JPS61158792A - Dna probe for detecting blue tong virus - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention This invention relates to the field of molecular diagnostics. More particularly, the invention relates to genes obtained from bluetongue virus, methods for synthesizing said genes, and the use of said genes in detection of bluetongue virus and production of viral proteins.

BACKGROUND OF THE INVENTION Brown tongue is an arthropod-borne disease of sheep and cattle. Bluetongue virus (BTV), the causative agent of this disease, is caused by the Reov-iridae mite (Reov-iridae).
) is classified as a member of the family. The name these so-called reoviruses is derived from Respiratory-Enterprise Orphans.
It was named after the initials of Eric-Orphan.

The latter designation is associated with a disease that does not infect humans. An important criterion for membership in this family is the double-stranded R
It has a genome consisting of 10, 11 or 12 segments of NA (dsRNA). More specifically, since BTV has a circular capsomere,
It is classified as one of the genus orbivirus. The BTV genome consists of 10 double-stranded RNA segments wrapped in a two-layered protein shell. The protein is antigenic and serves as the basis for serological typing of this group of viruses. The four common serotypes in the United States are BTV-10, 11°13
and 17. The serotypes were found to be genetically related. That is, they each have a common fingerprint, and based on this, viral RNA species can be reclassified (Sugyama et al., Virology, Vol. 114, pp. 210-217 (Summer 198)).

IO RNA segments can be electrophoretically separated on an agarose gel [Sugyama, American
Journal of Evidencemiology (Amer, J.
, Epidemfol, ), vol. 115, 332-3
47 pages (1981)]. In the gel system used, 1
Zero segments appear as a linear band from the top to the bottom of the gel. The three most dominant bands are Ll, L2, and L3 based on their relatively large molecular weights.
It is named. The next three bands are Ml, M2 and M
3, and the remaining four are St, S2, S3 and S.
It is named 4. Thanks to its small molecular weight, Sl
- Two bands of S4 may move together, resulting in one thick band instead of a real separate band. For example, in Seven Rotary Type 17, Sl and RN of 62
The A segment moves together.

In certain instances, the protein function encoded by the corresponding RNA is known. As mentioned earlier, the genetic information of the virus is contained within the two-layered protein shell, and in the case of serotype 17, the RN
A segments L2 and M2 encode the proteins that constitute the outermost part of the protein shell, while L3, LIS
Ml and Sl encode components of the inner protein shell.

In addition to the four BTV serotypes being common in the United States, these and other BTV serotypes are known to be endemic in other countries. For example, BTV-1 to 15 are known to occur in South Africa, and BTV-1 to West Pakistan.
16 is common, and in Australia BTV-20
and −21 are found.

As mentioned above, bluetongue is an arthropod-borne disease of sheep and cattle. This disease is characterized by high morbidity and variable but significant mortality in sheep. Although the disease causes many complications in cattle, mortality rates are generally lower than in sheep. The disease not only affects reproduction in sheep and cattle, but also causes weight loss and reduced wool and milk production, even when the animals recover. Cows can become continuously infected and the virus can be transmitted to the fetus. Semen can also be a reservoir for this virus.

From this point of view, bluetongue virus is an important problem related to the import and export of animals and their egg traits.

Serological tests are extensively performed to detect the presence of serum-specific antibodies to BTV.

Unfortunately, however, serological methods have limitations.

It may cross-react with other Orbis viruses such as Epizootic Hemorrhagic Disease Virus (EHDV), Euvenangi (EUB) virus, and Pallium (PAL) virus [Mohr, American Journal of Veterinary Research ( Amer, J., Ve.
t, Res, ), vol. 35, p. 1109 (1974)]. In particular, cross-reactivity to EHDV is important.

Another drawback is that the currently used serological tests are also not sufficiently sensitive or accurate [Brown et al., Proceedings of the 8th National Animal Breeders. Technical Conference (Proc, 8th, Nat'1.A
nimal BreedersTech, Conf,
), pp. 79-83 (1980)].

Recent developments in recombinant DNA technology and methods for probing the subcellular localization of specific genes have facilitated the development of sensitive and specific nucleic acid probes for diagnostic purposes.

It is an object of the present invention to provide a detection method based on nucleic acids that enables both serotype-specific and group-specific detection of bluetongue virus.

[Description of the Figures] Figure 1 shows the migration pattern of clone 7 plasmid DNA cut with Hind and run on a 1% acarose gel.

A is the migration pattern of the HindlII fragment of clone 7 DNA. pBr (322 vector and BTV-
The 2/3 complementary DNA of the 17 segments is marked on the left side of the figure. B is the Hind[lI control fragment of complementary DNA, the size of the fragment is expressed in kilobases on the left side of the figure.

Figure 2 shows the hybridization of clone 7 putesmid DNA that underwent nick translation.
This is an RNA migration pattern of NA. B is BTV-17
Figure 2 is an autoradiogram of clone 7 DNA hybridized to segment 3 RNA of . The numbers on the left side of the figure represent the numbering system of BTV genomic RNA. Segments 7 and 8 run together through the agarose gel.

FIG. 3 shows the sequence strategy used to sequence the cloned BTV-17 segment 3 gene. Restriction enzymes used for restriction fragment generation are shown on the left side of the figure. The chain length and direction in which the individual chains are arranged are indicated by solid arrows. The codes of the restriction sites used are as follows. H
3, HindIII; N, NcoI; DSDde[:
H, HinfI; PlPstl; B, BamHr;
ESEcoRI; Bl, BglII.

Figure 4a14b shows the cloned L of BTV-17.
The nucleotide sequences of the three genes are shown. The reading frame starting at positions 18-20 is the TAG located at positions 2721-2723.
Terminates with a codon.

