PL235253B1 - Method for differentiation, diagnostic test for the differentiation of the rabies vaccine strains from its field strains and application of the diagnostic test - Google Patents

Description

Description of the invention

The invention relates to a method of differentiation, a diagnostic test for differentiating between "vaccine" strains from rabies virus field strains, and the use of the diagnostic test. More specifically, the solution relates to a method of differentiating and identifying rabies virus field strains from rabies virus strains contained in oral vaccines for foxes against rabies called "vaccine" strains.

Differentiation of rabies virus strains into "vaccine" and field strains is an important element in the monitoring of the action of oral immunization of foxes against rabies. Since oral rabies vaccines contain live, attenuated (devoid of pathogenic properties) strains of rabies virus, there is a risk that these strains may revert to the pathogenic form. Therefore, all positive rabies cases diagnosed in the area where the vaccine is distributed should be differentiated to verify that the rabies case is not caused by rabies virus strains contained in the oral vaccines delivered in the field.

In the publication of A. Orłowska et al. (on-line, November 19, 2014), "Genetic characterization of the rabies virus vaccine strains used for oral immunization of foxes in Poland to estimate the effectiveness of vaccination" reveals the results of research that confirmed the similarity of vaccine strains and rabies virus strains circulating in the environment . The differential studies were based on RFLP analyzes.

Moreover, in A. Metlin et al., 2008, "Restriction enzyme technique for rapid discrimination of attenuated and field rabies viruses in the Russian Federation" discloses a quick and simple technique based on restriction enzyme digestion of RT-PCR products that has been tested to distinguish field viruses from vaccine strains.

The patent application WO2011062537 (published 2011-05-26) describes a detection method using molecular biology methods and a method of identifying classical swine fever virus (CSFV) strains. The invention relates to a method of detecting the presence of at least one classical swine fever virus (CSFV) vaccine strain in a biological sample and comprises the steps of: a) preparing a reaction mixture containing viral RNA from the biological sample; b) providing at least one vaccine specific probe designed to hybridize to nucleotides in the N-terminal coding region of the CSFV autoprotease (N pro) of the genome (s) of the vaccine strain under stringent conditions; c) performing a nucleic acid amplification reaction under stringent conditions, yielding a product; and d) determining from the product of step c) whether hybridization of the vaccine specific probe has occurred with the amplified viral vaccine DNA, and if so, indicating the presence of the CSFV vaccine strain (s) in the sample. The invention furthermore relates to primers, probes and kits for the detection and identification by molecular biology of CSFV vaccine strains and their differentiation from field strains.

Patent application UA69088 (published 2004-08-16) discloses a method of differentiating Marek's disease virus strains and isolates using strains FC-126 (serotype III), SBG (serotype II) and isolates of strain CVI / Rispens (serotype I). According to the invention, the presence and morphological changes of cells are recorded and compared in the process of cytopathic action in the culture of embryonic fibroblasts of different species of poultry.

Patent description RU2360252 (published 2009-06-27) describes a diagnostic kit and test for determining the activity of anti-rabies sera and a method of in vitro preparation of heterologous anti-rabies immunoglobulins. The test designed to determine the specific activity of anti-rabies sera and immunoglobulins includes a nitrocellulose membrane with superimposed positive (standard anti-rabies immunoglobulin samples with specific activity) and negative (normal horse serum) controls, deionized wash water, sample dilution solution, conjugate and layout for development (visualization). The invention aims to broaden the possibility of determining the specificity of sera and immunoglobulins for the production of heterologous anti-rabies immunoglobulins.

In the patent application KR20100006868 (published 2010-01-22), a diagnostic kit and a method for the rapid detection of anti-rabies antibodies using immunochromatography. The kit includes a nitrocellulose membrane containing a test line and a control line. The conjugate pad overlaps the nitrocellulose membrane on one side. The sample pad overlaps the conjugate pad. A pad with an adsorbent absorbing blood serum is placed on the other side of the nitrocellulose membrane.

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The patent application CN102721816 (published 2012-10-10) describes a diagnostic kit for the diagnosis of rabies virus. The operating conditions of the diagnostic kit are optimized such that the coating solution is 50 mmol / L carbonate buffer, pH 9.4; the optimal BSA-PBS breakpoint is 1%; the optimal concentration of the coating antigen is 8g / mL; optimal serum dilution 1: 300; optimal duration of action of the serum 1 hour; optimal dilution of the second antibody 1: 5000; optimal duration of action of the second antibody 1 hour; optimal development of the substrate color - 25 minutes. The reagent kit has high sensitivity, high accuracy and low production cost, is simple, fast and convenient to operate.

