Vaccines based on porcine torque teno virus and methods of diagnosing infections caused thereby
FIELD: medicine, pharmaceutics.
SUBSTANCE: inventions deal with infectious molecule of nucleic acid, coding infectious porcine Torque teNO viruses (PTTV), which contains at least one copy of genome sequence, selected from the group, consisting of sequences, corresponding to genotypes or subtypesPTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, as well as to biologically functional plasmid or viral vector, containing such infectious nucleic genome sequence, and host-cell, containing such plasmid or vector. In addition claimed inventions include live, attenuated expressible with vector application and purified recombinant capsid subunit or killed viral vaccines for protection against PTTV infection, as well as methods of immunisation of pigs against PTTV viral infection by said vaccine introduction.
EFFECT: characterised inventions can be used to prevent infection, caused by porcine Torque teNo virus.
23 cl, 53 dwg, 5 tbl, 24 ex
The present application claims the benefits of provisional applications U.S. patent No. 61/235,833, filed August 21, 2009, and provisional application U.S. patent No. 61/316,519, filed March 23, 2010, the description of which is included in this application by reference.
The technical field TO WHICH THIS INVENTION RELATES
This invention relates to vaccines for protection against infections caused by the swine virus Torque TeNO (Torque teno) (TTV), and infections caused by infectious DNA clones of porcine TTV (PTTV), and to their use. The present invention also relates to the diagnosis of infections caused by the swine virus Torque TeNO (PTTV), particularly infections caused by different types and type-specific viruses PTTV, and infections caused by many strains of different genotypes.
The LEVEL of TECHNOLOGY
Virus Torque TeNO (TTV) was first discovered in 1997 in Japan in a patient with post-transfusion hepatitis non-A and non-E type (Nishizawa, T., Okamoto, H., Konishi, K., coupled amplifier, H., Miyakawa, Y. and Mayumi, M. (1997). A NOvel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unkNOwn etiology. Biochem Biophys Res Commun 241(1), 92-7).
Since it was identified a large number of human TTV strains and two groups of TTV - related viruses, identified as virus Torque teNO mini (TTMV) and the virus Torque teNO midi (TTMDV) with high prevalence in serum and other tissues of healthy people (HiNO, S., and Miyata, H. (2007). Torque teNO virus (TTV): urrent status. Rev. Med. Virol. 17(1), 45-57; Okamoto, H. (2009a). History of discoveries and pathogenicity of TT viruses. Curr. Top. Microbiol. ImmuNOl 331, 1-20). Human TTV, TTMV and TTMDV are spherical viruses without the shell, containing a genome of circular single-stranded DNA (ndnc) length 3,6-3,9, 2,8-and 2,9 3,2 Sciences, respectively, and they are now classified in a new family of viruses Anelloviridae in Intemationa Committee on TaxoNOmy of Viruses (ICTV; http://www.ictvonline.org/virusTaxoNOmy.asp?bhcp=1) (Biagini, P. (2009). Classification of TTV and related viruses (anelloviruses). Curr. Top.Microbiol. ImmuNOl. 331, 21-33). These three groups of viruses that are related to TTV, exhibit a high degree of heterogeneity, with each group consists of a plurality of genogroups and genotypes (Biagini, P., Gallian, P., Cantaloube, J. F., Attoui, H., de Micco, P., and de Lamballerie, X. (2006). Distribution and genetic analysis of TTV and TTMV major phylogenetic groups in French blood doNOrs. J. Med. Virol. 78 (2), 298-304; Jelcic, I., Hotz-Wagenblatt, A., Hunziker, A., Zur Hausen, H., and de Villiers, E. M. (2004). Isolation of multiple TT virus geNOtypes from spleen biopsy tissue from a Hodgkin's disease patient: geNOme reorganization and diversity in the hypervariable region. J. Virol. 78(14),7498-507). It was reported on the distribution of multiple infections caused by TTV with different genotypes, as well as double or triple infections caused by TTV, TTMV and TTMDV, people, and these infections are regarded as normal events in healthy people (Niel, S., Saback, F. L., and Lampe, E. (2000)). Co-infection caused by many TTV strains belonging to different genotypes is common in healthy adults in Brazil (J. Clin. Microbiol. 38(5), 1926-30; NiNOmiya, M., Takahashi, M., HoshiNO, Y., Ichyama, K., Simmonds, P., and Okamoto, H. (2009). Analysis of the entire geNOmes of torque teNO midi virus variants in chimpanzees: infrequent cross-species infection between humans and chimpanzees. J. Gen. Virol. 90 (Pt 2), 347-58; Okamoto, H. (2009a). History of discoveries and pathogenicity of TT viruses. Curr. Top.Microbiol. ImmuNOl.331, 1-20; Takayama, S., Miura, T., Matsuo, S., Taki, M., and Sugii, S. (1999). Prevalence and persistence ofaNOvel DNA TT virus (TTV) infection in Japanese haemophiliacs. Br. J. Haematol. 104 (3), 626-9).
TTV cause infection not only in humans but also in different species of animals, including nonhuman primates, Tupaj, pigs, cattle, cats, dogs and sea lions (Biagini, R., Uch, R., Belhouchet, M., Attoui, H., Cantaloube, J. F., Brisbarre, N., and de Micco, P. (2007). Circular geNOmes related to anelloviruses identified in human and animal samples by using a combined rolling-circle amplification / sequence-independent single primer amplification approach. J. Gen. Virol. 88(Pt 10), 2696-701; Inami, T., Obara, T., Moriyama, M., Arakawa, Y., and Abe, K. (2000). Full-length nucleotide sequence of a simian TT virus isolate obtained from a chimpanzee: evidence for a new TT virus-like species. Virology 277(2), 330-5; Ng, T. F., Suedmeyer, W. K., Wheeler, E., Gulland, F., and Breitbart, M. (2009). NOvel anellovirus discovered from a mortality event of captive California sea lions. J. Gen. Virol.90 (Pt 5), 1256-61; Okamoto, H. (2009b). TT viruses in animals. Curr. Top.Microbiol. ImmuNOl. 331, 35-52; Okamoto, H., Nishizawa, T., Takahashi, M., Tawara, A., Peng, Y., Kishimoto, J., and Wang, Y. (2001). GeNOmic and evolutionary characterization of TT virus (TTV) in tupaias and comparison with species-specific TTV in humans and NOn-human primates. J Gen Virol. 82(Pt 9), 2041-50; Okamoto, H., Nishizawa, T., Tawara, A., Peng, Y., Takahashi, M., Kishimoto, J., Tanaka, T., Miyakawa, Y. and Mayumi, M. (2000a). Species-specific TT viruses in humans and NOnhuman primates and their phylogenetic relatedness. Virology 277(2), 368-78; Okamoto, H., Takahashi, M., Nishizawa, T., Tawara, A., Fukai, K., Muramatsu, U., Naito, Y., and Yoshikawa, A. (2002). GeNOmic characterization of TT vimses (TTV) in pigs, cats an dogs and their relatedness with species-specific TTV in primates and tupaias. J Gen. Virol. 83 (Pt 6), 1291-7). In addition, chimpanzees also become infected and TTMV TTMDV (NiNOmiya, M., Takahashi, M., HoshiNO, Y., Ichiyama, K., Simmonds, P., and Okamoto, H. (2009). Analysis of the entire geNOmes of torque teNO midi virus variants in chimpanzees: infrequent cross-species infection between humans and chimpanzees. J. Gen. Virol. 90(Pt 2), 347-58; Okamoto et al., 2000a, supra). Although the sizes of the genomes of strains identified TTV in animals, especially TTV in animals that are not primates, relatively smaller than in the strains of TTV person, they all have the same genome structure with at least two partially overlapping open reading frames (ORF1 and ORF2), translated from the negative DNA, and short cut retranslating plot (UTR) with a high content (about 90%) GC (guanine and cytosine) (Okamoto, 2009b, supra). Arrangement TTV ORF is very similar to the arrangement of anemia virus of chickens (CAV), belonging to the genus Gyrovirus in the family Circoviridae, but differs from the arrangement of pig circovirus (PCV) type 1 (PCV1) and 2 (PCV2), which is also classified in the same family (Davidson, I., and Shulman, L. M. (2008). Unraveling the puzzle of human anello virus infections by comparison with avian infections with the chicken anemia virus. Virus Res. 137(1), 1-15; HiNO, S., and Prasetyo, A. A. (2009). Relationship of Torque teNO virus to chicken anemia virus. Curr. Top Microbiol. ImmuNOl.331, 117-30). The genomes of PCV1 and PCV2 are ambisyllabic, and ORF1 encoded genomic floss and ORF2 encoded antigenome thread (HiNO and Miyata, 2007, supra). Recently, the template for transcription and the translated products of both genotype� 1 and 6 human TTV were identified by transfection of the respective infectious DNA the TTV clones in cultured cells (Mueller, B., Maerz, A., Doberstein, K., Finsterbusch, T., and Mankertz, A. (2008). Gene expression of the human Torque TeNO Virus isolate P/1C1. Virology 381(1), 36-45; Qiu, J., Kakkola, L., Cheng, F., Ye, C., Soderlund-Venermo, M., Hedman, K., and Pintel, D. J. (2005)). (Mueller, B., Maerz, A., Doberstein, K., Finsterbusch, T., and Mankertz, A. (2008). Gene expression of the human Torque TeNO Virus isolate P/1C1. Virology 381(1), 36-45; Qiu, J., Kakkola, L., Cheng, F., Ye, C., Soderlund-Venermo, M., Hedman, K., and Pintel, D. J. (2005). Human circovirus TTV genotype 6 expresses six proteins after transfection of the clone with full length (J. Virol. 79(10), 6505-10). The expression of at least six proteins, designated as ORF1, ORF2, ORF1/1, and ORF2/2, ORF1/2 and ORF2/3, three or more planned mRNA was described Kakkola, L., Hedman, K., Qiu, J., Pintel, D., and Soderlund-Venermo, M. (2009). Replication of and protein synthesis by TT viruses. Curr. Top Microbiol. ImmuNOl. 331, 53-64; Mueller et al., 2008, supra; Qiu et al., 2005, supra). Accordingly, it is probable that, when available, additional data concerning the animal TTV, intended structure of the genome of the animal TTV can be modified.
Although TTV was first identified in a patient with cryptogenic hepatitis, subsequent studies have not provided evidence of the significant role of TTV in the pathogenesis of hepatitis or other diseases (HiNO and Miyata, 2007, supra; Maggi, F., and Bendinelli, M. (2009). ImmuNObiology of the Torque teNO viruses and other anelloviruses. Curr. Top Microbiol. Immunol. 331, 65-90; Okamoto, 2009a, supra)). Although people believe that human TTV is not directly associated with the disease, recently belowstanding, that swine TTV (PTTV) partially contributes to the experimental induction of porcine dermatitis syndrome and neuropathy (PDNS) connected with the occurrence of reproductive/respiratory syndrome swine (PRRSV) (Krakowka, S. Hartunian, S., Hamberg, A., Shoup, D., Rings, M., Zhang, Y., Allan, G., and Ellis, J. A. (2008). Evaluation of induction of porcine dermatitis and nephropathy syndrome in gNOtobiotic pigs with negative results for porcine circovirus type 2. Am. J. Vet. Res. 69(12), 1615-22), and experimental induction of Multisystem syndrome of exhaustion after weaning from sows (PMWS), which is connected with infection caused by PCV2 in the model gebetically pigs (Ellis, J. A., Allan, G., Krakowka, S. (2008). Effect of coinfection with geNOgroup 1 porcine torque teNO vims on porcine circovirus type 2-associated postweaning multisystemic wasting syndrome in gNOtobiotic pigs. Am. J. Vet. Res. 69(12), 1608-14). These data suggest that the swine virus TTV is pathogenic for pigs. But a more in-depth research with a biologically pure form of the virus PTTV for full characteristics of diseases and pathological changes associated with infection caused by PTTV.
Compared to human TTV information on the human genome PTTV is very limited. Currently described only one genomic sequence with full length and two genomic sequences with an almost full length for PTTV in pigs in Japan and in Brazil, respectively (Niel, S., Diniz-Mendes, L., and Devalle, S. (2005). Rolling-circle amplification of Torque teNO virus (TTV) complete geNOmes from human and swine sera and dentification ofaNOvel swine TTV geNOgroup. J. Gen. Viro.l 86(Pt 5), 1343-7; Okamoto et al., 2002, supra.). Among the three known PTTV strains were grouped together strains Sd - TTV31 and TTV - 1P in genogroup 1 (PTTV1), while TTV - 2P was the only strain is classified in genogroup 2 (PTTV2) (Niel et al., 2005, supra). However, classification GeoGraph is a nebulous concept in the taxonomy of Virology and further and more accurate classification of PTTV, but it can be done only when it will be available to other genomic sequences with a total length of new PTTV strains representing many genotypes.
Previously it was shown that infections caused by PTTV, widely distributed in pigs in six countries, including the USA, Canada, Spain, China, Korea and Thailand (McKeown, N. E., Fenaux, M., Halbur, P. G., and Meng, X. J. (2004). Molecular characterization of porcine TT virus, an orphan virus, in pigs from six different countries. Vet. Microbiol. 104 (1-2), 113-7).
The question of whether TTV significant role in the pathogenesis of various diseases in pigs or not, still remains controversial. The example model gebetically pigs, it was shown that infection itself caused PTTV1, does not cause any clinical disease, but causes pathological changes (Krakowka, S. and Ellis, J. A., 2008. Evaluation of the effects of porcine geNOgroup 1 torque teNO virus in gNOtobiotic swine. Am. J. Vet. Res. 69, 1623-9). In pigs experimentally infected and PTTV1, and virus reproductive - REP�retornado syndrome swine (PRRSV), developed clinical porcine dermatitis and nephropathy syndrome (PDNS) (Krakowka, S. Hartunian, S., Hamberg, A., Shoup, D., Rings, M., Zhang, Y., Allan, G. and Ellis, J. A., 2008. Evaluation of induction of porcine dermatitis and nephropathy syndrome in gNOtobiotic pigs with negative results for porcine circovirus type 2. Am. J. Vet. Res. 69, 1615-22), while in pigs infected with the PTTV1 and porcine circovirus type 2 (PCV2), there has been a chronic Multisystem wasting syndrome after weaning piglets from sows (PMWS) (Ellis et al., 2008, supra). Although PCV2 is considered as an agent, basically calling clinical PMWS or illness associated with PCV (PCVAD), Spain has been a strong spread of infection caused PTTV2, in pigs suffering from PMWS with a low content or not with PCV2 PCV2 compared with pigs without PMWS (Kekarainen et al., 2006, supra). All these data suggest that swine TTV can serve as co-factors involved in launching and strengthening the development of diseases in pigs.
Porcine TTV was detected in samples of swine serum, feces, saliva, semen, and tissues from infected pigs, which shows that it is transmitted in various ways, including vertical and horizontal transmission (Kekarainen et al., 2007, supra; Pozzuto, T., Mueller, V., Meehan, B., Ringler, S. S., Mclntosh, K. A., Ellis, J. A., Mankertz, A. and Krakowka, S., 2009. In utero transmission of porcine torque teNO viruses. Vet. Microbiol. 137, 375-9; Sibila, M., Martinez-GuiNO, L., Huerta, E., Llorens, A., Mora, M., Grau-Roma, L., Kekarainen, T. and Segales, J., 2009. Swine torque teNO virus (TTV) infection and excretion dynamics in onventional pig farms. Vet. Microbiol. 139, 213-8). Currently, however, the detection of the presence of infection caused by TTV, based on the conventional PCR method. Thus, so far not been developed any method of serological analysis, no system viral culture. In particular, the methods of "nested PCR" amplification conservative sites in UTR PTTV1 and PTTV2, respectively, developed by a group of Spanish researchers have found wide spread (Kekarainen et al., 2006, supra). Since the rate of spread of the virus, apparently, is associated with severity of clinical disease, as shown by PCVAD caused by PCV2 (Opriessnig, T., Meng, X. J. & Halbur, P. G., 2007. Porcine circoviros type 2 associated disease: update on current termiNOlogy, clinical manifestations, pathogenesis, diagNOsis, and intervention strategies. J. Vet. Diagn. Invest. 19, 591-615), it would be important to determine the viral load of porcine TTV method of quantitative PCR in real time, than the presence of TTV DNA in conventional PCR method. In addition, PCR in real time is more reliable, fast and less expensive method compared to the conventional PCR method. Recently two methods have been described PCR in real time, based on the use of samples TaqMan for the detection and quantitative determination of two types of swine TTV (Brassard, J., Gagne, M. J., Houde, A., Poitras, E. and Ward, P., 2009. Development of a real-time TaqMan PCR assay for the detection of porcine and bovine Torque teNO virus. J. Appl. Microbiol. NOv 14, 2009, E pub. ahead of print; Gallei,A., Pesch, S., Esking, W. S., Keller, C. and Ohiinger, V. F., 2009. Porcine Torque teNO virus: Determination of viral geNOmic loads by geNOgroup-specific multiplex rt-PCR, detection of frequent multiple infections with geNOgroups 1 or 2, and establishment of viral full-length sequences. Vet. Microbiol. Dec 21, 2009, E pub. ahead of print). The main drawback of methods based on the use of samples is that can be obtained false negative results if the sample - binding sequences contain mutations (Anderson, T. P., WerNO, A. M., BeyNOn, K. A. and Murdoch, D. R., 2003. Failure to geNOtype herpes simplex virus by real-time PCR assay and melting curve analysis due to sequence variation within probe binding sites. J. din. Microbiol. 41,2135-7).
Given the high degree of heterogeneity among sequences of known strains of porcine TTV, expect variations of the sample - binding sequences in field PTTV strains. The PCR method in real time, based on the application of the dye SYBR Green is an alternative method of avoiding the occurrence of this problem despite its rather low specificity, providing a uniform way of detection and quantification of variants of porcine TTV. Moreover, melting curve analysis (MCA) after PCR in real time, based on the application of the dye SYBR Green, provides the specificity of the reaction and allows multiplex detection of different virus types (Ririe, K. M., Rasmussen, R. P. and Wittwer, C. T., 1997. Product differentiation by analysis of DNA elting curves during the polymerase chain reaction. Anal. Biochem. 245, 154-60). Methods of PCR in real time, based on the application of the dye SYBR Green, were applied successfully to different human and animal viruses (Gibellini, D., Gardini, F., Vitone, F., Schiavone, P., Furlini, G. and Re, M. C., 2006. Simultaneous detection of HCV and HIV-1 by SYBR Green real time multiplex RT-PCR technique in plasma samples. Mol. Cell Probes 20, 223-9; Martinez, E., Riera, P., Sitja, M., Fang, Y., Oliveira, S. and Maldonado, J., 2008. Simultaneous detection and geNOtyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green. Res. Vet. Sci. 85, 184-93; Mouillesseaux, P. K., Klimpel, K. R. and Dhar, A. K., 2003. Improvement in the specificity and sensitivity of detection for the Taura syndrome virus and yellow head virus of penaeid shrimp by increasing the amplicon size in SYBR Green real-time RT - PCR. J. Virol. Methods 111, 121-7; Wilhelm, S., Zimmermann, P., Selbitz, H. J. and Truyen, U., 2006. Real-time PCR protocol for the detection of porcine parvovirus in field samples. J. Virol. Methods 134, 257-60)
Currently on PTTV-specific humoral response, little is known. As methods using PCR do not reflect the development of infections caused by PTTV, in pigs to assess the prevalence of serotype PTTV and characteristics of the role of PTTV in the development of disease in pigs it is necessary to apply an efficient enzyme-linked immunosorbent assays(ELISA).