FIG. 5 shows hybridization of BTV clone 7 hybridized to BRV and EHP L3 RNA segments. Nick translated 32
Figure 2 is an autoradiogram of DNA purified from cells hybridized and infected with P-labeled clone 7 DNA to four US BTV serotypes and EHD serotype I. The numbers on the left side of the figure represent the numbering system for genomic RNA. Segments 7 and 8 of BTV and segments 7.8.9 of E HD run together in the agarose gel.

Figures 6a and 6b show detection of bluetongue virus by in situ hybridization with biotinylated BTV-17 probe DNA.

Figure 6a shows an uninfected control cell group, and Figure 6b shows a cell group infected with BTV for 20 hours.

FIG. 7 shows gel electrophoretic separation of BTV genomic RNA segments. BTV-1-15 (South Africa), -16 (West Pakistan), -17 (USA), -20
and -21 (Australia) were purified from infected BHK-21 cells and separated on a 1% agarose gel. RNA segments of each BTV serotype were identified by staining the gel with ethynium bromide.

Figure 8 shows segment 3 DNA of US BTV-17.
Northern blotting hybridization to corresponding RNA segments of other bluetongue viruses is shown. 19 B plotted on genetic screen paper
Autoradiogram of nick-translated 32p-labeled clone DNA hybridized to the TV serotype gene. The number of each BTV serotype is shown at the top of the autoradiogram.

Figure 9 shows the integration of cloned DNA into the viral gene,
In situ, indicating hybridization. Autoradiograms of nick-translated 32p-labeled clones hybridized to monolayers of infected or control cells are shown. Various BTV serotypes (1-17, 2
0 and 21) and control cells (c) are shown next to each chamber.

Figure 1'0 shows fluorescence detection of hybrids in cultured cells. In the chamber slide, lI, 17.20
Alternatively, nick-translate and biotinylated DUTP into the viral gene present in cells infected with 21.
The labeled DNA clone was hybridized.

After incubation of the hybrid with avidin fluorescent complex for 30 minutes at 18° C., it was detected using fluorescence microscopy. Two chambers containing uninfected control cells are indicated by rCJ.

DESCRIPTION OF THE INVENTION The present invention provides DNA sequences that are capable of hybridization with a portion of the RNA genome of at least one serotype of bluetongue virus.

Another aspect of the invention is to provide a cloning vector containing a DNA sequence capable of hybridizing with at least a portion of the RNA genome of the l serotype of bluetongue virus.

In yet another aspect, the invention provides for (a) isolating a double-stranded r(NA) from a bluetongue virus hepta rotib; (b) cleaving the RNA strand; and (c) cutting the 3° of each RNA strand. The terminal is polyadenylated and R
(d) complementary DNA in the presence of oligodeoxythymidine;
By contacting the polyadenylated RNA template described above with reverse transcriptase at time and temperature conditions suitable for the production of A, one complementary D is produced for each RNA template strand.
(e) removing the RNA template; and (f) self-annealing the complementary DNA product to generate a complementary DNA duplex. A method for producing a double-stranded DNA sequence capable of hybridization using a portion of the RNA genome of the L serotype is provided.

Yet another aspect of the invention provides a DNA sequence that is complementary to a portion of the r(NA) genome of the bluetongue virus and thereby capable of hybridization.
A DNA probe useful for detecting bluetongue virus is provided.

Another aspect of the present invention is to (a) supply probe DNA that is complementary to a portion of the RNA genome of bluetongue virus;
(c) detecting this DNA-RNA hybrid and thereby detecting the presence of virus in the sample. A method for detecting bluetongue virus in a sample containing bluetongue virus is provided.

The invention also provides a reagent kit useful for detecting BTV in a sample.

Finally, the invention provides a method for producing a viral protein comprising culturing a host cell under conditions promoting the synthesis of the viral protein, wherein said host cell is transformed with an expression vector containing a bluetongue virus DNA sequence. and the sequences, together with a promoter sequence, are oriented such that the viral DNA is expressed in a host to produce bluetongue virus proteins.

This invention relates to cloning the genetic information of bluetongue virus. Such information is not only useful as probes for detecting viral genomes, but also for recombinant D.
It is also useful for producing viral proteins by NA technology.

The principle used for cloning viral genetic information is as follows. After separation by electrophoresis, independent double-stranded RNAs were isolated from an agarose gel, the strands were cut, and their 3' ends were polyadenylated by reaction with polyadenylic polymerase. One copy of the complementary DNA was synthesized using reverse transcriptase with oligo(deoxy)thymidine as a primer. After removing the RNA template and self-annealing the complementary DNA products to ensure that all strands are of full length, the Klenow fragment of Escherichia coli (E, colt) DNA polymerase I was isolated. to repair its 3° ends, and the resulting complementary DNA duplex was transformed into Escherichia
The vector was cloned into the E. coli plasmid pBR322.

In the examples below, specific RNA segments (L3RN
Although the cloning of complementary DNA for A segment) is shown, this invention can be used to
It is intended to encompass the use of any or all segments obtained from any BTV serotype. For example, probes generated from the L3 gene containing the clone described in Example I may be used for serotypes 10111115 and 17 common in the United States, serotypes 10111115 and 17 common in South Africa, 15 TV-1 to 15 common in West Pakistan, -16, and popular TV in Australia.
It reacts in the hybridization assay with all BTV serotypes tested, such as -20 and -21. Therefore, this probe is particularly useful as a "group specific probe" capable of detecting a wide range of BTV serotypes.