Patent application CN102492042 (published 2012-06-13) describes the recombinant fusion protein 3F3Rmg and the method of its production, as well as the use for the detection of canine anti-rabies antibodies. The recombinant fusion protein is encoded by the 3F3Rmg fusion gene obtained by splicing the 3F3ScFv gene and the Rmg gene, and has the base sequence specified as SEQ. ID. Well. 1. Due to the bifunctional characteristics of the recombinant 3F3Rmg fusion protein, the fusion protein can bind to human red blood cells without agglutination, it can react with anti-rabies G antibodies and is subject to an agglutination phenomenon that can be observed with the naked eye. The diagnostic kit is easy to use, highly sensitive and specific. In particular, it is designed for the detection of canine anti-rabies antibodies and meets the criteria of speed, ease and convenience of handling.

Patent application US6015663 (published 2000-01-18) describes an enzymatic restriction test for the differentiation of porcine reproductive and respiratory syndrome virus (PRRSV) strains. The amplified cDNA from the ORF region 5 of the viral genome was analyzed for the identification of unique restriction regions that allow the differentiation of vaccine strains from field strains by using selected restriction enzymes. These tests are applicable to both clinical diagnosis of PRRSV field viruses and epidemiological studies to assess the source and spread of PRRS field viruses.

Patent RU2196992 (Published 2003-01-20) describes a method for identifying and differentiating vaccine and field strains of Hog's cholera virus. The method comprises synthesizing non-specific and CHCV-specific oligonucleotide primers, isolating viral RNA from vaccine or pathological material, synthesizing cDNA from viral RNA as a template by a reverse transcription reaction. PCR is performed and the first step is to amplify a fragment of the viral genome by using non-specific oligonucleotide primers. The second step of amplification should be performed using PCR and using CHCV-specific oligonucleotide primers. Subsequently, cleavage of the obtained amplified cDNA fragments should be performed with Taq I endonuclease. Differentiation of vaccine strain isolates and CHCV field strains should be performed on the basis of the data of the number and mass of the obtained restriction fragments after polyacrylamide gel electrophoresis. The method according to the invention makes it possible to obtain higher specificity and sensitivity of the techniques used, as a result of which a higher yield is obtained.

The method of differentiating rabies virus 'vaccine' strains to date has been based on the use of monoclonal antibodies and the technique of indirect immunofluorescence. The fixed impressions from the brain examined on the slides were reacted with a panel of monoclonal antibodies allowing for the differentiation of field strains from strains contained in oral vaccines.

Despite existing solutions, there is a continuing need to find an effective method of differentiating rabies virus field strains from rabies virus strains contained in oral vaccines for foxes. The indirect immunofluorescence method makes it difficult to read the results, as the lights are often non-specific and therefore the reading of the preparations causes diagnostic difficulties and requires a lot of experience. For this reason, very often this method requires prior virus multiplication in cell culture. If the viral load in the test material is very low, and in addition, the distribution of the virus in the brain tissue is uneven, often the virus may be below the detection limit, which may result in a false-negative result.

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The object of the present invention is to develop an efficient differentiation method and to develop a diagnostic test for differentiating rabies virus field strains from rabies virus strains contained in oral vaccines for fox against rabies called "vaccine" strains.

The present invention has achieved the accomplishment of the objective so defined and the solution of the problems described in the prior art related to the development of the invention, which enables the differentiation of field strains from rabies virus vaccine strains. For the above reasons, a fast, cheap and easy to interpret PCR-RFLP method was developed to differentiate between "vaccine" and field strains using restriction enzymes.

According to the invention, the amplification products of viral RNA (a segment of the gene encoding a fragment of the rabies virus nucleoprotein protein characterized by high conservation of the nucleotide sequence) are cleaved by restriction enzymes. With the help of the NebCutter 2 computer program, restriction enzymes were selected that allow easy strain differentiation by recognizing a restriction site, a short, specific nucleotide sequence, and then digesting it, resulting in DNA fragments of various lengths. The identification and differentiation of rabies virus strains into field and "vaccine" strains is done by comparing the lengths of the DNA fragments obtained against the restriction template obtained from digesting the DNA of the field and "vaccine" strains.

