 AAGAGGCAGT TGGGAAAGTG TGCACAGTTC TAAAAAGCAA AGGATATAGC ATGAACTCTG 780

TTGAAATTGG CAAGGCTAAA CAATATGCAG ATATACTAAA GGCTTGTAGT CCGAAAGCTA 840 *

AAGGACTTGC TGCAATGGAC CACTACAAAG AAGGGCTTAC ATCCATTTAC AGCATGTTTA 900

ATGCTACTAT TGATTTTGGG AAAAATGATT CTATTTAAAA GAGATTTGAG CTTGGTTTGC 960

TTTGTGTTGT GTTAAATGCA ΤΤΤΑΑΑΤΑΑΑ ΤΑΑΑΑΑΤΑΑΑ ΑΑΤΑΑΤΑΑΑΑ ΑΤΑΑΑΑΑΑΤΑ 1020

ΑΑΑΤΑΑΑΑΑΑ ΤΑΑΑΑΤΑΑΤΤ AATTATTGTT TGAGTGTATT AAGTGTGCTT ΤΤΑΤΑΑΑΤΤΑ 1080

TTTGAAATAA GTTTAATGTG TGTTTTAATA TATAAACTTG AAAGTGTGTT TGCGTGTGAA 1140

ATAAACAAGT AAAAAAGCTA CAAAAAATAA TAAAAACAAT AAAAACCACT ACAAAATATA 1200

ΤΑΤΑΤΑΤΑΤΑ ΤΑΤΑΤΑΑΑΤΑ ΑΑΑΤΑΑΑΑΑΑ CAAAAAACAA AAAACAAAAT AAAAAACAAA 1260

TAAAACAAAA CAAAAACAAA AAACAAAAAA CAAAAACAAA AACAAATACA AATTTTTGGC 1320 CAATTTGTGA GTAGATTGGA TTTAAAGTTT ATAATTTACT AATACATTCA TTTTAAAGCA 13 80

TTATTAAAGC AACCAAATTG TGCCCGAAGG CCATCCTTTT TTGGTTTTTA ΤΤΤΑΤΤΤΤΑΤ 1440

GATTTTTTTT GTATTTTTTA ΤΑΤΑΤΤΤΤΑΤ GTTTATTTAT TTATTTCATA TGATTGTTTT 1500 TGGTTTATCT GTTATTAGAT TTGTTTAATC TGATCTAAAT GCATGTAAAC AAACTTTACT 1560

TAGGGACAAG TTTAACCAAG GTTAATTTAA ATCTAATTTA GAAATAGCTT TCAAAACATC 1620

CTCTGATCTA GGAGAAATTT TTATTTCACC TGCAGCATAG TCAAATTTCC AATCGGGCAT 1680 CTGGATGCTA TCCAAATAAG ACATTGTTTG TGAGTTTCTC GGAAGACTTT TCAAGGTCTG 1740 AGAAAGGAAA TATTTTCTCC CATAACCATC TTCTGAATAA TTTAAAGTCT TTTTCCCTAG 1800 CTCAAACTTT TTTGGCTTTA GATCTGGCTC ATTTATAGTG AAGACAATCA TGTCTTGCTG 1860 AACACTGTCA CAGACAGTTT GAGCAAGTTC TGTGTGGATT TCAAGAAGTG AATGAACAAT 1920 TCTTTGAGGA TACGACATAT CATGGATAAC ATTGTAAGGT TCTTTTAGAT CAGAAAAATT 1980 CATCTCAATT TTATGAATCA CTGCTAGAAG GAGTTGATTC AAGAAGAGGT TTGACTGATC 2040 AAAGTTATAT TCAATGACTG TTTCAACATT GTTGTGATTC GGAAGACATT GAATGCTTAT 2100 ATAATACTCT TTAGGAGAAG ATTCATCAGA CAGGGTGAAG AACCGGCTGT ATGTGTTTGA 2160 TTTCATTATT TTAGGGATGC GCATCCAAAG GCTGATTTTA AAAGGCTTGT TATTATTGAG 2220 CCTGACAAAC GAATGAGTAT CTGACATTAA ATCAGAAGTT GCAGACATTG CCAGTGATTT 2280 TACAGCCAAA GTGTTTACTG TCCTGTTGTT GGTCTGGGAT TGGTTAAAAA CAGGCTTAAG 2340 TGTGTACAGC CATTCATGAA CACTTCTGGT AGGAGAAAGA ACACTGACTT TGCCCATGGA 2400

AP Ο Ο Ο 4 Ο 3

TTGGTTGTTT GAATACTTGA CATCAAAAAG GACTTCTTTG ACACAAGACA ATGATCCTTT 2460

ATTTGCAGCT TCAATGAAAT TATTTGGAGG GAAGATGTCA GATTCTTTCA GACCGAATCT 2520 TGAAGTAATT TCAGGCACTG CTATTTGCTC TTCAAGCATT CTGAGCAACT GAGAGGATAA 2580 CACTTTGAGA TGACCTTGAT GTTTAACACC GTTTGTGTTT GTTGTGTTAG ATCTGACCAG 2640 AATTTCGACA ACATTCCCTG AAAAGGAGAA ATTCAGATCA ATGTCATCAA AAATAGGCAC 2700 AAAACATCTC ACTATAATTT CTTTTTCTTC TGTTGGGAGA AAGCCAACTT TACCAGTGTA 2760 ACAGAAACTG GTCTTGCTCC TGGAGTCAGA ATAGAGTTGA GCTTCGACCA ACTTCTTTCC 2820 GGAAGATTGA TCATGAATCC AATAACTATC TACTACTGCT TTACCCGAAG ATGTTGTTCC 2880 CCAGATTGAG GACTTCGATT TGATAATTGT TTCATACATT GCACTAGACA TGTTAAAATG 2940 GAAGTAGTAA TGTAATTGAC AATATTGTAA GATTTGTTGT AGCTTGGTTG TTC 2993

SEQ ID NO: 11

Sequence type: Nucleotide

Sequence length: 789 nucleotides

Strandedness: Single stranded

Molecule type: Viral complementary S RNA nucleotide sequence coding for the nucleocapsid (N) protein from position 150 to position 938 of

SEQ ID NO:9

Original Source Organism: Impatiens Necrotic Spot Virus

ATGAACAAAG	CAAAGATTAC	CAAGGAGAAC	ATAGTCAAGC	TTTTGACTCA	ATCTGATTCC	60
TTAGAATTTG	AGGAAACTCA	AAATGAAGGG	TCTTTCAATT	TCACTGACTT	CTTTACCAAC	120
AACCGTGAAA	AGATTCAGAA	CATGACTACT	GCATCCTGCC	TATCCTTCCT	CAAGAATAGG	180
CAATCTATAA	TGAGAGTCAT	TAAGAGTGCT	GACTTCACTT	TTGGATCAGT	CACAATTAAG	240
AAAACTAGAA	ACAATTCAGA	AAGAGTTGGG	GTCAATGATA	TGACTTTCCG	AAGACTTGAT	300
GCAATGGTGA	GAGTTCATCT	TGTTGGAATG	ATAAAGGACA	ATGGATCTGC	TCTGACTGAA	360
GCTATAAATA	GCCTTCCAAG	TCACCCTCTG	ATTGCCTCAT	ATGGTCTTGC	AACCACAGAT	420
TTGAAATCCT	GTGTCTTGGG	TGTTCTCTTA	GGAGGAAGTC	TTCCTCTGAT	AGCATCTGTA	480
CTGAATTTTG	AAATAGCTGC	ACTAGTTCCG	GCTATTTATC	AAGATGCTAA	ACATGTTGAG	540
CTTGGAATTG	ACATGTCTAA	GTTTAGCACT	AAAGAGGCAG	TTGGGAAAGT	GTGCACAGTT	600
CTAAAAAGCA	AAGGATATAG	CATGAACTCT	GTTGAAATTG	GCAAGGCTAA	ACAATATGCA	660
GATATACTAA	AGGCTTGTAG	TCCGAAAGCT	AAAGGACTTG	CTGCAATGGA	CCACTACAAA	720
GAAGGGCTTA	CATCCATTTA	CAGCATGTTT	AATGCTACTA	TTGATTTTGG	GAAAAATGAT	780

AP Ο Ο Ο 4 Ο 3

SEQ ID NO: 12

Sequence type: Nucleotide

Sequence length: 1350 nucleotides

Strandedness: Single stranded ·

Molecule type: Complementary S RNA nucleotide sequence (to the viral S RNA nucleotide sequence) from position 1581 to position 2930 of SEQ ID NO:9

Original Source Organism: Impatiens Necrotic Spot Virus

TTAATTTAAA	TCTAATTTAG	AAATAGCTTT	CAAAACATCC	TCTGATCTAG	GAGAAATTTT	60
TATTTCACCT	GCAGCATAGT	CAAATTTCCA	ATCGGGCATC	TGGATGCTAT	CCAAATAAGA	120
CATTGTTTGT	GAGTTTCTCG	GAAGACTTTT	CAAGGTCTGA	GAAAGGAAAT	ATTTTCTCCC	180
ATAACCATCT	TCTGAATAAT	TTAAAGTCTT	TTTCCCTAGC	TCAAACTTTT	TTGGCTTTAG	240
ATCTGGCTCA	TTTATAGTGA	AGACAATCAT	gtcttgctga	ACACTGTCAC	AGACAGTTTG	300
AGCAAGTTCT	GTGTGGATTT	CAAGAAGTGA	ATGAACAATT	CTTTGAGGAT	ACGACATATC	360
ATGGATAACA	TTGTAAGGTT	CTTTTAGATC	AGAAAAATTC	ATCTCAATTT	TATGAATCAC	420
TGCTAGAAGG	AGTTGATTCA	AGAAGAGGTT	TGACTGATCA	AAGTTATATT	CAATGACTGT	480
TTCAACATTG	TTGTGATTCG	GAAGACATTG	AATGCTTATA	TAATACTCTT	TAGGAGAAGA	540
TTCATCAGAC	AGGGTGAAGA	ACCGGCTGTA	TGTGTTTGAT	TTCATTATTT	TAGGGATGCG	600
CATCCAAAGG	CTGATTTTAA	AAGGCTTGTT	ATTATTGAGC	CTGACAAACG	AATGAGTATC	660
TGACATTAAA	TCAGAAGTTG	CAGACATTGC	CAGTGATTTT	ACAGCCAAAG	TGTTTACTGT	720
CCTGTTGTTG	GTCTGGGATT	GGTTAAAAAC	AGGCTTAAGT	GTGTACAGCC	ATTCATGAAC	780
ACTTCTGGTA	GGAGAAAGAA	CACTGACTTT	GCCCATGGAT	TGGTTGTTTG	AATACTTGAC	840
ATCAAAAAGG	ACTTCTTTGA	CACAAGACAA	TGATCCTTTA	TTTGCAGCTT	CAATGAAATT	900
ATTTGGAGGG	AAGATGTCAG	ATTCTTTCAG	ACCGAATCTT	GAAGTAATTT	CAGGCACTGC	960
TATTTGCTCT	TCAAGCATTC	TGAGCAACTG	AGAGGATAAC	actttgagat	GACCTTGATG	1020
TTTAACACCG	TTTGTGTTTG	TTGTGTTAGA	TCTGACCAGA	ATTTCGACAA	CATTCCCTGA	1080
AAAGGAGAAA	TTCAGATCAA	TGTCATCAAA	AATAGGCACA	AAACATCTCA	CTATAATTTC	1140
TTTTTCTTCT	GTTGGGAGAA	AGCCAACTTT	ACCAGTGTAA	CAGAAACTGG	TCTTGCTCCT	1200
GGAGTCAGAA	TAGAGTTGAG	CTTCGACCAA	CTTCTTTCCG	GAAGATTGAT	CATGAATCCA	1260
ATAACTATCT	ACTACTGCTT	TACCCGAAGA	TGTTGTTCCC	CAGATTGAGG	ACTTCGATTT	1320
GATAATTGTT	TCATACATTG	CACTAGACAT				1350