Also see the examples below! There are 272 clones described in
Although a fully replicative clone of a 2 base pair long L3 gene segment is represented, use as a source of probe DNA does not necessarily require the complete L3 gene segment, if desired. That is, fragments of the L3 gene sequence can also be used if desired. The L3 clone, or any other clone containing BTV-DNA, can be fragmented into small clonal fragments, e.g. by reaction with an endonuclease, and used as group-specific or hepta-rotib-specific probes as described above. Fragmentation techniques, such as testing whether they work, are a matter of routine experience for those skilled in the art. Therefore, for purposes of this invention, the term probe used herein is
In assays based on hybridization of the probe with viral nucleic acids, it refers to a nucleic acid molecule representing a complete gene segment or a fragment thereof useful for virus detection.

When used for BTV detection, the probe is labeled with an analytically detectable reagent. Example 1 employs a radioactive label for detection, whereas Example 2 employs a biotin-avidin detection assay as a non-radioactive labeling method. Other assay methods are well known in the art and any can be easily substituted. Although not limited to,
Alternative methods include immunoassays such as ELISA, which use antibodies or DNA-r (NA-hybrid antibodies) guided by fluorescent dyes, proteins, or probe molecules. Competitive assays consisting of labeling the probe with an inactive subunit and labeling the probe with a second subunit such that the subunits are religated upon hybridization to restore enzymatic activity are also included.

The invention is particularly useful for detecting BTV in samples obtained from infected cells or microorganisms using in situ hybridization techniques. Samples that can be used include cell culture, tissue samples from the brain or lungs, fluids, nasal secretions, urine, uterine canal or vaginal secretions, semen, pleural fluid, peritoneal fluid, serum, plasma, and brain or lung fluid. fluid, middle ear fluid, synovial fluid, gastric aspirate, or feces;
Since TV is known to be an arthropod-borne disease, the presence of the virus can also be monitored in the arthropod vector, the swamp mosquito.

The high sensitivity of this assay method reduces the number of false negative results, and the specificity of hybridization reduces the number of false positive results. Finally, because the element being tested is a viral nucleic acid rather than a protein, the deleterious effects of mutations are reduced. That is, when translated into a protein, a single base change may result in the loss of an antigenic determinant;
In the case of multiple incompatible base pairs, the fidelity of the hybridization reaction is not affected.

This method has been successfully used to recover RNA from infected cells or to identify viral RNA in cell culture using in situ cell hybridization techniques. Regardless of how the cloned probe can be used to detect the viral genome, it is clear that this probe can provide a diagnostic tool for all BTV infections. By using in situ hybridization methods, large numbers of samples can be
They can be processed simultaneously in a fairly short time. Furthermore, this method has sufficient sensitivity to be used even when using a small amount of DNA, such as several μ99 parts.

The invention also includes a kit consisting of a transport container that is sectioned to closely accommodate a series of containers. The first container contains a DNA probe complementary to a portion of the bluetonguewill RNA genome, the second container contains a non-bluetonguewill control RNA, and the third container contains a DNA probe complementary to a portion of the bluetonguewill RNA genome. The container contains control Bluetongue Will RN.
Contains A.

Furthermore, the DNAs of the present invention are useful as a source of genetic information for BTV protein synthesis using recombinant DNA techniques. Once properly linked to an expression vector so as to be under the control of the expression vector's promoter sequence, the vector containing the inserted viral DNA segment can be used to introduce a suitable host microorganism.

The transformed host is then cultured under conditions that promote BTV protein synthesis.

[Examples] The examples shown below are exemplified to explain the present invention in more detail, but the scope of the invention is not limited thereby.

Example I This example describes the cloning and sequencing of the L3 gene from BTV serotype-17.

(Virus growth and isolation of double-stranded RNA) Sugyama et al. [
American Journal of Evidermology (
Amer, J., Epidermol, ), 1
5, pp. 332-347 (1981)], BTV serotype = 17 was changed to BHV-17 to BTV.
-21 cells and purified the 2. , Virol, )].

Each RNA species was isolated using the method described by Maniatis et al.
ng)], Cold Spring Harbor Laboratory (cold Spring Harbor L
laboratory), pp. 168-169 (1982) was modified as described below and electroeluted from the gel.

Electroelution was performed using only a dialysis membrane to capture the eluting RNA. 2mM Tris (pH 8,0)
RNA was harvested by washing the membrane three times using 200 μσ each of mother liquor containing 100 μι, 2 mM EDTA, and 0.4 M NACQ. BTV serotype 17 and BHK-21 cells were cultured with American Type culture coleccidin [(American Type
Culture Co11ec-tion), l 20
31, Park Lawn Drive, o-7 Kubiru, Maryland.

Rockville, Maryland), 20
852-1976] was publicly available. BT
The V strain has an accession number of ATCC VR-875, while the well-known Syrian or Gorean hamster kidney cell line B) (K-21 has an accession number of ATCC CCL 10).

(2 wood mr (polyadenylene of NA)) A double-stranded RNA preparation is left to denature in the presence of Q, C11M methylmercury at room temperature for 10 minutes. Then, by adding the same amount of 2-mercaptoethanol. , inactivates methylmercury. Schitzher [European Journal of Biochemistry (Eur, J
, Biochem, ), volume 37, pages 31-40 (1
ATP and Escherichia coli (E. coli) polyadenyl polymerase [Bethesda Research Laboratories (Bethesda Research Laboratories, 973)]
Research Laboratories)
, Dr. Gaithersberg
, M.

The 3° ends of both strands of the double-stranded RNA were polyadenylated using [obtained from D.]. The terminally polyadenylated RNA was purified by applying a Sephadex G-50 column to 10n+ prior to use in complementary DNA synthesis.
Modified with M methylmercury.

