U.S. patent application number 13/840805 was filed with the patent office on 2013-11-28 for infectious genomic dna clone and serological profile of torque teno sus virus 1 and 2.
This patent application is currently assigned to VIRGINIA TECH INTELLECTUAL PROPERTIES, INC.. The applicant listed for this patent is Virginia Tech Intellectual Properties, Inc.. Invention is credited to Yaowei HUANG, Xiang-Jin MENG.
Application Number | 20130315944 13/840805 |
Document ID | / |
Family ID | 49621789 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130315944 |
Kind Code |
A1 |
MENG; Xiang-Jin ; et
al. |
November 28, 2013 |
INFECTIOUS GENOMIC DNA CLONE AND SEROLOGICAL PROFILE OF TORQUE TENO
SUS VIRUS 1 AND 2
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.
Inventors: |
MENG; Xiang-Jin;
(Blacksburg, VA) ; HUANG; Yaowei; (Blacksburg,
VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Virginia Tech Intellectual Properties, Inc. |
Blacksburg |
VA |
US |
|
|
Assignee: |
VIRGINIA TECH INTELLECTUAL
PROPERTIES, INC.
Blacksburg
VA
|
Family ID: |
49621789 |
Appl. No.: |
13/840805 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12861378 |
Aug 23, 2010 |
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13840805 |
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Current U.S.
Class: |
424/186.1 ;
435/235.1; 435/320.1; 435/5; 536/23.72 |
Current CPC
Class: |
G01N 2469/20 20130101;
G01N 2333/01 20130101; A61K 39/12 20130101; C07K 14/005 20130101;
C12N 2750/00022 20130101; C12N 2750/00021 20130101; C12N 2750/00034
20130101; A61K 2039/53 20130101; C12N 15/86 20130101; C12N
2750/00043 20130101; C12Q 1/701 20130101; C12N 7/00 20130101; G01N
33/56983 20130101; A61K 2039/552 20130101 |
Class at
Publication: |
424/186.1 ;
536/23.72; 435/320.1; 435/235.1; 435/5 |
International
Class: |
C07K 14/005 20060101
C07K014/005; C12N 7/00 20060101 C12N007/00; G01N 33/569 20060101
G01N033/569; C12N 15/86 20060101 C12N015/86 |
Claims
1. An infectious nucleic acid molecule of Torque teno sus virus
(TTsuV) comprising a nucleic acid molecule encoding an infectious
TTsuV which contains at least one copy of genomic sequence having
at least 85% homology to a genomic sequence of TTsuV2.
2. The infectious nucleic acid molecule of claim 1, wherein the at
least one copy of genomic sequence having at least 95% homology to
the genomic sequence of TTsuV2.
3. The infectious nucleic acid molecule of claim 1, wherein the
genomic sequence of TTsuV2 is of genomic clone of PTTV2c-VA.
4. The infectious nucleic acid molecule of claim 1, wherein the
genomic sequence is selected from sequences set forth in SEQ ID
NO:1.
5. The infectious nucleic acid molecule of claim 1, wherein the
genomic sequence of TTsuV2 is of genomic clone of TTV2-#471942.
6. The infectious nucleic acid molecule of claim 1, wherein the
genomic sequence is selected from sequences set forth in SEQ ID
NO:2.
7. The infectious nucleic acid molecule of claim 1, wherein the
genomic sequence of TTsuV2 comprising at least one genetic marker
in intron 1.
8. The infectious nucleic acid molecule of claim 1, wherein the
genetic marker in intron 1 is an artificially introduced
restriction site.
9. A biologically functional plasmid or viral vector containing the
infectious nucleic acid molecule according to claim 1.
10. The biologically functional plasmid or viral vector of claim 9,
containing more than one copy of the infectious nucleic acid
molecule.
11. The biologically functional plasmid or viral vector of claim 9,
containing tandem copies of genomic clone of PTTV2c-VA.
12. A suitable host cell transfected by the vector comprising the
infectious nucleic acid molecule according to claim 1.
13. An infectious TTsuV produced by cells containing the infectious
nucleic acid molecule according to claim 1.
14. An infectious TTsuV produced by cells containing the infectious
nucleic acid molecule according to claim 7.