Isolated rabies virus RNA (using a commercial kit, e.g. from Qiagen: "Qiamp Viral RNA Mini Kit", or by Chomczyński's method) from 20% of homogenates in the brain of sick animals or from the contents of oral rabies vaccine blisters, in the first stage, the amplification reaction is preceded by reverse transcription (RT-PCR). The fragment of the gene encoding the rabies virus nucleoprotein corresponding to position 55-660 nt of the genome of the reference PV strain (Acc. No. M 13215) is amplified. JW12 primers with the sequence SEQ are used for amplification. ID No. 1 (ATGTAACACCYC TACAATG) and JW6DPL with the sequence SEQ. ID No. 2 (CAA TTC GCA CAC ATT TTG TG) designed by Heaton et al. (1997). RT-PCR is performed using a commercial kit, for example from Invitrogen ("Superscript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase"). 2 μΙ RNA is added to the reaction mixture containing 7 μΙ DNase / RNase free water; 12.5 μΙ 2x buffer reaction mix; 1 μΙ of Superscriptlll / Platinum Taq enzyme mixture and 1 μΙ of JW12 primer (20 μΜ) and 1 μΙ JW6DPL (20 μΜ). The reaction is performed in a thermocycler under the following time-temperature conditions:

RT-PCR time-temperature profile Temperature Time Number of cycles 50 ° C 30 minutes 1 95 ° C 15 minutes 1 95 ° C 30 seconds 35 cycles 49 ° C 30 seconds 72 ° C 1 minute 72 ° C 10 minutes 1 4 ° C Up to the electrophoretic separation step

In the next step, the products of RT-PCR amplification are cut by restriction enzymes. For this purpose, 2 μΙ of RT-PCR product is added to the mixture containing the restriction enzyme, the reaction buffer appropriate for the enzyme and water. Cutting conditions are as recommended by the enzyme manufacturer, most often at 37 ° C for 2 hours.

The subject of the invention is a method of differentiating "vaccine" strains from rabies field virus, characterized in that the amplification products of viral RNA are cleaved by restriction enzymes selected from restriction endonuclease, and then the lengths of the obtained DNA fragments are compared against the restriction template resulting from digesting DNA field and "vaccine" strains, in which a JW12 primer defined by SEQ is used for amplification. Well. 1 and the JW6DPL primer identified by the sequence SEQ. Well. 2, wherein the fragment of the gene encoding the rabies virus nucleoprotein corresponding to position 55-660 nt is amplified.

From the genome of the reference PV strain (Acc. No. M 13215), and wherein the restriction enzymes are Dra I, Msp I or Nla IV.

Preferably, the method comprises the following steps:

(a) the isolated rabies virus RNA from homogenates in the brain of diseased animals or from oral rabies vaccines is subjected to a reverse transcription preceded amplification reaction;

b) the RT-PCR amplification products are cleaved by restriction enzymes selected from among the restriction endonuclease;

c) after digestion of the amplicon with restrictionases and electrophoretic separation, DNA fragments of different lengths are obtained, and then the identification and differentiation of rabies virus strains into field and "vaccine" strains by comparing the length of the obtained DNA fragments against the restriction template resulting from the digestion of DNA of the field strains and "Vaccines".

Preferably, the Dra I enzyme which recognizes the TTT / AAA sequence is used. Preferably the enzyme Msp I is used which has a restriction site for the C / CGG sequence.

Preferably the enzyme Nla IV having a restriction site for the GGN / NCC sequence is used.

Preferably 2 µl RNA is added to the reaction mixture containing 7 µl DNase / RNase free water; 12.5 pl 2x buffer reaction mix; 1 µl of the SuperscriptIII / Platinum Taq enzyme mixture and 1 µl of a JW12 primer (20 µM) determined by the sequence SEQ. Well. 1 and 1 pl JW6DPL (20 pM) determined by the sequence SEQ. Well. 2, where the reaction is performed in a thermocycler.

Preferably it is used to differentiate rabies virus field strains from rabies virus "vaccine" strains contained in oral rabies vaccines for foxes.

Preferably, the method comprises oral rabies vaccines for red foxes.

Another object of the invention is a diagnostic test for the differentiation and identification of "vaccine" strains from rabies field virus, characterized in that the amplification products of viral RNA are cleaved by restriction enzymes selected from restriction endonuclease, and the lengths of the obtained DNA fragments are compared against the template restriction digestion derived from DNA digestion of rabies virus field and "vaccine" strains, in which a JW12 primer defined by SEQ is used for amplification. Well. 1 and the JW6DPL primer identified by the sequence SEQ. Well. 2, wherein the fragment of the gene encoding the rabies virus nucleoprotein corresponding to position 55-660 nt of the genome of the reference PV strain (Acc. No. M 13215) is amplified, and wherein the restriction enzymes are Dra I, Msp 1 or Nla IV.