(Synthesis of Complementary DNA) Complementary DNA of polyadenylated BTV RNA was synthesized using reverse transcriptase of avian myeloblastomatosis virus [Life Sciences, St. Petersburg;
FL)] and ]5°-phosphorylated oligodeoxythymidine)+1~Ill [Collaborative Research (co
Laborative Research), Waltham, MA] was used as a primer (Maniades et al.
page). Reaction conditions were similar to those described by Buell et al. [Buell et al., Journal of Biol Chem.
, vol. 253, pp. 2471-2482 (1978)],
RNA-cDNA was purified from nucleotides on a G-50 Sephadex column and precipitated with ethanol. The RNA template is an RNA-DNA hybrid.
It was removed from the hybrid by incubating overnight at room temperature in 3M K'OH. After neutralization, the product was passed through Sephadex G-50 and the resulting complementary D
NA was precipitated with ethanol.

(Production of full-length double-stranded complementary DNA) Complementary DNA consisting of bipolar transcripts was mixed with 10 mM Tris-HC (
! (+)H8,O), 0.4M NaCL1mMEDT
It was dissolved in 50ml of Mixture A and incubated at 60°C for 24 hours to allow self-annealing. Complementary DNA after annealing was collected by ethanol precipitation. While cloning the complementary DNA preparation, to ensure that it is fully double-stranded, the product was cloned with the Klenow fragment of Escherichia coli (E. coli) DNA polymerase I [new
New England Biology
[Nucleic Acid Research (Nucleic Acid Research)].
Ac1d Res, ), vol. 10, 1335-1
343 (1982)].

(Molecular cloning) Escherichia coli (E, colD plasmid
nd I[I, the ends were repaired with the Klenow fragment of Escherichia coli (E. coli) DNA polymerase, and dephosphorylated with alkaline phosphatase. T, DNA ligase [New England Biolab
land B 1olabo)] using the complementary DN
A was blunt-end ligated to this plasmid. By this means, the HindI[1 site was effectively reconstituted with the terminal AT of the BTV Aiura DNA. This hybrid plasmid was used in Escherichia coli (E.

coli) strain MC1061 competent cells [Casadaban et al., Journal of Molecular Biology (J, Mo1. Biol, ), vol. 138, 179
207 (1980)] and colonies were selected on the basis of ampicillin resistance.

The obtained transformed cells were subjected to Grunstein-Hognes
Colony hybridization assay [Grunstein, Proceedings of National Academy of Sciences (Proc.

Nat'l Acad, Sci, )USA,
72, pp. 3961-3965 (1978)] using an oligodeoxythymidine primer and a short replication probe of 1P-labeled complementary DNA made with polyadenylated virus r(NA) (Bishop et al., 1978). A group of hybridization-positive colonies were screened for the presence of BTV-CDNA insertion sites.
They were characterized and compared in size by comparison with the Hinf I restriction fragment in pBR322.

The largest clones were run on a “northern” plot;
The nucleotide sequence was analyzed.

(RNA gel electrophoresis and plotting) BTV-
RNA was extracted from BHK-21 cells infected with P. 17 and separated by electrophoresis on a 1.0% agarose gel as previously described. Following electrophoresis, Alwin et al.
cad, sci, ) USA, vol. 74, 5350
-5354 (1977)], gels were prepared for plotting. Briefly, gels were prepared with 14mM mercaptoethanol and 1μ9/MQ.
Soaked in 50mM NaOH containing ethidium bromide for 20 minutes. The gel was washed four times with 12 mM sodium phosphate buffer, pH 6.5 (for 5 minutes each time), and then transferred to a nitrocellulose hybridization transfer membrane [New England Nuclear
grand Nuclear), Boston (BO8tO
n, MA)] was plotted by osmotic pressure for 2 hours.

After plotting, the membrane was air-dried and further roasted in an oven to 80-100°C for at least 2 hours.

Hybridization to DNA probes was performed as described by Anhart [Biochemical and Biophysical Research Communications (Biochem, B.
iophys, Res, Comm, ) vol. 23, 6
41-652 (1966)].

RNA: 5XSSCS 1% SDS, 100μ7/R
(1 denatured salmon sperm DNA, and 0.04% each of polyvinylpyrrolidone, Ficoll, and bovine serum albumin [Sigma Chemical Company (S i
GMA Chemical Company, St. Louis, MO) in 50% formamide for 16 hours at 42°C. The membrane was then added to the nick-translated 5tp-labeled DNA sample in the same mixture for 4
Hybrid generation was carried out for 16 hours at 2°C. After hybridization, the membranes were incubated at 25°C for 5 min, twice in 2x SSC, at 65°C for 30 min, twice in 2xSSC containing 0.05% SDS, and then at 25°C for 30 min at 0. lX
Washed twice with 5SC. After cleaning, the membrane is air-dried.
Autoradiography was performed.

(DNA Sequence Analysis) The method of Maxam and Gilbert [Methods, Talents, Enzymology]
(Methods Enzmol, ), 65.49
7-559 (1980). The DNA nucleotide sequence was determined using the previously described formic acid protocol for the adenine + guanine reaction (Bishop et al., supra). .

(Result) The size of one of the L gene clones (clone 7) was
Measurements were made as shown in the figure. D in recombinant clones
The NA insert was excised from the plasmid DNA with Hindll and run in a 1% agarose gel in parallel with the DNA marker. The size of the DNA fragment is 2.8Kb
This suggests that it contains the entire L3 gene.