15. A method for diagnosing TTsuV infection, comprising
immobilizing an immunogentic fragment or a complete protein of a
polypeptide sequence of ORF1 protein of TTsuV 1 or 2; contacting a
serum sample from a pig suspected of TTsuV infection with the
immobilized immunogentic fragment or complete protein; and
detecting captured antibody specific to the immunogentic
fragment.
16. The method of claim 15, wherein the polypeptide sequence is
selected from the group consisting of ORF1 proteins of TTsuV
genotypes or subtypes TTsuV1a, TTsuV1b or TTsuV2.
17. The method of claim 15, wherein the polypeptide sequence is
selected from the group consisting of N-terminal truncated ORF1
proteins of TTsuV genotypes or subtypes TTsuV1a or TTsuV1b.
18. The method of claim 16, wherein the polypeptide sequence is
amino acid No. 317-635 of ORF1 protein of TTsuV1a.
19. The vaccine of claim 16, wherein the polypeptide sequence is
amino acid No. 322-639 of ORF1 protein of TTsuV1b.
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 including pigs (Hino, S., and H. Miyata. 2007.
Torque teno virus (TTV): current status. Rev Med Virol 17:45-57;
Okamoto, H. 2009. TT viruses in animals. Curr Top Microbiol Immunol
331:35-52). Most recently, all human and other animal anelloviruses
have been assigned into a newly established family Anelloviridae
that includes nine genera (Biagini, P., M. Bendinelli, S. Hino, L.
Kakkola, A. Mankertz, C. Niel, H. Okamoto, S. Raidal, C. G. Teo,
and D. Todd. 2011. Anelloviridae, p. 331-341. In A. M. Q. King, M.
J. Adams, E. B. Carstens, and E. J. Lefkowitz (ed.), Virus
Taxonomy, 9th Report of the ICTV. Elsevier Academic Press, London).
Human anelloviruses include Torque teno virus (TTV), Torque teno
mini virus (TTMV) and Torque teno midi virus (TTMDV) that belong to
three different genera. Human TTV, TTMV and TTMDV are non-enveloped
spherical viruses with DNA genomes of 3.6-3.9, 2.8-2.9 and 3.2 kb
in length, respectively (Okamoto, H. 2009. History of discoveries
and pathogenicity of TT viruses. Curr Top Microbiol Immunol
331:1-20). These three groups of human anelloviruses show a high
degree of genetic diversity, and infections of TTV, TTMV and TTMDV
at a high prevalence in human populations have been documented
worldwide (Ninomiya, M., M. Takahashi, T. Nishizawa, T.
Shimosegawa, and H. Okamoto. 2008. Development of PCR assays with
nested primers specific for differential detection of three human
anelloviruses and early acquisition of dual or triple infection
during infancy. J Clin Microbiol 46:507-14; Okamoto, H. 2009.
History of discoveries and pathogenicity of TT viruses. Curr Top
Microbiol Immunol 331:1-20). On the other hand, porcine
anelloviruses or Torque teno sus viruses (TTSuV) is assigned into a
new genus Iotatorquevirus comprising two species (TTSuV1 and
TTSuV2), each also characterized by high genetic diversity with a
genomic size of approximately 2.8 kb (Huang, Y. W., Y. Y. Ni, B. A.
Dryman, and X. J. Meng. 2010. Multiple infection of porcine Torque
teno virus in a single pig and characterization of the full-length
genomic sequences of four U.S. prototype PTTV strains: implication
for genotyping of PTTV. Virology 396:287-97, Niel, C., L.
Diniz-Mendes, and S. Devalle. 2005. Rolling-circle amplification of
Torque teno virus (TTV) complete genomes from human and swine sera
and identification of a novel swine TTV genogroup. J Gen Virol
86:1343-7). TTSuV1 and TTSuV2 are highly prevalent in pig
populations in many countries (Gallei, A., S. Pesch, W. S. Esking,
C. Keller, and V. F. Ohlinger. 2010. 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 143:202-12; Kekarainen, T., M. Sibila, and
J. Segales. 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 87:833-7; McKeown, N.
E., M. Fenaux, P. G. Halbur, and X. J. Meng. 2004. Molecular
characterization of porcine TT virus, an orphan virus, in pigs from
six different countries. Vet Microbiol 104:113-7).