Preferably, the Dra I enzyme recognizes the TTT / AAA sequence.

Preferably, the Msp I enzyme has a restriction site for the C / CGG sequence.

Preferably, the Nla IV enzyme has a restriction site for the GGN / NCC sequence.

Another object of the invention is the use of the diagnostic test as defined above for the differentiation and identification of "vaccine" strains contained in oral rabies vaccines for red foxes used in oral immunization of foxes and rabies virus strains isolated from rabid field animals.

The attached figures allow for a better explanation of the essence of the invention, where:

Figure 1 shows the results of RFLP analysis of 17 rabies virus field strains (lanes 1-17) and the "vaccine" strains of SAD B19 (lane 18) and SAD Bem (lane 19) after digestion with the restriction enzyme Dra I. M - DNA mass size marker;

Figure 2 shows the results of RFLP analysis of 17 rabies virus field strains (lanes 1-17) and "vaccine" strains (lanes 18-19) after digestion with Msp I. M - DNA mass size marker;

Figure 3 shows the results of RFLP analysis of 17 rabies virus field strains (lanes 1-17) and "vaccine" strains (lanes 18-19) after digestion with the restriction enzyme Nla IV. M - DNA mass size marker.

For a better understanding of the invention, exemplary solutions are presented below. The following descriptions for the cleavage of RT-PCR amplification products with the respective restrictionases illustrate the invention.

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Examples

P r z k ł a d I

The most vividly differentiating enzyme is Dra I restrictionase. This enzyme recognizes a restriction site, TTT / AAA six nucleotide sequence, where DNA cleavage occurs. 2 μΙ of RT-PCR product is added to the reaction mixture containing 1 μL of restriction enzyme, 8 μL of Tango reaction buffer, and 71 μL of molecular biology water. Incubation with the enzyme lasts 2 hours at 37 ° C. As a result, amplicons of rabies virus "vaccine" strains (600 bp) are cleaved into two fragments of 450 bp and 150 bp (Fig. 1, lanes 18-19). The restriction products are separated in a 1.5 - 2% agarose gel with the addition of 5 μl of ethidium bromide at a concentration of 10 mg / ml. For this purpose, 1.5-2 g of agarose are dissolved in 100 ml of 1x concentrated electrophoresis buffer, eg TAE, in a microwave oven. The dissolved agarose is poured onto a comb table and allowed to set for 20-30 minutes. 8 µl of Dra I cleavage product is mixed with 1 µl of 6x Loading Buffer and then the whole is applied to the wells of the solidified gel. The electrophoresis is performed for 40 minutes at a voltage of 90-100 V. After completion of the electrophoresis, the gel is viewed under UV light on a transilluminator or in an image analysis camera. As the amplicons of the field strains lack a Dra I restriction site, as a result, the gel shows one 600 bp band after electrophoretic separation of the cleavage products (Figure 1, lanes 1-17).

P r z x l a d II

The enzyme allowing for the differentiation of "vaccine" and field strains is also Msp I, for which the restriction site is the C / CGG sequence. The RT-PCR amplification product (2 μΓ) is added to the reaction mixture containing 0.8 μL of the Msp I enzyme; 2.2 μl of buffer and 5 μl of water for molecular biology. Digestion is carried out for 2 hours at 37 ° C. The cut products are then separated on an agarose gel and viewed under UV light analogously to the previous example. The DNA fragments (amplicons) obtained as a result of digestion create patterns characteristic of a specific rabies virus strain. The "vaccine" strains (Figure 2, lanes 18-19) have a different electrophoretic pattern in RFLP analysis than the field strains (Figure 2, lanes 1-17).

P r x l a d III

The enzyme Nla IV can also be used to differentiate between "vaccine" and field strains. This enzyme has a cleavage site in the GGN / NCC sequence. 2 μl of the amplification product are cut analogously to the examples I and II. DNA is added to 8 µl of reaction mixture containing 0.1 µl of enzyme; 0.8 μl buffer and 7.1 μl molecular biology water. Cutting is carried out for 2 hours at 37 ° C. The products are then separated by electrophoresis and analyzed in a camera under UV light. The "vaccine" strains have a different cutting pattern (pattern) than the field strains (Figure 3).

The test according to the invention, regardless of the restriction enzyme used, enables a quick, easy and, above all, cheap way to differentiate between "vaccine" and field strains of rabies virus. According to the restriction enzyme used in Examples 1 to 3, the "vaccine" strains have a different electrophoretic pattern in RFLP analysis of the nucleoprotein gene fragment gene fragment than the field strains.