Sequence analysis determined that the length of this fragment, excluding the homopolymer portion at the end, was 2772 base pairs.

Competitive DNA L2 and L3 of BTV move together through the agarose gel, which causes the induced BTV D
The identity of the NA clones was confirmed by measuring the hybridizable double-stranded RNA genome segments from them. Figure 2 (A) is
Total BTV-17RN after electrophoresis in 1% agarose gel
Figure 2 (B) shows the Northern plot of A.
A nick-translated 3tp-labeled probe specifically hybridized from the clone is shown, revealing that this is the only RNA segment of BTV-17L3.

rgyric acid i1i, 1. One L3-specific clone (clone 7) was strand-cleaved by standard techniques using restriction endonuclease fragments as shown in Figure 3. and end-labeled restriction DNA
Regarding sample A, the sequence of the cloned L3 gene was determined. The distance between each sequenced DNA strand is indicated by a solid arrow. The complete nucleotide sequence of the DNA clone of the L3 gene is shown in Figures 4a and 4b. About 80 genes
% was analyzed from both directions. Excluding the homopolymer portion at the end, the entire sequence is 2772 nucleotides long.

The corresponding size of L3 double-stranded RNA is 178×10
6 Daltons, which is in good agreement with previous reports [Berwoard et al., Journal of Pyrology, Vol. 10, 783]
-794 pages (1972)]. Nucleotides 18-2
A single open reading frame starting at 0 and ending at nucleotides 2721-2723 is calculated to be 103°412 Daltons in size and has a net charge of -4 9011
It encodes a primary gene product consisting of 11 amino acids.

Table 1 BTV-12 segment 3 gene production Alanine (A) 65 Arginine (R) 69 Aspartic acid (D) 59 Asparagine (N)
42 Cysteine (c) 5 Glutamic acid (E) 50 Glutamine (Q) 42 Glycine (G) 42 Histidine (H) 16 Isoleucine (r) 650 Isine (L)
82 Lysine (K)
28 Medionine (M)
37 Phenylalanine (F)
39 Proline (P)'
46 serine (S)
41 Threonine (T)
51 tryptophan (W)
10 Tyrosine (Y)
39 Valine (V)
73 effective charge −
4 molecular weight 103,412 (hybridization ability of clone L3 segment of American bluetongue virus and livestock epidemic hemorrhagic disease (EHD) virus (hybridizing ability with equivalent segment of related orbivirus)) Upper Rotaib, BTV-
10, -11, -13 and -17 have been shown to be genetically related (e.g. have a common fingerprint, allowing for a new reclassification of the NA species). Therefore, the L of these viruses
The question of whether there is any sequence homology among the 33 segments was investigated. To detect common sequences, BTV
- Nick translated the L3 segment of 17''
'P-labeled cDNA probe was prepared, and this was transformed into BTV-
10 or -11 or -13 or -17, or E
Plots containing purified genomic RNA from cells infected with either HD-1 virus were hybridized under stringent conditions. As shown in Figure 5, the 32P-labeled probe hybridizes to the L3 RNA of all four US serotypes, but not to the covalent segment of the EHD of related orbisviruses. The results revealed that this gene has a nucleotide sequence common to all American bluetongue viruses.

(Deposited to A, T, C, C,) Escherichia coli (E, coli) clone L-
3 transductants were from the American Type Culture Collection (American Type Cu1).
atureCo+ 1ection), 12301 Park Lawn Drive, Rockville, Maryland (P
arklawndrive, rockville,
Maryland) (September 24, 1984) with accession number ATCC 39875,
Paid the prescribed fee. During the prosecution of this application, this culture was approved by the Commissioner at 370. F, R'! 1
．． 14 and 35 U, S, C, 'J 122. If the patent based on this application is approved, all restrictions on the public availability of this culture will finally be lifted and the culture will be available forever during the term of the patent. Also, if this culture becomes non-viable or conservatively killed, it can be replaced with a living culture having the same taxonomic description.

Example 2 This example demonstrates the use of the BTV-17L3 clone as a probe to detect BTV in infected cells in situ.

Acid-cleaned coverslips are treated with denatured egg white and PVP [Brigalati et al., Pyrology.
ogy), vol. 126, pp. 32-50 (1983)].

This cover glass was sterilized with ultraviolet light, and 20% of BT infection was detected.
This is placed on a tissue culture dish in which a monolayer of BHK-21 cells was previously grown overnight. Several coverslips are left uninfected and used as negative controls.

Immediately after infection, coverslips are collected, rinsed with PBS, and fixed in Carnoy's B fixative (60% native ethanol, 30% chloroform, 10% acetic acid) for 5 minutes at room temperature. Air-dried coverslips can be stored at room temperature for a minimum of 2 weeks and at -70°C for 3 months.

By Nick translation, L3 of BTVI7
Escherichia coli (E. coli) containing pBR322 with complementary DNA replication of RNA.

Biotinylated deoxyUTP [Enzo, Biochem (Enz.

A hybrid bioprobe is created by incorporating a biochem r nc, )]. This biotinylated probe was precipitated with ethanol, and after drying,
50% deionized formamide, 10% high molecular weight dextran sulfate, 2×SSC, and 0.4 m
g/mura of denatured salmon sperm DNA was dissolved in a hybridization solution containing 1 g/mura of denatured salmon sperm DNA.

25 μC of the above solution was dropped onto a microscope slide glass, the cell side of the cover glass was placed over this, sealed with rubber cement, and then heated with steam at 80° C. for 3 minutes.