[0004] Human and porcine anelloviruses share the same genomic
structure, which consists of at least four presumed open reading
frames (ORFs), ORF1, ORF2, ORF1/1 and ORF2/2, as well as a short
stretch of high GC content in the untranslated region (UTR) (Huang,
Y. W., Y. Y. Ni, B. A. Dryman, and X. J. Meng. 2010. Multiple
infection of porcine Torque teno virus in a single pig and
characterization of the full-length genomic sequences of four U.S.
prototype PTTV strains: implication for genotyping of PTTV.
Virology 396:287-97; Okamoto, H., M. Takahashi, T. Nishizawa, A.
Tawara, K. Fukai, U. Muramatsu, Y. I Saito, and A. Yoshikawa. 2002.
Genomic characterization of TT viruses (TTVs) in pigs, cats and
dogs and their relatedness with species-specific TTVs in primates
and tupaias. J Gen Virol 83:1291-7; 39. Qiu, J., L. Kakkola, F.
Cheng, C. Ye, M. Soderlund-Venermo, K. Hedman, and D. J. Pintel.
2005. Human circovirus TT virus genotype 6 expresses six proteins
following transfection of a full-length clone, J Virol 79:6505-10).
The transcription pattern and related translational products of
human TTV genogroup 1 have been experimentally determined by using
two full-length TTV DNA clones (Mueller, B., A. Maerz, K.
Doberstein, T. Finsterbusch, and A. Mankertz. 2008. Gene expression
of the human Torque Teno Virus isolate P/1C1. Virology 381:36-45;
39. Qiu, J., L. Kakkola, F. Cheng, C. Ye, M. Soderlund-Venermo, K.
Hedman, and D. J. Pintel. 2005. Human circovirus TT virus genotype
6 expresses six proteins following transfection of a full-length
clone, J Virol 79:6505-10). It was shown that the human TTV genome
expresses three or more spliced mRNAs encoding at least six
proteins, ORF1, ORF2, ORF1/1, ORF2/2, ORF1/2 and ORF2/3 (Mueller,
B., A. Maerz, K. Doberstein, T. Finsterbusch, and A. Mankertz.
2008. Gene expression of the human Torque Teno Virus isolate P/1C1.
Virology 381:36-45). The transcriptional analysis and protein
expression profile using cloned full-length genomic DNA have not
been reported for TTSuV.
[0005] The ORF1 of TTSuV is believed to encode a viral capsid and
replication-associated protein (Huang, Y. W., Y. Y. Ni, B. A.
Dryman, and X. J. Meng. 2010. Multiple infection of porcine Torque
teno virus in a single pig and characterization of the full-length
genomic sequences of four U.S. prototype PTTV strains: implication
for genotyping of PTTV. Virology 396:287-97; Okamoto, H., M.
Takahashi, T. Nishizawa, A. Tawara, K. Fukai, U. Muramatsu, Y. I
Saito, and A. Yoshikawa. 2002. Genomic characterization of TT
viruses (TTVs) in pigs, cats and dogs and their relatedness with
species-specific TTVs in primates and tupaias. J Gen Virol
83:1291-7). IgG antibodies against the ORF1 of TTV and TTSuV have
been detected in human and pig sera, respectively (15. 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; Kakkola, L., H.
Boden, L. Hedman, N. Kivi, S. Moisala, J. Julin, J.
Yla-Liedenpohja, S. Miettinen, K. Kantola, K. Hedman, and M.
Soderlund-Venermo. 2008. Expression of all six human Torque teno
virus (TTV) proteins in bacteria and in insect cells, and analysis
of their IgG responses. Virology 382:182-9; 38. Ott, C., L. Duret,
I. Chemin, C. Trepo, B. Mandrand, and F. Komurian-Pradel. 2000. Use
of a TT virus ORF1 recombinant protein to detect anti-TT virus
antibodies in human sera. J Gen Virol 81:2949-58).