BAD ORIGINAL

APO00403

SEQ ID NO :13

Sequence type: Nucleotide

Sequence length: 642 nucleotides

Strandedness: single stranded

Molecule type: Viral complementary S RNA strand of the intergenic region from position 939 to position 1580 of SEQ ID NO:9

Original Source Organism:Impatiens Necrotic Spot Virus

AAGAGATTTG	AGCTTGGTTT	GCTTTGTGTT	GTGTTAAATG	CATTTAAATA	AATAAAAATA	60
AAAATAATAA	AAATAAAAAA	TAAAATAAAA	AATAAAATAA	TTAATTATTG	TTTGAGTGTA	120
TTAAGTGTGC	TTTTATAAAT	TATTTGAAAT	AAGTTTAATG	TGTGTTTTAA	TATATAAACT	180
TGAAAGTGTG	TTTGCGTGTG	AAATAAACAA	GTAAAAAAGC	TACAAAAAAT	AATAAAAACA	240
ATAAAAACCA	CTACAAAATA	TATATATATA	TATATATAAA	TAAAATAAAA	AACAAAAAAC	300
AAAAAACAAA	ATAAAAAACA	AATAAAACAA	AACAAAAACA	AAAAACAAAA	AACAAAAACA	360
AAAACAAATA	CAAATTTTTG	GCCAATTTGT	GAGTAGATTG	GATTTAAAGT	TTATAATTTA	420
CTAATACATT	CTTTTAAAGC	ATTATTAAAG	CAACCAAATT	GTGCCCGAAG	GCCATCCTTT	480
TTTGGTTTTT	ΑΤΤΤΑΤΊΤΤΑ	TGATTTTTTT	tgtatttttt	ATATATTTTA	TGTTTATTTA	540
TTTATTTCAT	ATGATTGTTT	ttggtttatc	TGTTATTAGA	TTTGTTTAAT	CTGATCTAAA	600
TGCATGTAAA	CAAACTTTAC	TTAGGGACAA	GTTTAACCAA	GG		642

SEQ ID NO :14 ( Sequence type:Nucleotide

Sequence length: 4970 nucleotides Strandedness: single stranded Molecule type: Viral M RNA

Original Source Organism: Impatiens Necrotic Spot Virus

AGAGCAATCA GTGCATCAAA ATTATATCTA GCCGAATTCA ATCATTATCT TCTCAATATT 60 TTAATTCTTA ATCTACCGTC CAGAGATGAA TAGTTTTTTC AAATCACTCA GATCATCTAG 120 CAGCAGGGAG CTAGATCACC CTAGGGTTAC AACTACCCTC TCTAAACAAG GAGCAGACAT 180 TGTTGTACAC AATCCTTCTG CTAATCACAA CAACAAGGAA GTTCTCCAAA GAGCCATGGA 240 TAGCTCTAAA GGGAAGATTT TGATGAACAA TACAGGCACC TCATCACTAG GCACATATGA 300 GTCTGACCAG ATATCTGAAT CAGAGTCTTA TGATCTTTCT GCTAGAATGA TTGTTGATAC 360 AAATCATCAT ATCTCCAGCT GGAAAAATGA TCTTTTTGTA GGTAATGGTG ATAAAGCTGC 420 AACCAAGATA ATTAAGATAC ATCCAACCTG GGATAGCAGA AAACAATACA TGATGATCTC 480

AP Ο 00 4 Ο 3

TTTAATTGAT	CCTAACAAGA	GTGTTAATGC	CAGAACTGTT	TTGAAAGGGC	AAGGAAGCAT	600
TAAAGATCCT	ATATGTTTTG	TTTTTTATCT	AAATTGGTCC	ATTCCAAAAG	TTAACAACAC	660
TTCAGAGAAT	TGTGTTCAGC	TTCATTTATT	ATGTGATCAA	GTTTACAAGA	AAGATGTTTC	720
TTTTGCTAGT	GTCATGTATT	CTTGGACAAA	AGAATTCTGT	GATTCACCAA	GAGCAGATCT	780
GGATAAAAGC	TGCATGATAA	TACCCATCAA	TAGGGCTATT	AGAGCCAAAT	CGCAAGCCTT	840
CATTGAAGCC	TGCAAGTTAA	TCATACCTAA	AGGCAATTCT	GAAAAGCAAA	TTAGAAGACA	900
ACTTGCAGAG	CTAAGTGCTA	ATTTAGAGAA	ATCTGTTGAA	GAAGAGGAGA	ATGTTACTGA	960
TAACAAGATA	GAGATATCAT	TTGATAATGA	AATCTAAATA	TGTTTTCATT	TAATAATAAA	1020
TAATATATAT	TGTTCATAAT	ATTTTGAATG	TTTAAGTAAA	AAATAAAGCA	AGATAAAAAA	1080
CTATATATAT	ATATATATAT	AGAAGTATAA	AATATATATG	TATTTGTGTT	TAAAAACAAA	1140
TCAAAAACCA	AAAAAGAAAA	AAGAAAAAAT	AAACAAAAAA	CAAAAACAAA	AACAAAAACA	1200
AACAAAAAGC	AAAAAATAGA	AAAAAGTTGA	AAAAAACCAA	AAAAATTTTT	TTTGTAAATA	1260
AATAAGGCTC	CGGCCAGATT	TGGTCTAAGA	CCTTTTTATT	TGTTTTTATA	CATTTTATTT	1320
GTTTTTGTTG	ATTTTTATTT	TTATTATTTT	ΤΑΤΑΤΤΤΓΤΤ	ATATAGTTTG	CTTATTTAAC	1380
ACTTATTTAG	ACAAATTAAA	TTTATTTGAT	TACAATCATT	CTGCCTTATT	TAATTTAAAA	1440
CACATTTGGT	GTATATTCCA	ATGAATTTAA	TCATATACCG	CTGAAGTCTA	GAGGAGGTCT	1500
TCTTCTAGTG	ATGGTGTCTT	TACCAGAAGA	CGTGGAAACC	AAAGAATAAT	CATTAGTGTC	1560
TTCAATATAT	TTTGTCTTGT	AAGACTTGTT	TCTAACATAG	CCTCTACACA	TTGTGGCAAC	1620
AATAGAGCAG	AGGTAAGCAA	GAGCAAATAC	AAAGAGTATG	AGCAATACTA	CTCTGACTGT	1680
ATCAAAGAAG	GATCCAAAGT	GGCTTGCTAT	AAAGTTAAAA	GGGCTTTTAA	CATAGTCCCA	17 4 0
AAAGCTCCAA	ACTGATGTGT	CAGAATTATA	TTGCTGTTCC	TCGTGTGCAT	GTTGGTCATT	1800
TTGATCAATT	ATGTTTTCTG	GTTCCAGCAC	AGCAACAGAA	TCTACAAGTG	CCTCAACTGA	1860
GTATGATTTG	TCTCCTTCTG	GTTCTATAAT	CATTTTTTGT	TTTTCTGGGT	TAGAAGTGCA	1920
GAACATTGTC	AAGTTATACT	TATTAGCACC	TTTCTTTACT	GCTATCTGGT	ATGTTGACAA	1980
TGAACATTGT	TTCATGGTTA	ACCTTGCAGA	AAAAGTTATG	TCTGATATAA	ATGAGGCAGC	2040
ACACCTCAGC	CCTTGGCTAC	ATAAGAAACA	TCCCTTACAG	CTTAAAGAGA	CAGAACTCAA	2100
TATAGGCTTT	TTTGGTACAG	TTTTAAACAA	TTCAGAAGGT	AGATCCAAAA	CAATTTTAAG	2160
CTTACCTAGA	CTAAAGATCT	TTTCCATATA	AAAACTATTC	TGGTCAGTAA	ACTGAACTGG	2220
AATGTCCGAT	atttggttca	AACCTGTTTT	AAATCTGTAT	GTGTCATAAC	CACATGATTT	2280
TATCGTAATT	GTTTTTTTAC	CAATTGCTGA	ACAATCCCAG	GACAGATCGT	TTGTATCTAA	2340
TGTTTTCTTA	GAGAAAATGG	GATCACCTTG	GTGTGAAAGT	TGAGGATGAC	CAAACATTTT	2400
TGATGGATTA	TTTAATCTAG	CTATGTTTCC	CGCATATACG	TGACTATCAG	GTCCATGAGC	2460
TATCAGCTGG	CCTATTGTTA	AGCCATCATT	ATGGAAATCC	GCTAATATAT	CAGCCTGGAA	2520
ATATCCTGAT	TCAGATGGGA	CTTCCTCAGA	TACAGTGAAA	CACTTTGCTC	CCACAAATCC	2580
AGATATACAT	ACTTCAGACT	TGATTGTTGA	TTTAATAACA	GAATAAATCC	TGAAAGATTG	2640
ATCCATATCA	TACACATTTC	TACAAAACCC	ACAAGTGGCT	CCTTCATTGA	TAGCCAAACA	2700
CCAAACCTCT	TCACAACCCC	AGTAAGATGT	TGGTGTTATG	CAGAAATCTT	GATACCCAGT	2760

AP000403 )

TATCGGTTGT TCTTTTCTGC AATCTGAGCA TTTACCTGTG CATGTTGAAA AGAAATCAGT 2820 GTGGGTGCTT TGTATAGGAG CTGTAGTGTA TTGTTCAGAA ACATCATACT GTATTCTAAC 2880 ΤΤΤΤΤΤΑΑΤΑ TAAACAACAA ACTTCTGAGC AGTGCTAGAA CTTTTGTCAT TAAGAGAGAA 2940 AACTGTGCCC CCACCTGATA ATAAAGATTC TTCTATCATG TATCTATATT TTCCATCTAT 3000

CACCGAGTCA AATATGAGAG ATTTTCTTGG AAAAATGCTT TCAGGTATGT CTGATTCATT 3060 AGATTTAAGT GCATCTCCAG AAATGTATCC ATATTTTTCA GTTTTATTGT AGAAATCAAT 3120 TATACCATTC CTAAGCCTTT TCATGAAGTG TAGATTCACA GCATTCAATC CCAATGTGTC 3180 ACCAGAATAT TCTAAGAACC CATTATCTAA AGGCTTGCTT TGGAAAATAG AGGCATACTC 3240 ACAACCAAAT CTGCATTTGA CAAAAGTTAC TAAAGCATTT TCAGTTATCC TGCCTTTGCA 3300 TTCTTGATAA GGTATACAAT CCATAGGACC TTCTGTCACA ACATTGGTTA GAAAGTTAGA 3360 TTCTACAATA GAATTTTCTT TAATAGCACA GAAGCATTGG TCTTTTTCAG GACATTTGTC 3420 *' ATATCTGTTT GTAACAAAGC GGTCACAACC AGGGACATAA TAACAGCTAT CCAAACACTG 3480 AGCAGTTTGA GCCATAGACA TAGGCATCTG TGACAAAATC AGAAATCCTA TCAAAGTTTC 3540 TGTGACTGCT TTTAGGAAAG AGAGGCCTAT TTTTGTATTA ACTATCAAAT GGAACCATTC 3600 AATGCTAGCC CAGTTGTATT TTTTATTCTT CTCTGCTGTT CTAGTTATTA TAGGACATTC 3660 TTCTGAGTGT TCTTCAGAGG CTTTGTTTTT GTTACAAATG CATAATTTTG AGCATTCATG 3720 GGTTACCAAA CATAAATTTC CACAGACCTT ACATTTCAAG GGAAAATAAG ACCATAAATA 3780 ATTTATCAGT AGTAGTATAG GATACGTTAT CAATCCCAGA AGATCATACC CATAGAACAG 3840 TGTTTTAGAT GTTTTGTTTA CCAAGTACCT TATAGGGAAA TAGACAATCA GAGCAATCAT 3900