Then, immediately place the slide glass on ice for 3 minutes.
Then in a wet incubator for at least 4 hours, 3
The mixture was left at 7°C to generate hybrids. Next, collect the coverslip from the slide, use 2xSSC,
4 min in 2x SSC containing 0.1% SDS, twice for 4 min.
once for 20 min at 2°C, and finally with PBS, once at room temperature.
Wash once for 5 minutes. After the final wash, excess PBS was blotted from the coverslip, and the coverslip was placed with the cell side inwards using fluorescein-conjugated avidin [Pectron,
Dickinson
n)] O, 1m? /ff72Hm and leave it at room temperature for 2
Leave for 0 minutes. Finally, the coverslips are washed with PBS at room temperature for 30 minutes and observed under a fluorescence microscope. 6th a.
Figure 6b is a micrograph of uninfected cells (6a) and BTV infected cells (6b).

Example 3 This example shows how the BTV
-17L3 clone reactivity is shown.

Northern blot hybridization techniques were used to determine whether the cloned DNA of segment 3 of BTV-17 shared homologous gene sequences with BTV serotypes isolated from other South African and Australian origins. Two purified bottles of each serotype of bluetongue virus! The lRNA is subjected to electrophoresis,
Birdy et al. [Journal of Pyrology (J
, of Virol, ), Volume 51, No. 3, 754-79
5 (1984), this was carried out in a genetic screen [NEN, Boston, M.
A) Blow 7 to Ko. FIG. 7 shows electrophoretic images of 10 RNA segments of 19 BTV serotypes.

Four American serotypes (BTV-10, -11, -
13 and -17), the 19 different viruses show surprisingly similar RNA segment patterns in this gel system. Figure 8 is BT
This shows the results of hybridization of the plotted viral RNA to the nick-translated P-labeled DNA of segment 3 of V. As shown in FIG.
h H1l i11. It becomes Pik ee + +-nNA.

Initial experiments to detect viral gene sequences in infected cell cultures used nick-translated 3-containing P-labeled D
NA probe was used. Hamster lungs (HmLu)
Cells were grown on 4-8 compartment slides [Miles Laboratories] and! Each BTV serotype was infected at a multiplicity of 0.1-IPFU per cell. 16-20 after infection
At time, slides containing infected cell cultures were collected from the growth medium and rinsed three times with phosphate buffered saline (PBS). Cells were cultured in Kalony B fixative (60% ethanol,
30% chloroform, 10% acetic acid) for 5 minutes according to Enzo Biochem's instructions (1983). The samples were then pulled up, air dried, and stored at 4°C.

Each chamber containing dehydrated cells was incubated at 32P-D.
Cover with 25-50 μQ of hybridization mixture containing the NA probe. The lid of the chamber slide was sealed with tape, placed in a container, and heated to 80° C. for 5 minutes in a water bath as described by Brigalati et al., supra. Transfer the slides to a 37°C incubator for 12
The ink was incubated for ~16 hours. After hybrid generation,
Remove the liquid, store it at 4°C, and use it again. The chamber and plastic support were removed from the chamber slides and each slide was washed with 2X SSC and PBS as specified in Enzo Biochem's instructions.
Washed with. To generate hybrids and detect radioactive DNA, slides were briefly dried and
subjected to autoradiography. The results are shown in Figure 9. In some wells, cells were detached before 16 hours (e.g., BTV-2, BTV-6), so the analysis was repeated at a lower multiplicity of infection (0.01~O, IP
fu/cell).

With this method, the presence of viral infection was detected in each monolayer containing infected cells. It was not detected in uninfected cells used as a control.

Similar hybridization experiments were performed using nick-translated and biotin-labeled DNA probes instead of 32p-labeled probes.

To perform fluorescence detection of hybridized DNA containing biotin, probes were recovered as described above, slides were washed and air-dried, and then fluorescein-conjugated avidin (0, 1, 11I/mQ, Becton. 25 to 50 µg of PET (manufactured by Knockinson) were added to each chamber. After incubating the sample at 18°C for 30 min, P
Washed three times for 5 minutes each in BS, plots were dried, mounted on glass slides with glycerin, and examined for fluorescence. 1st
Figure O shows representative results for hybrids identified by incubation with the avinone-fluorescein complex.

Again, signs of strong fluorescence were observed in infected cells.

In contrast, nothing was observed in control cells.

To obtain a measure of the relative sensitivity of the in situ hybridization method, HmLu cells were infected with BTV-2 at various multiplicities of virus infection.

After infection, chamber slides were fixed at various time intervals and treated with in situ hybridization with a nick-translated 3tp-labeled DNA probe. The genome could already be detected from about 8 hours after infection, and it became clear that nucleic acid hybridization was directly proportional to the multiplicity of infection for the cells. The results showed that moderately high virus titers can be detected very quickly by this method.

[Brief explanation of the drawing]