So, still no subunit, inactivated or live vaccine against porcine TTV. It is desirable and preferable to carry out the expression of recombinant capsid proteins PTTV from different genotypes to create subunit vaccines and get info�klonnie molecular DNA clones from PTTV different genotypes for distribution of organic forms PTTV in the cell culture system, which is used to create inactivated or live vaccines.
Disclosure of the INVENTION
The present invention provides a molecule of infectious nucleic acid ("infectious clone DNA) swine virus Torque teNO (PTTV), which is a nucleic acid molecule encoding an infectious PTTV, which contains at least one copy of genomic sequence having a degree of homology is at least equal to 80% compared to the genomic sequence selected from the group consisting of PTTV genotypes 1A-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA.
According to one aspect of this invention provides infectious clones PTTV DNA according to claim 1 of the claims, characterized by the fact that a genomic sequence selected from the sequences described in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.
The present invention also provides a biologically functional plasmid or viral vector containing the infectious genomes PTTV the Present invention provides a suitable host cell, transfected with a plasmid or viral vector of the infectious clone DNA.
The present invention provides infectious PTTV produced by their cells, transfected clones infectious PTTV DNA. The present invention also relates to a viral vaccine, containing non-toxic, no�th physiologically acceptable carrier and an immunogenic amount of a member, selected from the group consisting of (a) nucleic acid molecule containing at least one copy of genomic sequence having a degree of homology is at least equal to 80% compared to the genomic sequence selected from the group consisting of PTTV genotypes Ia-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA or its complementary thread, (b) a biologically functional plasmid or viral vector containing a nucleic acid molecule containing at least one copy of genomic sequence having a degree of homology is at least equal to 80% in relation to the genomic sequence selected from the group consisting of PTTV genotypes 1A-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA or its complementary thread, and (C) an avirulent, infectious nonpathogenic PTTV, which contains at least one copy of genomic sequence having a degree of homology that is equal to at least 80% relative to the genomic sequence selected from the group consisting of genotypes PTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA.
In accordance with one aspect of this invention, the vaccine contains a live virus PTTV obtained from clones of infectious PTTV. According to another aspect of this invention, the vaccine contains inactivated virus PTTV obtained from clones of infectious PTTV.
The present invention predusmatriva�et purified recombinant proteins, expressed capsid gene of the ORF1 of PTTV genotypes or subtypes PTTV1a-VA, PTTV1b-VA and PTTV2c-VA in a bacterial system for the expression and application of recombinant capsid proteins as subunit vaccines against infections caused by PTTV. According to one variant of the invention recombinant capsid proteins used in the composition of subunit vaccines expressed in the baculovirus expression in other vector systems for the expression.
According to another aspect of this invention, the vaccine further comprises an adjuvant.
The present invention also provides a method of immunization of pigs against viral infection caused by PTTV, comprising administering to the pig an immunologically effective amount of a viral vaccine.
According to another aspect of the present invention said method includes the introduction of the pig recombinant subunit capsid protein molecules infectious nucleic acid, or a live virus PTTV.
According to another aspect of the present invention said method comprises parenteral, intranasal, intradermal or transdermal administration of the vaccine the swine.
The present invention provides also highlighted the polynucleotide consisting of the nucleotide sequence PTTV1a-VA, presented in SEQ ID NO:9.
This from�the acquisition also provides the polynucleotide selected, consisting of the nucleotide sequence PTTV1b-VA, presented in SEQ ID NO:10.
The present invention provides also highlighted the polynucleotide consisting of the nucleotide sequence PTTV2b-VA, presented in SEQ ID NO:11.
The present invention provides also highlighted the polynucleotide consisting of the nucleotide sequence PTTV2c-VA, presented in SEQ ID NO:12.
The present invention also provides subunit vaccine containing an immunogenic fragment of a polypeptide sequence or a complete protein, translated according to a polynucleotide sequence selected from the group consisting of ORF1, ORF2, ORF1/1, and ORF2/2 of PTTV genotypes or subtypes PTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, in particular ORF 1 that encodes a capsid protein.
According to another aspect of the present invention polynucleotide sequence selected from the group consisting of ORF1 of PTTV genotypes or subtypes PTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA.
According to another aspect of the present invention the polynucleotide ORF sequence is a 1 genotypes PTTV1a.
According to another aspect of the present invention the polynucleotide ORF sequence is a 1 genotypes PTTV1b.
According to one aspect of the invention a polypeptide sequence selected from the group consisting of after�of euteleostei, presented in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.
According to another aspect of the present invention the polypeptide sequence represents SEQ ID NO:13. According to another aspect of the present invention the polypeptide sequence represents SEQ ID NO:14. According to another aspect of the present invention the polypeptide sequence represents SEQ ID NO:16. According to one specific embodiment of this invention the polypeptide sequence represents the C-terminal section (as.to. 310-625) SEQ ID NO:16. According to another aspect of the present invention the polypeptide sequence represents SEQ ID NO:20.
According to another aspect of this invention, the vaccine further comprises an adjuvant.
The present invention also provides a method of immunization of pigs against viral infection caused by PTTV, comprising administering to the pig an immunologically effective amount of a viral vaccine containing an immunogenic fragment of a polypeptide sequence or a complete protein, translated according to a polynucleotide sequence selected from the group consisting of ORF1, ORF 2, ORF1/1, and ORF2/2 of PTTV genotypes or subtypes PTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA.
According to one aspect of the d�steering of the invention said method includes the introduction of the pig immunogenic fragment or recombinant capsid protein.
According to another aspect of the present invention said method comprises parenteral, intranasal, intradermal or transdermal administration of the vaccine pig. According to another aspect of the present invention said method includes the introduction of the pig vaccine lymph, or intramuscular injection.
The present invention relates also to method of diagnosis of infection. caused PTTV1 and quantitative determination of viral load PTTV1, including extraction of the DNA from the sample, presumably infected PTTV1, conducting polymerase chain reaction (PCR) using primers containing the sequence presented in SEQ ID NO:29 and SEQ ID NO:30, and the detection of specific amplification PTTV1. According to one aspect of the invention polymerase chain reaction is a PCR in real time using the dye SYBR Green.
The present invention also provides a method for diagnosing infections caused PTTV2, and quantitative determination of viral load PTTV2, including extraction of the DNA from the sample, presumably infected PTTV2, conducting polymerase chain reaction (PCR) using primers containing the sequence presented in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, and the detection of specific amplification PTTV2. According to one ASPE�of this invention tov polymerase chain reaction is a PCR in real time using the dye SYBR Green.
The present invention also provides a method of simultaneous detection and diagnosis of infections caused PTTV1 and PTTV2, including extraction of the DNA from the sample, presumably infected PTTV, conducting polymerase chain reaction (PCR) using primers containing the sequence presented in SEQ ID NO:31 and SEQ ID NO:32, and the detection of specific amplification PTTV1 and PTTV2. According to one aspect of the invention polymerase chain reaction is a PCR in real time using the dye SYBR Green.
In addition, the present invention provides also a method of simultaneous detection and diagnosis of infections caused PTTV1a and PTTV1b, including extraction of the DNA from the sample, presumably infected PTTV1, the first polymerase chain reaction (PCR) using primers containing the sequence presented in SEQ ID NO:33 and SEQ ID NO:34, the second polymerase chain reaction (PCR) using primers containing the sequence presented in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, and detection of specific amplification PTTV1a and PTTV1b.
The present invention also provides a method of diagnosing infection caused by PTTV, including immobilization immunogenic fragment of a polypeptide sequence, Translia�according to this polynucleotide sequence, selected from the group consisting of ORF1, ORF2, ORF1/1, and ORF2/2 of PTTV genotypes or subtypes PTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA; contacting the serum sample of a pig, presumably infected PTTV, the immobilized immunogenic fragment and detecting captured antibody specific for the immunogenic fragment.
According to one aspect of the present invention polynucleotide sequence selected from the group consisting of ORF1 of PTTV genotypes or subtypes PTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA.
According to one aspect of the present invention the polynucleotide sequence is ORF1 represents genotype PTTV1a-VA virus PTTV.
According to another aspect of the present invention the polynucleotide sequence is ORF1 represents genotype PTTV1b-VA virus PTTV.
According to another aspect of the present invention the polynucleotide sequence is ORF1 is a subtype PTTV2c-VA virus PTTV.
According to another aspect of the invention a polypeptide sequence selected from the group consisting of the sequences represented by SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28.
According to one embodiment of this invention the polypeptide sequence presented in SEQ ID NO:13. According to another embodiment, yeah�tion of the invention the polypeptide sequence presented in SEQ ID NO:14. According to one embodiment of this invention the polypeptide sequence presented in SEQ ID NO:16. According to one specific embodiment of this invention the polypeptide sequence represents the C-terminal section (as.to. 310-625) SEQ ID NO:16. According to another aspect of the present invention the polypeptide sequence represents SEQ ID NO:20.
The present invention provides three standardized method enzyme-linked immunosorbent assay (ELISA) for the diagnosis of infections caused by PTTV and detection of antibodies in serum of pigs infected with genotypes PTTV1-VA, PTTV1b-VA virus PTTV and all known subtypes of PTTV type 2.
Diagnostic tests ELISA based on the application expressed by bacteria or expressed by baculovirus ORF1 capsid protein of genotypes PTTV1a-VA, PTTV1b-VA and PTTV2c-VA virus PTTV.
According to another aspect of this invention the detection of the captured antibody by Western blotting. According to another aspect of this invention the detection of the captured antibody by enzyme-linked immunosorbent assay (ELISA).
BRIEF description of the DRAWINGS
The above features of the invention will be more apparent from the following detailed description of the invention in conjunction with the drawings, n�:
Figures 1A and 1B represent a schematic diagram of the genomic structures, strategies, genomic cloning and Assembly of the four strains of swine virus TTV group 1 (type 1) and group 2 (type 2), the prototype U.S. strain.
Figure 2 presents PASC (pairwise comparison of sequences), comparison of nucleotide sequences 121 of TTV strain, which are available in GenBank. Shows genus, species, types, subtypes, variants and the respective percent identity of nucleotide sequences.
Figure 3A illustrates the phylogenetic tree constructed by the method of Association of neighbors based on the use of full-genome nucleotide sequences.
Figure 3B illustrates the phylogenetic trees constructed based on the predicted amino acid sequences of ORF1 using seven strains of porcine TTV.
Figure 3C illustrates the phylogenetic trees constructed based on the predicted amino acid sequences of ORF1/1 with seven strains of porcine TTV.
Figure 3D illustrates the phylogenetic trees constructed based on the predicted amino acid sequences of ORF2 using seven strains of porcine TTV.
Figure 3E illustrates the phylogenetic trees constructed based on the predicted AMI�kislotnyh sequences ORF2/2 using seven strains of porcine TTV.
Figure 4 shows the alignment of the complete amino acid sequences of ORF1 among seven strains of TTV.
Figure 5 shows the alignment of the complete amino acid sequences of ORF2 among seven strains of TTV.
Figure 6A illustrates melting curves of PCR products PTTV1 in real-time after 40 cycles of amplification standard matrix samples (shown in blue) and 20 samples of swine serum.
Figure 6B illustrates melting curves of PCR products PTTV2 in real-time after 40 cycles of amplification standard matrix samples and 20 samples of swine serum.
In the figures 7A-7B shows the results of melting curve analysis (MCA) duplex PCR products PTTV1/PTTV2 using the dye SYBR Green in real-time.
Figure 8 is an alignment of the nucleotide sequences located HaN - terminal section intended for ORF1 seven PTTV strains.
In the Figures 9A and 9B shows the profiles hydrophilicity and conservative areas of the four known porcine TTV2.
Figures 10A and 10B illustrate the expression and purification of recombinant capsid protein ORF1 PTTV2c.
In the Figures 11A and 11B shows representative results of the analysis of seven samples of swine serum by the method of Western blotting.
Figure 12 illustrates the consistency of the results of the analysis PTTV2-ORF1 m�methods Western blotting and ELISA.
Figure 13 shows plots Box - Whisker showing the dependence of the number of antibodies to PTTV2 in the serum of pigs from viral load for 138 pigs from different sources.
Figure 14A shows a retrospective assessment of viral load PTTV2.
Figure 14C shows the number of antibodies to capsid protein PTTV2-ORF1 to 10 pigs grown before reaching 2 months. after the arrival.
Figures 15A-15C illustrate the stages of expression and purification of recombinant capsid protein ORF 1 in PTTV1a and PTTV1b and
Figure 16 shows examples of the results of analyses PTTV1a - ORF1 by the method of Western blotting in serum samples collected from pigs on a farm in Wisconsin.
The IMPLEMENTATION of the INVENTION
In accordance with this invention in one specific example of the above four new subtype of swine virus TTV were allocated one hog in Virginia.
In Figure 1A, both of the genome PTTV1 and PTTV2 shown in bold and the number and directions of the four alleged ORF (ORF1, ORF2, ORF1/1, and ORF2/2) indicated by arrows. Also shown are areas enriched with GC. The dotted lines in the groups A and D show the plots used for the detection PTTV1 and PTTV2, respectively, in samples of serum and semen nested PCR. Circles b and C, dashed lines show two overlapping PCR fragment for genomic kleinova�Oia PTTV1, and the dotted circles E and F show two overlapping PCR fragment for genomic cloning PTTV2. Locations of primers used in this experiment (see Table 1), also shown in the respective positions.
A single serum sample taken from hog (SR # 5), gave a positive result for PTTV1 and PTTV2 in the first round of PCR, which indicates a greater viral load, it was used for subsequent cloning of the full genomes of PTTV. Surprisingly, initial attempts using two sets of primers (NG372 / NG373 and NG384 / NG385) in the case of inverted PCR for cloning of the first strain Sd - TTV31 PTTV for the purpose of amplification of viral genomic DNA were unsuccessful (Okamoto et al., 2002, supra). After several attempts, the PCR product was not obtained.
Based on the original phase sequence AND PTTV1 and plot D PTTV2, got two new pairs of primers (TTVl-If(SEQ ID NO:1)/TTV1-2340R (SEQ ID NO:2) and TTV1-2311F(SEQ ID NO:3)/TTV1-IR(SEQ ID NO:4)) for the purpose of amplification plots b and C, overlapping the estimated genome PTTV1, and two additional pairs of primers (TTV2-IF(SEQ ID NO:5)/TTV2-2316R(SEQ ID NO:6) and TTV2-GCF(SEQID NO:7)/TTV2-IR(SEQ ID NO:8)) for the purpose of amplification plots E and F, covering the estimated genome PTTV2, respectively (Figure 1A and table 1). Primers TTV1-2340R (SEQ ID NO:2) and TTV1-231 IF (SEQ ID NO:3) were obtained from the total sequence in strains Sd-TTV31 (Okamoto et al., 2002, supra) and TV-lp (Niel et al., 2005), which is absent in strain TTV-2p PTTV2 (Niel et al., 2005, supra), while primers TTV2-2316R(SEQID NO:6) and TTV2-GCF(SEQID NO:7) were obtained from the sequence of TTV strain - 2P, which is absent in the two strains PTTV1. Each of the resulting four different PCR products with the expected size was insertion in the cloning vector with a blunt end and the obtained recombinant plasmids were transformed into Escherichia coli. Were identified from eight to fifteen positive (white) clones for each construct of fragments b, C, E and F, then they were sequenced.
Unexpectedly it turned out that the same pigs there are two types of PTTV in genogroup 1 and in genogroup 2, which showed the presence of sequences in each construct. For the differentiation and Assembly of four PTTV strains we compared the sequences with three known PTTV strains, Sd - TTV31, TTV - 1P and TTV - 2P (Figures 1B and 1C).
Figure 1B shows the stage of differentiation and Assembly of the complete genomic sequences of PTTV strains 1A - VA and PTTV1b - VA virus PTTV1 with PCR fragments b and C, which are then subjected to cloning. Initiating codons ORF1 and ORF2 in the fragment, and the termination codons in the fragment of ORF1 With indicated by "^" or "*". The corresponding sequences et�known strains PTTV1, Sd TTV31 and TTV - 1P, is also shown. Conserved sequences are shaded, and the dashed line shows deletions of nucleotides.
In case PTTV1 the initiating codon ATG and the termination codon TGA alleged ORF1 is located in fragments b and C, respectively (Figure 1B). The location of the codons in the two groups PTTV1 was different, the first codon is identical to the Sd TTV31 and the second is identical to the TTV - 1P (Figure 1B). In addition, the initiating codon in ORF2 two groups also were located in different places, in line with ORF1. Moreover, phylogenetic analyses for four different sequences plot (two according to sequencing and in two strains Sd - TTV31 and TTV - 1P) and four different sequences on a site With confirmed that the first sequence was clustertools with Sd TTV31, while the second clustertools with TTV - 1P (these data not shown). Consequently, we were able to differentiate and assemble two groups of libraries of sequences from both fragments b and C in the two genomes PTTV1 full length, which were designated as strains PTTV1a-VA (SEQ ID NO:9) and PTTV1b-VA (SEQ ID NO:10), respectively (see Figure 1).
Figure 1C shows a stage of differentiation and Assembly of the complete genomic sequences of strains PTTV2b-VA and PTTV2c-VA with PCR fragments E and F, which are then subjected to cloning. Shows the relevant sequence of TTV strain - R, and conserved sequences are shaded. The dotted lines show the deletion in nucleotides. Respectively shown unique nucleotides overlapping plot (dotted lines) for each strain ("continuous", "AG" nucleotide " in PTTV2b-VA (SEQ ID NO:11) and two single nucleotide "A" and "G" in PTTV2c-VA (SEQ ID NO:12)).
The process of differentiation of the two strains PTTV2 easier. Unique continuous "AG" nucleotide, located on the overlapping area of the two PCR fragments were common to the two groups of sequences from fragments E and F, respectively (Figure 1). "Collected" complete genomic sequence was represented by strain PTTV2 and was designated as PTTV2b-VA (SEQ ID NO:11). In a similar way on the basis of two unique single nucleotide "A" and "G" for General overlapping section of another set of sequences from fragments E and F, respectively, was collected complete genomic sequence of a second strain, designated as PTTV2c - VA (SEQ ID NO:12) (see Figure 1C). Phylogenetic analyses using four sequences from fragments E and F, together with two corresponding sequences of TTV - 2P confirmed this pattern (data not shown).
The present invention provides for four selected genotypes or subtypes of swine virus TTV associated with in�rosnay infection in pigs. The present invention includes, but is not limited to, genotypes or subtypes of swine virus TTV PTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, with these genotypes or subtypes of the virus have nucleotide sequences represented in SEQ ID NO: 9 (PTTV1a-VA), SEQ ID NO:10 (PTTV1b-VA), SEQ ID NO:11 (PTTV2b-VA) and SEQ ID NO:12 (PTTV2c-VA), their functional equivalent or complementary strands. It should be borne in mind that the specific nucleotide sequence, derived from any swine TTV will have slight differences that naturally exist between individual viruses. These differences in the sequences can touch deletions, substitutions, insertions, etc.
Genomic structure proposed for each of the four PTTV strains, was analyzed in detail and are shown in Table 2 together with data for three known PTTV strains, Sd-TTV31, TTV-1P and TTV - 2P. All four American PTTV strains have similar size of genomes, constituting 2,878 p. O. (PTTV1a-VA SEQ ID NO:9), 2,875 p. O. (PTTV1b-VA SEQ ID NO:10), 2,750 p. O. (PTTV2b-VA SEQ ID NO:11) and 2,803 p. O. (PTTV2c-VA SEQ ID NO:12), respectively. Both strains PTTV1a - VA (SEQ ID NO:9) and Sd TTV31 had the same length of the genome. The previously described sequence in strains of TTV-1P and TTV-2P all contain numerous non-characterized nucleotides at the site UTR enriched in GC. After artificial fill those nucleotide consensus sequences corresponding to PTTV1 and PTTV2, would�about shows what TTV-1p is more closely related to PTTV1b-VA (SEQ ID NO:10) and that TTV-2p more closely associated with PTTV2b-VA (SEQ ID NO:11) in the genome, respectively (data not shown).