GATCAATCTA AACCATGAGA AGTTGATGCA AGCAGTTTGT TTGTAAATAT TTTTGGAGTA 3960 CTTTATAATA CAATCTCTAA CTCTTTTGTC CACTAAAGGA ACTTTAGAAG ACTTGTCACC 4020

GCACAATAGG TTATGCTTAC CATCCATATT TTCTTCTGTG AAAGTCAAAC TAACTGAGCC 4080

AGAGAAGCTT ATTATGGAAT GGCTCATGTC ACTTCCTTCT CTTTTGACGA CGTAACCCAT 4140 GATTTTCTCA GGTGTAGTTA ATGAAACTGT ATAAGAATTA ACTATGTTTG TTTTTGATAT 4200

TTTACAATCA CCTGAGAATT TCACACTCTG GAGAGAGACT GTGCCATTAG TTGGTCTAGA 4260

ATTGTACATG ATTGGATAAT TGTAATTCTC CAAACTTTCA ATTATATAGA ATTTAGTTCC 4320

TATAGATAAT TTCCTTTTGT TATCGATTTT TGTTATTGGT ACAACTGGAA CAGTTTCAAA 4380 GCTTCTTGGC AATTCAGAAG ATCCTTCACA GTTTCCCAAT TTAGTTATAG TGTCACTGAT 4440 ACATGAATAT ATAACACCAT TGCTTTCTAC TTGGTAATAA ACATTGAATG TTGAAACTCC 4500 TTTAATGCTA CAAGTCAAAC TTGAAGCATT TAGGCATGGA TTTGGTAAAT CCATAACTGA 4560 TATAGTTGTT GGTGTAGAAG ACAATCCACT TGGAGATTGA GGTACCTCAT TATTGGCAAG 4620 AACAGTTTGA GTATCTCGTG TTGGTCTAAG GGTTTTACCT GTTGCATTCT GGAGCATTTC 4680 AGCCAAAGTA TCTAGAATTT CATTTTTATG ATCTACAGAA CGGTCATAAT AAGCTTCATC 4740 ATAAATTTCT GGATGATCGC CCCTTTCAAC ATGAATCTTT GCATCTGTCT CCTTTAATGC 4800 CATAAAGGAT AAGATAACAG AAGTAACAAC TAGTGTACAT ACACTAATTT TAACAAGTAA 4860 CTCGCACATC TTTAGAATTT TCATTCTAAA AAGTCGAATA ACACTAGTTC TAAAATTGCT 4910 TTATGAGTTT GATCTGTTGT ATGTAGAGTT TTGTTTGCAC TGATTGCTCT 4970

BAD ORIGINAL

AP 0 00 4 0 3

SEQ ID NO» 15

Sequence type: Nucleotide

Sequence length: 912 nucleotides

Strandedness: single strand

Molecule type: Viral M RNA nucleotide sequence coding for the NSm protein gene from position 86 to position 997 of SEQ ID NO:14

Original Source Organism: Impatiens Necrotic Spot Virus

ATGAATAGTT	TTTTCAAATC	ACTCAGATCA	TCTAGCAGCA	GGGAGCTAGA	TCACCCTAGG	60
GTTACAACTA	CCCTCTCTAA	ACAAGGAGCA	GACATTGTTG	TACACAATCC	TTCTGCTAAT	120
CACAACAACA	AGGAAGTTCT	CCAAAGAGCC	ATGGATAGCT	CTAAAGGGAA	GATTTTGATG	180
AACAATACAG	GCACCTCATC	ACTAGGCACA	TATGAGTCTG	ACCAGATATC	TGAATCAGAG	240
TCTTATGATC	TTTCTGCTAG	AATGATTGTT	GATACAAATC	ATCATATCTC	CAGCTGGAAA	300
AATGATCTTT	TTGTAGGTAA	TGGTGATAAA	GOTGCAACCA	AGATAATTAA	GATACATCCA	360
ACCTGGGATA	GCAGAAAACA	ATACATGATG	ATCTCAAGGA	TAGTTATCTG	GATATGCCCT	420
ACTATAGCTG	ATCCTGATGG	GAAATTGGCT	GTAGCTTTAA	TTGATCCTAA	CAAGAGTGTT	480
AATGCCAGAA	CTGTTTTGAA	AGGGCAAGGA	AGCATTAAAG	ATCCTATATG	TTTTGTTTTT	540
TATCTAAATT	GGTCCATTCC	AAAAGTTAAC	AACACTTCAG	AGAATTGTGT	TCAGCTTCAT	600
TTATTATGTG	ATCAAGTTTA	CAAGAAAGAT	GTTTCTTTTG	CTAGTGTCAT	GTATTCTTGG	6 6 0
ACAAAAGAAT	TCTGTGATTC	ACCAAGAGCA	GATCTGGATA	AAAGCTGCAT	GATAATACCC
ATCAATAGGG	CTATTAGAGC	CAAATCGCAA	GCCTTCATTG	AAGCCTGCAA	GTTAATCATA	~30
CCTAAAGGCA	attctgaaaa	GCAAATTAGA	AGACAACTTG	CAGAGCTAAG	TGCTAATTTA	34 0
GAGAAATCTG	TTGAAGAAGA	GGAGAATGTT	ACTGATAACA	AGATAGAGAT	ATCATTTGAT	9 0 0
AATGAAATCT	AA					? 1 2

SEQ ID No »16

Sequence type: nucleotide

Sequence length: 473 nucleotides

Strandedness: single stranded

Molecule type: Intergenic region of viral M RNA from position 998 to position 1470 of SEQ ID NO:14

Original Source Organism: Impatiens necrotic spot virus

ATATGTTTTC ATTTAATAAT AAATAATATA TATTGTTCAT AATATTTTGA ATGTTTAAGT -?

AAAAAATAAA GCAAGATAAA AAACTATATA TATATATATA TATAGAAGTA TAAAATATAT 120

ATGTATTTGT GTTTAAAAAC AAATCAAAAA CCAAAAAAGA AAAAAGAAAA AATAAACA-A I 60

BAD ORIGINAL

AP Ο Ο Ο 4 Ο 3

AAACAAAAAC AAAAACAAAA ACAAACAAAA AGCAAAAAAT AGAAAAAAGT TGAAAAAAAC 240 CAAAAAAATT TTTTTTGTAA ATAAATAAGG CTCCGGCCAG ATTTGGTCTA AGACCTTTTT 300 ATTTGTTTTT ATACATTTTA TTTGTTTTTG TTGATTTTTA ΤΤΙΤΤΑΤΤΑΤ ΊΤΤΤΑΤΑΤΤΤ 360 TTTATATAGT TTGCTTATTT AACACTTATT TAGACAAATT ΑΑΑΤΤΤΑΤΤΤ GATTACAATC 420 ATTCTGCCTT ΑΤΤΤΑΑΤΤΤΑ AAACACATTT GGTGTATATT CCAATGAATT ΤΑΑ 473

SEQ ID Νο. 17

Sequence type: Nucleotide

Sequence length: 3414 nucleotides

Strandedness: single stranded

Molecule Type: Viral M RNA from 1471 - 4884

Original Source Organism: Impatiens Necrotic Spot Virus

TCATATACCG	CTGAAGTCTA	GAGGAGGTCT	TCTTCTAGTG	ATGGTGTCTT	TACCAGAAGA	60
CGTGGAAACC	AAAGAATAAT	CATTAGTGTC	TTCAATATAT	TTTGTCTTGT	AAGACTTGTT	120
TCTAACATAG	CCTCTACACA	TTGTGGCAAC	AATAGAGCAG	AGGTAAGCAA	GAGCAAATAC	180
AAAGAGTATG	AGCAATACTA	CTCTGACTGT	ATCAAAGAAG	GATCCAAAGT	GGCTTGCTAT	240
AAAGTTAAAA	GGGCTTTTAA	CATAGTCCCA	AAAGCTCCAA	ACTGATGTGT	CAGAATTATA	300
TTGCTGTTCC	TCGTGTGCAT	GTTGGTCATT	TTGATCAATT	ATGTTTTCTG	GTTCCAGCAC	360
AGCAACAGAA	TCTACAAGTG	CCTCAACTGA	GTATGATTTG	TCTCCTTCTG	GTTCTATAAT	420
CATTTTTTGT	TTTTCTGGGT	TAGAAGTGCA	GAACATTGTC	AAGTTATACT	TATTAGCACC	480
TTTCTTTACT	GCTATCTGGT	ATGTTGACAA	TGAACATTGT	TTCATGGTTA	ACCTTGCAGA	540
AAAAGTTATG	TCTGATATAA	ATGAGGCAGC	ACACCTCAGC	CCTTGGCTAC	ATAAGAAACA	600
TCCCTTACAG	CTTAAAGAGA	CAGAACTCAA	TATAGGCTTT	TTTGGTACAG	TTTTAAACAA	660
TTCAGAAGGT	AGATCCAAAA	CAATTTTAAG	CTTACCTAGA	CTAAAGATCT	TTTCCATATA	720
AAAACTATTC	TGGTCAGTAA	ACTGAACTGG	AATGTCCGAT	ATTTGGTTCA	AACCTGTTTT	780
AAATCTGTAT	GTGTCATAAC	CACATGATTT	TATCGTAATT	GTTTTTTTAC	CAATTGCTGA	840
ACAATCCCAG	GACAGATCGT	TTGTATCTAA	TGTTTTCTTA	GAGAAAATGG	GATCACCTTG	900
GTGTGAAAGT	TGAGGATGAC	CAAACATTTT	TGATGGATTA	TTTAATCTAG	CTATGTTTCC	960
CGCATATACG	TGACTATCAG	GTCCATGAGC	TATCAGCTGG	CCTATTGTTA	AGCCATCATT	1020
ATGGAAATCC	GCTAATATAT	CAGCCTGGAA	ATATCCTGAT	TCAGATGGGA	CTTCCTCAGA	1080
TACAGTGAAA	CACTTTGCTC	CCACAAATCC	AGATATACAT	ACTTCAGACT	TGATTGTTGA	1140
TTTAATAACA	GAATAAATCC	TGAAAGATTG	ATCCATATCA	TACACATTTC	TACAAAACCC	1200
ACAAGTGGCT	CCTTCATTGA	TAGCCAAACA	CCAAACCTCT	TCACAACCCC	AGTAAGATGT	1260
TGGTGTTATG	CAGAAATCTT	GATACCCAGT	TATCGGTTGT	TCTTTTCTGC	AATCTGAGCA	1320
TTTACCTGTG	CATGTTGAAA	AGAAATCAGT	GTGGGTGCTT	TGTATAGGAG	CTGTAGTGTA	1380