FIG. 1 shows the migration pattern of clone 7 plasmid DNA cut with Hindnl and run on a 1% agarose gel. A is the Hind■ fragment of clone 7 DNA, and B is the HindI [I restriction fragment of the cDNA. Figures are expressed in kilobases. FIG. 2 shows hybridization of clone 7 plasmids that underwent nick translation. A is BTV
-17 genomic RNA agarose migration pattern,
B is an autoradiography of clone 7 DNA hybridized to segment 3 RNA of BTV-17. Figure 3 shows cloned BTV-17 segment 3.
This is the sequence strategy used to sequence the gene. The codes of the restriction sites used are as follows. H3, Hind
m: N, NcoI; D, Ddel; H, Hin
f I; P, Pst I: B, Bam HI:
E, EcoRI: B 11pgl II. Figures 4a and 4b show the nucleotide sequence of the cloned L3 gene of BTV. Note that FIG. 4b is a continuation of FIG. 4a. Figure 5 shows hybridization of BTV clone 7 hybridized to BVR and EHD L3 segments. The numbers on the left represent the number notation of the genomic RNA. Figures 6a and 6b show the results of in situ hybridization detection (fluorescence labeling) of biotinylated BTV-17 using probe DNA; Figure 6a shows uninfected control cells, Figure 6b shows BTV-infected cells. shows. FIG. 7 shows separation of BTV genomic RNA segments by 1% agarose gel electrophoresis. BTV-1-15 (
South Africa) 5, -16 (Pakistan), -17 (USA), -20 and -21 (Australia). FIG. 8 is an autoradiogram of nick-translated 32p-labeled DNA from hybridization in which BTV-1777) tF) component 3 DNA was Northern blotted to the corresponding RNA segment of other bluetongue viruses. Figure 9 shows in situ hybridization of cloned DNA to viral genes. Autoradiograms of 32p-labeled clones hybridized to monolayers of infected and control cells are shown. Figure 10 shows fluorescence detection of hybrids in infected or uninfected cells. Uninfected control chambers are designated rcJ. In addition, No. 1, 2.5.6a, 6b, 7.8.9 and 1
Figure 0 is a reproduction of the photograph. Patent applicant Research Corporation agent
Patent attorney Aoyama Aoyama and 1 other personFIG, 1 FIG, 2 〒ToA ATT TCC0% Gee 8TG
GCT GCT CAG MT GAG
CM CGT CCG~ CGA AHA MG
ACG ACG CCG TAT TTA
GM GGG CAT (1; TG T'rA
TCA: GACTCA GGG CCA C
TG CCT TCCGTG TTCGCG T
TG CAA GAG AHAKO CAA HeheC
GTG AGG CM GTG CM GC
T GAT TAT ATG ACA GC
A ACA% GAG C'l'T CAT
TTT ACA CTA CCG GAT G
TA CAA AAG 八T'r CT'I'
GAT: ATT 8AA ACG T”r8
GCT GCA GAA CM GTG TA
C He He He 8TCGTCAAA% CCT He He T
ATr 'rCA 'rTCAGA CAT A
TCGTA ATG CAG TCA AGA G
ATA GTT TTA CGA GTG GAT
ACC 'l'Ac GAA GAG
ATG 'rCA CAG GTTI GAT'
GT'l' ATA ACG GAA GAT
GAA CCA GAA AAA TTCT
kT TCA ACTATTCAAG AAA G
TG CGG'! ”TT AHA CGCGGA A
AA GGA TCCTTT A? A＾CAT G
AT ATT CCG ACCAGA GAT
CAT CGCGGCAT GAG GTT
GC? ':; CCA Gkk GTG 'f'TA
GGA GTCGAA TTCAAG AAT GT
A CTA CCT CTC3ACA GCCGAG
CAT CGCGCA ATG AT? '
CAG AACGCA TG GAT GGA
: ATA ATT GAG MCGGA
AACGTA GCT ACA CGA GAC
GTT GACGTA; to τ Gce GCCT
GT TCCGAA CCA ATCTAT
CGC 8TA 'rACAA'! 'ACA:l;
CAA GGG TAT AT'l' GAG Ge
A GTG CAA TTA CAA GAG TTA
AGG AATA ATT GGG TGG TTA
GAG AGG TTA GGG CAG AGG
AAA AGA ATCACGτTCG CAA GA
G GTT CTG ACT GAT TTT AGG
AGG CM GACACA ATT: GTC'
rTA GCT TTA CAG CTA C
CG GTCAAT CCA CAG GTA
GTG TGGT GTG CCG CGCAGC
TCT ATCGCCMCTA ATCATG AA
T A'l”A GCA:; TGCTTA CCCA
CA GGG GM TACATCGCG CM MC
CM AGA ATTA TCG ATT ACG C
TG ACCCAA AGA ATA ACA ACA
ACG GGG CCA TT'TT AT'T
CTA ACT GGA TCA AcCCC
A ACT GCA CAG CAA CTT
AAT GATl AGOAAG ATC'rA
'l' TTA GCG CTA ATG TTT C
CT GGA CAG ATT AHAGO G'T
CTA AAA ATCGAT CCT G
GCGAG kGG ATG GAT CCG
GCk GTAAA ATG C"l'CGC
TGGCG? f' GTA GGT CAT
TTG CTCTTT ACA GCG
GGTA AGA TTCACG AAT TT
A ACA CCA MT ATG GCG AGA
CAG CTCGATA GCCCTA AAC
GA'l'TA'rT'rA CTT TAT A
TG TAT AACAce AGA GTT50TC
MT TAT GG CCA ACG GC; T GA
G CCG T”l”A GAT TTCCAG AT
TA AGG MT GAG TAT GACTGT
MT CTT TTT AGA GCA GAT TT
ACA to CGC CGAACA GGA TACMCG
GA TGG GCT ACA AT'f' GAT
GTT GAG TAτAGA GACCTG G
CCCCT TACGTG CAT GCG
CAG CGC1rAc ATA CGT τ8T
Ding GT GGT To Ding CGAT TCG CGC
GAG TTCATT AAT CC to ACA
ACA? A'r GGC8T to CG
A'l'G 8CT TAT CAT TCT
TACM'! 'GAG ATC'! '? A
C(:A ATc Tete^GT? GCC:
CCA GGG AAA CAT TCT
GAG GCG GCG TM: TTT
CGCACCATOCTG CCCTTT CkC
: ATG GTA AAG 'ffT GCT
AGG ATA AAT CAA AT(:
Ate^heheCGAA GAT 〒τAC80 heheT
C'l'C;TGT'rCTCGT'TG
CCG GA'l' CAT ATC? To C88 ccA TTA TTA C:CCGACC
TA Eight? T GCT GGG CCG CA
T CAG Me GeeGAC(:CA GTT
GTG CTA CAT G? 'G AC
T TGG 8TA TcG CTG ↑Ca
TeTCGCteTTCAACAGG TCG TTT
GAA CCA kCG CA? 'AGG AAT
GAG kTG CTCCAAATCGCT CCA
CTG ATCGAG Dingcc GTT TkT
GCG 'rcG GAG CτT? Cτ GT
GATCAAG GTA GA'r 8TG
CGA CACTTG TCA TTA ATG
CAG AGA AGATTCCCJ CAT G
TT TTA ATCCAA GCG AGG CCC
; TCCCAT Tel” 'l'GG A^^GC
AGT? CTG M? GACAGCCCCG
OAG GCG GAG AAA GCA
CTT ATC entered AC TATCG CAT TCC
CAT enter A'r'! 'TCAT(: input τ
Entering A AGG GAT ATG AτGCG?
τCaGTA ATG CTCCCA TCA
CTG CAA CCA TCG TTA
AAG CTCCTA TTA GAAGAG
GAG GCA TGG Gee GCT GCA A
ACGA'l' TTC (JA GAT CTG AT
CCTTACT OAT CAA CTT TA'l'
ATG CkT CGA GA'l' ATCTTC
CCA GAA CCA CGGTTG CAT C
AT GTT GAG AGG T'rCAG
A CAG GAA CGT T'TCTAT
TACACGAACATOTTA GAG G
CCCCA CCA GAA ATA CAT
CGT GTA Gff CAG TATAC
T TAT GAG AT'T GCA CGT TT
A CAA GCG AACATCGGA C-entering ATT
T αtGCA GCT CTA All:A
CGCATT A'rG GAT GAT G
A'l' GACTGG GTG AGA T
'! 'TGGCGGT GTCTTA CGCACT
GTA CGCGTT λ^A ding'rCTTT CA
T GCG CG included CCCCCA GACGAT
ATT CTA CAG GGC'r'rA Cc
T'r? c AG(TA'r CAT A(JAA
CGAG AAA GCT GGA'! TA T
cA TA'l' GCG ACG A'PT AA
G 〒八でOCT AC'l'GAG Ace A
CA ATT TTT TkT CTG ATA
TAT AAT GTCGAA TTCTCCAAC
kCG CCCGAT TCT TTG GTG T
TG ATT AAT CCA GCk TACk
cG ATCACTAAA CTT TTCATT
AACAAG AGA ATT GTr
GAG CGA GTA CGG CTT AG
ACM A'! 'T TTG GCCGTA
TTG AACAGA AGA TTCG'rG
GCA TACAAA GGAkfi), ATC
AGA ATT ATG GACATCACT
CAA'TCG CTCAAG ATCGGCG
CCMG CTG GCT GCG CCA
ACT CTCTAG AT CGCGACC
AA TCT kTG CACT? CGTA
GCG GCA GCCGM A'l'A C
ACT'rA CFIG, 6゜FIG, 6゜t匡FIG, IO Procedural Amendment (-# <) December 20, 1985 1 Case Indication 1985 Patent Application No. 193004 2, Name of Invention Fruitang Relationship to the Case of Person Who Corrects DNA Probe 3 for Virus Detection Patent Applicant Address 25, Suite 853, Broadway, New York, New York, United States of America Name: Research Houbolayon 4, Agent