Assembled genomic sequences of genotypes and subtypes of swine virus TTV, PTTV1a-VA (SEQ ID NO:9), PTTV1b-VA (SEQ ID NO:10), PTTV2b-VA (SEQ ID NO:11) and PTTV2c-VA (SEQ ID NO:12) were registered in Genbank® (Nucleic Acids Research, Jan 2008, 36 (Database issue): D25-30) under the access numbers GU 456383, GU 456384, GU456385 and GU456386, respectively.
|A comparison of the organization of genomes and ORF in seven strains of swine TTV|
|Virus||Porcine TTV species 1||Porcine TTV species 2|
|Type 1A||Type 1b||Subtype 2A||Subtype 2b||Subtype 2C|
|Full length (u.)||2878||2878||2875||Not Montblanc.||He Montblanc.||2750||2803|
|The access number in Genbank||GU456383||AB076001||GU456384||AY823990||AY823991||GU456385||GU456386|
|The size (and.to.)||635||635||639||637||624||625||625|
|The size (and.to.)||73||73||72||72||68||68||68|
|The size (and.to.)||174||174||182||182||178||178||178|
|The size (and.to.)||224||224||228||228||199||199||199|
|The polyadenylation signal|
(A. K. TA.K.A)
|The numbers (in addition to the full size of the genome, ORF and numbers of exons) indicate positions of nucleotides (u.) in the genome of the respective PTTV strains|
During the two recent studies were identified transcribed viral mRNA and the expression of at least six viral proteins during replication of human TTV (Mueller et al., 2008, supra; Qiu et al., 2005, supra), which is more than the predicted number of ORFS encoded by the human TTV (Okamoto, H., Nishizawa, T., Tawara, A., Takahashi, M., Kishimoto, J., Sai, T., & Sugai, Y. (2000b). TT virus mRNAs are detected in the bone marrow clls from an infected individual. Biochem. Biophys. Res. Commun. 279 (2), 700-7), so we have included information about a new TTV person for comparison with sequences PTTV. 5' -ends of montillot were mapped at the nucleotide "A" which is located at below 25 nucleotides during transcription of the TATA-box (Mueller et al., 2008, supra). It's the starting point, its neighboring sequence (CGA.K.TGGCTGAGTTTATGCCGC (SEQ ID NO:39); the starting point is underlined) and located at a distance against the direction of transcription of the TATA-box (24 nucleotide; table 2) are very conservative in all seven PTTV strains, suggesting that PTTV and human TTV can use a common 5'-end of mRNA for translation.
Near TATA-box were identified five additional fully conservative plot in all seven PTTV strains. Two plots with a length of 11 nucleotides each (AGTCCTCATTT (SEQ ID NO:40) and A. K. CCA.K.TCAGA (SEQ ID NO:41)) are located upstream of TATA-box, while the other three plots (CTGGGCGGGTGCCGGAG of 17 nucleotides (SEQ ID NO:42); CGGAGTCA.K.GGGGC of 14 nucleotides (SEQ ID NO:43); TATCGGGCAGG of 11 nucleotides (SEQ ID NO:44)) are located between the 5'-end of mRNA and initiator to don ORF2. These conservative PTTV-conserved sequences may contain common elements that regulate expression of a viral gene.
Earlier three ORF (ORF 1-3) have been proposed in the genome of three known PTTV strains, respectively (Niel et l., 2005, supra, Okamoto et al., 2002, supra).
Four PTTV strains from the USA, which are the prototypes in this study, have this structure. Corresponding to ORF3 in human TTV was renamed in ORF2/2, as it initiates the same ATG codon in ORF2 and it remains in the same ORF (extending it) after splicing (Figure 1A) (Mueller et al., 2008, supra, Qiu et al., 2005, supra). We follow the nomenclature of human TTV for the revision of the classification PTTV in this study. ORF1 / 1 human TTV represents the first identified viral protein that is encoded by two exons in ORF1 (Qiu et al., 2005, supra). ORF1/1 and ORF1 have common identical N - and C-part. The opposite part of ORF1/1 in PTTV easily identified in all seven PTTV strains.
In human TTV ORF1 and ORF2 encoded viral mRNA with approximately 2.8 T. D. (thousands of nucleotides), while ORF1/1 and ORF1/2 are encoded split viral mRNA with about 1.2 T. D. (Mueller et al., 2008, supra; Qiu et al., 2005, supra). As these four ORFS in the genomes of PTTV were decoded and since the sequence and position of the alleged donor and acceptor splicing sites in seven PTTV strains are very conservative (see Table 2), believe that swine TTV, apparently, also encodes two corresponding mRNA.
Many strains of human TTV genetically similar to CAV encoding TTV protein, inducing apoptosis (TAIP), in which the anti - �Apolonia part CAV was named apoptin (de Smit, M. N., and NOtebom, M. N. (2009). Apoptosis-inducing proteins in chicken anemia virus and TT virus. Curr Top Microbiol ImmuNOl 331, 131-49). ORF TAIP was introduced in ORF2. However, in porcine TTV does not exist corresponding to TAIP. A recent study showed that the expression of apoptin or TAIP required for CAV replication in cultured cells (Prasetyo, A. A., Kamahora, T., Kuroishi, A., Murakami, K., and HiNO, S. (2009). Replication of the virus anemia chickens requires apoptin and supplemented VP3 human virus torque teno (TTV) (Karpova et al. 385(1), 85-92).
Pairwise comparison of sequences (PASC) is a useful method that allows to build a graph of the frequency distribution of the percent identity of the pair of nucleotide sequences from all available genomic sequences of viruses in the same family (Bao, Y., Kapustin, Y., and Tatusova, T. (2008). Virus Classification by Pairwise Sequence Comparison (PASC). In "Encyclopedia of Virology, 5 vols." (B. W. J. Mahy, and M. N. V. Van Regenmortel, Eds.), Vol.5, pp.342-8. Elsevier, Oxford). Different peaks generated by the PASC program will generally reflect groups of genera, species, types, subtypes and strains (see Figure 2).
In this study, we conducted PASC TTV using 121 full genomic sequences of human and animal strains related to TTV, available in GenBank (Figure 2). Assuming that the members of TTV are classified in a separate family Anelloviridae, two peaks corresponding to 36-55% and 55-7% identity of nucleotide sequences, represent groups of genera and species, respectively (Figure 2). Accordingly, the type TTV is defined as a group of TTV with 67-85% identity of nucleotide sequences and the subtype TTV is defined as a group of TTV with 85-95% identity of nucleotide sequences. The TTV strains with more than 95% identity of nucleotide sequences can further be classified on the options (see Figure 2). Similar classification for sequences of isolates 103 TTV was proposed Jelcic et al (Jelcic, I., Hotz-Wagenblatt, A., Hunziker, A., Zur Hausen, H., and de Villiers, E. M. (2004). Isolation of multiple TT virus geNOtypes from spleen biopsy tissue from a Hodgkin's disease patient: geNOme reorganization and diversity in the hypervariable region. J Virol 78(14), 7498-507).
These proposed criteria for the classification of TTV were applied when conducting phylogenetic analyses of genomic sequences 4 prototype strains from the USA and 3 other known PTTV strains. Paired comparison of complete nucleotide sequences of these strains showed that four strains PTTV1 have to 54.0-56.4 percent identity of nucleotide sequences compared with three strains PTTV2 (table 3). Consequently, previously shown in the literature "genogroup" PTTV probably should be called "views", and PTTV1 and PTTV2 will be the types 1 and 2 porcine TTV, respectively. View 1 PTTV consists of two types of viruses, designated as type 1A (including TTV - 1P and PTTV1b-VA (SEQ ID NO:10)), COO�, respectively since the identity of the nucleotide sequences in these two types of viruses is 69,8 with 70.7% (see Table 3). Sd TTV31 and TTV 1a - VA (SEQ ID NO:9) are variants of strains of the same species due to the high value of percent identity of their nucleotide sequences (95,1%). However, two strains of type 1b, TTV - 1P and PTTV1b-VA (SEQ ID NO:10), can belong to two different subtypes (identity of nucleotide sequences is 86.4%). For type 2 PTTV three strains, likely to be classified in a separate subtypes (TTV - 2P for subtype 2A, PTTV2b-VA SEQ ID NO:11) for subtype 2b and PTTV2c-VA (SEQ ID NO:12) for subtype 2C, respectively, based on the magnitude of the percent identity of their nucleotide sequences are equal and 86.5-90.9 per cent. This proposed new classification system PTTV is clearly visible in the phylogenetic tree (see Figure 3).
Phylogenetic trees constructed on the basis of an assumed amino acid sequence in ORF1, ORF1/1, and ORF2 and ORF2/2 in PTTV, also consistent with this proposed classification (see Figures 3B-3E).
|Pairwise comparison of sequences in the complete genomes of seven strains of swine TTV|
|View 1 with�other TTV||Type 2 porcine TTV|
|Type 1a||Type 1b||Subtype 2A||Subtype 2b||Subtype 2C|
These data were obtained using the program PASC, and the numbers show the % identity of nucleotide sequences.
Unique mutations, deletions and/or insertions scattered among all genomes in various kinds, types and subtypes of PTTV. For example, the position of the initiating and terminating codons ORF1 and initiating codons ORF2, shown in Figure 1, differs in the types 1A and 1b PTTV. Two strains PTTV1b also contain a deletion of 2-after the initiation codon in ORF2 contrast PTTV1 a (see Figure 1B).
It should be noted that both TTV - 2P and PTTV2b - VA charcterised a deletion of 52 so-called, which is lo� at the distance of 39 so-called upstream of the first conserved sequences of length 11 T. N. (AGTCCTCATTT (SEQ ID NO:40)) in UTR, compared to PTTV2c - VA. Because of this deletion, the size of the genome PTTV2b-VA (probably TTV - 2P) was significantly smaller than the size of the genome PTTV2c-VA (see Table 2). A number of clones subversive human TTV was isolated from serum samples. They are treated as full TTV genomes, as ORF in most of these subversif molecules remain intact (de Villiers et al., 2009; Leppik et al., 2007). They are variable in length in UTR and fully or partially deleteroute. The status of TTV - 2P and PTTV2b-VA resembles the state subversif molecules TTV in humans, which means that the subtypes PTTV2a and PTTV2b can be subbaratnam molecules derived from subtype PTTV2c. It should be noted that the 3'-end sequence of primer TTV2-nF with nested PCR (see Table 1), which is normally used for the detection PTTV2 in field samples by other groups (Ellis et al., 2008, supra; Kekarainen et al., 2007, supra; Kekarainen et al., 2006, supra; Krakowka et al., 2008, supra) is located on both sides of the deletion. Consequently, currently used for the detection PTTV2 method nested PCR, apparently, is not sufficient to identify genetically different different genetically different strains of subtype PTTV2c.
Source of selected viral strains are serum samples, feces, �Luna, semen and tissues of pigs infected with the virus TTV. However, it is anticipated that the method of using recombinant DNA can be used for replication and chemical synthesis of the nucleotide sequence. Therefore, the present invention provides the polynucleotide selected that includes, but without limitation, the nucleotide sequence represented in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, or its complementary strands; the polynucleotide. which hybridise and complementary at least 67% of the nucleotide sequence presented in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, preferably complementary to 85% or more, preferably complementary to 95%; or immunogenic fragment selected from the group consisting of amino acid sequences of ORF1 protein represented in SEQ ID NO:13 (PTTV1a-VA), SEQ ID NO:14 (PTTV1b-VA), SEQ ID NO:15 (PTTV2b-VA), SEQ ID NO:16 (PTTV2c-VA), the amino acid sequence of ORF2 protein represented in SEQ ID NO:17 (PTTV1a-VA), SEQ ID NO:18 (PTTV1b-VA), SEQ ID NO:19 (PTTV2b-VA), SEQ ID NO:20 (PTTV2c-VA), the amino acid sequence of the protein ORF1/ 1, is presented in SEQ ID NO:21 (PTTV1a-VA), SEQ ID NO:22 (PTTV1b-VA), SEQ ID NO:23 (PTTV2b-VA), SEQ ID NO:24 (PTTV2c-VA), the amino acid sequence of the protein ORF2/2, is presented in SEQ ID NO:25 (PTTV1a-VA), SEQ ID NO:26 (PTTV1b-VA), SEQ ID NO:27 (PTTV2b-VA), SEQ ID NO:28 (PTTV2c-VA).
Immunogenic and antigenic coding areas can be determined by methods�AMI, known in the prior art, and then they can be applied for production of monoclonal or polyclonal antibodies for screening of immunoreactivity or for other diagnostic purposes. The present invention also provides a purified immunogenic protein encoded allocated by the polynucleotides. It is desirable that the protein was isolated or recombinant protein ORF1 or ORF2 protein contained in at least one of the above-mentioned selected subtypes TTV, more preferably such protein was protein ORF1.
I believe that ORF1 porcine TTV encodes a structural protein associated with replication (Maggi, F., and Bendinelli, M. (2009). InunuNObiology of the Torque teNO viruses and other anelloviruses. Curr Top Microbiol Immunol 331, 65-90). ORF1 - coding products seven PTTV strains have a length equal 624-635. K. and contain large amounts of arginine residues at its N-end, which are believed to possess DNA-binding activity (see Figure 4). Figure 4 conserved sequences are shaded. The dotted lines indicate amino acid deletions. The RCR motifs (type replication - replication-type rolling ring) shown in solid lines. Three HVRs (hypervariable region) (PTTV1 - HVRs 1, 2 and 3) in strains PTTV1 and two HVRs (PTTV2 - HVRs 1 and 2) in strains PTTV2 shown by dotted lines. Border connections ORF1/1 indicated by arrows. Predicted motifs Rapley�ation by type rolling ring (RCR) (Ilyina, T. V., and Koonin, E. V. (1992). Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucl Acids Res. 20(13), 3279-85) are in different positions in different types and subtypes of PTTV, which may be type - or subtype-specific. Motif III RCR is conservative in PTTV strains of type 1A (regulation and. K. 14-17 in PTTV1a - VA SEQ ID NO:13) and in strains of type 1b (position and. K. 379-382 in PTTV1b - VA SEQ ID NO:14), respectively, while the same conservative motif, identified in all three strains PTTV2, is located in the a position.to. 482-485 PTTV2b - VA SEQ ID NO:15 (Figure 4). Both PTTV2b - VA SEQ ID NO:15 and PTTV2c - VA SEQ ID NO:16 also include the conserved motif II RCR (HxQ) in the a position.to. 331-333 in PTTV2b-VA, which is missing in TTV-2p (see Figure 4).
Proteins ORF in PTTV strains of types 1 and 2 have a very low percentage identity of sequences constituting only 22.4 and 25.8%, which makes difficult the identification of a very conserved amino acid sequences in the two species (see Figure 4). The percentage of identity and.to. ORF1 in PTTV, type 1, type strains 1A and 1b, is 50,3 was 52.7%. In four strains PTTV1 were identified three main hypervariable region (HVR), PTTV1 - HVRs 1-3, with quite a large number of amino acid substitutions, while in the three strains PTTV2 observed two HVRs (PTTV2 - HVRs 1 and 2) (see Figure 4). Three strains PTTV2 have about 20 amino acid de�of ECI in the relevant field PTTV1 - HVR1. Moreover, two HVRs PTTV2 are located in the relevant field PTTV1-HVR3 (see Figure 4). Three HVRs are only located in ORF1, but not in the truncated ORF1/1. They probably play a role in escape from immunological surveillance of the host and contribute to sustainable PTTV infection, as evidenced by the study of human TTV.
The amino acid sequence of the ORF2 significantly different in the four strains PTTV1 (PTTV1a-VA SEQ ID NO:17; PTTV1b-VA SEQ ID NO:18) and three strains PTTV2 (PTTV2b-VA SEQ ID NO:19; PTTV2c-VA SEQ ID NO:20) (see Figure 5). However, they contain a common conservative protein-tyrosine-phosphatase-like (PTPase) motif (Wx7Hx3CxCx5H) N-end (see Figure 4). This motif is also conservative in all strains of TTV, TTMV and TTMDV person, as well as CAV. Protein ORF2 TTMV or CAV were also active serine/threonine/phosphatase (S/T Ppase) (Peters, M. A., Jackson, D. S., Crabb, B. S., and Browning, G. F. (2002). Chicken anemia virus VP2 is a NOvel dual specificity protein phosphatase. J. Biol. Chem. 277(42), 39566 - 73). Believed to be a dual specificity protein ORF2 regulates host gene transcription, signal transduction and the reaction of cytokines during viral replication. Recently, the analysis of mutagenesis of two conservative basic amino acid residues before the last histidinol the remnant motif in CAV showed that these two balance affect virus replication, cytopathology in vitro and attenuation in vivo (Peters, M. A., Crabb, B. S., E. Washington A., and Browning, G. F. (2006). Site-directed mutagenesis of the VP2 gene of Chicken anemia virus affects virus replication, cytopathology and host-cell MHC class I expression. J. Gen. Virol. 87(Pt 4), 823-31; Peters, M. A., Crabb, B. S., Tivendale, K. A., and Browning, G. F. (2007). Attenuation of chicken anemia virus by site-directed mutagenesis of VP2. J. Gen. Virol, 88(Pt 8), 2168-75). Two basic amino acid residues ("KK") are conservative in three strains PTTV2. However, only the first principal balance ("R") is stored in two strains PTTV2, while both the major residue in strains PTTV1b replaced (see Figure 5). In Figure 5 the dotted lines indicate amino acid deletions. Five conservative amino acids in the overall motive Wx7Hx3CxCx5H (underlined) identified in TTV, TTMV and CAV, shaded. Position two amino acid residues before the last histidinol the remainder of the specified motif, which has been shown to affect viral replication, cytopathology of virus and in vivo attenuation in CAV, marked with the symbol "^".
In summary, it should be noted that the present invention has allowed to define in the serum sample of one hog from Virginia complete genomic sequences of the four strains of swine TTV representing different genotypes or subtypes of the virus. Data resulting from this study clearly show that, as in the case of human TTV, there are numerous types of infection caused by different g�a low tol erant genotypes and subtypes of PTTV, probably common to pigs. We also received new information concerning the organization of genomes, the degree of variability and characteristics of conservative nucleotide and amino acid motifs PTTV, which will improve the present method of detection with the use of PCR, will help to get the reagents for serological diagnosis and initiate structural and functional study of PTTV. Also proposed a new classification of PTTV, based on phylogenetic and genetic analyses of genomic sequences of the seven known PTTV strains.
The present invention also provides methods for diagnosing infections caused by swine TTV, by detection of viral DNA in samples taken from pigs or other mammals infected with porcine TTV. One preferred embodiment of this invention relates to methods of detection of nucleic acid sequences in swine TTV in pigs and other mammalian species using oligonucleotide primers for polymerase chain reaction (PCR) for the purpose of diagnosis of a viral infection or disease. Diagnostic tests that are suitable for detecting the presence or absence of sequences of viral nucleic acids in swine TTV in pigs or other species of mammals, �fied selection of viral DNA from samples selected in pigs infected with porcine TTV, or in pigs with suspected presence of such infections, and conducting a quantitative PCR in real time in the presence of the dye SYBR Green and RTT VI-specific (SEQ ID NO:29/SEQ ID NO:30) or PTTV2-specific (SEQ ID NO:31/SEQ ID NO:32) primers.