BAD ORIGINAL

APO00403

TTGTTCAGAA ACATCATACT GTATTCTAAC TTTTTTAATA TAAACAACAA ACTTCTGAGC 1440 AGTGCTAGAA CTTTTGTCAT TAAGAGAGAA AACTGTGCCC CCACCTGATA ATAAAGATTC 1500 TTCTATCATG TATCTATATT TTCCATCTAT CACCGAGTCA AATATGAGAG ATTTTCTTGG 1560 AAAAATGCTT TCAGGTATGT CTGATTCATT AGATTTAAGT GCATCTCCAG AAATGTATCC 1620 ATATTTTTCA GTTTTATTGT AGAAATCAAT TATACCATTC CTAAGCCTTT TCATGAAGTG 1680 TAGATTCACA GCATTCAATC CCAATGTGTC ACCAGAATAT TCTAAGAACC CATTATCTAA 1740 AGGCTTGCTT TGGAAAATAG AGGCATACTC ACAACCAAAT CTGCATTTGA CAAAAGTTAC 1800 TAAAGCATTT TCAGTTATCC TGCCTTTGCA TTCTTGATAA GGTATACAAT CCATAGGACC 1860 TTCTGTCACA ACATTGGTTA GAAAGTTAGA TTCTACAATA GAATTTTCTT TAATAGCACA 1920 GAAGCATTGG TCTTTTTCAG GACATTTGTC ATATCTGTTT GTAACAAAGC GGTCACAACC 1980 AGGGACATAA TAACAGCTAT CCAAACACTG AGCAGTTTGA GCCATAGACA TAGGCATCTG 2040’ TGACAAAATC AGAAATCCTA TCAAAGTTTC TGTGACTGCT TTTAGGAAAG AGAGGCCTAT 2100 TTTTGTATTA ACTATCAAAT GGAACCATTC AATGCTAGCC CAGTTGTATT TTTTATTCTT 2160

CTCTGCTGTT CTAGTTATTA TAGGACATTC TTCTGAGTGT TCTTCAGAGG CTTTGTTTTT 2220

GTTACAAATG CATAATTTTG AGCATTCATG GGTTACCAAA CATAAATTTC CACAGACCTT 2280 ACATTTCAAG GGAAAATAAG ACCATAAATA ATTTATCAGT AGTAGTATAG GATACGTTAT 2340

CAATCCCAGA AGATCATACC CATAGAACAG TGTTTTAGAT GTTTTGTTTA CCAAGTACCT 2400

TATAGGGAAA TAGACAATCA GAGCAATCAT GATCAATCTA AACCATGAGA AGTTGATGCA 2460

AGCAGTTTGT TTGTAAATAT TTTTGGAGTA CTTTATAATA CAATCTCTAA CTCTTTTGTC 2520 CACTAAAGGA ACTTTAGAAG ACTTGTCACC GCACAATAGG TTATGCTTAC CATCCATATT 2580

TTCTTCTGTG AAAGTCAAAC TAACTGAGCC AGAGAAGCTT ATTATGGAAT GGCTCATGTC 2540

ACTTCCTTCT CTTTTGACGA CGTAACCCAT GATTTTCTCA GGTGTAGTTA ATGAAACTGT 2700

ATAAGAATTA ACTATGTTTG TTTTTGATAT TTTACAATCA CCTGAGAATT TCACACTCTG 2760 GAGAGAGACT GTGCCATTAG TTGGTCTAGA ATTGTACATG ATTGGATAAT TGTAATTCTC 2820

CAAACTTTCA ATTATATAGA ATTTAGTTCC TATAGATAAT TTCCTTTTGT TATCGATTTT 2880 TGTTATTGGT ACAACTGGAA CAGTTTCAAA GCTTCTTGGC AATTCAGAAG ATCCTTCACA 2940 GTTTCCCAAT TTAGTTATAG TGTCACTGAT ACATGAATAT ATAACACCAT TGCTTTCTAC 3000 TTGGTAATAA ACATTGAATG TTGAAACTCC TTTAATGCTA CAAGTCAAAC TTGAAGCATT 3060 TAGGCATGGA TTTGGTAAAT CCATAACTGA TATAGTTGTT GGTGTAGAAG ACAATCCACT 3120 TGGAGATTGA GGTACCTCAT TATTGGCAAG AACAGTTTGA GTATCTCGTG TTGGTCTAAG 3180 GGTTTTACCT GTTGCATTCT GGAGCATTTC AGCCAAAGTA TCTAGAATTT CATTTTTATG 3240 ATCTACAGAA CGGTCATAAT AAGCTTCATC ATAAATTTCT GGATGATCGC CCCTTTCAAC 3300 ATGAATCTTT GCATCTGTCT CCTTTAATGC CATAAAGGAT AAGATAACAG AAGTAACAAC 3360

AP Ο Ο Ο 4 Ο 3

SEQ ID NO: 18

Sequence type: nucleotide

Sequence length: 36 nucleotides

Strandedness: single stranded

Molecule type: 5' end of M RNA pan-handle

Original Source Organism: Impatiens Necrotic Spot Virus

AGAGCAATCA GTGCATCAAA ATTATATCTA GCCGAA 36

SEQ ID NO: 19

Sequence type: Nucleotide

Sequence length: 36 nucleotides

Strandedness: single stranded

Molecule type: 3* end of M RNA pan-handle '

Original Source Organism: Impatiens Necrotic Spot Virus

CTGTTGTATG TAGAGTTTTG TTTGCACTGA TTGCTC 36

SEQ ID NO: 20

Sequence type: Nucleotide

Sequence length: 4970 nucleotides

Strandedness: single stranded

Molecule type: Complementary M RNA

Original Source Organism: Impatiens Necrotic Spot Virus

AGAGCAATCA	GTGCAAACAA	AACTCTACAT	ACAACAGATC	AAACTCATAA	AGCAATTTTA	60
GAACTAGTGT	TATTCGACTT	TTTAGAATGA	AAATTCTAAA	GATGTGCGAG	TTACTTGTTA	120
AAATTAGTGT	ATGTACACTA	GTTGTTACTT	CTGTTATCTT	atcctttatg	GCATTAAAGG	180
AGACAGATGC	AAAGATTCAT	GTTGAAAGGG	GCGATCATCC	AGAAATTTAT	GATGAAGCTT	240
ATTATGACCG	TTCTGTAGAT	CATAAAAATG	AAATTCTAGA	TACTTTGGCT	GAAATGCTCC	300
AGAATGCAAC	AGGTAAAACC	CTTAGACCAA	CACGAGATAC	TCAAACTGTT	CTTGCCAATA	360
ATGAGGTACC	TCAATCTCCA	AGTGGATTGT	CTTCTACACC	AACAACTATA	TCAGTTATGG	420
ATTTACCAAA	TCCATGCCTA	AATGCTTCAA	GTTTGACTTG	TAGCATTAAA	GGAGTTTCAA	480
CATTCAATGT	TTATTACCAA	GTAGAAAGCA	ATGGTGTTAT	ATATTCATGT	ATCAGTGACA	540
CTATAACTAA	ATTGGGAAAC	TGTGAAGGAT	CTTCTGAATT	GCCAAGAAGC	TTTGAAACTG	600

BAD ORIGINAL &

AP Ο Ο Ο 4 Ο 3 ι

TTCCAGTTGT	ACCAATAACA	AAAATCGATA	ACAAAAGGAA	ATTATCTATA	GGAACTAAAT	660
TCTATATAAT	TGAAAGTTTG	GAGAATTACA	ATTATCCAAT	CATGTACAAT	TCTAGACCAA	720
CTAATGGCAC	AGTCTCTCTC	CAGAGTGTGA	AATTCTCAGG	TGATTGTAAA	ATATCAAAAA	780
CAAACATAGT	TAATTCTTAT	ACAGTTTCAT	TAACTACACC	TGAGAAAATC	ATGGGTTACG	840
TCGTCAAAAG	AGAAGGAAGT	GACATGAGCC	ATTCCATAAT	AAGCTTCTCT	GGCTCAGTTA	900
GTTTGACTTT	CACAGAAGAA	AATATGGATG	GTAAGCATAA	CCTATTGTGC	GGTGACAAGT	960
CTTCTAAAGT	TCCTTTAGTG	GACAAAAGAG	TTAGAGATTG	TATTATAAAG	TACTCCAAAA	1020
ATATTTACAA	ACAAACTGCT	TGCATCAACT	TCTCATGGTT	TAGATTGATC	ATGATTGCTC	1080
TGATTGTCTA	TTTCCCTATA	AGGTACTTGG	TAAACAAAAC	ATCTAAAACA	CTGTTCTATG	1140
GGTATGATCT	TCTGGGATTG	ATAACGTATC	CTATACTACT	ACTGATAAAT	TATTTATGGT	1200
CTTATTTTCC	CTTGAAATGT	AAGGTCTGTG	G.AAATTTATG	TTTGGTAACC	CATGAATGCT	1260
CAAAATTATG	CATTTGTAAC	AAAAACAAAG	CCTCTGAAGA	ACACTCAGAA	GAATGTCCTA	1320
TAATAACTAG	AACAGCAGAG	AAGAATAAAA	AATACAACTG	GGCTAGCATT	GAATGGTTCC	1380
ATTTGATAGT	TAATACAAAA	ATAGGCCTCT	CTTTCCTAAA	AGCAGTCACA	GAAACTTTGA	1440
TAGGATTTCT	GATTTTGTCA	CAGATGCCTA	TGTCTATGGC	TCAAACTGCT	CAGTGTTTGG	1500
ATAGCTGTTA	TTATGTCCCT	GGTTGTGACC	GCTTTGTTAC	AAACAGATAT	GACAAATGTC	1560
CTGAAAAAGA	CCAATGCTTC	TGTGCTATTA	AAGAAAATTC	TATTGTAGAA	TCTAACTTTC	1620
TAACCAATGT	TGTGACAGAA	GGTCCTATGG	ATTGTATACC	TTATCAAGAA	TGCAAAGGCA	1680
GGATAACTGA	AAATGCTTTA	GTAACTTTTG	TCAAATGCAG	ATTTGGTTGT	GAGTATGCCT	1740
CTATTTTCCA	AAGCAAGCCT	TTAGATAATG	GGTTCTTAGA	ATATTCTGGT	GACACATTGG	1800
GATTGAATGC	TGTGAATCTA	CACTTCATGA	AAAGGCTTAG	GAATGGTATA	ATTGATTTCT	1860
ACAATAAAAC	TGAAAAATAT	GGATACATTT	CTGGAGATGC	ACTTAAATCT	AATGAATCAG	1920
ACATACCTGA	AAGCATTTTT	CCAAGAAAAT	CTCTCATATT	TGACTCGGTG	ATAGATGGAA	1980
AATATAGATA	CATGATAGAA	GAATCTTTAT	TATCAGGTGG	GGGCACAGTT	TTCTCTCTTA	2040
ATGACAAAAG	TTCTAGCACT	GCTCAGAAGT	TTGTTGTTTA	TATTAAAAAA	GTTAGAATAC	2100
AGTATGATGT	TTCTGAACAA	TACACTACAG	CTCCTATACA	AAGCACCCAC	ACTGATTTCT	2160
TTTCAACATG	CACAGGTAAA	TGCTCAGATT	GCAGAAAAGA	ACAACCGATA	ACTGGGTATC	2220
AAGATTTCTG	CATAACACCA	ACATCTTACT	GGGGTTGTGA	AGAGGTTTGG	TGTTTGGCTA	2280
TCAATGAAGG	AGCCACTTGT	GGGTTTTGTA	GAAATGTGTA	TGATATGGAT	CAATCTTTCA	2340
GGATTTATTC	TGTTATTAAA	TCAACAATCA	AGTCTGAAGT	ATGTATATCT	GGATTTGTGG	2400
GAGCAAAGTG	TTTCACTGTA	TCTGAGGAAG	TCCCATCTGA	ATCAGGATAT	TTCCAGGCTG	2460
ATATATTAGC	GGATTTCCAT	AATGATGGCT	TAACAATAGG	CCAGCTGATA	GCTCATGGAC	2520
CTGATAGTCA	CGTATATGCG	GGAAACATAG	CTAGATTAAA	TAATCCATCA	AAAATGTTTG	2580
GTCATCCTCA	ACTTTCACAC	CAAGGTGATC	CCATTTTCTC	TAAGAAAACA	TTAGATACAA	2640
ACGATCTGTC	CTGGGATTGT	TCAGCAATTG	GTAAAAAAAC	AATTACGATA	AAATCATGTG	2700
GTTATGACAC	ATACAGATTT	AAAACAGGTT	TGAACCAAAT	ATCGGACATT	CCAGTTCAGT	2760
TTACTGACCA	GAATAGTTTT	TATATGGAAA	AGATCTTTAG	TCTAGGTAAG	CTTAAAATTG	2820