Bibliography:

1. Heaton P.R., Johnstone P "McElhinney L.M., Cowley R., O'sullivan E., Whitby J.E .: Heminested PCR Assay for Detection of Six Genotypes of Rabies and Rabies-Related Viruses. J. Clin. Microbiol. 1997, 35, 2762-2766.

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Sequence Listing

SEQ. ID No. 1 starter JW12

ATGTAACACCYC TACAATG

SEQ. ID No. 2 starter JW6DPL

CAA TTC GCA CAC ATT TTG TG

Claims (13)

1. A method of differentiating "vaccine" strains from rabies field virus, characterized in that the amplification products of the viral RNA are cleaved by restriction enzymes selected from among the restriction endonuclease, and the lengths of the obtained DNA fragments are compared against the restriction template obtained from digesting the DNA of the field strains and "vaccines", which use a JW12 primer defined by SEQ for amplification. Well. 1 and the JW6DPL primer identified by the sequence SEQ. Well. 2, wherein the fragment of the gene encoding the rabies virus nucleoprotein corresponding to position 55-660 nt of the genome of the reference PV strain (Acc. No. M 13215) is amplified, and wherein the restriction enzymes are Dra I, Msp I or Nla IV. 2. The differentiation method according to claim The process of claim 1, comprising the following steps: (a) the isolated rabies virus RNA from homogenates in the brain of diseased animals or from oral rabies vaccines is subjected to a reverse transcription preceded amplification reaction; b) the RT-PCR amplification products are cleaved by restriction enzymes selected from among the restriction endonuclease; c) after digestion of the amplicon with restrictionases and electrophoretic separation, DNA fragments of different lengths are obtained, and then the identification and differentiation of rabies virus strains into field and "vaccine" strains by comparing the length of the obtained DNA fragments against the restriction template resulting from the digestion of DNA of the field strains and "Vaccines". 3. The differentiation method according to claim The process of claim 1, wherein the Dra I enzyme recognizes the TTT / AAA sequence. 4. The differentiation method according to claim The process of claim 1, wherein the enzyme Msp I has a restriction site for the C / CGG sequence. 5. The method of differentiation according to claim The method of claim 1, wherein the enzyme Nla IV has a restriction site for the GGN / NCC sequence. 6. The differentiation method according to claim The method of claim 2, wherein 2 µ RNA is added to the 7 µΙ DNase / RNase free water reaction mixture; 12.5 μΙ 2x buffer reaction mix; 1 μΙ of the SuperscriptII / Platinum Taq enzyme mixture and 1 μΙ of JW12 primer (20 μΜ) of the specified SEQ sequence. Well. 1 and 1 μΙ JW6DP1, (20 μΜ) determined by the sequence SEQ, No. 2, where the reaction is performed in a thermocycler. 7. The method of differentiation according to claim The method of claim 2, which is used to differentiate rabies virus field strains from rabies virus "vaccine" strains contained in oral rabies vaccines for foxes. 8. The method of differentiation according to claim A method according to claim 2, comprising oral rabies vaccines for red foxes. PL 235 253 B1 9. A diagnostic test for the differentiation and identification of vaccine strains from rabies field virus, characterized in that the amplification products of viral RNA are cleaved by restriction enzymes selected from among the restriction endonuclease, and the lengths of the obtained DNA fragments are compared against the restriction template resulting from digestion DNA of rabies virus field and "vaccine" strains in which the JW12 primer is used for amplification as defined by SEQ, No. 1 and the JW6DPL primer identified by the sequence SEQ. Well. 2, wherein the fragment of the gene encoding the rabies virus nucleoprotein corresponding to position 55-660 nt of the genome of the reference PV strain (Acc. No. M 13215) is amplified, and wherein the restriction enzymes are Dra I, Msp 1 or Nla IV. 10. A diagnostic test according to claim 1, The method of claim 6, wherein the Dra I enzyme recognizes the TTT / AAA sequence. 11. A diagnostic test according to claim 1, The method of claim 9, wherein the enzyme Msp I has a restriction site for the C / CGG sequence. 12. A diagnostic test according to claim 1, 9. The process of claim 9, wherein the Nla IV enzyme has a restriction site for the GGN / NCC sequence. 13. Use of a diagnostic test as defined in claims 9 to 12 for the differentiation and identification of "vaccine" strains contained in oral rabies vaccines for red foxes used in the oral immunization of foxes and rabies virus strains isolated from rabid animals from the field.