In accordance with another variant of the invention diagnostic method can be adapted for the simultaneous detection PTTV1 and PTTV2 by application of duplex PCR method in real time. More specifically, the method includes isolating viral DNA from samples taken from pigs infected with porcine TTV, or in pigs with suspected presence of such infections, the implementation of PCR in real time using both PTTV1-specific (SEQ ID NO:29/SEQ ID NO:30) or PTTV2-specific (SEQ ID NO:31/SEQ ID NO:32) primers in the same PCR reaction in real time. Since the value of Tm at PTTV1 and PTTV2 may be determined by the method of the ISA, the presence of DNA PTTV1 and PTTV2 can be determined simultaneously.
According to another embodiment of this invention a method of diagnosing may use the duplex nested PCR. The method includes isolating viral DNA from samples taken from pigs infected with porcine TTV, or in pigs with suspected presence of such infection on the implementation of the first round P�R with one pair of primers P1ab-mF (SEQ ID NO:33)/P1ab-mR (SEQ ID NO:34) and a second round of PCR using a mixture of two pairs of primers, P1a-nF (SEQ ID NO:35)/P1a-nR (SEQ ID NO:36) for detecting PTTV1a and P1b-nF (SEQ ID NO:37)/P1b-nR (SEQ ID NO:38) for detecting PTTV1b and visualization of PCR products.
The above methods of diagnosis can be optimized by the person skilled in the art with methods well known in the art.
Accordingly, one of the variants of this invention relates to two new odnopalatnim methods of analysis by PCR in the presence of the dye SYBR Green in real-time to quantify viral loads in two types of swine TTV, respectively. PTTV1 - and PTTV2-specific primers were obtained for targeting (targeting) on a very conservative area in the six genomes PTTV1 and four genomes PTTV2, therefore, the available currently. Another variant of this invention combines two odnoletnih method of analysis in duplex PCR method in real time with the subsequent ISAS viral amplicon, which can be identified by their different melting points, with the aim of simultaneous detection of two types of swine TTV, PTTV1a and PTTV1b. According to a third embodiment of a method of analysis using a duplex nested PCR for simultaneous amplification of viral DNA from two types PTTV1 in the first round of PCR and differential detection types 1A and 1b in the second round�e PCR was designed to identify two types of swine TTV, PTTV1a and PTTV1b, in one sample. These tests are simple and practical methods of diagnosing type-specific and type-specific porcine TTV.
A sequence of potential primers were identified by multiple alignment of 10 sequences of complete genomes available porcine TTV. PTTV1-specific primers TTV1F (SEQ ID NO:29) and TTV1R (SEQ ID NO:30) were designed based on two conservative regions of the genome, located immediately before the alleged ORF2 in the six genomes PTTV1, while PTTV2 - specific primers TTV2F4 (SEQ ID NO:31) and TTV2R4 (SEQ ID NO:32) were constructed on the basis of two conservative regions of the genome, located immediately behind the alleged ORF2/2 in the four genomes PTTV2 (see Table 4). The primers showed no propensity to samovlastii and to the cross-coupling. The expected amplicon sizes were 118 p. O. fragment from primers PTTV1 corresponding to the genome PTTV1b-VA and the 200 BP fragment from primers PTTV2 corresponding to the genome PTTV2c-VA, respectively.
Accordingly, one particular embodiment of this invention the PCR method in real time with the use of the dye SYBR Green, using PTTV1 - and RTTA-specific primers can be used specifically for the detection of DNA from porcine TTV1 and TTV2, respectively. In case PTTV1 was �of ustroena standard curve in the range of concentrations of target DNA in a 25 µl. For the interval of 4.4×101-4,4×108copies, was shown a linear relationship. The minimum limit of detection (44 copies) had met the threshold cycle reactions (Ci) equal to 33,57. In case PTTV2 was also constructed standard curve and used for the detection of DNA concentration in the range from 8.6×100to 8.6×108copies in 25 µl of reaction medium. The corresponding Ci minimum limit of detection (copies of 8.6) was 36,53.
According to another particular variant of this invention a method of duplex nested PCR with the use of SYBR Green is used for the simultaneous detection of DNA from porcine TTV1 and TTV2. The difference of values of TmI PTTV1 (87,0°C) and PTTV2 (80,0°C), 7°, gives the opportunity to distinguish one from another using ISA. Therefore, two methods odnoblochnogo analysis can be combined into a single duplex PCR method in real time for simultaneous detection PTTV1 and PTTV2. The positive sample was a sample that had a symmetrical peak melting among Tmfor this product. This new method of analysis was first confirmed using a 10-fold dilution of the mixture of standards PTTV1 and PTTV2. Nematicidal negative controls using sterile water as template, showed the occurrence of non-specific amplification caused �Ross-dimerization between the primers PTTV1 and PTTV2, not observed when conducting odnoletnih analyses (see Figure 7A). This has led to a distinct melting peak between 72,0°C and 76,0°C. Figure 7A shows the peak melting temperature of the standard PTTV1 (red; Tm=87,0°C), standard PTTV2 (green; Tm=80,0°C) and amatrices negative control (caused by the cross dimerization of primers; black). In the Figures 7B-7E shows the peak melting temperature of representative serum samples with different viral loads PTTV1 and PTTV2. Figure 7B shows the sample No. 5 serum hog: a relatively high load PTTV1 and PTTV2, but PTTV2>PTTV1; Figure 7C shows a sample No. 12 serum hog: a relatively high load PTTV1 and PTTV2, but PTTV1>PTTV2; Figure 7D shows a sample No. 14 serum hog: low load PTTV1 and PTTV2; Figure 7E shows a sample No. 10 serum hog: PTTV1 positive, but PTTV2 negative. Viral load (unit: genomic copies/ml) PTTV1 and PTTV2 in each sample, which were determined by the method anaplectes PCR in real time is indicated above the corresponding peak melting temperature.
In one example, when the analysis of 20 serum samples collected from adults hogs, used the method of duplex analysis in real time, samples with relatively high viral loads both PTTV1 and PTTV2 characterized�camping two distinct melting curves, relevant PTTV1 and PTTV2 without nonspecific peak melting temperature (Figures 7B-7C), whereas samples with a low viral load or PTTV1, or PTTV2 had the virus-specific and non-specific melting curves (see Figures 7D-7E). Although the two peak melting temperature for the sample No. 14, were low, they were considered as positive, as they were characterized visually distinct and symmetrical rise and fall corresponding to TmPTTV1 and PTTV2 (see Figure 7D). In contrast, the sample No. 10 was considered positive only in respect PTTV1, as a symmetrical peak melting temperature PTTV2 was absent (see Figure 7E). These results are consistent with the results of the two odnoletnih analyses (see Table 5). Moreover, the size and shape of the melting peaks qualitatively reflect the appropriate viral load in detektiruya the sample.
According to another aspect of the present invention a method of duplex nested PCR for differential detection of two types of swine TTV, PTTV1 and PTTV2.
The inventor of this invention has demonstrated the existence of two different genotypes of porcine TTV species 1 identified as PTTV1 and PTTV1b. In order to establish whether co-infection caused PTTV1 and PTTV1b, a common disease in pigs was developed by the new JV�sob duplex nested PCR for the rapid determination of differences between the two genotypes. Alignment of genomic DNA sequences of porcine TTV allowed us to identify a conservative region of the genome, located in the N-terminal part of the alleged ORF1 that encodes the viral capsid protein (see Figure 8). This area also contains a complete ORF2 and incomplete UTR upstream. Primers were designed P1ab-mF (SEQ ID NO:33)/P1ab-mR (SEQ ID NO:34) for simultaneous DNA amplification and PTTV1 a, and PTTV1b in the first round of PCR. For differential amplification of each genotype during the second round of PCR was used a mixture of PTTV la-specific primers P1a-nF (SEQ ID NO:35)/P1a-nR (SEQ ID NO:36) and PTTV1b-specific primers P1b-nF (SEQ ID NO:37)/P1b-nR (SEQ ID NO:38). The final PCR products for PTTV1a and PTTV1b size was 162 and 96 p. O. p. O., accordingly, they can very easily distinguished from each other by a method of gel electrophoresis on 1% agarose gel, stained aticipation. Don't expect this method of analysis allows to detect DNA PTTV2 due to the specificity of the primers (see Figure 8). Figure 8 conserved sequences are shown by dots and shaded. Dashed lines indicate nucleotide deletions. The location and direction of three pairs of primers used for duplex nested PCR, were marked with arrows.
In one example, 20 serum samples collected from adults hogs, was subjected to analysis by means of the attached duplex� PCR and were positive for PTTV1a and PTTV1b, as evidenced by visualization of two bands of the expected size, with subsequent confirmation by sequencing of PCR products (data not shown). 19 semen samples were not amplified PCR products, which was in agreement with the results of conventional nested PCR for PTTV1 and PCR in real time, as described above.
In the retrospective studies (Segales et al., 2009, supra), it was found that infection caused by two species of swine TTV, was in pigs in 1985 in Spanish pig farms. However, the question of whether porcine TTV with any disease in pigs, remains unresolved. Since both types of porcine TTV is widely distributed among domestic pigs, the definition of TTV viral loads is probably more important than establishing the presence of TTV DNA. It was shown that the level of viral load in the serum samples and sperm is an important marker for PCVAD during infection with PCV2 (Opriessnig et al., 2007, supra). Thus, the availability of quantitative methods PTTV-specific analysis with the use of PCR in real time will help identificirati terms of potential disease associated with porcine TTV.
Were recently described two methods of analysis using PCR in real time, based on the samples TaqMan. Method odnoblochnogo analysis, developed by the canadian group, has not been type-spec�cal and was only designed for the quantitative determination of total viral loads of two types PTTV (Brassard et al., 2009, supra). Method duplex analysis, proposed by the German group, allowed to carry out the specific and simultaneous detection of both species (Gallei et al., 2009, supra). Target sequences of the primers used in the conduct of these tests were determined by alignment of the three genomic sequences of porcine TTV (Sd TTV31, TTV-1p and TTV-2p) and were located in the UTR. In the present study with 7 additional available complete genomic sequences PTTV (4 sequences PTTV1 and 3 sequences PTTV2) we analyzed and again identified conservative region 10 complete genomes of PTTV. On the basis of the corrected result of this alignment, obtained in the course of the study, we have developed two species-specific methods anaplectes PCR in real time in the presence of the dye SYBR Green to quantify viral loads PTTV1 and PTTV2, respectively. The primers used in our methods, which can increase the accuracy of the quantification, have been engineered to bind to a conservative genomic regions different from the regions in known methods. Our analyses showed a significant specificity and sensitivity of detection 44 of genomic copies for PTTV1 and 8.8 genomic copies / 25 µl environment for PTT2, while the reported limit of detection equal to 10 genomic copies when performing duplex PCR in real time using sample TaqMan (Gallei et al., 2009, supra). In addition, PCR in real time with the dye SYBR Green is a flexible and inexpensive method that can be performed directly without the need for fluorescently labeled tags. Finally, given the high genetic diversity of porcine TTV results of analyses using SYBR Green hardly influenced by the different known variants of swine TTV, which, apparently, contain mutations to the probe-binding sequences in the test using sample TaqMan.
Despite the presence of TTV DNA all serum samples collected from healthy pigs tested in this study contained low quantities PTTV1 and PTTV2 that were equal to less than 2 x 10 copies/ml. moreover, three semen samples was found to have extremely low titer DNA PTTV2. A large portion of serum samples tested were positive for PCV2 DNA that was identified by conventional nested PCR (data not shown). Many PCV2 - positive pigs with a low viral load, did not develop clinical PCVAD (circovirus infection). The proposed threshold for the development of PCVAD is equal to 107or more genomic copies of PCV2 per ml syware�Ki (Opriessnig et al., 2007, supra). In addition, DNA-positive semen against PCV2 is also the sole marker of disease status (Opriessnig et al., 2007, supra; Pal, N., Huang, Y. W., Sharma, D. M., Kuster, C., Meng, X. J., Halbur, P. G. and Opriessnig, T., 2008. Development and validation of a duplex real-time PCR assay for the simultaneous detection and quantification of porcine circovirus type 2 and an internal control on porcine semen samples. J. Virol. Methods 149, 2 IP 25). The situation with species-specific PTTV can be a similar situation with PCV2 and to induce diseases of pigs may require a high titer of PTTV, amounting to more than 107copies/ml. Species-specific PCR methods, real-time developed in this study are simple and practical tools for future studies of communication PTTV disease with the use of a large number of clinical samples in the case of various diseases.
In addition, by combining two kinds of species-specific odnoblochnogo analysis we have developed and proved a fast, inexpensive and reliable screening method for simultaneous detection and differentiation of two types of swine TTV, PTTV1 and PTTV2, consisting of duplex PCR in real time, based on ISA. Although this method is not intended for precise quantification of both types of PTTV, it is a more convenient approach, which could replace conventional nested PCR detection. Compared to the PC� in real time, the conventional method of nested PCR for detection of porcine TTV is lengthy (requiring 4 rounds of PCR), time-consuming and prone to contamination of images occurring during the multiple rounds of PCR. Due to the difference of values of TmPTTV1 and PTTV2 method ISAS held after amplification with the use of duplex PCR, allows to provide a clear specificity of the reaction. Another advantage of this duplex method in real time is that the inclusion of standards PTTV1 and PTTV2 is not significant when carrying out the required Protocol, which makes this method more applicable in all diagnostic laboratories, equipped with automatic devices for PCR in real time.
Have been described many cases of infection caused by porcine TTV with different genotypes or subtypes of the same species (Gallei et al., 2009, supra). In particular, our earlier study showed that type 1 porcine TTV consists of two different types, PTTV1 (including strains Sd-TTV31 and PTTV1 a-VA) and PTTV1b (including strains of TTV-1p and PTTV1b-VA). Two recently described isolate PTTV1 with complete genomes, swSTHY-TT27 (GQ120664) from Canada and TTV1 #471819 (GU188045) from Germany, on the basis of phylogenetic analysis (data not shown) both were classified as representatives of type 1b. Duplex nested PCR described by us, confirmed that dual infection caused by two genotypes PTTV1, met in pigs often. This new method of analysis still assetserver diagnostic approach using PCR to determine the differences between infections, caused PTTV1a and PTTV1b. As in not currently known, one or both types of infections caused PTTV1a and PTTV1b, represent a relevant factor associated with the disease, our differential PCR method should be of great importance for potential future relationship of these two types of PTTV.
In accordance with another aspect of the present invention ORF proteins of swine TTV were expressed and used for immunological analysis for the detection of the presence of specific antibodies to porcine TTV. According to one embodiment of this invention three shortened and histidine tagged protein ORF1 PTTV1a, PTTV1b and PTTV2 were expressed and purified in Escherichia coli (E. coli), respectively. Next, were developed and proved two methods for serum analysis, Western blotting and ELISA based on the recombinant antigens using samples of swine serum, obtained from different sources. In particular, serological determination using PTTV1a-, PTTV1b - and PTTV2-specific ELISA method provides an accurate and simple tool to detect communication infection caused by porcine TTV with diseases.
In accordance with a further aspect of this invention ORF proteins of swine TTV were expressed and purified as recombinant capsid proteins of ORF1 in the E. Coli system (see Figure 10, Figure 15). These shortened and IU�ene histidine capsid proteins ORF1 PTTV1a, PTTV1b and PTTV2, were expressed and purified in Escherichia coli (E. coli), respectively, and served as a recombinant capsid subunit vaccines against infections caused by PTTV.
Four strains TTV2, TTV-2p, TTV2#472142, PTTV2b-VA and PTTV2c-VA, contained available to date, complete genome sequences. Although from a phylogenetic point of view, they are classified in three proposed subtypes, a comparative analysis of the hydrophilicity profiles ORF1 that encodes amino acids of the four PTTV2 showed that they contained three hydrophilic region arginine-enriched region and from.to. 1-49 on the N-end and two special areas (I and II), located in the middle and C-terminal parts, respectively (Figure 9A). The C-terminal region used for expression ukorochenniy ORF1 PTTV2c-VA and related fields that are contained in the other three strains PTTV2 shown by the dotted line. Alignment of amino acid sequences showed high levels of conservativeness of the sequences of areas I (and.to. 322-349) and II (and.to. 536-625) in four strains PTTV2 (Figure 9C).
Because I believe that hydrophilic region are important for the antigenicity of many proteins, the C-terminal region (a.to. 310-625) in ORF1 PTTV2c-VA, SEQ ID NO:16, containing two domains was selected for protein expression and was used as antigen for the detection PTTV2 - specific�of antibodies in swine serum. According to one aspect of the present invention, the expression ukorochenniy ORF1 PTTV2c was sufficient for detection of all subtypes PTTV2 (2A, 2b, and 2C; see also Figure 3A).
According to one of the variants of this invention was obtained and expressed in E. coli C-terminal region of ORF1 gene PTTV2c, merged with 8 x His-tags. Recombinant protein was insoluble and expressively in the inclusion bodies of bacteria. Figure 10 A shows the results of SDS - PAGE of unpurified products 2C - ORF1. Figure 10 shows the results of SDS - PAGE of purified products 2C - ORF1. Figure 10C shows the results of Western blotting of the purified products 2C - ORF1 using an anti - His-tagged mAb. Arrow with tip white show ORF1 protein of the expected size and its "shortened" products while with black arrows show the probable edge dimmers expected and truncated proteins. M: protein markers. Figure 10A shows two important polypeptide (arrows pointing in white) obtained in the untreated sample 2C - ORF1, compared with the control. Band around 40 kDa consistent with the expected size 2C - ORF1, while the polypeptide with a M. W. of about 30 kDa was, apparently, truncated at N-end product obtained from the first product. After purification on a column of Nickel SDS - PAGE showed the presence of four polypeptides, including two �painted stripes (see Figure 10B). They were also detected by the method of Western blotting using mAb against the His tag (see Figure 10). Two bands, indicating high molecular weight (arrow with black edge) denote homodimer formed by two polypeptides with M. W. of about 40 kDa and about 30 kDa, respectively, based on the predicted size (approximately 80 kDa and about 60 kDa).
The results showed that the purified C-terminal PTTV2c - ORF1 was successfully obtained and could be used for detection of antibodies porcine TTV2 in swine serum.
In accordance with another variant of the invention antibodies to porcine TTV2 in various serum samples can be detected by the method of Western blotting using the purified C-terminal PTTV2c - ORF1. Arrows with white edges indicate ORF1 protein with the expected size and its processionary product. It should be noted that only a strip of green color were considered positive. Conventional pigs (healthy and sick), pigs of the breed CD/CD and hebetechnik pigs from different sources were selected from more than 200 serum samples. Then arbitrarily chose the samples for the detection of immunoglobulin antibodies to PTTV2c - ORF1 using the purified C-terminal PTTV2c - ORF1 as the antigen. Figure 11 shows the results of Western blotting of selected samples of swine serum normal �viney, Figure 11 In - for pigs CD/CD and Figure 11C - hebetechnik pigs. Purified products PTTV2c - ORF1 were used as antigens. In most samples of normal pigs (Figure 11A) and pigs CD/CD (Figure 11B) were detektirovanie two prominent bands of approximately 40 kDa and about 30 kDa, indicating the presence of these pigs strong infections caused PTTV2. However, all hebetechnik pigs from two different sources (Blacksburg, VA and Ames, IA) antibodies to PTTV2 were detected (Figure 11). There was also an extra band with low molecular weight. They are probably caused by nonspecific reactivity when performing Western blotting.