BAD ORIGINAL

AP Ο Ο Ο 4 Ο 3

	TTTTGGATCT	ACCTTCTGAA	TTGTTTAAAA	CTGTACCAAA	AAAGCCTATA	TTGAGTTCTG	2880
	TCTCTTTAAG	CTGTAAGGGA	TGTTTCTTAT	GTAGCCAAGG	GCTGAGGTGT	GCTGCCTCAT	2940
	TTATATCAGA	CATAACTTTT	TCTGCAAGGT	TAACCATGAA	ACAATGTTCA	TTGTCAACAT	3000
	ACCAGATAGC	AGTAAAGAAA	GGTGCTAATA	AGTATAACTT	GACAATGTTC	TGCACTTCTA	3060
	ACCCAGAAAA	ACAAAAAATG	ATTATAGAAC	CAGAAGGAGA	CAAATCATAC	TCAGTTGAGG	3120
	CACTTGTAGA	TTCTGTTGCT	GTGCTGGAAC	CAGAAAACAT	AATTGATCAA	AATGACCAAC	3180
	ATGCACACGA	GGAACAGCAA	TATAATTCTG	ACACATCAGT	TTGGAGCTTT	TGGGACTATG	3240
	TTAAAAGCCC	TTTTAACTTT	ATAGCAAGCC	ACTTTGGATC	CTTCTTTGAT	ACAGTCAGAG	3300
	TAGTATTGCT	CATACTCTTT	GTATTTGCTC	TTGCTTACCT	CTGCTCTATT	GTTGCCACAA	3360
)	TGTGTAGAGG	CTATGTTAGA	AACAAGTCTT	ACAAGACAAA	ATATATTGAA	GACACTAATG	3420
	ATTATTCTTT	GGTTTCCACG	TCTTCTGGTA	AAGACACCAT	CACTAGAAGA	AGACCTCCTC	3480 *'
	TAGACTTCAG	CGGTATATGA	TTAAATTCAT	TGGAATATAC	ACCAAATGTG	TTTTAAATTA	3540
	AATAAGGCAG	AATGATTGTA	ATCAAATAAA	TTTAATTTGT	CTAAATAAGT	GTTAAATAAG	3600
	CAAACTATAT	AAAAAATATA	AAAATAATAA	AAATAAAAAT	CAACAAAAAC	AAATAAAATG	3660
	TATAAAAACA	AATAAAAAGG	TCTTAGACCA	AATCTGGCCG	GAGCCTTATT	TATTTACAAA	3720
	AAAAATTTTT	TTGGTTTTTT	TCAACTTTTT	TCTATTTTTT	GCTTTTTGTT	TGTTTTTGTT	3730
	TTTGTTTTTG	TTTTTTGTTT	ATTTTTTCTT	TTTTCTTTTT	TGGTTTTTGA	TTTGTTTTTA	3840
	AAC AC AAAT A	CATATATATT	TTATACTTCT	ATATATATAT	ATATATATAG	TTTTTTATCT	3900
	TGCTTTATTT	TTTACTTAAA	CATTCAAAAT	ATTATGAACA	ATATATATTA	TTTATTATTA	3960
	AATGAAAACA	TATTTAGATT	TCATTATCAA	ATGATATCTC	TATCTTGTTA	TCAGTAACAT	4020
	TCTCCTCTTC	TTCAACAGAT	TTCTCTAAAT	TAGCACTTAG	CTCTGCAAGT	TGTCTTCTAA	4090
	TTTGCTTTTC	AGAATTGCCT	TTAGGTATGA	TTAACTTGCA	GGCTTCAATG	AAGGCTTGCG	4140
	ATTTGGCTCT	AATAGCCCTA	TTGATGGGTA	TTATCATGCA	GCTTTTATCC	AGATCTGCTC	4200
	TTGGTGAATC	ACAGAATTCT	TTTGTCCAAG	AATACATGAC	ACTAGCAAAA	GAAACATCTT	4250
	TCTTGTAAAC	TTGATCACAT	AATAAATGAA	GCTGAACACA	ATTCTCTGAA	GTGTTGTTAA	4320
	CTTTTGGAAT	GGACCAATTT	AGATAAAAAA	CAAAACATAT	AGGATCTTTA	ATGCTTCCTT	4330
	GCCCTTTCAA	AACAGTTCTG	GCATTAACAC	TCTTGTTAGG	ATCAATTAAA	GCTACAGCCA	4440
	ATTTCCCATC	AGGATCAGCT	ATAGTAGGGC	ATATCCAGAT	AACTATCCTT	GAGATCATCA	4500
	TGTATTGTTT	TCTGCTATCC	CAGGTTGGAT	GTATCTTAAT	TATCTTGGTT	GCAGCTTTAT	4550
	CACCATTACC	TACAAAAAGA	TCATTTTTCC	AGCTGGAGAT	ATGATGATTT	GTATCAACAA	4520
	TCATTCTAGC	AGAAAGATCA	TAAGACTCTG	ATTCAGATAT	CTGGTCAGAC	TCATATGTGC	4530
	CTAGTGATGA	GGTGCCTGTA	TTGTTCATCA	AAATCTTCCC	TTTAGAGCTA	TCCATGGCTC	4^40
	TTTGGAGAAC	TTCCTTGTTG	TTGTGATTAG	CAGAAGGATT	GTGTACAACA	ATGTCTGCTC	4300
	CTTGTTTAGA	GAGGGTAGTT	GTAACCCTAG	GGTGATCTAG	CTCCCTGCTG	CTAGATGATC	4 ^s50
	TGAGTGATTT	GAAAAAACTA	TTCATCTCTG	GACGGTAGAT	TAAGAATTAA	AATATTGAGA	4'-20
	AGATAATGAT	TGAATTCGGC	TAGATATAAT	TTTGATGCAC	TGATTGCTCT		4-70

bad ORIGINAL

AP Ο Ο Ο 4 Ο 3

SEQ ID NO: 21

Sequence type: Nucleotide

Sequence length: 3414 nucleotides

Strandedness: Single stranded

Molecule type: Complementary M RNA coding for the G1/G2 precursor proteins from position 87 to position 3500 of SEQ ID NO: 20

Original Source Organism:

ATGAAAATTC	TAAAGATGTG	CGAGTTACTT	GTTAAAATTA	GTGTATGTAC	ACTAGTTGTT	60
ACTTCTGTTA	TCTTATCCTT	TATGGCATTA	AAGGAGACAG	ATGCAAAGAT	TCATGTTGAA	120
AGGGGCGATC	ATCCAGAAAT	TTATGATGAA	GCTTATTATG	ACCGTTCTGT	AGATCATAAA	180
AATGAAATTC	TAGATACTTT	GGCTGAAATG	CTCCAGAATG	CAACAGGTAA	AACCCTTAGA	240
CCAACACGAG	ATACTCAAAC	TGTTCTTGCC	AATAATGAGG	TACCTCAATC	TCCAAGTGGA	300
TTGTCTTCTA	CACCAACAAC	TATATCAGTT	ATGGATTTAC	CAAATCCATG	CCTAAATGCT	360
TCAAGTTTGA	CTTGTAGCAT	TAAAGGAGTT	TCAACATTCA	ATGTTTATTA	CCAAGTAGAA	420
AGCAATGGTG	TTATATATTC	atgtatcagt	GACACTATAA	CTAAATTGGG	AAACTGTGAA	480
GGATCTTCTG	AATTGCCAAG	AAGCTTTGAA	ACTGTTCCAG	TTGTACCAAT	AACAAAAATC	540
GATAACAAAA	GGAAATTATC	TATAGGAACT	aaattctata	TAATTGAAAG	TTTGGAGAAT	600
TACAATTATC	CAATCATGTA	CAATTCTAGA	CCAACTAATG	GCACAGTCTC	TCTCCAGAGT	660
GTGAAATTCT	CAGGTGATTG	TAAAATATCA	AAAACAAACA	TAGTTAATTC	TTATACAGTT	720
TCATTAACTA	CACCTGAGAA	AATCATGGGT	TACGTCGTCA	AAAGAGAAGG	AAGTGACATG	780
AGCCATTCCA	TAATAAGCTT	CTCTGGCTCA	GTTAGTTTGA	CTTTCACAGA	AGAAAATATG	840
GATGGTAAGC	ATAACCTATT	GTGCGGTGAC	AAGTCTTCTA	AAGTTCCTTT	AGTGGACAAA	900
AG AGTTAGAG	ATTGTATTAT	AAAGxAClCC	AAAAATATTT	ACAAACAAAC	TGCTTGCATC	960
AACTTCTCAT	GGTTTAGATT	GATCATGATT	GCTCTGATTG	TCTATTTCCC	TATAAGGTAC	1020
TTGGTAAACA	AAACATCTAA	AACACTGTTC	TATGGGTATG	ATCTTCTGGG	ATTGATAACG	1080
TATCCTATAC	TACTACTGAT	AAATTATTTA	TGGTCTTATT	TTCCCTTGAA	ATGTAAGGTC	1140
TGTGGAAATT	TATGTTTGGT	AACCCATGAA	TGCTCAAAAT	TATGCATTTG	TAACAAAAAC	1200
AAAGCCTCTG	AAGAACACTC	AGAAGAATGT	CCTATAATAA	CTAGAACAGC	AGAGAAGAAT	1260
AAAAAATACA	ACTGGGCTAG	CATTGAATGG	TTCCATTTGA	TAGTTAATAC	AAAAATAGGC	1320
CTCTCTTTCC	TAAAAGCAGT	CACAGAAACT	TTGATAGGAT	TTCTGATTTT	GTCACAGATG	1380
CCTATGTCTA	TGGCTCAAAC	TGCTCAGTGT	TTGGATAGCT	GTTATTATGT	CCCTGGTTGT	1440
GACCGCTTTG	TTACAAACAG	ATATGACAAA	TGTCCTGAAA	AAGACCAATG	CTTCTGTGCT	1500
ATTAAAGAAA	ATTCTATTGT	AGAATCTAAC	TTTCTAACCA	ATGTTGTGAC	AGAAGGTCCT	1560
ATGGATTGTA	TACCTTATCA	AGAATGCAAA	GGCAGGATAA	CTGAAAATGC	TTTAGTAACT	1620
TTTGTCAAAT	GCAGATTTGG	TTGTGAGTAT	GCCTCTATTT	TCCAAAGCAA	GCCTTTAGAT	1680