Claims (1)

Claims: 1. A DNA sequence hybridizable by the RNA genome of at least one serotype of bluetongue virus. 2. The arrangement according to claim 1, wherein the serotype is BTV-17 and the arrangement is substantially in the following 5' to 3' direction. [There is a gene sequence] 3. A cloning vector containing the DNA sequence described in claim 1 or 2. 4. A host microorganism transformed with the vector according to claim 3. 5. The host according to claim 4, wherein the host has a trait identified as ATCC39875. 6. (a) Isolate double-stranded RNA species from bluetongue virus serotypes, (b) separate the RNA strands, and (c) polyadenylate the 3' end of each RNA strand to form RN.
(d) generating a complementary DN in the presence of oligodeoxythymidine;
(e) synthesizing a complementary DNA copy of each RNA template strand by contacting the polyadenylated RNA template strand with reverse transcriptase at time and temperature conditions suitable to produce A; (f) double-stranded DNA hybridizable from the RNA genome of at least one serotype of bluetongue virus, comprising self-annealing the complementary DNA products to produce a complementary DNA duplex; manufacturing method. 7. A DNA probe useful for the detection of bluetongue virus, comprising a DNA sequence that is complementary to and thereby hybridizable to a portion of the RNA genome of bluetongue virus. 8. (a) An analytically detectable probe DNA that is complementary to a portion of the RNA genome of bluetongue virus.
(b) contacting the probe with the RNA under conditions that promote the formation of a DNA-RNA hybrid; (c) detecting the DNA-RNA hybrid and thereby detecting the virus present in the sample. A method for detecting bluetongue virus in a bluetongue virus-containing sample comprising: 9. The method of claim 8, comprising performing step (b) in situ. 10. A first container containing a DNA probe complementary to a portion of the bluetongue virus RNA genome; a second container containing control non-bluetongue virus RNA;
A kit comprising a transport container partitioned to tightly accommodate a series of containers, including a container and a third container containing control bluetongue virus RNA. 11. The kit according to claim 10, wherein the probe includes the sequence according to claim 1 or 2.