In accordance with another alternative of this invention RTU-specific ELISA analysis can be used as a serologic test for porcine TTV2. For multiple samples taken from normal pigs on a farm in Wisconsin were shown seronegative results (see Figure 12). These negative results were collected and used as a negative standard in the development RTO-specific ELISA. The remaining samples from this source were positive (Figure 12, four lines on the left). In addition, porcine serum from commercial companies used in the cell culture (it is assumed that pigs no disease recorded OIE), also had strong anti - PTTV2 - RF2 positivity (Figure 12) and was used as a positive control for ELISA. The concentration of purified antigen 2C - ORF1, porcine serum and IgG conjugate were determined by checkerboard titration to determine the lowest background signal and obtain the most big difference between OD405 for positive and negative control samples. The optimum amount of antigen was 69 ng per well, and the optimal results of the ELISA were obtained using a dilution of 1: 100 serum samples and diluted 1:4000 IgG conjugates. Critical values for ELISA were in the range of 0.25 to 0.5 in every experience. Figure 4 shows a representative result, reflecting the consistency of the results of Western blotting of serum and developed ELISA method.
To correlate communications viral load PTTV2 by PCR in real time and the level of IgG antibodies to PTTV2 in serum collected from 10 pigs in the same herd after their arrival in the new economy and 2 months. after arrival, analyzed the serum. 9 of 10 pigs in 2 months. were reduced viral load (three animals virus was not detected), while the titers of antibodies to PTTV2 were increased in 9 of 10 pigs (Figures 14A-14C). These results suggest that 10 pigs had the infection at an early stage caused PTTV2 that caused humoral response and had produced a progressive IgG antibody to capsid protein ORF1. IgG and�titlo to PTTV2 - ORF1 was able to neutralize it and even clean the virus indicating that ORF1 really encodes the viral capsid protein and may contain neutralizing epitopes PTTV2.
According to one variant of the invention the C-terminal proteins PTTV1a - and PTTV1b - ORF1 were expressed and purified in E. coli system, respectively. Electrophoresis SDS - PAGE and Western blotting using using mAb against the His tag showed that both products, 1A - and 1b-ORF1, contained two polypeptide, one with the expected size (about 40 kDa) and the other in the form of the alleged homodimer (about 80 kDa) (see Figures 15A-C). Figure 15A shows the results of SDS - PAGE of crude and refined products 1a - ORF1. Figure 15B shows the results of refined products 1b - ORF1 and 1b - ORFlctruc. Figure 15C shows the results of Western blotting of the purified products 1A - and 1b - ORF1 using mAb against the His-tag. Arrow with tip white show ORF1 protein of the expected size, while the arrows with the tip black color shows the estimated dimer proteins ORF1. Compared with the expression of 2C - ORF1 processionary polypeptide was observed. As a comparative control was carried out the expression of C-terminal processioning phase 1b - ORF1 (1b - ORF1ctruc), which resulted in a polypeptide of lower molecular weight compared to its C - terminal reprezentirovannoe opposite part 1b - ORF1 (see Figure 15B).>
In accordance with one variant of the invention the purified proteins C-terminal PTTV1a - and PTTV1b-ORF1 was used to develop a method genotype-specific Western blotting of serum and ELISA method, as described above for PTTV2. Figure 16 shows the negative (columns 1-2) and positive (columns 3-5) results of Western blotting and ELISA of serum samples with the use of 1a - ORF1 as the antigen. When conducting PTTV1a - and PTTV1b-specific ELISA method was used the same amount of antigen (69 ng) serum dilution of 1:100 and the conjugate dilution IgG 1:4000 (results not shown).
In addition, the present invention provides a diagnostic reagent for the detection of infections caused by TTV, which includes monoclonal or polyclonal antibody, purified from the natural host, for example, by the inoculation of the pigs swine TTV or immunogenic composition according to the invention in immunogenic amount for producing a viral infection and to isolate the antibody from the serum of infected pigs. Or in experimental animals can be induced by antibodies to natural or synthetic polypeptides derived or expressed from amino acid sequence or immunogenic fragments encoded by the nucleotide sequence selected porcine TTV. For example, monoclonal� antibodies can be obtained from cells of hybridomas, which are obtained from the mice, such as, for example, mice of the line Balb/c, immunitary a polypeptide antigen derived from a nucleotide sequence selected porcine TTV. Breeding cells of hybridomas is performed by cultivation in the system containing hypoxanthine, thymidine and aminopterin, in standard culture medium, for example in modified according to the method of Dulbecco environment Needle (DMEM) or minimal supporting environment. Cells of hybridomas that produce the antibody may be cloned by methods known in the art. Then discrete colonies that formed, can be placed in individual wells of culture plates for cultivation in a suitable culture medium. Identification of cells secreting antibodies produced by conventional methods of screening using the corresponding antigen or immunogen. The cell culture of hybridomas in vitro or in vivo by obtaining ascitic fluid in mice after injection of cells of hybridomas produces the desired monoclonal antibody by known methods.
According to another alternative method capsid protein of porcine TTV can be expressed in the expression system based on the baculovirus or in the system based on the E. coli according to known methods. Expressed recombinant capsid protein of Saint�tion TTV can be used as antigen for the diagnosis of a solid-phase enzyme immunoassay (ELISA). For example, the ELISA based on the recombinant capsid antigen, can be used for detection of antibodies to swine TTV in pigs and other mammals. Although the ELISA method is preferred, can be used other known diagnostic tests, such as immunofluorescence analysis (IFA), immunoperoxidase method (IPA), etc.
When this is desirable, commercial ELISA diagnostic method according to the present invention can be applied for the diagnosis of infections in pigs caused by porcine TTV. Examples illustrate the application of the purified proteins ORF1 and ORF2 porcine TTV for development of an ELISA for detection of antibodies to TTV in pigs. Serum collected from pigs infected with porcine TTV, and negatine serum samples collected from control animals, were used to confirm this method. It was shown that PTTV2-specific, PTTV1a-specific and PTTV1b-specific antibodies specifically recognize proteins PTTV - ORF. Further standardization of the test methods known to the person skilled in the art can optimize the commercialization of the method of diagnosis of swine TTV.
Another aspect (object) of the present invention is unique immunogenic composition containing the selected porcine TTV or antigenic protein encoded by the selected Poliny�jothidam, as described above, and its application to induction or production of antibodies. The composition comprises a non-toxic physiologically acceptable carrier and optionally one or more adjuvants. Suitable carrier materials such as water, saline, ethanol, ethylene glycol, glycerol, etc., chosen out of the ordinary excipients, and you can add other components (co-formulants). In order to ensure physical compatibility and stability of the final composition, it is possible to conduct standard tests.
In addition, according to the present invention provides a molecular vaccine against swine hepatitis b virus Torque teNO (TTV) (infectious molecular) and nucleotide molecules of swine hepatitis b virus Torque teNO (TTV) (virus thin necklace"), live viruses produced using the nucleotide molecules, and veterinary vaccines to protect pigs from swine TTV viral infection or disease caused by infection of swine TTV and other viruses. In addition, the invention provides immunogenic polypeptide products of the expression that can be used as vaccines.
Molecule new infectious DNA from porcine TTV is a nucleotide molecule encoding at least the section of infectious PTTV1a-VA (SEQ ID NO:9), PTTV1b-VA (SEQ ID NO:10), PTTV2c-VA (SEQ ID NO:11) or PTTV2c-VA (SEQ ID NO:12) of the genome. Clone in�actionnow PTTV DNA preferably, contains at least one of ORF1 (open reading frame), ORF2, ORF1/1, and ORF2/2 genes PTTV1 or PTTV2. To create tandem repeats infectious PTTV clones in a single DNA molecule can be incorporated (insertion) multiple copies PTTV1a-VA (SEQ ID NO:9), PTTV1b-VA (SEQ ID NO:10), PTTV2c-VA (SEQ ID NO:11) or PTTV2c-VA (SEQ ID NO:12) of the genome.
It is shown that the cloned genomic DNA PTTV, in particular PTTV1a-VA, PTTV1b-VA, PTTV2c-VA and tandem repeats PTTV2b-RR, PTTV2c-RR, as described herein, an in vitro or in vivo infectious, if transferout in cells PK-15 and give to the pigs. This new, easily reproducible pathogenic agent itself is ideal for the development of an appropriate programme of vaccination for the prevention of PTTV infection in pigs.
According to another embodiment of the present invention received three PTTV DNA clone with one copy of PTTV DNA genome when using the prototype isolates US PTTV1a-VA, PTTV1b-VA and PTTV2c-VA using "fusion" PCR (PCR with stitching), respectively. Each of the full-size genomic DNA inserted into the cloning vector pSC-B-amp/kan by ligation of blunt ends. The restriction site BamH I is a unique site on three PTTV genomes, which were created at both ends of the three genomes with the aim of promoting the education of concatemers and thereby simulate TTV circular genome. A single cleavage of the selected plasmid DNA of each clone using amH I clearly have resulted in two different fragment size 4.3 T. p. O. and 2.8 - T. p. O. (Fig.18A). Fragments of 4.3 T. p. O. was a frame vector, whereas fragments of 2.8 T. p. O. was a built-in genomic DNA PTTV. In the case of an empty vector pSC-B-amp/kan, split by the same enzyme, is observed only band 4.3 T. p. O. (Fig.18A). The resulting PTTV clones represent pSC-PTTV1a, pSC-PTTV1b and pSC-PTTV2c, respectively (Fig.17A-C).
Further, two copies of the genome of a full-sized PTTV2c - VA formed using a clone pSC-PTTV2c, was ligated in tandem repeats in the vector pSC-B-amp/kan, getting the clone pSC-2PTTV2c - RR (Fig.17D). Comparison of patterns of a single cleavage pSC-PTTV2c and pSC-2PTTV2c-RR using Af1 II showed that the latter plasmid has an additional fragment of 2.8 T. p. O., representing a second copy PTTV2c genome (Fig.18V, right panel). Then we used the same cloning strategy to obtain the tandem-dimeric form PTTV2b DNA clone formed from TTV clone TTV2-#471942-full (Germany). Additional fragment size 2.8 T. p. O., representing a second copy PTTV2b genome, is present in this design, designated pSC-2PTTV2b-RR (Fig.17F), which were digested only with Hind III, in comparison with the genomic counterpart with one copy (Fig.18V, left panel), confirming successful design.
Competence in relation to replication is designed PTTV infectious clones was checked by in vitro Tran�the infection of the cell line PK-15. The IFA) using the selling rabbit polyclonal antibodies against PTTV2c ORF1 confirmed that concatemer clones as TTV2-#471942 - full and pSC-PTTV2c competent for replication, respectively (Fig.19A and Fig.20A). Passaging transfected cells does not eliminate or weaken the fluorescent signals (Fig.19B and Fig.20V), this suggests that the expression of ORF1 proteins due PTTV2 concatemers that mimic natural PTTV2b or PTTV2c ring of the molecule. No fluorescent signals were observed in cells with simulated DNA transfection with the use of non-immune rabbit serum as the antibody for ELISA (IFA) detection (data not shown). It is also shown that concatemer pSC-PTTV1a clone are competent for replication with the use of antibodies against PTTV1a ORF1 (Fig.21). Positive fluorescent signals were localized in the nucleus of transfected or passaged cells, this indicates that swine TTV, apparently, is replicated in the nucleus of cells. This is not unexpected, as the circovirus pigs (PCV, PCV) has a similar expression pattern in vitro.
Direct transfection of tandem-dimeric form of the clone pSC-2PTTV2b-RR or pSC-2PTTV2c-RR cells PK-15 leads to viral replication and produces ORF1 capsid antigen. The IFA using antibodies against PTTV2 ORF1 confirmed that both clones are unique� competent for replication, and positive ORF1 antigens localized in the nuclei (Fig.22A and b).
According to one embodiment of the present invention to inoculate pigs can be applied infectious clones of porcine TTV, which then reveal the immune response of the animal host and stimulate the production (production) of neutralizing antibodies. In one specific embodiment of the present invention, two tandem-dimeric PTTV2 clone were infectious (contagious) when they were injected into the lymph nodes and muscles of normal pigs.
To test in vivo infective potential (infectivity) PTTV2 molecular clones of conventional pigs inoculated with clone pSC-2TTV2b-RR or pSC-2TTV2c-RR. Serum samples take in animals at 0, 7, 14, 21 and 28 days after inoculation (DPI). PTTV2 DNA was detected in pSC-2TTV2c-RR-inoculated pigs, starting at 7 DPI (#92), 14 DPI (#188 and #191) and 21 DPI (#180), respectively (Fig.23A, right panel). PTTV viremia appeared later in pigs inoculated with the clone pSC-2TTV2b-RR: the two pigs she has appeared on 14 DPI (#189 and #192), one at 21 DPI (#181) and one at 28 DPI (#193) (Fig.23A, left panel). Viral load increased during the course of all inoculated pigs, higher viral load was observed on 28 DPI before opening, as determined using PTTV2-specific real-time PCR (Fig.23A). Products real-time PCR, amplified with the use�Denmark selected pigs sequenced and found that they have sequences identical to the sequences of the relevant regions pSC-2TTV2b-RR or pSC-2TTV2c-RR (data not shown).
All inoculated pigs were negative to PTTV2 ORF1 antibodies at 0 and 7 DPI. At 14 DPI, all four pSC-2TTV2b-RR-inoculated pigs were seroconverters (changed the profile of antibodies in serum) in anti-PTTV2 ORF1 IgG, whereas pSC-2TTV2c-RR-inoculated group had seroconvertion 14 (#92 and #180), 21 (#191) and 28 (#188) DPI, respectively (Fig.23V). The results show that there has been an active infection of swine virus TTV2b or TTV2c.
The infectious viral vaccine and infectious molecular DNA clones, and methods of their use are also included in the scope of the present invention. Inoculated pigs protect against viral infections and associated diseases caused TTV2 infection or co-infection. A new method protects pigs in need of protection against viral infection by introducing the pig immunologically effective amount of the vaccine according to the invention, for example, such as a vaccine containing an immunogenic amount of the infectious PTTV DNA plasmid or viral vector containing the infectious clone of PTTV DNA, recombinant DNA PTTV, expression products of polypeptides expressed in bacteria or in baculovirus purified recombinant ORF1 capsid�CSOs protein, etc. To provide broad-spectrum protection against viral infections, pigs can simultaneously give other antigens, such as PRRSV, PPV, other infectious swine agents and Immunostimulants.
Vaccines contain, for example, viral and infectious molecular DNA clones, cloned infectious PTTV DNA genome in suitable plasmids or vectors, such as the pSC - a vector, an avirulent live virus, inactivated virus, subunit vaccine based on the expressed recombinant capsid protein, etc. in combination with a non-toxic physiologically acceptable carrier and optionally with one or more adjuvants. The vaccine may also contain infectious TTV2 molecular DNA clone as described herein. Infectious PTTV DNA, plasmid DNA containing the infectious viral genome, and live viruses are preferred, with live viruses are the most preferred. Avirulent live virus vaccine of the present invention has the advantage over traditional viral vaccines, which use either a weakened (attenuated) live viruses that are at risk to return to the virulent state, or dead cells in the culture will spread a virus, which may not allow sufficient to induce an immune response antibodies to �protection against viral diseases.
Vaccines and methods of their use are also included in the scope of the present invention. Inoculation protects mammals against serious viral infection, can also provide protection against diseases associated with HCV co-infection PTTV, such as a syndrome of dermatitis and nephropathy piglets (PDNS), Multisystem wasting syndrome after weaning piglets from sows (PMWS) and other related diseases. Vaccines contain, for example, be inactivated or attenuated porcine TTV virus, non-toxic, physiologically acceptable carrier and optionally one or more adjuvants.
Adjuvant that can be administered in combination with the vaccine of the present invention, is a substance that increases the immunological response of the pig to the vaccine. An adjuvant may be administered simultaneously with the vaccine and in the same region as that of the vaccine, or at different times, for example, as a booster vaccination. Preferably also enter pig adjuvants in a manner different from the method of introduction of the vaccine, or in an area different from the area of introduction of the vaccine. Suitable adjuvants include, but are not limited to, aluminum hydroxide (alum), immunostimulatory complexes (ISCOMS), non-ionic block polymers or copolymers, cytokines (such as IL-1, IL-2, IL-7, IFN-α, IFN-β, IFN-γ, etc.), saponins, monophosphorylated A (MLA, IFF), muramyldipeptide (MDP), etc. Other suitable adjuvants include, for example, aluminum potassium sulfate, heat-labile or heat-stable enterotoxin isolated from Escherichia coli, cholera toxin or its B-subunit, diphtheria toxin, tetanus toxin, pertussis toxin, incomplete or complete adjuvant freind etc Adjuvants based toxins such as diphtheria toxin, tetanus toxin and pertussis toxin can be inactivated before use, for example, by treatment with formaldehyde.
In addition, the vaccine may contain additional antigens for the initiation of the immunological activity of infectious PTTV DNA clones, such as a virus porcine reproductive and respiratory syndrome (PRRSV), porcine parvovirus (PPV), other infectious agents and immune stimulants for pigs.
New vaccines of the present invention is not limited to any particular type or method of cooking. Cloned viral vaccines include, but are not limited to, infectious DNA vaccines (i.e. using plasmids, vectors or other common carriers for direct injection of DNA to pigs), live vaccines, modified live vaccines, inactivated vaccines, subunit vaccines, attenuated vaccines, genetically engineered vaccines, etc. These vaccines are prepared by standard methods known in the prior art.
Another advantage is that �repectfully live virus of the present invention provides a genetically stable vaccine which is easier to prepare, store and deliver than other types of attenuated vaccines.
In another preferred vaccine of the present invention are suitable plasmids for delivery of non-pathogenic DNA-clone pigs. Unlike traditional vaccines that use the whole virus, transmitted live or dead cell culture, the present invention involves direct inoculation of pigs plasmid DNA containing the infectious viral genome.
Additional genetically engineered vaccines, desirable in the present invention, receive by methods known in the prior art. Such methods include, but without limitation, further manipulation of recombinant DNA, modification or replacement of the amino acid sequences of recombinant proteins, etc.
Genetically engineered vaccines based on recombinant DNA get, for example, identifying alternative sites viral gene encoding proteins responsible for the induction of stronger immune or protective (protective) response in pigs (e.g., proteins, obtained using ORF1, ORF1/1, and ORF2, ORF2/2, etc.). Such identified genes or immunodominant fragments can be cloned into standard vectors for expression of proteins, such as baculovirus vector, and used to infect suitable�appropriate host cells (see, for example, O'reilly et al., "Baculovirus Expression Vectors: A Lab Manual," Freeman & Co., 1992). The host cell is cultivated so that they expressively specified vaccine proteins that can be purified to the desired degree of purity and cook in the form of a suitable vaccine product. Recombinant subunit vaccines based on a murine bacteria (Fig.10, Fig.15) or expressed by baculovirus ORF1 capsid proteins PTTV1a, PTTV1b and PTTV2.
If the clones retain any unwanted natural properties to cause disease, it is also possible to specify precisely the nucleotide sequence in the viral genome responsible for any residual virulence, and using genetic engineering to make the avirulent virus, for example, by the method of site-directed mutagenesis. Site-directed mutagenesis allows you to add, remove or replace one or more nucleotides (see, for example, Zoller et al., DNA 3: 479-488, 1984). Synthesized oligonucleotide containing the desired mutation, and hybridized plot with single-strand (single-stranded) of viral DNA. Hybrid molecule obtained by this procedure is used to transform bacteria. Then selected dvuhlitrovuyu DNA containing the mutation, are used to obtain full DNA ligation to the restriction fragment of the latter, and its further transferout in podhodyashiy� cell culture. Ligation of the genome to a suitable vector for the transfer can be performed by any standard method known to the ordinary skilled in the art. Transfection of the vector into the host cell for the production of viral progeny can be done by any conventional methods, such as transfection mediated by calcium phosphate or DEAE-dextran, electroporation, fusion of protoplasts, and other well-known methods (see, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press, 1989). Further cloned virus detects a given mutation. Or it is possible to synthesize two oligonucleotide, containing the appropriate mutation. They can be hybridizati education dvuhruchevoj DNA that can be embedded in the viral DNA, getting a full-sized DNA.