APO0040 3

C'·

	AATGGGTTCT	TAGAATATTC	TGGTGACACA	TTGGGATTGA	ATGCTGTGAA	TCTACACTTC	1740
	ATGAAAAGGC	TTAGGAATGG	TATAATTGAT	TTCTACAATA	AAACTGAAAA	ATATGGATAC	1800
	ATTTCTGGAG	ATGCACTTAA	ATCTAATGAA	TCAGACATAC	CTGAAAGCAT	TTTTCCAAGA	1860
	AAATCTCTCA	TATTTGACTC	GGTGATAGAT	GGAAAATATA	GATACATGAT	AGAAGAATCT	1920
	TTATTATCAG	GTGGGGGCAC	AGTTTTCTCT	CTTAATGACA	AAAGTTCTAG	CACTGCTCAG	1980
	AAGTTTGTTG	TTTATATTAA	AAAAGTTAGA	ATACAGTATG	ATGTTTCTGA	ACAATACACT	2040
	ACAGCTCCTA	TACAAAGCAC	CCACACTGAT	TTCTTTTCAA	CATGCACAGG	TAAATGCTCA	2100
	GATTGCAGAA	AAGAACAACC	GATAACTGGG	TATCAAGATT	TCTGCATAAC	ACCAACATCT	2160
	TACTGGGGTT	GTGAAGAGGT	TTGGTGTTTG	GCTATCAATG	AAGGAGCCAC	TTGTGGGTTT	2220
Θ	TGTAGAAATG	TGTATGATAT	GGATCAATCT	TTCAGGATTT	ATTCTGTTAT	TAAATCAACA	2280
	ATCAAGTCTG	AAGTATGTAT	ATCTGGATTT	GTGGGAGCAA	AGTGTTTCAC	TGTATCTGAG	2340 ’
	GAAGTCCCAT	CTGAATCAGG	ATATTTCCAG	GCTGATATAT	TAGCGGATTT	CCATAATGAT	2400
	GGCTTAACAA	TAGGCCAGCT	GATAGCTCAT	GGACCTGATA	GTCACGTATA	TGCGGGAAAC	2460
	ATAGCTAGAT	TAAATAATCC	ATCAAAAATG	TTTGGTCATC	CTCAACTTTC	ACACCAAGGT	2520
	GATCCCATTT	TCTCTAAGAA	AACATTAGAT	ACAAACGATC	TGTCCTGGGA	TTGTTCAGCA	2580
	ATTGGTAAAA	AAACAATTAC	GATAAAATCA	TGTGGTTATG	ACACATACAG	ATTTAAAACA	2640
	GGTTTGAACC	AAATATCGGA	CATTCCAGTT	CAGTTTACTG	ACCAGAATAG	TTTTTATATG	2700
	GAAAAGATCT	TTAGTCTAGG	TAAGCTTAAA	ATTGTTTTGG	ATCTACCTTC	TGAATTGTTT	2760
	AAAACTGTAC	CAAAAAAGCC	TATATTGAGT	TCTGTCTCTT	TAAGCTGTAA	GGGATGTTTC	2820
	TTATGTAGCC	AAGGGCTGAG	GTGTGCTGCC	TCATTTATAT	CAGACATAAC	TTTTTCTGCA	2880
	AGGTTAACCA	TGAAACAATG	TTCATTGTCA	ACATACCAGA	TAGCAGTAAA	GAAAGGTGCT	2940
)	AATAAGTATA	ACTTGACAAT	gttctgcact	TCTAACCCAG	AAAAACAAAA	AATGATTATA	3000
a > t - .	GAACCAGAAG	GAGACAAATC	ATACTCAGTT	GAGGCACTTG	TAGATTCTGT	TGCTGTGCTG	3060
( ’	GAACCAGAAA	ACATAATTG ATCAAAATGAC	CAACATGCAC	ACGAGGAACA	GCAATATAAT	3120
	TCTGACACAT	CAGTTTGGAG	CTTTTGGGAC	TATGTTAAAA	GCCCTTTTAA	CTTTATAGCA	3180
	AGCCACTTTG	GATCCTTCTT	TGATACAGTC	AGAGTAGTAT	TGCTCATACT	CTTTGTATTT	3240
	GCTCTTGCTT	acctctgctc	TATTGTTGCC	ACAATGTGTA	GAGGCTATGT	TAGAAACAAG	3300
	TCTTACAAGA	CAAAATATAT	TGAAGACACT	AATGATTATT	CTTTGGTTTC	CACGTCTTCT	3360
	GGTAAAGACA	CCATCACTAG	AAGAAGACCT	CCTCTAGACT	TCAGCGGTAT	ATGA	3414

SEQ ID No. 22

Sequence types Nucleotide

Sequence length: 912 nucleotides

Strandedness: single stranded

Molecule type: Complementary viral M RNA from 3974 to 4885 of SEQ ID No. 20

AP Ο Ο Ο 4 Ο 3

I

TTAGATTTCA	TTATCAAATG	ATATCTCTAT	CTTGTTATCA	GTAACATTCT	CCTCTTCTTC	60
AACAGATTTC	TCTAAATTAG	CACTTAGCTC	TGCAAGTTGT	CTTCTAATTT	GCTTTTCAGA	120
ATTGCCTTTA	GGTATGATTA	ACTTGCAGGC	TTCAATGAAG	GCTTGCGATT	TGGCTCTAAT	180
AGCCCTATTG	ATGGGTATTA	TCATGCAGCT	TTTATCCAGA	TCTGCTCTTG	GTGAATCACA	240
GAATTCTTTT	GTCCAAGAAT	ACATGACACT	AGCAAAAGAA	ACATCTTTCT	TGTAAACTTG	300
ATCACATAAT	AAATGAAGCT	GAACACAATT	CTCTGAAGTG	TTGTTAACTT	TTGGAATGGA	360
CCAATTTAGA	TAAAAAACAA	AACATATAGG	atctttaatg	CTTCCTTGCC	CTTTCAAAAC	420
AGTTCTGGCA	TTAACACTCT	TGTTAGGATC	AATTAAAGCT	ACAGCCAATT	TCCCATCAGG	480
ATCAGCTATA	GTAGGGCATA	TCCAGATAAC	TATCCTTGAG	ATCATCATGT	ATTGTTTTCT	540
GCTATCCCAG	GTTGGATGTA	TCTTAATTAT	CTTGGTTGCA	GCTTTATCAC	CATTACCTAC	600
AAAAAGATCA	TTTTTCCAGC	TGGAGATATG	ATGATTTGTA	TCAACAATCA	TTCTAGCAGA	660
AAGATCATAA	GACTCTGATT	CAGATATCTG	GTCAGACTCA	TATGTGCCTA	GTGATGAGGT	720
GCCTGTATTG	TTCATCAAAA	TCTTCCCTTT	AGAGCTATCC	ATGGCTCTTT	GGAGAACTTC	780
CTTGTTGTTG	TGATTAGCAG	AAGGATTGTG	TACAACAATG	TCTGCTCCTT	GTTTAGAGAG	840
GGTAGTTGTA	ACCCTAGGGT	GATCTAGCTC	CCTGCTGCTA	GATGATCTGA	GTGATTTGAA	900
AAAACTATTC	AT					912

SEQ ID No. 23

Sequence type: Nucleotide

Sequence length: 446 nucleotides

Strandedness: Single stranded

Molecule type: CaMV 35S' promoter and Ω sequence of tobacco mosaic virus Original Source Organism:

GGATCCGGAA CATGGTGGAG CACGACACGC TTGTCTACTC CAAAAATATC 50 AAAGATACAG TCTCAGAAGA CCAAAGGGCA ATTGAGACTT TTCAACAAAG 100 TTATTGTGAA GATAGTGGAA AAGGAAGGTG GCTCCTACAA ATGCCATCAT 150 TGCGATAAAG GAAAGGCCAT CGTTGAAGAT GCCTCTGCCG ACAGTGGTCC 200 CAAAGATGGA CCCCCACCCA CGAGGAGCAT CGTGGAAAAA GAAGACGTTC 250 CAACCACGTC TTCAAAGCAA GTGGATTGAT GTGATATCTC CACTGACGTA 300 AGGGATGACG CACAATCCCA CTATCCTTCG CAAGACCCTT CCTCTATATA 350 AGGAAGTTCA TTTCATTTGG AGAGGACTTT TTACAACAAT TACCAACAAC 400

AP Ο Ο Ο 4 Ο 3

SEQ ID NO :24

Sequence type:Amino acid

Sequence length: 303 amino acids (NSm protein)

Original Source Organism: Impatiens Necrotic Spot Virus c

Met

Asn

Ser

Phe

Phe 5

Lys

Ser

Leu

Arg

Ser 10

Ser

Arg

Glu 15

15

Leu

Asp

His

Pro

Arg 20

Val

Thr

Leu 25

Ser

Lys

Gin

Gly

Ala 30

30

Asp

lie

Val

His 35

Asn

Pro

Ser

Ala

Asn 40

His

Asn

Lys

Glu 45

45

Val

Leu

Gin

Arg

Ala 50

Met

Asp

Ser

Lys 55

Gly

Lys

He

Leu

Met 60

60

Asn

Thr

Gly

Thr 65

Ser

Leu

Gly

Thr 70

Tyr

Glu

Ser

Asp

Gin 75

75

lie

Ser

Glu

Ser

Glu 80

Ser

Tyr

Asp

Leu

Ser 85

Ala

Arg

Met

lie

Val 90

90

Asp

Thr

Asn

His

His 95

lie

Ser

Trp

Lys 100

Asn

Asp

Leu

Phe

Val 105

105

BAD ORIGINAL £

AP Ο Ο Ο 4 Ο 3

Gly Asn Gly Asp Lys

Ala Ala Thr Lys

lie 115

lie

Lys

He

His

Pro 120

120

110

Thr

Trp

Asp

Ser

Arg

Lys

Gin

Tyr

Met

lie

Ser

Arg

He

Val

135

125

130

135

lie

Trp

lie

Cys

Pro

Thr

lie

Ala

Asp

Pro

Asp

Gly

Lys

Leu

Ala

150

140

145

150

Val

Ala

Leu

lie

Asp

Pro

Asn

Lys

Ser

Val

Asn

Ala

Arg

Thr

Val

165

155

160

165

Leu

Lys

Gly

Gin

Gly

Ser

He

Lys

Asp

Pro

He

Cys

Phe

Val

Phe

180

170

175

180

Tyr

Leu

Asn

Trp

Ser

He

Pro

Lys

Val

Asn

Thr

Ser

Glu

Asn

195

185

190

195

Cys

Val

Gin

Leu

His

Leu

Cys

Asp

Gin

Val

Tyr

Lys

Asp

210

200

205

210

Val

Ser

Phe

Ala

Ser

Val

Met

Tyr

Ser

Trp

Thr

Lys

Glu

Phe

Cys

225

215

220

.

225

Asp

Ser

Pro

Arg

Ala

Asp

Leu

Asp

Lys

Ser

Cys

Met

He

Pro

240

230

235

240

He

Asn

Arg

Ala

He

Arg

Ala

Lys

Ser

Gin

Ala

Phe

He

Glu

Ala

255

245

250

255

Cys

Lys

Leu

He

Pro

Lys

Gly

Asn

Ser

Glu

Lys

Gin

lie

Arg

270

260

265

270

Arg

Gin

Leu

Ala

Glu

Leu

Ser

Ala

Asn

Leu

Glu

Lys

Ser

Val

Glu

285

275

280

285

Glu

Asn

Val

Thr

Asp

Asn

Lys

lie

Glu

lie

Ser

Phe

Asp

300

290

295

300

Asn

Glu

lie

3 03

SEQ ID NO :25

Sequence types Amino acids

Sequence length: 262 amino acids (N protein)

Original Source Organism: Impatiens Necrotic Spot Virus

Met

Asn

Lys

Ala

Lys 5

He

Thr

Lys

Glu

Asn 10

lie

Val

Lys

Leu

Leu 15

15

Thr

Gin

Ser

Asp

Ser 20

Leu

Glu

Phe

Glu

Glu 25

Thr

Gin

Asn

Glu

Gly 30

3 0

Ser

Phe

Asn

Phe

Thr 35

Asp

Phe

Thr

Asn 40

Asn

Arg

Glu

Lys

lie 45

45

Gin

Asn

Met

Thr

Thr 50

Ala

Ser

Cys

Leu

Ser 55

Phe

Leu

Lys

Asn

Arg 60

6 ·?