[0006] The pathogenic potential of anellovirus is still
controversial. Currently, human TTV is not considered to be
directly associated with a particular disease, although recent
studies suggested TTV may serve as an immunological trigger of
multiple sclerosis (Maggi, F., and M. Bendinelli. 2010. Human
anelloviruses and the central nervous system. Rev Med Virol
20:392-407). Similarly, whether TTSuV is associated with a swine
disease is still debatable. TTSuV1 was shown to partially
contribute to the experimental induction of porcine dermatitis and
nephropathy syndrome (PDNS) and postweaning multisystemic wasting
syndrome (PMWS or porcine circovirus associated disease, PCVAD) in
a gnotobiotic pig model (Ellis, J. A., G. Allan, and S. Krakowka.
2008. Effect of coinfection with genogroup 1 porcine torque teno
virus on porcine circovirus type 2-associated postweaning
multisystemic wasting syndrome in gnotobiotic pigs. Am J Vet Res
69:1608-14; 22. Krakowka, S., C. Hartunian, A. Hamberg, D. Shoup,
M. Rings, Y. Zhang, G. Allan, and J. A. Ellis. 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). PMWS-affected pigs in Spain had a
higher prevalence and viral loads of TTSuV2 than the
PMWS-unaffected pigs (Aramouni, M., J. Segales, M. Sibila, G. E.
Martin-Valls, D. Nieto, and T. Kekarainen. 2011. Torque teno sus
virus 1 and 2 viral loads in postweaning multisystemic wasting
syndrome (PMWS) and porcine dermatitis and nephropathy syndrome
(PDNS) affected pigs. Vet Microbiol 153:377-81; 21. Kekarainen, T.,
M. Sibila, and J. Segales. 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
87:833-7). Moreover, a significantly lower level of anti-TTSuV2
antibody was found in PCVAD-affected pigs than in PCVAD-unaffected
pigs (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). 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. Postweaning multisystemic wasting syndrome
produced in gnotobiotic pigs following exposure to various amounts
of porcine circovirus type 2a or type 2b. Vet Microbiol 153:229-39;
Huang, Y. W., K. K. Harrall, B. A. Dryman, T. Opriessnig, E. M.
Vaugh, M. B. Roof, and X. J. Meng. 2012. Serological profile of
Torque teno sus virus species 1 (TTSuV1) in pigs and antigenic
relationships between two TTSuV1 genotypes (1a and 1b), between two
species (TTSuV1 and 2), and between porcine and human
anelloviruses. J. Virol. Submitted Manuscript; Lee, S. S., S.
Sunyoung, H. Jung, J. Shin, and Y. S. Lyoo. 2010. Quantitative
detection of porcine Torque teno virus in Porcine
circovirus-2-negative and Porcine circovirus-associated
disease-affected pigs. J Vet Diagn Invest 22:261-4; Ritterbusch, G.
A., C. A. Sa Rocha, N. Mores, N. L. Simon, E. L. Zanella, A.
Coldebella, and J. R. Ciacci-Zanella. 2011. Natural co-infection of
torque teno virus and porcine circovirus 2 in the reproductive
apparatus of swine. Res Vet Sci.
doi:10.1016/j.rvsc.2011.04.001).
[0007] Due to the lack of a cell culture system to propagate
anelloviruses, little is known regarding the molecular biology and
pathogenesis of anelloviruses. In order to definitively
characterize diseases associated with anellovirus infection, an
appropriate animal model is needed. Since multiple infections of
different genotypes or subtypes of human TTV or TTSuV are common
events (Gallei, A., S. Pesch, W. S. Esking, C. Keller, and V. F.
Ohlinger. 2010. 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
143:202-12; Huang, Y. W., Y. Y. Ni, B. A. Dryman, and X. J. Meng.
2010. Multiple infection of porcine Torque teno virus in a single
pig and characterization of the full-length genomic sequences of
four U.S. prototype PTTV strains: implication for genotyping of
PTTV. Virology 396:287-97; Ninomiya, M., M. Takahashi, T.
Nishizawa, T. Shimosegawa, and H. Okamoto. 2008. Development of PCR
assays with nested primers specific for differential detection of
three human anelloviruses and early acquisition of dual or triple
infection during infancy. J Clin Microbiol 46:507-14), a
biologically pure and isolated form of a specific anellovirus
generated from full-length infectious DNA clone is also required
for a pathological study of a single phenotype. Although infectious
DNA clones of human TTV in cultured cells have been reported (de
Villiers, E. M., S. S. Borkosky, R. Kimmel, K. Gunst, and J. W.