Immunologically effective amount of the vaccine of the present invention is administered to the pig in need of protection against viral infection. Immunologically effective amount, or an immunogenic amount by which the inoculated pig, you can easily identify or simply be titrated by means of standard testing. An effective amount is an amount, which is achieved through immunological response to the vaccine is sufficient to protect pigs exposed (exposed) PTTV virus. �predpochtitelno, the pig is protected to the extent in which significantly decreases, becoming less intense or completely warned from one to all of the adverse physiological symptoms or effects of a viral disease.
The vaccine can be administered in a single dose or as repeated doses. The spacing of doses can be, for example, about 1-1000 micrograms of plasmid DNA containing the infectious chimeric DNA genome (depending on the concentration of immunoactive component of the vaccine), preferably 100-200 micrograms TTV DNA clone pigs, but the dose should not contain the amount of viral antigen, sufficient to cause side (pathological) reaction or physiological symptoms of viral infection. In the prior art discloses methods for determining or titrating suitable doses of active antigenic agent to determine the minimum effective dose considering the weight of the pig, the concentration of the antigen and other standard factors. Preferably, as infectious vaccines use viral DNA clone, or live infectious virus can be obtained in vitro, and then live virus used as a vaccine. In this case, the pig can be given, for example, about 50-10000 TCID50, (dose infecting 50% tissue culture) of live viruses.
New vaccines according to the invention are not limited as�m any particular type or method of obtaining. Vaccines include, but are not limited to, modified live vaccines, inactivated vaccines, subunit vaccines, attenuated vaccines, genetically engineered vaccines, etc.
The advantage of live vaccines is that the recipient of the vaccine activates all possible immune reactions, including systemic, local, humoral and cellular immune responses. The disadvantages of live virus vaccines, which may take precedence over the advantages is the possibility of contamination alive random viral agents or increased the risk that the virus can restore virulence.
For the preparation of inactivated virus vaccines increase of viruses and producing virus may occur in cultured cell lines of pigs, for example, but without limitation, cells such as PK-15. Then the inactivation of viruses using serial passaging optimize protocols, well known to ordinary skilled in the art, or, preferably, in accordance with the methods as described herein.
Inactivated virus vaccines can be prepared by treatment of swine TTV inactivating agents such as formalin or hydrophobic solvents, acids, etc., by irradiation with ultraviolet light or x-rays, by heating, etc. the Inactivation is carried out with�special, known in the prior art. For example, chemical inactivation of the corresponding virus sample or serum sample containing the virus, within sufficient time to process the inactivating agent, in sufficient quantity or with sufficient concentration, for a sufficiently high (or low, depending on the inactivating agent) the values of temperature or pH, inaktivera virus. Inactivation by heating is carried out at such temperature and for such time sufficient to inactivate the virus. Inactivation by irradiation was carried out using the wavelength of light or other energy source for a time sufficient to inactivate the virus. The virus is believed an inactivated, if he is not able to infect a cell susceptible to infection.
The preparation of subunit vaccines is usually different from the preparation of a modified live vaccine or inactivated vaccine. Before the preparation of subunit vaccines, you need to identify protective (protective) or antigenic components of the vaccine. In the present invention the antigenic components PTTV were identified as ORF1 capsid proteins of viruses PTTV1a, PTTV1b and PTTV2 that the present invention expressively and purified in Escherichia coli (E. coli), and the other expression system such as baculovirus system stock�these, for use as a recombinant subunit vaccine based on the core proteins. Such protective or antigenic components include some amino acid segments, or fragments of viral capsid proteins that cause particularly strong or protective immune response in pigs; choose single or multiple viral capsid proteins, oligomers and higher-order Association of viral capsid proteins that form the substructure of viruses or identifiable part, or links of such substructures; oligoglycosides, glycolipids or glycoproteins, such as lipoproteins or lipid groups associated with the virus, present on or near the surface of the virus or viral substructures, etc. Preferably, as the antigenic component subunit vaccines use ORF1 protein. Can also be used for other proteins, for example, those that are encoded by a nucleotide sequence in ORF2, ORF1/1, and ORF2/2 gene. These immunogenic components are easily identified by methods known in the prior art. After identifying protective or antigenic sites of the virus (i.e., "subunit") are purified and/or cloned by methods known in the prior art. Subunit vaccine provides an advantage over other vaccines based on alive�th virus since the subunit, such as a purified subunit of the virus, less toxic than the whole virus.
If subunit vaccine produced using recombinant DNA, expression of cloned subunits, such as genes ORF1, ORF2. ORF1/1, and ORF2/2, can be realized, for example, by the method described above and can also be optimized by methods known to those skilled in the art (see, for example, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, Mass. (1989)). On the other hand, if the applied subunit is an intact structural symptom of the virus, such as a capsid protein, then the method of its separation from the virus can be optimized. In any case, after optimization of the inactivation Protocol Protocol clean subunit can be optimized before receiving.
For the preparation of attenuated live vaccines pathogenic virus first attenuatot (doing non-pathogenic or harmless) by methods known in the prior art, or preferably, as described herein. For example, attenuated viruses can be prepared by the method of the present invention, which includes a new serial passaging on embryos of pigs. An attenuated (weakened) viruses can be found in nature, and they can contain naturally occurring gene deletion or, alternatively, pathogenic Viru�s can be attenuate, carrying deletions or mutations in genes. Attenuated and inactivated viral vaccines contain preferred vaccine of the present invention.
Genetically engineered vaccines are also desirable in the present invention, receive by methods known in the prior art. Such methods include, but without limitation, the use of RNA, recombinant DNA, recombinant proteins, live viruses, etc.
For example, after cleaning, the wild-type virus can be isolated from appropriate clinical biological samples such as serum, feces, saliva, semen, and tissue by methods known in the art, preferably methods disclosed herein, using infected pigs or infected with the corresponding cell line. DNA is extracted from the biologically pure virus or infectious agent by methods known in the art, and purified by methods known in the art, preferably by ultracentrifugation in a CsCl gradient. cDNA of the viral genome cloned in an appropriate host by methods known in the prior art (Maniatis et al., see above), and then analyze the viral genome, identifying the regions of the genome that are essential for the production of antigenic sites of the virus. The further procedure is generally the same as for a modified live vaccine or subunit VA�care.
Genetically engineered vaccines based on recombinant DNA get, for example, identifying the plot of a viral gene, which encodes proteins responsible for the induction of stronger immune or protective response in pigs (e.g., proteins, obtained using ORF1, ORF2, ORF1/1, and ORF2/2, etc.). Such identified genes or immunodominant fragments can be cloned into standard vectors for expression of proteins, such as baculovirus vector, and used to infect appropriate host cells (see, e.g., O'reilly et al., "Baculovirus Expression Vectors: A Lab Manual," Freeman & Co., 1992). The host cell is cultivated so that they expressively specified vaccine proteins that can be purified to the desired degree of purity and cook in the form of a suitable vaccine product.
Genetically engineered proteins that are applicable in vaccines, it is possible to Express, for example, insect cells, yeast cells or mammalian cells. Genetically engineered proteins that can clear or select the conventional methods, can be directly inoculate pigs or other mammals in order to protect from swine TTV.
Line of insect cells (such as sf9, sf21 or HIGH FIVE) can be transformed using a vector for the transfer, containing polynucleotide acid obtained through the use of a virus or Skopje�created with the viral genome, which encodes one or more immunodominant proteins of the virus. A vector for the transfer includes, for example, linearization of baculovirus DNA and a plasmid containing polynucleotides specified. Line host cells can be jointly transliterate with linearized DNA of baculovirus and plasmid to obtain a recombinant baculovirus.
Or DNA from selected swine TTV, which encodes one or more capsid proteins, can be embedded in live vectors, such as poxvirus (virus group pox) or adenovirus, and be used as a vaccine.
Immunologically effective amount of the vaccine of the present invention is administered to pigs or other mammals that need protection against specified infection or syndrome. "Immunologically effective amount" can be easily identified or tested with standard testing. An effective amount means such amount, which is achieved through immunological response to the vaccine is sufficient to protect the pig or other mammal having been exposed to swine or TTV co-infection of swine TTV, which can cause a syndrome of dermatitis and nephropathy piglets (PDNS), Multisystem wasting syndrome after weaning piglets from sows (PMWS) or related disease. Preferably, a pig or MLC�supply other kind of protect to the extent which significantly decreases, becoming less intense or completely warned from one to all of the adverse physiological symptoms or effects of a viral disease.
The vaccine can be administered in a single dose or as repeated doses. Dose can be, for example, about 1-1,000 micrograms of viral antigen (depending on the concentration of immunoactive component of the vaccine), but must not contain the amount of viral antigen, sufficient to cause an adverse reaction or physiological symptoms of viral infection. In the prior art discloses methods for determining or titrating suitable doses of active antigenic agent with regard to the weight of the bird or mammal, the concentration of the antigen and other standard factors.
The vaccine can be administered to the pigs. The vaccine also can be given to people, such as the pig (pig farms workers) who are at increased risk of infection by a viral agent. It is assumed that it is possible to create a vaccine based on the TTV of the pig to ensure the overall protection against swine and human TTV. In other words the vaccine on the basis of porcine TTV can preferably be constructed in such a way as to protect from infection with the human TTV, through the use of so-called "method Jenner" (i.e. by the method of Edward Jenner can� to use the vaccine of cowpox virus against human smallpox). Preferably, the vaccine was injected directly to pigs or other mammals species, not exposed to TTV virus. The vaccine conveniently be administered orally, intrabuccal, intranasal, transdermal, parenteral, etc. Parenteral route of administration includes, but is not limited to, intramuscular, intravenous, intraperitoneal and subcutaneous route.
For introducing a liquid vaccine of the present invention can be prepared in aqueous solution, syrup, elixir, tinctures, etc. Such dosage forms are known in the prior art, and is usually prepared by dissolving the antigen and other conventional additives in the respective systems of the carrier or solvent. Suitable carriers or solvents include, but are not limited to, water, physiological saline, ethanol, ethylene glycol, glycerol, etc. Standard additives are, for example, approved colors, corrigent (gives taste and flavour), sweeteners and antimicrobial substances - preservatives, such as thimerosal (utilitytrailer sodium). Such solutions can be stabilized, for example by adding partially hydrolyzed gelatin, sorbitol, or cell culture medium, and aboveright by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, dihydro�ofat sodium, the potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, etc.
Liquid dosage forms may include suspensions and emulsions, which contain suspendresume or emulsifying agents in combination with other standard components. These types of liquid dosage forms can be prepared by conventional methods. For example, the suspension can be prepared using a colloid mill. Emulsions can be prepared using, for example, a homogenizer.
Parenteral dosage forms for administration in the form of an injection system in body fluids, require proper isotonicity and pH buffering to levels corresponding to levels in body fluids of a mammal. If necessary izotonichnost can be suitably adjusted with sodium chloride or other salts. To increase the solubility of the ingredients in the dosage form and stability of the liquid drug can be appropriate solvents, such as ethanol or propylene glycol. Other additives that can be used in the vaccine of the present invention include, but are not limited to, dextrose, conventional antioxidants and conventional chelating agents such as ethylenediaminetetraacetic acid (EDTA). Parenteral dosage forms should also be sterilized before use.
Negative�enny examples demonstrate certain aspects of the present invention. However, it is clear that these examples are given for illustration only and is not intended to be exhaustive with regard to conditions and scope of this invention. It should be understood that when given the typical reaction conditions (e.g., temperature, reaction time, etc.), we can also apply the conditions both above and below the interval, although they are generally less suitable. The reactions in the examples is carried out at room temperature (about 23°C to about 28°C) and at atmospheric pressure. Unless otherwise specified, in this specification, parts and percentages are given as parts by weight and weight percentages, and all temperatures are indicated in degrees Celsius.
Extraction of viral DNA, nested PCR and genomic PCR
The study used appropriate samples of serum and seminal fluid from 20 normal adults hogs from hog farms in Virginia. Total DNA was isolated from 20 samples of serum and 19 samples of semen, using a mini kit for DNA isolation QIA.K.mp DNA mini kit (Qiagen). To screen for positive PTTV - containing samples were first carried out the amplification of conservative regions in the UTR PTTV1 and PTTV2 the method of "nested" PCR using polymerase AmpliTag Gold (Applied Biosystems). Two pairs of primers used for amplification of the fragment And in PTTV1, was a TTV1 - mF (SEQ ID NO: 45)/TTV1-mR (SEQ ID NO:46) (for men� round PCR) and TTV1-nF (SEQ ID NO:47)/TTV1-nR (SEQ ID NO:48) (second round PCR), while two pairs of primers used for amplification of fragment D in PTTV2, was a TTV2-mF (SEQ ID NO:49)/TTV2-mR (SEQ ID NO:50) (for first round PCR) and TTV2-nF (SEQ ID NO:51)/TTV2-nR (SEQ ID NO:52) (for the first round PCR; Fig.1A and table 1).
In order to amplificatoare full genomic sequence as PTTV1 and PTTV2, we first carried out the converted genomic PCR, using a pair of conservative gene - specific primers TTV1-IF (SEQ ID NO:1)/TTV1-IR (SEQ ID NO:4) localized in the region And for PTTV1, and another pair of gene-specific primers TTV2-IF (SEQ ID NO:5)/TTV2-IR (SEQ ID NO:8), localized in the area D to PTTV2, respectively, with merged with Herculite II DNA polymerase (Stratagene) according to manufacturer's instructions. There were no PCR products of the expected size. Then we created new sets of primers for the amplification of two regions, covering full PTTV1 and PTTV2 genomes, in the second round PCR, respectively (Fig.1A). The pair of primers used for amplification of fragments b and C in PTTV1, was a TTV1-IF (SEQ ID NO:1)/TTV1-2340R (SEQ ID NO:2) and TTV1-231 IF (SEQ ID NO:3)/TTV1-IR (SEQ ID NO:4), respectively, while the pair of primers used for amplification of fragments E and F in PTTV2, was a TTV2-IF (SEQ ID NO:5)/TTV2-2316R (SEQ ID NO:6) and TTV2-GCF (SEQ ID NO:7)/TTV2-IR (SEQ ID NO:8), respectively (Fig.1A and table 1). Fragments C and F contain GC - rich region PTTV1 and PTTV2, ACC�tstone. Amplified PCR products individually excised, purified and sequentially cloned into vector pSC-B-amp/kan (Stratagene), using a set of PCR-cloning StrataClone Blunt PCR cloning kit according to the manufacturer's instructions (Stratagene) followed by DNA sequencing.
Screening of samples positive for the swine TTV, taken from pigs on a farm in Virginia
Recently in pigs in different geographic regions were detected DNA from porcine TTV method of "nested" PCR-based UTR (untranslated) sequence Japanese PTTV1 strain Sd - TTV31 (McKeown et al., 2004, see above). Given the recent identification PTTV2 two different sets of primers for nested PCR were used to amplify A region PTTV1 and region D PTTV2, respectively (Fig.1A) (Ellis et al., 2008, see above; Kekarainen, T., Sibila, M., and Segales, J. (2006). Prevalence of swine Torque teNO virus in post-weaning multisystemic wasting syndrome (PMWS)-affected and NOn-PMWS-affected pigs in Spain. J Gen Virol 87(Pt 4), 833-7; Krakowka et al., 2008, supra). A similar detection method was also applied in this study to identify the PTTV strains from pigs in the United States. To perform the screening on samples positive for the domestic PTTV1 - or PTTV2-virus, for subsequent use to determine the full genomic sequences of nested PCR analyze 20 samples of serum (SR#1-20) and 19 samples CE�internal fluid (SM#1-18 and SM#20), taken from 20 pigs (hogs) on a farm in Virginia. To our surprise, all of the 20 serum samples were positive in relation PTTV1, and 19 were also positive in relation PTTV2 (except SR#18). In contrast, only one sample of semen (SM#6) was PTTV1-positive and 3 samples of seminal fluid (SM#8, 9 and 20) were PTTV2-positive. This result is consistent with the results of a recent study in Spain, where it was shown that the samples of semen of pigs are positive for the PTTV DNA (Kekarainen, T., Lopez-Soria, S., and Segales, J. (2007). Detection of swine Torque teNO virus geNOgroups 1 and 2 in boar sera and semen. TheriogeNOlogy 68(7), 966- 71), and thus suggests the idea of a possible vertical transmission of PTTV. However, the velocity of propagation as PTTV1 and PTTV2 in the seminal fluid are significantly lower than in serum, this suggests that there is no direct Association between the presence of PTTV DNA in serum and seminal fluid from the same pigs.
Sequence and phylogenetic analysis
Generic analysis and alignment of DNA and amino acid sequences was conducted using the software package Lasergene (DNASTAR Inc., Madison, WI). Genomic sequences of the three known PTTV strains and their corresponding registration numbers in GenBank used for alignment and comparison, this Sd-TTV31 (AV), TTV - 1p (AY823990) and TTV - 2p (AY823991). Pairwise comparisons were performed�of Teleostei (PASC) was performed, using 121 full genomic sequence of TTV strains of human and animal from GenBank, using online programs PASC (http://www.ncbi.nlm.nih.gov/sutils/pasc/viridty.cgi?textpage=overview) (Bao et al., 2008).
Phylogenetic trees are built by combining nearest neighbors of binding using the program PAUP 4.0 (David Swofford, Smithsonian Institute, Washington, DC, distributed Sinauer Associate Inc.) on the basis of full genomic sequences and predicted amino acid sequences of ORF 4 seven PTTV strains. Data were obtained by 1000-fold resampling.
Design PCR primers for diagnosis of infection with swine PTTV Analysis and alignment of DNA sequences was performed using the software package Lasergene (DNASTAR Inc., Madison, WI). Further, given the full genomic sequences of ten strains of porcine TTV and their corresponding registration numbers in GenBank used for alignment. View PTTV1: Sd TTV31 (AV), PTTV1a - VA (GU456383), TTV - 1p (AY823990), PTTV1b - VA (GU456384), swSTHY - TT27 (GQ120664) and TTV1 #471819 (GU188045). View PTTV2: PTTV2b - VA (GU456385), PTTV2c - VA (GU456386), TTV - 2p (AY823991) and TTV2 #472142 (GUI 88 046). Were identified conserved sequences among 6 PTTV1 and 4 PTTV2 genomes, respectively, and then used to control the selection of primers for real-time PCR using the software Beacon Designer (PREMIER Biosoft International, Palo Alto, CA). Primer�, used for duplex nested PCR for PTTV1 were created using the software package Lasergene.
Standard curves of real-time PCR for PTTV1 and PTTV2 Region 2091 p. O., the corresponding PCR fragment In the genome PTTV1b - VA, re were amplified using the same PCR fragment using primers TTV1-IF (5'-CATAGGGTGTA.K.CCA.K.TCAGATTTA.K.GGCGTT-3') and TTV1-2340R (5'-GGTCATCAGACGATCCATCTCCCTCAG - 3') as described previously (Huang et al., 2010). The resulting amplicon is purified by extraction in the gel using the kit QIAquick Gel Extraction Kit (Qiagen), and quantitatively determined on a NaNODrop spectrophotometer, which was used as a standard template for real-time PCR porcine TTV species 1. A clone of the full-size DNA of strain PTTV2c - VA, pSC-PTTV2c, designed, assembling PCR fragments E and F from PTTV2c - VA in vector pSC-B-amp/kan (Huang et al., unpublished data). The plasmid pSC-PTTV2c (7082 p. O.) was used as a standard template for real-time PCR porcine TTV species 2, and the concentration of plasmid DNA was measured using NaNODrop spectrophotometer. To construct standard curves of real-time PCR used 10-fold serial dilutions of two matrices, respectively.