BAD ORIGINAL

AP Ο Ο Ο 4 Ο 3 οο

Gin

Ser

He

Met

Arg 65

Val

He

Lys

Ser

Ala 70

Asp

Phe

Thr

Phe

Gly 75

75

Ser

Val

Thr

He

Lys 80

Lys

Thr

Arg

Asn

Asn 85

Ser

Glu

Arg

Val

Gly 90

90

Val

Asn

Asp

Met

Thr 95

Phe

Arg

Leu

Asp 100

Ala

Met

Val

Arg

Val 105

105

His

Leu

Val

Gly

Met 110

lie

Lys

Asp

Asn

Gly 115

Ser

Ala

Leu

Thr

Glu 120

120

Ala

lie

Asn

Ser

Leu 125

Pro

Ser

His

Pro

Leu 130

lie

Ala

Ser

Tyr

Gly 135

135

Leu

Ala

Thr

Asp 140

Leu

Lys

Ser

Cys

Val 145

Leu

Gly

Val

Leu

Leu 150

150

Gly

Ser

Leu

Pro 155

Leu

He

Ala

Ser

Val 160

Leu

Asn

Phe

Glu

He 165

165

Ala

Leu

Val

Pro 170

Ala

He

Tyr

Gin

Asp 175

Ala

Lys

His

Val

Glu 180

180

Leu

Gly

He

Asp

Met 185

Ser

Lys

Phe

Ser

Thr 190

Lys

Glu

Ala

Val

Gly 195

195

Lys

Val

Cys

Thr

Val

Leu

Lys

Ser

Lys

Gly

Tyr

Ser

Met

Asn

Ser

210

200

205

210

Val

Glu

He

Gly

Lys

Ala

Lys

Gin

Tyr

Ala

Asp

lie

Leu

Lys

Ala

225

215

220

225

Cys

Ser

Pro

Lys

Ala

Lys

Gly

Leu

Ala

Met

Asp

His

Tyr

Lys

240

230

235

240

Glu

Gly

Leu

Thr

Ser

lie

Tyr

Ser

Met

Phe

Asn

Ala

Thr

lie

Asp

255

245

250

255

Phe

Gly

Lys

Asn

Asp

Ser

lie

262

260

SEQ ΙΟ NO: 26

Sequence type: Amino acid

Sequence length: 449 amino acids

Molecule type: NSs protein

Original Source Organism: Impatiens Necrotic Spot Virus

Met Ser Ser Ala Met lyr Glu Thr lie He Lys Ser Lys Ser Ser 15 5 10 15 lie Trp Gly Thr Thr Ser Ser Gly Lys Ala Val Val Asp Ser Tyr 30

AP Ο Ο Ο 4 Ο 3 (?)

	Trp	Ile	His	Asp	Gin 35	Ser	Ser	Gly
	Leu	Tyr	Ser	Asp	Ser 50	Arg	Ser	Lys
	Lys	Val	Gly	Phe	Leu 65	Pro	Thr	Glu
	Cys	Phe	Val	Pro	Ile 80	Phe	Asp	Asp
&	Ser	Gly	Asn	Val	Val 95	Glu	Ile	Leu
C	Thr	Asn	Gly	Val	Lys 110	His	Gin	Gly
	Gin	Leu	Leu	Arg	Met 125	Leu	Glu	Glu
	Thr	Ser	Arg	Phe	Gly 140	Leu	Lys	Glu
	Asn	Phe	Ile	Glu	Ala 155	Ala	Asn	Lys
	Glu	Val	Leu	Phe	Asp 170	Val	Lys	Tyr
	Lys	Val	Ser	Val	Leu 185	Ser	Pro	Thr
4	Tyr	Thr	Leu	Lys	Pro 200	Val	Phe	Asn
(	Thr	Val	Asn	Thr	Leu 215	Ala	Val	Lys
	Ser	Asp	Leu	Met	Ser 230	Asp	Thr	His
	Asn	Lys	Pro	Phe	Lys 245	Ile	Ser	Leu
	Met	Lys	Ser	Asn	Thr 260	Tyr	Ser	Arg
	Ser	Ser	Pro	Lys	Glu 275	Tyr	Tyr	Ile
	His	Asn	Asn	Val	Glu 290	Thr	Val	Ile
	Asn	Leu	Phe	Leu	Asn 305	Gin	Leu	Leu
	Glu	Met	Asn	Phe	Ser 320	Asp	Leu	Lys

Lys	Lys 40	Leu	Val	Glu	Ala	Gin 45	45
Thr	Ser 55	Phe	Cys	Tyr	Thr	Gly 60	60
Glu	Lys 70	Glu	Ile	Ile	Val	Arg 75	75
Ile	Asp 85	Leu	Asn	Phe	Ser	Phe 90	90
Val	Arg 100	Ser	Asn	Thr	Thr	Asn 105	105
His	Leu 115	Lys	Val	Leu	Ser	Ser 120	120
Gin	Ile 130	Ala	Val	Pro	Glu	Ile 135	135
Ser	Asp 145	Ile	Phe	Pro	Pro	Asn 150	150
Gly	Ser 160	Leu	Ser	Cys	Val	Lys 165	165
Ser	Asn 175	Asn	Gin	Ser	Met	Gly 180	180
Arg	Ser 190	Val	His	Glu	Trp	Leu 195	195
Gin	Ser 205	Gin	Thr	Asn	Asn	Arg 210	210
Ser	Leu 220	Ala	Met	Ser	Ala	Thr 225	225
Ser	Phe 235	Val	Arg	Leu	Asn	Asn 240	240
Trp	Met 250	Arg	Ile	Pro	Lys	Ile 255	255
Phe	Phe 265	Thr	Leu	Ser	Asp	Glu 270	270
Ser	Ile 280	Gin	Cys	Leu	Pro	Asn 285	285
Glu	Tyr 295	Asn	Phe	Asp	Gin	Ser 300	300
Leu	Ala 310	Val	He	His	Lys	Ile 315	315
Glu	Pro 325	Tyr	Asn	Val	Ile	His 330	330

AP Ο Ο Ο 4 Ο 3

Asp Met	Ser	Tyr Pro 335	Gin	Arg	Ila	Val His 340	Ser Leu Leu	Glu lie 345	345
His Thr	Glu	Leu Ala 350	Gin	Thr	Val	Cys Asp 355	Ser Val Gin	Gin Asp 360	360
Met He	Val	Phe Thr 365	He	Asn	Glu	Pro Asp 3.70	Leu Lys Pro	Lys Lys 375	375
Phe Glu	Leu	Gly Lys 380	Lys	Thr	Leu	Asn Tyr 385	Ser Glu Asp	Gly Tyr 390	390
Gly Arg	Lys	Tyr Phe 395	Leu	Ser	Gin	Thr Leu 400	Lys Ser Leu	Pro Arg 405	405
Asn Ser	Gin	Thr Met 410	Ser	Tyr	Leu	Asp Ser 415	lie Gin Met	Pro Asp 420	420
Trp Lys	Phe	Asp Tyr 425	Ala	Ala	Gly	Glu lie 430	Lys lie Ser	Pro Arg 435	435
Ser Glu Asp Val Leu Lys Ala lie Ser Lys Leu Asp Leu 440 445 . SEQ ID NO :27 Sequence type: Amino acid Sequence length: 1137 eunino acids (G1G2 precursor protein) Original Source Organism: Impatiens Necrotic Spot Virus	Asn 1	449
Met Lys	lie	Leu Lys 5	Met	Cys	Glu	Leu Leu 10	Val Lys lie	Ser Val 15	15
Cys Thr	Leu	Val Val 20	Thr	Ser	Val	lie Leu 25	Ser Phe Met	Ala Leu 30	30
Lys Glu	Thr	Asp Ala 35	Lys	lie	His	Val Glu 40	Arg Gly Asp	His Pro 45	45
Glu lie	Tyr	Asp Glu 50	Ala	Tyr	Tyr	Asp Arg 55	Ser Val Asp	His Lys 60	60
Asn Glu	lie	Leu Asp 65	Thr	Leu	Ala	Glu Met 70	Leu Gin Asn	Ala Thr 75	75
Gly Lys	Thr	Leu Arg 80	Pro	Thr	Arg	Asp Thr 85	Gin Thr Val	Leu Ala 90	90
Asn Asn	Glu	Val Pro 95	Gin	Ser	Pro	Ser Gly 100	Leu Ser Ser	Thr Pro 105	105
Thr Thr	lie	Ser Val 110	Met	Asp	Leu	Pro Asn 115	Pro Cys Leu	Asn Ala 120	120

