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Infectious genomic DNA clone and serological profile of torque teno sus virus 1 and 2

Active Publication Date: 2013-11-28
VIRGINIA TECH INTPROP INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides an infectious nucleic acid molecule of TTsuV, which allows for the diagnosis and treatment of TTsuV infections. These infectious nucleic acid molecules can be contained in biologically functional plasmid or viral vectors, which can be used to infect cells and produce live infectious TTsuV. The infectious TTsuV can be detected using an immunogentic fragment or a complete protein of TTsuV, which is immobilized on a solid surface and reacted with a serum sample from a pig suspected of TTsuV infection. The invention also provides a method for isolating and culturing TTsuV, which can be used to develop diagnostic tools and treatments for the virus.

Problems solved by technology

The pathogenic potential of anellovirus is still controversial.
Similarly, whether TTSuV is associated with a swine disease is still debatable.
However, results from other studies did not support a direct association of TTSuV1 or TTSuV2 with PCVAD or association of type 2 porcine circovirus (PCV2) and TTSuV with porcine reproductive failures (Gauger, P. C., K. M. Lager, A. L. Vincent, T. Opriessnig, M. E. Kehrli, Jr., and A. K. Cheung. 2011.
Due to the lack of a cell culture system to propagate anelloviruses, little is known regarding the molecular biology and pathogenesis of anelloviruses.

Method used

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  • Infectious genomic DNA clone and serological profile of torque teno sus virus 1 and 2
  • Infectious genomic DNA clone and serological profile of torque teno sus virus 1 and 2
  • Infectious genomic DNA clone and serological profile of torque teno sus virus 1 and 2

Examples

Experimental program
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example 1

Cell Lines and Cell Cultures

[0130]A total of twelve continuous cell lines were used in this study. A type 1 porcine circovirus (PCV1)-free porcine kidney epithelial cell line PK-15 (Fenaux, M., T. Opriessnig, P. G. Halbur, F. Elvinger, and X. J. Meng. 2004. A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs. J Virol 78:6297-303), a swine testis cell line ST (ATCC CRL-1746, passage 6), a baby hamster kidney fibroblast cell line BHK-21 (ATCC CCL-10, passage 62), and an African green monkey kidney epithelial Vero cell (ATCC CCL-81, passage 95) were each grown in modified Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. A porcine monocytic cell line 3D4 / 31 (ATCCCRL-2844, passage 8), a porcine small intestinal epithelial cell line IPEC-J2 (a gift from Dr. Anthony Blikslager at North ...

example 2

Analysis of TTSuV1 or TTSuV2 Contamination in Cultured Cells by Real-Time Quantitative PCR (qPCR)

[0131]To ensure that the porcine-derived cell lines used in the study were free of TTSuV contamination, five cell lines, PCV1-free PK-15, 3D4 / 31, IPEC / J2, BHK-21 and MARC-145, were tested for TTSuV1 or TTSuV2 DNA by using two singleplex SYBR green-based real-time qPCR assays (Huang, Y. W., B. A. Dryman, K. K. Harrall, E. M. Vaughn, M. B. Roof, and X. J. Meng. 2010. Development of SYBR green-based real-time PCR and duplex nested PCR assays for quantitation and differential detection of species- or type-specific porcine Torque teno viruses. J Virol Methods 170:140-6). Briefly, total DNA was extracted from each cell line using the QIAamp DNA mini kit (Qiagen) and was subsequently subjected to TTSuV1 or TTSuV2 qPCR detection in a 25-0 PCR system using SensiMix SYBR & Fluorescein kit (Quantace Ltd) as described previously (Huang, Y. W., B. A. Dryman, K. K. Harrall, E. M. Vaughn, M. B. Roof, a...

example 3

Generation of a Rabbit Anti-TTSuV2 ORF1 Antiserum

[0132]The inventors have previously expressed and purified a recombinant truncated ORF1 protein of TTSuV2 (PTTV2c-VA strain) (Huang, Y. W., K. K. Harrall, B. A. Dryman, N. M. Beach, S. P. Kenney, T. Opriessnig, E. M. Vaughn, M. B. Roof, and X. J. Meng. 2011. Expression of the putative ORF1 capsid protein of Torque teno sus virus 2 (TTSuV2) and development of Western blot and ELISA serodiagnostic assays: correlation between TTSuV2 viral load and IgG antibody level in pigs. Virus Res 158:79-88). The purified protein products were used to immunize two New Zealand white rabbits as a custom antibody production service at Rockland Immunochemicals (Gilbertsville, Pa.). Serum samples from both rabbits were collected before immunization (pre-bleed) and at 45 days post-immunization.

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Abstract

The present invention also provides infectious DNA clones, biologically functional plasmid or viral vector containing the infectious nucleic acid genome molecule of Torque teno sus virus (TTsuV). The present invention also provides methods for diagnosing TTsuV infection via immunological methods, e.g., enzyme-linked immunoabsorbent assay (ELISA) and Western blot using PTTV specific antigens for detecting serum PTTV specific antibodies which indicate infections TTsuV1, TTsuV2, and individual TTsuV1 genotypes.

Description

REFERENCE TO RELATED APPLICATION[0001]This patent application in a continuation-in-part of U.S. patent application Ser. No. 12 / 861,378, which claims the benefit of U.S. Provisional Patent Application No. 61 / 235,833, filed on Aug. 21, 2009, and U.S. Provisional Patent Application 61 / 316,519, filed on Mar. 23, 2010. The disclosures of the above mentioned priority applications are hereby incorporated by reference in their entirety into the present disclosure.FIELD OF INVENTION[0002]The present invention relates to infectious DNA clones of Torque teno sus virus (TTsuV), also known as porcine Torque teno virus (PTTV), and diagnosis of Torque teno sus virus (TTsuV) infection, particularly diagnosis of species- or type-specific TTsuV infection, and simultaneous infection of multiple strains from different genotypes.BACKGROUND OF THE INVENTION[0003]Anelloviruses are small, single-stranded, circular DNA viruses that infect a wide range of animal species from humans to domestic animals includ...

Claims

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Application Information

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IPC IPC(8): C07K14/005C12N7/00G01N33/569C12N15/86
CPCC07K14/005C12N15/86C12N7/00G01N33/56983A61K39/12A61K2039/53A61K2039/552C12N2750/00021C12N2750/00022C12N2750/00034C12N2750/00043C12Q1/701G01N2333/01G01N2469/20
Inventor MENG, XIANG-JINHUANG, YAOWEI
Owner VIRGINIA TECH INTPROP INC
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