Extraction of viral DNA for PCR analysis
Total DNA was isolated from 20 samples of serum and 19 samples of semen taken from 20 conventional adult�x pigs (hogs) (without clinical syndromes) from the pig farm in Virginia using a mini kit for DNA isolation QIA.K.mp DNA mini kit (Qiagen) as described earlier (Huang et al., 2010). Sample volume used for extraction DNA was 400 µl for serum and seminal fluid, the latter eluate consisted of 50 µl of sterile water. All samples extracted DNA was stored at -20°C prior testing by RT real-time PCR. Detection of swine TTV in these samples standard nested PCR described previously (Huang et al., 2010). As a negative control using total DNA extracted from the serum sample of the goat the same method.
Quantitative PCR analyses in real time with the dye SYBR PTTV1 - and PTTV2-specific real-time PCR was performed, respectively, using a kit SensiMix SYBR &Fluorescein kit (Quantace Ltd) and device MyiQ iCYCLER for real-time PCR (BIO-RAD Laboratories). Each 25 - μl reaction mixture contained 12.5 µl of the dye SYBR green Master Mix, 4 μl of extracted DNA, 0.5 µl of each primer (10 nm) and 7.5 µl of sterile water. The PCR conditions for PTTV1 were as follows: 10 min at 95°C and then 40 cycles of amplification (15 sec at 95°C, 30 sec at 59.4°C, 10 s at 72°C). Then immediately determine the melting point, gradually increasing the temperature from 55°C to 95°C every 0.5°C measured fluorescent signal. The PCR conditions for PTTV2 were the same as for PTTV1, IP�the shutdown, the annealing temperature was 56°C. In each experience as a positive control includes the standard matrix for PTTV1 and PTTV2. The amplification and analysis of results is carried out using the MyiQ software System (BIO-RAD Laboratories). All the samples are put in a dual iteration on the same tablet.
Specificity and sensitivity monoplex analyses the Optimal annealing temperature for amplification in PTTV1 - and PTTV2-specific analyses accounted for 59.4°C and 56°C, respectively, as determined using 10-fold dilutions of amplification using a gradient of annealing temperatures. The amplification product 118-p. O. with the use of primers TTV1F/TTV1R managed to spend just PTTV1 matrix, while the amplification product of 200 BP using the matrix PTTV2 observed only when used primers TTVF4/TTVR4. In neither analysis did not observe any cross products of amplification, confirming the specificity of the primers and targets (data not shown).
Standard curve for PTTV1 built in the range of concentrations of target DNA per 25 µl. It was shown that the linear interval covers from 4.4×101to 4.4×108copies. The minimum detection limit (44 copies) had met the threshold cycle (Ct) Is at 37.57. It was assumed that the test specimens with Ct>is at 37.57 are below detection limit and are not amenable �kolichestvennomu definition. Similarly constructed standard curve PTTV2 and used for the detection of DNA concentration from 8.6×10° to 8.6×108copies per 25 μl reaction mixture. The threshold cycle Q corresponding to the minimum detection limit (8.6 copies), was 36.53. All the samples that were considered as PTTV1 - or PTTV2 - positive, had a number of copies below the maximum detection limit. Melting curves using a 10 - fold dilution PTTV1 or PTTV2 standard matrix (Fig.6A and 6B; blue curves), as well as 20 serum samples of pigs, give the melting temperature (Tm) 87.0°C for PTTV1 and 80.0°C for PTTV2, respectively (Fig.6A and 6B; red curves). There are no peaks for the negative control using sterile water or DNA goat serum as a matrix (Fig.6A and 7B; black lines).
Quantitative determination of porcine TTV1 and TTV2 in samples of serum or semen of pigs (hogs)
|Comparison of the detection of porcine TTV various methods of analysis of 20 serum samples and 19 samples of semen taken from adult pigs on a farm in Virginia|
|The number of positive value, and it./the total number, about�certain different methods of analysis|
|Samples||PTTV1 RT-PCR||PTTV1 nested PCR||PTTV2 RT-PCR||PTTV2 nested PCR||PTTV1/PTTV2 duplex RT-PCR|
Analysis PTTV1/PTTV2 by using duplex real-time PCR
Analysis PTTV1/PTTV2 by using duplex real-time PCR (RT-PCR) performed in a PCR system 25 µl containing 12.5 µl of the dye SYBR green Master Mix, 0.5 µl of each of PTTV1 primers, 0.5 µl of each of PTTV2 primers, 4 ál of DNA and 6.5 ál PT�sterile water. The duplex PCR conditions and method of determining the melting temperature were the same as in the case PTTV1, except that the annealing temperature was 58°C. the peak melting analyze to distinguish PTTV1 - and PTTV2-specific amplicons.
Duplex nested PCR
First round PCR was performed with a set of Platinum HiFi PCR Supermix (Invitrogen) using 4 µl of extracted DNA in a total volume of 50 µl. For PCR used the following conditions: 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec with an initial matrix denaturation of DNA at 94°C for 2 min, an Aliquot of the product of the first round PCR was used for second round PCR with the same PCR reagents and in the same conditions. One pair of primers, P1ab - mF/P1ab - mR was used in the first round of PCR, whereas in the second round of PCR used a mixture of two pairs of primers P1a-nF/P1a-nR to detect PTTV1a and P1b-nF/P1b-nR to detect PTTV1b (table 1). Amplification products were visualized by gel electrophoresis in 1% agarose gel, stained with ethidium bromide, and two lanes for each type distinguished in the UV rays.
Construction of expression plasmids PTTVI and PTTV2 ORF To C-terminal sections of the ORF1 PTTV1a, PTTV1b and PTTV2c were amplified using the respective full-sized clones DNA (pSC-PTTV1a, pSC-PTTV1b and pSC-PTTV2c; described elsewhere). It was assumed that amplificative�nye fragments encode protein products with 319 and.to. (amino acids and.to.) for PTTV1a (position ORF1 and.to. 317-635 (SEQ ID NO:13); registration number in GenBank GU 456383), 318.to. for PTTV1b (ORF1 and.to. the provisions 322-639 (SEQ ID NO:14); registration number in GenBank GU456384), and 316 a.to. for PTTV2c (ORF1 and.to. the provisions 310-625 (SEQ ID NO:16); registration number in GenBank GU456386), respectively. C-terminal truncated (processionary) fragment PTTV1b encoding 248 amino acids (a.to.) (ORF1 and.to. the provisions 322-569 (SEQ ID NO:14)), also were amplified and used as a control for comparison of results in SDS-PAGE analysis. All plasmids designed for cloning PCR products into a triple expression vector pTriEx1.1-Neo (NOvagen) in E. coli cells/baculovirus/mammalian between the restriction sites Ncol and Xhol to obtain protein, fused with a 8×His-tail on the C - end. Four recombinant plasmids were designated pTri - PTTV1a - ORF1, pTri - PTTV1b - ORF1, pTri - PTTV1b-ORF1ctruc and pTri - PTTV2c-ORF1. All cloned sequences were confirmed by DNA sequencing.
Expression of recombinant proteins PTTVI and PTTV2
Competent cells of strain Rosetta 2 (LUCKY) pLacI (NOvagen) transformed with four expression plasmids, respectively, and the bacteria were plated on plates with LB agar containing 100 μg/ml ampicillin, and grown overnight at 37°C. One who grew up in the result of the transformation of the colony for each construct were used to inoculate 3 ml of LB medium containing 100 µg�/ml ampicillin (LB/amp), and were grown for 6-8 hours at 37°C. Turbid culture - 3 ml for each design is then used to create the original bacterial solutions by adding sterilized by filtration, 25% glycerol solution and the freezing of the culture at a temperature below -80°C. Before cleaning 10 ál frozen initial bacterial solution for each construct were used to inoculate 3 ml of a primary culture of LB/amp and grown for 6-8 hours at 37°C. 100 ml stood with PM (Overnight) environment Express TV Media (NOvagen) was inoculable initial culture to induce protein expression, and grown for 16-18 hours at 37°C. After incubation the resulting self-induction the culture was centrifuged at 3400 rpm for 15 minutes at 4°C. the resulting supernatant for each construct was discarded and the bacterial precipitation was stored at -20°C until use.
Purification and dialysis of recombinant proteins
Recombinant proteins were insoluble and expressibility (and localized) in bacterial cells include. Each of the bacterial precipitation (pellets) were treated with BugBuster reagent for extraction of proteins and lizotsimom according to the manufacturer's Protocol, and for destruction of DNA and RNA were added to a nuclease - benzonase (NOvagen). Then, each of the precipitation cells activate sequentially resuspended�up to 840 ál of lysis buffer (6M guanidine hydrochloride, 0.1 M sodium phosphate, 0.01 M Tris-chloride, 0.01 M imidazole, pH 8.0) and frozen at -80°C for at least 30 minutes. Then the sediments were thawed, diluted in additional amounts (2.5 ml) of lysis buffer and gently stirred at a rotational movement for 30 minutes at room temperature. The lysate supernatants were collected by centrifugation with acceleration 15000×g for 30 minutes at room temperature. In each decanted supernatant was added a 50% slurry of Ni-NTA His-bind (NOvagen), and the mixture was shaken for 60 minutes at room temperature in order to promote the binding of his-tag. The mixture of the lysate/resin was loaded into an empty chromatography column. After the initial in the column eluate was added 7 ml of buffer for lysis, left to elyuirovaniya. Then each column was washed twice with wash buffer portions 7 ml (8M urea, 0.1 M sodium phosphate, 0.15 M sodium chloride, 0.02 M imidazole, pH 8.0). The elution of the target protein (target protein) was performed by adding in column (4) separate aliquots of elution buffer in portions of 1 ml (8M urea, 0.05 M sodium phosphate, 1M sodium chloride, 0.5 M imidazole, pH 8.0). Four fractions of the eluate were analyzed by the method of SDS-PAGE and staining Kumasi blue.
Eluates containing significant concentrations of target protein, was injected into a dialysis cassette with a volume of 0.5 ml to 3 ml with �cut (border transmission) molecular weight of 20000 (Pierce). For dialysis used a series of 4 buffers for dialysis (dialysis buffer): dialysis buffer 1 (6 M urea, 0.05 M sodium phosphate, 0.8 M sodium chloride, 0.3 M imidazole, pH 8.0), dialysis buffer 2 (4 M urea, 0.033 M sodium phosphate, 0.533 M sodium chloride, 0.2 M imidazole, pH 8.0), dialysis buffer 3 (2.67 M urea, 0.022 M sodium phosphate, 0.356 M sodium chloride, 0.133 M imidazole, pH 8.0) and dialysis buffer 4 (1.5 M urea, 0.0148 sodium phosphate, 0.237 M sodium chloride, 0.089 M imidazole, pH 8.0). The dialysis cassette was sequentially immersed in each dialysis buffer and rotated therein for 6 hours at 4°C. At the end of each dialysis of recombinant protein fused with His - tag (tail), extracted from the cassette, was quantified on the NaNODrop spectrophotometer and frozen at -80°C.
SDS - PAGE and Western blot (immune blotting using antibody to His-tag (tail)
Method of analysis the Western blot was developed for the detection of purified recombinant proteins using an anti-6×His - tagged monoclonal antibody (Rockland). Equal volumes of each of the purified protestirovannyx (truncated) ORF1 proteins and LDS/10% β-ME were mixed and boiled at 95°C for 10 minutes. In each corresponding cell with 4-12% Bis-Tris polyacrylamide gel (Invitrogen) was added to 10 ál of the boiled sample and passed current with a voltage of 200 volts for 43 minutes in 1×MES under�girdling buffer (Invitrogen). Proteins were transferred to PVDF membrane (Bio-Rad) using a device for semi-dry blot transfer Trans-blot semi-dry and 1 × buffer for transfer (Invitrogen). Upon completion of the transfer PVDF membrane were incubated in Odyssey blocking buffer (Li-Cor) at room temperature for 1 hour. Antibody against 6×His-tagged Mab were diluted in the ratio of 1:1000 in Odyssey blocking buffer / 0.2% tween 20 and transferred to the membrane after removal of the previous Odyssey blocking buffer. MAb was allowed to inkubirovanija with a membrane on rocking either for 2 hours at room temperature or overnight at 4°C, and then the membrane was washed three times with phosphate buffered using tris saline / 0.05% tween 20 (TBS - T, Sigma). Goat antibody against rabbit IgG IRDye 800 (Li-Cor) diluted in a ratio of 1:5000 in Odyssey blocking buffer / 0.2% tween 20 / 0.1% SDS. Transferred to savegeometry PVDF membrane and left to inkubirovanija for 1 hour at room temperature with gentle shaking on a rocking chair. The membrane was washed three times with TBS-T, 1x TBS and visualized using a Li-Cor Odyssey.
Western blot serum
Developed a Western blot of serum and applied it to identify positive and negative serum controls in ELISA (ELISA). After carrying out SDS - PAGE, as described above, proteins were transferred on PVDF membrane, which was then incubated in Odyssey blocking b�Ferre (Li-Cor) at room temperature for 1 hour. Selected serum sample was diluted at a ratio of 1:100 in Odyssey blocking buffer / 0.2% tween 20 and transferred to the membrane after removal of the previous Odyssey blocking buffer. Serum sample was left on the rocker to inkubirovanija with the membrane for 2 hours at room temperature, and then the membrane was washed three times with tris-buffered saline/ 0.05% tween 20 (TBS-T, Sigma). Goat antibody against swine IgG IRDye 800 (Rockland) was diluted in the ratio 1:2500 in Odyssey blocking buffer / 0.2% tween 20/0.1% SDS. Transferred to savegeometry PVDF membrane and left to inkubirovanija for 1 hour at room temperature with gentle shaking on a rocking chair. The membrane was washed three times with TBS - T, 1x TBS and visualized using a Li-Cor Odyssey.
Indirect PTTV1a; PTTV1b - and PTTV2-specific ELISA (ELISA) Optimal concentrations of antigens used for immobilization on tablets and dilution of antisera and conjugates were determined by checkerboard titration. ELISA (ELISA) started (initiated) breeding of each of the purified recombinant protein, fused to His-the end (PTTV1a, PTTV1b and PTTV2c, respectively), to a concentration of 680 ng/ml in 1 × carbonate buffer for sensitization of the surfaces (CERs) at pH 9.6, and coated tablets ELISA (Greiner) for immobilization (100 µl/well). Plates were sealed off and left to inkubirovanija at 37°C in the Techa�ie 2 hours. After immobilization divorced proteins was removed, and each well was washed 3 times with 300 μl 1 × TBS-T. Then added containing protein blocking buffer (Pierce) (300 μl/well) and the plates left to inkubirovanija at 37°C for 1 hour. At the same time, in 96 - well block for dilution of serum samples were diluted in the ratio of 1:100 in 150 μl containing proteins in blocking buffer. Then the block was removed and 100 ál of each dilution of the serum sample was transferred into each corresponding hole on the plates by ELISA. Plates were left to inkubirovanija at 37°C for 2 hours, then each well was washed 3 times with 300 μl of TBS-T. Further, anywhereman with HRP antibody against swine IgG (Rockland) was diluted in the ratio 1:4000 in 12 ml containing protein blocking buffer and 100 μl of this dilution was added to each well of the tablet. Incubated at 37°C for 1 hour, and then each well was washed 3 times with 300 μl of TBS-T. For the existence of ELISA to each well of the tablet was added 100 µl of the substrate is Sure Blue Reserve 1-Component (KPL). After 20 minutes, to each well was added 100 μl of IN HCL solution to stop the manifestation. Plates were then read at 450 nm.
Serum samples of pigs used in the study on cell cultures from trading company (obtained in New Zealand and considered to be free from all IE (OIE) diseases), was used as a positive control in the three protocols, ELISA (ELISA), as all serum samples were PTTV1a-, PTTV1b - and PTTV2-positive by definition by the method of Western blot for serum and showed high OD - optical density (>2.0). First, we used the pooled serum samples of sterile (bustronic, gnotobiology) pigs as a negative control, since they were negative in detection by the method of Western blot. Then negative serum samples sterile pigs we compared with some serum samples of pigs from a conventional pig farm in Wisconsin. They were also negative in the detection method, the Western blot (immunoblotting) and OD values corresponded to the values OD negative serum sterile (gnotobiology) pigs. This serum conventional pigs, we merged and used as a negative control. Limit value (threshold) for each analysis, ELISA (ELISA) was calculated as the mean OD of the negative control group (n=4) plus three times the standard deviation.
The design of the full-sized clones of genomic DNA from porcine TTVIa, lb and 2 with PCR fragments b and C from US (USA) isolate PTTV1a - VA (registration No.. in GenBank GU456383) were amplified again using the previously described constructions, and then collected in panoram�RNA genomic DNA with the site BamH I at both ends of the genome using PCR with overlapping, using DNA polymerase Herculase II Fusion (Stratagene) vector pSC-B-amp/kan (Stratagene). The resulting construction was designated pSC-PTTV1a (Fig.17A). Using the same strategy, designed clone pSC-PTTV1b (Fig.17B), originating from the US isolate PTTV1b-VA (registration NO. in GenBank GU456384), and the clone pSC-PTTV2c (Fig.17C), rising to US to isolate PTTV2c-VA (registration number in GenBank GU456386) with the same restriction sites (Vatn (I) on the vector with the same core. Plasmid TTV2-#471942-full (Fig.E) containing a full-sized genomic DNA obtained from a pathogenic isolate of porcine TTV2 from Germany. TTV2-#471942 was donated by Dr. Andreas Gallei (BIVI, Germany). On the basis of phylogenetic analysis (data not shown) TTV2-#471942 was attributed to swine TTV subtype 2b along with the US isolate of PTTV1b-VA.
Design tandem-dimeric DNA clones of porcine TTV2b and 2 with Full size PTTV2c genome is excised from the clone pSC-PTTV2c splitting using VAT I, was purified and was ligated with the formation of concatemers. Legirovannye concatemer cloned into vector pSC-B-amp/kan, who previously digested with BamH I, received a tandem dimeric form DNA clone pSC-2PTTV2c-RR (Fig.ID). Similarly, the tandem-dimeric DNA clone pSC-2PTTV2b-RR, obtained from the clone TTV2-#471942-full using EcoR V restriction sites (Fig.IF).
Getting PTTV1a-, PTTV1b - and PTTV2-specific policealne antibodies against ORF1
Product, Cody�following ORF1, represents the estimated capsid protein of the virus TTV. To generate PTTV1a-, PTTV1b - and PTTV2-specific anti-ORF1 polyclonal antibodies for the detection of expression of PTTV ORF1 proteins and to determine the infectivity of PTTV DNA clones three ORF1 protein from PTTV1a, PTTV1b and PTTV2c expressible in E. coli, purified, and then used to immunize new Zealand white rabbits, respectively, as special services (service) for obtaining antibodies in Rockland ImmuNOchemicals (Gilbertsville, PA). Each polyclonal antibody against ORF1 were obtained using serum from immunized rabbits.
In vitro transfection of infectious PTTV clones
Cells PK-15 were seeded at a density of 2×105cells per well in 6-well plate and before transfection were grown to 60% -70% of a monolayer. DNA clones pSC-2PTTV2b-RR and pSC-2PTTV2c-RR was transferrable directly into the cells PK-15, respectively, using lipofectamine LTX (Invitrogen) according to the manufacturer's Protocol. Accordingly, in the case of clones pSC-PTTV1a, pSC-PTTV2c and TTV2-#471942-full for transfection used them subsidized concatemer obtained as described above. Cells were cultured for 3-5 days and then used immunofluorescence analysis (IFA) for the detection of expression of TTV ORF1 pigs. Or transfected cell-passaged (passively) to new 6-well plates and continued to cultivate in tech�of 3 days before IFA detection.