BAD ORIGINAL

AP Ο Ο Ο 4 Ο 3

Ser

Leu

Thr

Cys 125

Ser

He

Lys

Gly Val 130

Ser

Thr

Phe

Asn

Val 135

135

Tyr

Gin

Val

Glu 140

Ser

Asn

Gly

Val lie 145

Tyr

Ser

Cys

He

Ser 150

150

Asp

Thr

He

Thr

Lys 155

Leu

Gly

Asn

Cys Glu 160

Gly

Ser

Glu

Leu 165

165

Pro

Arg

Ser

Phe

Glu 170

Thr

Val

Pro

Val Val 175

Pro

lie

Thr

Lys

lie 180

180

Asp

Asn

Lys

Arg

Lys 185

Leu

Ser

lie

Gly Thr 190

Lys

Phe

Tyr

lie

He 195

195

Glu

Ser

Leu

Glu

Asn 200

Tyr

Asn

Tyr

Pro He 205

Met

Tyr

Asn

Ser

Arg 210

210

Pro

Thr

Asn

Gly

Thr 215

Val

Ser

Leu

Gin Ser 220

Val

Lys

Phe

Ser

Gly 225

225

Asp

Cys

Lys

He

Ser 230

Lys

Thr

Asn

lie Val 235

^sn

Ser

Tyr

Thr

Val 240

240

Ser

Leu

Thr

Pro 245

Glu

Lys

lie

Met Gly 250

Tyr

Val

Lys

Arg 255

255

Glu

Gly

Ser

Asp

Met 260

Ser

His

Ser

lie lie 265

Ser

Phe

Ser

Gly

Ser 270

270

Val

Ser

Leu

Thr

Phe 275

Thr

Glu

Asn Met 280

Asp

Gly

Lys

His

Asn 285

285

Leu

Cys

Gly

Asp 290

Lys

Ser

Lys Val 295

Pro

Leu

Val

Asp

Lys 300

300

Arg

Val

Arg

Asp

Cys 305

lie

Lys

Tyr Ser 310

Lys

Asn

He

Tyr

Lys 315

315

Gin

Thr

Ala

Cys

He 320

Asn

Phe

Ser

Trp Phe 325

Arg

Leu

He

Met

lie 330

330

Ala

Leu

lie

Val

Tyr 335

Phe

Pro

lie

Arg Tyr 340

Leu

Val

Asn

Lys

Thr 345

345

Ser

Lys

Thr

Leu

Phe 350

Tyr

Gly

Tyr

Asp Leu 355

Leu

Gly

Leu

lie

Thr 360

360

Tyr

Pro

lie

Leu

Leu 365

Leu

He

Asn

Tyr Leu 370

Trp

Ser

Tyr

Phe

Pro 375

375

Leu

Lys

Cys

Lys

Val 380

Cys

Gly

Asn

Leu Cye 385

Leu

Val

Thr

His

Glu 390

390

Cys

Ser

Lys

Leu

Cys 395

lie

Cys

Asn

Lys Asn 400

Lys

Ala

Ser

Glu

Glu 405

405

His

Ser

Glu

Cys 410

Pro

He

Thr Arg 415

Thr

Ala

Glu

Lys

Asn 420

420

BAD ORIGINAL &

AP Ο Ο Ο 4 Ο 3 ) 72

Lys

Tyr

Asn

Trp 425

Ala

Ser

lie

Glu

Trp 430

Phe

His

Leu

lie

Val 435

435

As η

Thr

Lys

He

Gly 440

Leu

Ser

Phe

Leu

Lys 445

Ala

Val

Thr

Glu

Thr 450

450

Leu

He

Gly

Phe

Leu 455

lie

Leu

Ser

Gin

Met 460

Pro

Met

Ser

Met

Ala 465

465

Gin

Thr

Ala

Gin

Cys 470

Leu

Asp

Ser

Cys

Tyr 475

Tyr

Val

Pro

Gly

Cys 480

480

Asp

Arg

Phe

Val

Thr 485

Asn

Arg

Tyr

Asp

Lys 490

Cys

Pro

Glu

Lys

Asp 495

495

'\

Gin

Cys

Phe

Cys

Ala 500

lie

Lys

Glu

Asn

Ser 505

He

Val

Glu

Ser

Asn 510

510

<·

Phe

Leu

Thr

Asn

Val 515

Val

Thr

Glu

Gly

Pro 520

Met

Asp

Cys

lie

Pro 525

525

Tyr

Gin

Glu

Cys

Lys 530

Gly

Arg

lie

Thr

Glu 535

Asn

Ala

Leu

Val

Thr 540

540

Phe

Val

Lys

Cys

Arg 545

Phe

Gly

Cys

Glu

Tyr 550

Ala

Ser

lie

Phe

Gin 555

555

Ser

Lys

Pro

Leu

Asp 560

Asn

Gly

Phe

Leu

Glu 565

Tyr

Ser

Gly

Asp

Thr 570

570

Leu

Gly

Leu

Asn

Ala 575

Val

Asn

Leu

His

Phe 580

Met

Lys

Arg

Leu

Arg 585

585

Asn

Gly

lie

He

Asp 590

Phe

Tyr

Asn

Lys

Thr 595

Glu

Lys

Tyr

Gly

Tyr 600

600

C

lie

Ser

Gly

Asp

Ala 605

Leu

Lys

Ser

Asn

Glu 610

Ser

Asp

He

Pro

Glu 615

615

<

Ser

lie

Phe

Pro

Arg 620

Lys

Ser

Leu

lie

Phe 625

Asp

Ser

Val

He

Asp 630

630

Gly

Lys

Tyr

Arg

Tyr 635

Met

He

Glu

Ser 640

Leu

Ser

Gly

Gly 645

645

Gly

Thr

Val

Phe

Ser 650

Leu

Asn

Asp

Lys

Ser 655

Ser

Thr

Ala

Gin 660

660

Lys

Phe

Val

Tyr 665

lie

Lys

Val

Arg 670

Ila

Gin

Tyr

Asp

Val 675

675

Ser

Glu

Gin

Tyr

Thr 680

Thr

Ala

Pro

He

Gin 685

Ser

Thr

His

Thr

Asp 690

590

Phe

Ser

Thr

Cys 695

Thr

Gly

Lys

Cys

Ser 700

Asp

Cys

Arg

Lys

Glu 705

7 05

Gin

Pro

lie

Thr

Gly 710

Tyr

Gin

Asp

Phe

Cys 715

lie

Thr

Pro

Thr

Ser 720

<» 1 1 4. V

BAD ORIGINAL &

AP Ο Ο Ο 4 Ο 3

Ο ™

Tyr Trp Gly Cys Glu Glu Val Trp Cys Leu Ala lie Asn Glu Gly 735 725 730 735

Ala Thr Cys Gly Phe Cys Arg Asn Val Tyr Asp Met Asp Gin Ser 7 50 740 745 750

Phe Arg lie Tyr Ser Val lie Lys Ser Thr lie Lys Ser Glu Val 765 755 760 765

Cys He Ser Gly Phe Val Gly Ala Lys Cys Phe Thr Val Ser Glu 780 770 775 780

Glu Val Pro Ser Glu Ser Gly Tyr Phe Gin Ala Asp He Leu Ala 795 785 790 795

Asp Phe His Asn Asp Gly Leu Thr lie Gly Gin Leu lie Ala His 810 800 805 810 /', Gly Pro Asp Ser His Val Tyr Ala Gly Asn lie Ala Arg Leu Asn 825

815 820 825

Asn Pro Ser Lys Met Phe Gly His Pro Gin Leu Ser His Gin Gly 840 830 835 840

Asp Pro He Phe Ser Lys Lys Thr Leu Asp Thr Asn Asp Leu Ser 855 845 850 855

Trp Asp Cys Ser Ala He Gly Lys Lys Thr He Thr He Lys Ser 870 860 865 870

Cys Gly Tyr Asp Thr Tyr Arg Phe Lys Thr Gly Leu Asn Gin He 385 875 880 885

Ser Asp lie Pro Val Gin Phe Thr Asp Gin Asn Ser Phe Tyr Met 900 890 895 900

Glu Lys He Phe Ser Leu Gly Lys Leu Lys He Val Leu Asp Leu 915 905 910 915 ( Pro Ser Glu Leu Phe Lys Thr Val Pro Lys Lys Pro He Leu Ser 930

920 925 930

Ser Val Ser Leu Ser Cys Lys Gly Cys Phe Leu Cys Ser Gin Gly 945 935 940 945

Leu Arg Cys Ala Ala Ser Phe He Ser Asp He Thr Phe Ser Ala 960 950 955 960

Arg Leu Thr Met Lys Gin Cys Ser Leu Ser Thr Tyr Gin He Ala 975 965 970 975

Val Lys Lys Gly Ala Asn Lys Tyr Asn Leu Thr Met Phe Cys Thr 990 980 985 990

Ser Asn Pro Glu Lys Gin Lys Met He He Glu Pro Glu Gly Asp 1005 995 1000 1005

Lys Ser Tyr Ser Val Glu Ala Leu Val Asp Ser Val Ala Val Leu 1020

AP Ο Ο Ο 4 Ο 3

Glu

Pro Glu Asn lie 1025

He Asp Gin Asn Asp Gin His Ala His Glu 1035

1030

1035

Glu

Gin

Tyr Asn

Ser

Asp

Thr

Ser Val

Trp

Ser

Phe

Trp Asp

1050

1040

1045

1050

Tyr

Val

Lys

Ser Pro

Phe

Asn

Phe

lie Ala

Ser

His

Phe

Gly Ser

1065

1055

1060

1065

Phe

Asp

Thr Val

Arg

Val

Leu Leu

He

Leu

Phe

Val Phe

1080

1070

1075

1080

Ala

Leu

Ala

Tyr Leu

Cys

Ser

lie

Val Ala

Thr

Met

Cys

Arg Gly

1095

1085

1090

1095

Tyr

Val

Arg

Asn Lys

Ser

Tyr

Lys

Thr Lys

Tyr

He

Glu

Asp Thr

1110

1100

1105

1110

ρ

Asn

Asp

Tyr

Ser Leu

Val

Ser

Thr

Ser Ser

Gly

Lys

Asp

Thr He

1125

1115

1120

1125

Thr

Arg

Arg Pro

Pro

Leu

Asp

Phe Ser

Gly

lie

1137

1130

1135

BAD ORIGINAL

AP Ο Ο Ο 4 Ο 3

HAVING NOW PARTICULARIV DFSCRI8FD AND ASCFR?*·—rf ·\ ΛΗΑΤ MA.V.·.» Ή· :... · ·,

ΤΟ βν ri.nl OrtiVlEL;. I, Μ DlClARL lhAI rthAi * Λ, Ci-,· .· ·,

Claims

CLAIMS: 1. Recombinant INSV DNA constructs comprising a DNA sequence coding for transcription into

a) an RNA sequence of an INSV or an RNA sequence homologous thereto ;

b) an RNA sequence of an INSV or an RNA sequence homologous thereto capable of encoding for an INSV protein or a part thereof , in which one or more codons have been replaced by synonyms , or an RNA sequence homologous thereto ; or

c) an RNA sequence complementary to an RNA sequence according to a) or b), which INSV DNA is under expression control of a promoter and a terminator capable of functioning in plants.
2. A DNA construct according to Claim 1, wherein the INSV DNA sequences code for transcription into:

i) the viral S RNA nucleotide sequence from 1 to 3017 (SEQ. ID No.l) ii) the viral S RNA nucleotide sequence from position 25 to 3017 (SEQ. ID No.2);

iii) the viral S RNA nucleotide sequence from 87 to 1436 (SEQ. ID No.3);

iv) the viral S RNA nucleotide sequence from 2080 to 2868 (SEQ. ID No.4);

BAD ORIGINAL

APΟ Ο ο 4 Ο 3

ν) the viral S RNA * pan-handle · structure comprising

a) a first nucleotide sequence of from about 30 to about 36 nucleotides in length from the 5' end of the viral S RNA and

b) a second nucleotide sequence of from about 30 to about 36 nucleotides in length from the 3' end of the viral S RNA vi) the viral S RNA nucleotide sequence from 1437 to 2079; (SEQ ID No. 7) vii) the viral S RNA nucleotide sequence from 1440 to 2041; (SEQ ID No.8) viii) the viral complementary S RNA nucleotide sequence from 1 to about 3017; (SEQ ID No.9) ix) the viral complementary S RNA nucleotide sequence from 1 to 2993; (SEQ ID No.10)

x) the viral complementary S RNA nucleotide sequence from 150 to 938; (SEQ ID No.11) xi) the S RNA nucleotide sequence from 1581 to 2930 of the viral complementary S RNA strand; (SEQ ID No.12);

xii) the viral complementary S RNA secondary structure having a nucleotide sequence of 642 nucleotides from 939 to 1580; (SEQ ID No.13)

BAD ORIGINAL ft

AP Ο Ο Ο 4 Ο 3

Ο xiii) xiv) χν) xvi) xvii) xviii) xix) t

xx) xxi)

S RNA nucleotide sequence from 87 to 1436 in which one or more codons have been replaced by their synonyms;

S RNA nucleotide sequence from 2080 to 2868 in which one or more codons have been replaced by their synonyms;

the M RNA nucleotide sequence from 1 to 4970 (SEQ ID NO.14);

the M RNA sequence from 86 to 997 (SEQ ID No.15);

the M RNA sequence of the intergenic region from 993 to 1470 (SEQ ID No.16);

the M RNA sequence from 1471 to 4884; (SEQ ID No. 17) the M RNA pan-handle structure comprising : a) a first nucleotide sequence of from about 30 to about 36 nucleotides in length from the 5' end of the viral M RNA and

b) a second nucleotide sequence of from about 30 to about 36 nucleotides in length from the 3' end of the viral M RNA;

the complementary viral M RNA sequence from 1 to 4970; (SEQ ID No.20) the complementary viral M RNA sequence from position 87 to position 3500 of the complementary viral M RNA sequence; ( SEQ ID No.21)

BAD ORIGINAL

AP Ο Ο Ο 4 Ο 3 xxii) the complementary viral M RNA sequence from position 3974 to 4385 (SEQ ID No.22) xxiii) RNA sequences homologous to the nucleotide sequences defined under i) to xii) and xv) to xxii) hereinabove.

xxiv) fragments of sequences defined under i) to xxii) hereinabove.
3. A DNA construct according to Claim 1, wherein the DNA sequence codes for transcription into INSV-RNA sequences of a pan-handle, or into RNA sequences homologous thereto.
4. A DNA construct according to Claim 1 wherein the DNA sequence codes for transcription into INSV-RNA sequences of a pan-handle wherein the pan-handle structure comprises two complementary strands comprising 36 nucleotides in length.
5. A DNA construct according to Claim 1, wherein the DNA sequence codes for transcription into INSV RNA sequences of an open reading frame in viral complementary sense, or into corresponding RNA sequences in which one or more codons have been replaced by synonyms thereof, or into RNA sequences homologous thereto.
6. A DNA construct according to Claim 1, wherein the the DNA sequence codes for transcription into INSVRNA sequences of a secondary structure, or into RNA