Immunofluorescence analysis (IFA)
Transfetsirovannyh or passaged cells were washed twice in PBS and fixed with acetone. Five hundred microliters of antibody, specific to PTTV1a or PTTV2, at a dilution of 1:500 in PBS, was added to cells and incubated for 1 hour at room temperature. The cells was washed three times with PBS, and then added 500 µl labeled with Texas red or Alexa Fluor 488 goat antibody against rabbit IgG (Invitrogen) at a dilution of 1:200. After incubation at room temperature for 1 hour and washing with the use of PBS cells were stained with 500 µl of DAPI (KPL, Inc.) at a dilution of 1:1000 and visualized under fluorescent microscope.
In vivo inoculation of conventional pigs the tandem-dimeric porcine TTV2 clones
The study of inoculation of pigs was carried out to determine the infectivity of the two tandem-dimeric clones TTV2 pigs: pSC-2TTV2b-RR and pSC-2TTV2c-RR. Briefly, eight conventional pigs aged 4 weeks, were seronegative and virus DNA-negative for TTV2 pigs were randomly divided into two groups of four animals each. Each group, animals were placed separately and kept in conditions that meet the requirements of the institutional Committee on animal care and use.
All pigs in each group did a combined injection as to whether�factual node, and intramuscularly. Each of the four pigs (No. 181, 189, 192 and 193) were injected with 200 μg pSC-2TTV2b-RR plasmid DNA, whereas the other four pigs (No. 92, 180, 188 and 191) were inoculable 200 mcg pSC-2TTV2c-RR clone. In pigs checked daily for clinical symptoms of the disease within 28 days. On day 28 after inoculation performed these autopsies all the pigs.
Although the present invention is illustrated by description of several of its variants, and illustrative cases are described in detail, the applicants have no intention in any way to limit the appended claims to such an extent. Specialists in the art can easily make modifications. Therefore, the invention in its broader aspects is not limited to the shown and described specific details, representative devices and methods, and illustrative examples. Accordingly, it is possible to make deviations from such details without derogating from the spirits or scope of the basic idea of the invention.
1. The infectious nucleic acid molecule of the virus Torque teNO pigs (PTTV) comprising a nucleic acid molecule that encodes an infectious PTTV, which contains at least one copy of genomic sequence selected from the group consisting of sequences corresponding to the genotypes eliotian PTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, represented by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
2. Infectious molecule by the nucleic acid of claim 1, in which at least one copy of genomic sequence selected from the group consisting of sequences PTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, represented by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
3. Expression plasmid containing the infectious nucleic acid molecule of claim 1.
4. Plasmid according to claim 3, containing more than one copy of the infectious nucleic acid molecule.
5. Gene expression viral vector containing the infectious nucleic acid molecule of claim 1.
6. Viral vector according to claim 5, containing more than one copy of the infectious nucleic acid molecule.
7. A host, producing an avirulent infectious PTTV, transfairusa the expression plasmid according to claim 3 or viral gene expression vector according to claim 5, containing the infectious nucleic acid molecule of claim 1.
8. Avirulent infectious PTTV, intended for the manufacture of a vaccine for immunization of pigs against PTTV viral infection produced by the host cell according to claim 7.
9. Vaccine for immunization of pigs against PTTV viral infections, containing non-toxic physiologically acceptable carrier and an immunogenic amount of an avirulent infectious nonpathogenic virus Toque teNO pigs containing a genomic sequence selected from the group consisting of sequences corresponding to genotypes or subtypes PTTV1a-VA, PTTV1b-VA, PTTV2b-VA or PTTV2c-VA, represented by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
10. The vaccine according to claim 9, which contains live virus PTTV.
11. The vaccine according to claim 9, which contains killed virus PTTV.
12. The vaccine according to claim 9, which further comprises an adjuvant.
13. The method of immunization of pigs against PTTV viral infection, consisting in the introduction of the pig immunologically effective amount of the vaccine according to claim 9.
14. A method according to claim 13, comprising administering to the pig vaccine parenterally, intranasally, intradermally or transdermally.
15. A method according to claim 13, comprising administering to the pig vaccine inside the lymph node or intramuscularly.
16. The selected polynucleotide encoding an infectious PTTV, consisting of the polynucleotide sequences PTTV1a-VA, represented by SEQ ID NO: 9.
17. The selected polynucleotide according to claim 16, polynucleotide sequence which comprises the nucleotide sequence PTTV1a-VA, represented by SEQ ID NO: 9.
18. The selected polynucleotide encoding an infectious PTTV, consisting of the polynucleotide sequences PTTV1b-VA, represented by SEQ ID NO: 10.
19. The selected polynucleotide according to claim 18, polynucleotide the sequence of which is �W nucleotide sequence PTTV1b-VA, represented by SEQ ID NO: 10.
20. The selected polynucleotide encoding an infectious PTTV, consisting of the polynucleotide sequences PTTV2b-VA, represented by SEQ ID NO: 11.
21. The selected polynucleotide according to claim 20, polynucleotide sequence which comprises the nucleotide sequence PTTV2b-VA, represented by SEQ ID NO: 11.
22. The selected polynucleotide encoding an infectious PTTV, consisting of the polynucleotide sequences PTTV2c-VA, represented by SEQ ID NO: 12.
23. The selected polynucleotide according to claim 22, polynucleotide sequence which comprises the nucleotide sequence PTTV2c-VA, represented by SEQ ID NO: 12.
SUBSTANCE: characterised strain was isolated from diseased pigs and produced by serial passages on sensitive hetero- and homologous cell cultures and deposited in the collection of the FSBI "Federal Animal healthcare centre" under the registration number of FMD virus strain A No.2187/Kuti/2013 (production). The presented strain is reproduced in monolayer cell culture of porcine kidney (PK), passaged cell cultures of kidney of Siberian mountain ibex (SMIK-30), VPK-21 and IB-RS-2. During 18÷24 hours of incubation the virus yield in the said cell cultures reaches the values of 6.0÷7.0 lg TCD50/cm3. At high multiplicity of infection (1÷10 TCD/cell) causes TCID50 after 5 hours, while maintaining the original characteristics when passaging in cell cultures for 5 passages.
EFFECT: invention can be used to monitor the antigenic and immunogenic activity and for producing biological products for diagnostics and specific prophylaxis of FMD of type A.
6 tbl, 6 ex
SUBSTANCE: invention refers to medicine, namely to oncology. The subject of the invention is a new strain of the Sendai virus Sen293nsk1 adapted to effective replication in the human cell culture HEK293. The produced strain of the Sendai virus possesses lower virulence for laboratory mice and higher ability to destroy human tumour cells. The strain is supposed to be used experimentally as a therapeutic oncolytic preparation for treating malignant diseases. The invention can be used in treating oncologic diseases.
EFFECT: invention enables providing higher clinical effectiveness in oncologic diseases by using the murine Sendai virus non-pathogenic for humans, possessing an increased oncolytic activity and intensifying anti-tumour immunity.
FIELD: medicine, pharmaceutics.
SUBSTANCE: claimed invention relates to such compositions and pharmaceutical compositions, which include poxviruses, and namely to those, which include extracellular enveloped viruses. Claimed invention also relates to such method, which is intended for production of poxviruses, as well as poxviruses, obtained in accordance with claimed invention. In addition, claimed invention also relates to application of claimed poxviruses and said composition for medication preparation.
EFFECT: obtaining pharmaceutical compositions, which include poxviruses.
11 cl, 3 dwg
SUBSTANCE: method of detection and differentiation of a genome of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates comprises: extraction of the DNA from the biological material, posing a multiplex polymerase chain reaction using synthesised primers complementary to regions of genes M130R and M151R of the myxoma virus of rabbit, and having the following nucleotide composition: MF 5'TGG-AGC-TTT-TCA-AGC-ATT 3', MR 5'ATA-TCT-CGG-CTC-TAG-GGC-GAG 3', MZ 5' [FAM]-AG-CGT-CGG-ACG-TCT-TCG-TT-[RTQ1] 3', VF 5'AGC-CCT-ATA-AAC-CCG-TAG-ACG-AAC 3', VR 5'CAA-GCT-TTT-TTT-TAT-CCT-CGT-CCG 3', VZ 5' [R6G]-TCG-ACG-GTT-TCG-TCC-GCC-TTC-TTG-[BHQ2] 3', DNA amplification of the virus and evaluation of the reaction. The present inventions may be used in veterinary virology for the detection and differentiation of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates.
EFFECT: increase in accuracy.
2 cl, 6 tbl, 6 ex
SUBSTANCE: invention refers to biotechnology, virology and immunology. Particularly, the present invention refers to a new avian astrovirus; to antibodies and their fragments targeted to the above new virus; to antigen preparations, proteins and DNA molecules of new avian astrovirus; to vaccines based on the above new virus or to its antigen preparations, protein or DNA; to methods for producing such vaccines and to diagnostic kits. The present invention can be used in veterinary science.
EFFECT: preparing the new avian astrovirus.
11 cl, 5 dwg, 4 tbl, 12 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for preparing influenza A or B viruses in a cell culture, and a composition of the cell culture for preparing influenza A or B viruses.
EFFECT: invention provides the serum-free culture medium, avoids the need for the stage of cell culture medium replacement, prepares the influenza viruses with the high live virus recovery and can be used for the active immunisation of individuals and for producing the antibodies for various applications, including passive immunisation and diagnostic immunoassays.
22 cl, 24 dwg, 47 tbl, 1 ex
SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for producing viral-like influenza virus particles (IVP) in a plant or a part thereof. The method involves the expression of a new influenza virus protein HA in plants and purification thereof. The invention also aims at IVPs containing the influenza virus protein HA and herbal lipids. The invention also refers to nucleic acids coding an improved influenza virus HA, as well as to vectors. The IVPs can be used in developing the influenza vaccines or for the treatment of existing vaccines.
EFFECT: presented group of inventions can be used in medicine.
18 cl, 44 dwg, 1 ex
SUBSTANCE: invention relates to medical virology and deals with an influenza virus strain. The vaccine strain B/60/Massachusetts/2012/10 is a reassortant, obtained by crossing the "wild" virus B/Massachusetts/2/2012 with the cold-adapted temperature-sensitive virus B/USSR/60/69 -attenuation donor. The strain B/60/Massachusetts/2012/10 is deposited in the State Collection of Viruses FSBI D.I.Ivanovsky Scientific Research Institute of Virology Russian Ministry of Health under No 2740, actively reproduces in developing chicken embryos at the optimal temperature of 32°C, is characterised by temperature sensitivity and cold-adaptedness and safety for laboratory animals. Reassortant inherited genes, which code surface antigens of virus hemagglutinin (HA) and neuraminidase (NA), from the epidemic parent virus and remaining six genes, which code internal non-glycosylated proteins, from the attenuation donor.
EFFECT: claimed invention can be applied in practical healthcare for the prevention of influenza morbidity among adults and children.
FIELD: veterinary medicine.
SUBSTANCE: presented subline of cells A4C2/9k is highly sensitive to the ASF virus. The growth medium is used as medium Needle-MEM with 10% blood serum of swine. The inoculating concentration is 80-100 thousand cells/ml. The mitotic index to 2-3 days of cultivation is 25-35. The subline of cells is deposited at the Specialized collection of cell cultures of agricultural and game animals at the All-Russian research institute of experimental veterinary medicine under the number of 87.
EFFECT: invention enables to isolate the African swine fever virus without prior adaptation in reaction of hemadsorption and provides its accumulation in titre.
1 tbl, 3 ex
SUBSTANCE: invention relates to medical virology and deals with a strain of influenza virus. The vaccine strain A/17/Indiana/2011/72 (H3N2v) - reassortant, obtained by crossing of the "wild" virus A/Indiana/10/2011 (H3N2v) with the cold-adapted temperature-sensitive virus A/Leningrad/13 4/17/57 (H2N2) - attenuation donor. The strain A/17/Indiana/2011/72 (H3N2v) is deposited in the State Collection of Viruses FSBI D. I. Ivanovsky Scientific Research Institute of Virology Russian Ministry of Health, under No 2739, actively reproduces in developing chicken embryos at the optimal temperature of 32°C, is characterised by temperature-sensitivity and cold-adaptedness and safety for laboratory animals. Reassortant inherited genes, which code surface antigens of virus hemagglutinin (NA) and neuramindase (NA), from the epidemic parent virus and remaining six genes, which code internal non-glycosylated proteins, from the attenuation donor.
EFFECT: claimed invention can be applied in practical healthcare for the prevention of influenza disease morbidity among adults and children.
SUBSTANCE: invention relates to the field of biotechnology, in particular to the lentiviral delivery of apoptin into tumour cells, and can be used in medicine. The method includes obtaining a lentiviral construct, expressing modified apoptin, fused with a sectretory signal of lactotransferrin and a transduction signal (ST-CTP-apoptin), with the further obtaining of recombinant lentiviral particles, defective by replication and carrying the modified apoptin, which are later introduced into T-lymphocytes (TILs), obtained in the surgical ablation of the tumour or in the process of obtaining a biopsy, possessing the ability to penetrate into tumour cells. After that, obtained TILs are autotransplanted to the said patient.
EFFECT: invention makes it possible to increase the ability of apoptin to penetrate into tumour cells and produce an oncolytic effect with respect to all the tumour cells.
2 dwg, 3 ex
SUBSTANCE: invention relates to biotechnology and gene engineering. A method for selecting at least one transfected eukaryotic host cell expressing a target product, the eukaryotic host cells comprise at least an introduced polynucleotide encoding the target product, an introduced polynucleotide encoding a DHFR enzyme using at least one expression vector, providing a plurality of eukaryotic host cells, whose viability is dependent upon folate uptake, wherein the said host cells comprise at least a foreign polynucleotide encoding the target product, a foreign polynucleotide encoding a DHFR enzyme, culturing the said plurality of the eukaryotic host cells in a selective culture medium comprising folic acid in a concentration of 12.5-50 nM combined with a concentration of MTX of 2.3-500 nM, selecting at least one eukaryotic host cell expressing the target product.
EFFECT: described is a method of the target product and culture medium preparation.
11 cl, 2 tbl, 2 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to immunology. What is presented is a completely human monoclonal antibody, which binds insulin-like growth factor-II (IGF-II) and has a cross responsiveness to IGF-I, as well as its antigen-binding fragment. There are disclosed a nucleic acid molecule coding an antibody according to the invention, a vector and a host cell for the expression of the antibody according the invention. There are described a pharmaceutical composition, as well as conjugates for treating and diagnosing malignant tumour, using the antibody according to the invention in preparing the therapeutic agent and a method for determining IGF-II and IGF-I levels in a patient's sample.
EFFECT: present invention can find further application in cancer therapy.
16 cl, 27 ex, 18 tbl
SUBSTANCE: invention relates to field of immunology. Claimed is isolated antibody to ICOS protein of people with increased effector function. Also described are cell and method of obtaining antibody in accordance with claimed invention, pharmaceutical composition, method of treating autoimmune disease or disorder, transplant rejection and malignancy of human T-cells, as well as method of depletion of ICOS-expressing T-cells, method of destroying germ centre structure in secondary lymphoid organ of primates, methods of depleting B-cells of germ centre of secondary lymphoid organ and circulating B-cells, which have undergone class switching, in primates.
EFFECT: invention can be further applied in therapy of diseases, mediated by T-cells.
33 cl, 21 dwg, 3 tbl
SUBSTANCE: invention refers to biotechnology and immunology. There are presented optimised genes of light and heavy chains of Infliximab, an anti-tumour necrosis factor alpha (TNF-alpha) antibody, as well as a cell line VKPM-N-131, and a method for antibody biosynthesis. Nucleotide sequences of the genes coding the light and heavy chains of Infliximab are optimised in order to provide the content of codones most specific for mammals; the G/C content is expected to make 50-60% of the total composition; the absence of expanded tracts of a degenerate composition and the absence of RNA secondary structures.
EFFECT: Chinese hamster ovary cell line (CHO) produced by transfection by expression structures containing the genetic sequences according to the invention, enables producing at least 50 mg/l of the monoclonal antibody Infliximab.
4 cl, 3 dwg, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to biotechnology and represents anti-nerve growth factor (NGF) antibodies. The present invention also discloses a pharmaceutical composition for relieving pain associated with a disease or a condition, wherein pain progression or persistence is mediated by NGF, containing the above antibodies, as well as a kit for treating a HGF-related disease, such as e.g. osteoarthritis, nucleic acids coding a heavy or light chain of the antibody, an expression vector, a host cell for preparing the above antibodies, a method for expressing the above anti-NGF antibodies, as well as using the above antibodies in managing pain and for preparing a therapeutic agent for managing pain associated with the disease or condition, wherein pain progression or persistence is mediated by NGF.
EFFECT: present invention enables producing the anti-NGF antibodies characterised by high stability in vivo.
16 cl, 7 dwg, 13 tbl, 8 ex
SUBSTANCE: present invention refers to immunology. Presented is an antibody able to bind to an amplified epidermal growth factor receptor (EGFR) and to de2-7 EGFR, a truncated version of EGFR, and characterised by sequences of variable domains. There are also disclosed a kit for diagnosing a tumour, an immunoconjugate, pharmaceutical compositions and methods of treating a malignant tumour based on using the antibody according to the invention, as well as a single-cell host to form the antibody according to the present invention.
EFFECT: invention can find further application in diagnosing and treating cancer.
43 cl, 98 dwg, 20 tbl, 26 ex
SUBSTANCE: synthetic DNA is proposed, encoding human erythropoietin, having the sequence Seq ID No. 1, comprising its expression vector, the method of production of erythropoietin producer strain, and a strain of a Chinese hamster ovary cells - producer of recombinant human erythropoietin, deposited under the number RKKK(P) 761 D.
EFFECT: invention enables to increase the expression level of recombinant human erythropoietin.
5 cl, 1 tbl, 8 dwg, 4 ex
SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.
EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.
23 cl, 74 dwg, 17 tbl, 33 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to antibodies including human antibodies and their antigen-binding portions, which specifically bind to CCR2, in particular to human CCR2, and can act as CCR2 inhibitors. Anti-CCR2 antibodies are those binding to first and/or second extra-cellular CCR2 loops. The present invention also refers to human anti-CCR2 antibodies and to their antigen-binding portions. The present invention refers to the recovered heavy and light chains of immunoglobulin initiated from human anti-CCR2 antibodies, and to nucleic acid molecules coding such immunoglobulins. The present invention also refers to methods for preparing human anti-CCR2 antibodies and their antigen-binding portions, to compositions containing such antibodies or their antigen-binding portions, and to methods for using antibodies and their antigen-binding portions, and compositions for diagnosing and treating.
EFFECT: invention refers to methods for gene therapy with the use of nucleic acid molecules coding molecules of heavy and light chains of immunoglobulin, wherein the above molecules contain anti-CCR2 antibodies and their antigen-binding portions.
25 cl, 24 dwg, 8 tbl, 17 ex
SUBSTANCE: invention relates to biochemistry, particularly to use of a protein-coding gene, which consists of an amino acid sequence represented in SEQ ID NO.2, to obtain transgenic rice with long, large and multiple grains and increased yield, more grains per panicle and rolled leaves. Disclosed is use of a transgenic cell line of rice and a recombinant vector to obtain transgenic rice with long, large or multiple grains and increased yield, more grains per panicle and rolled leaves.
EFFECT: invention enables to efficiently obtain transgenic rice with long, large or multiple grains and increased yield, more grains per panicle and rolled leaves.
5 cl, 16 dwg, 4 tbl, 2 ex