Fei. 2011. The diversity of torque teno viruses: in vitro
replication leads to the formation of additional
replication-competent subviral molecules. J Virol 85:7284-95;
Kakkola, L., J. Tommiska, L. C. Boele, S. Miettinen, T. Blom, T.
Kekarainen, J. Qiu, D. Pintel, R. C. Hoeben, K. Hedman, and M.
Soderlund-Venermo. 2007. Construction and biological activity of a
full-length molecular clone of human Torque teno virus (TTN)
genotype6. FEBS J 274:4719-30; Leppik, L., K. Gunst, M. Lehtinen,
J. Dillner, K. Streker, and E. M. de Villiers. 2007. In vivo and in
vitro intragenomic rearrangement of TT viruses. J Virol
81:9346-56), it is important to construct an infectious TTSuV DNA
clone so that TTSuV can be used as a useful model to study the
replication and transcription mechanisms and to dissect the
structural and functional relationships of anellovirus genes. More
importantly, the availability of a TTSuV infectious DNA clone will
afford us an opportunity to use the pig as a model system to study
the replication and pathogenesis of TTSuV or even human TTV.
[0008] Multiple infections of human TTV with different genotypes in
a single human individual or TTSuV with different genotypes or
subtypes in a single pig have been documented (Ball, J. K., R.
Curran, S. Berridge, A. M. Grabowska, C. L. Jameson, B. J. Thomson,
W. L. Irving, and P. M. Sharp. 1999. TT virus sequence
heterogeneity in vivo: evidence for co-infection with multiple
genetic types. J Gen Virol 80 (Pt 7): 1759 68; Forms, X., P.
Hegerich, A. Darnell, S. U. Emerson, R. H. Purcell, and J. Bukh.
1999. High prevalence of TT virus (TTV) infection in patients on
maintenance hemodialysis: frequent mixed infections with different
genotypes and lack of evidence of associated liver disease. J Med
Virol 59:313-7; Gallei, A., S. Pesch, W. S. Esking, C. Keller, and
V. F. Ohlinger. 2010. 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
143:202-12; Huang, Y. W., Y. Y. Ni, B. A. Dryman, and X. J. Meng.
2010. Multiple infection of porcine Torque teno virus in a single
pig and characterization of the full-length genomic sequences of
four U.S. prototype PTTV strains: implication for genotyping of
PTTV. Virology 396:289-97; Jelcic, I., A. Hotz-Wagenblatt, A.
Hunziker, H. Zur Hausen, and E. M. de Villiers. 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 78:7498-507; Niel, C., F. L.
Saback, and E. Lampe. 2000. Coinfection with multiple TT virus
strains belonging to different genotypes is a common event in
healthy Brazilian adults. J Clin Microbiol 38:1926-30; Ninomiya,
M., M. Takahashi, T. Nishizawa, T. Shimosegawa, and H. Okamoto.
2008. Development of PCR assays with nested primers specific for
differential detection of three human anelloviruses and early
acquisition of dual or triple infection during infancy. J Clin
Microbiol 46:507-14). These findings raise the question whether the
anti-ORF1 capsid antibodies recognized by the antigen from a
particular TTV or TTSuV species/geno types also comprise anti-ORF1
antibodies against other distinct TTV or TTSuV species/genotypes
and whether the anti-ORF1 antibodies from one TTV or TTSuV genotype
can cross-protect against infection with another genotype. To our
knowledge, for human TTV or TTSuV infection there is no information
on this topic available to date. Furthermore, the antigenic
diversity and relationship of anelloviruses have never been
assessed (Maggi, F., and M. Bendinelli. 2009. Immunobiology of the
Torque teno viruses and other anelloviruses. Curr Top Microbiol
Immunol 331:65-90). It is reasonable to speculate that there is
little, if any, antigenic cross-reactivity between different
anellovirus species/genotypes, due to the fact that concurrent
infections with multiple anelloviruses in a single individual or
animal exist.
[0009] The inventors have previously developed and validated serum
Western blot (WB) and indirect ELISA assays for detection of the
IgG antibody against TTSuV2 in porcine sera using the purified
recombinant TTSuV2-ORF1 protein expressed in E. coli (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). By using
TTSuV2-specific real-time quantitative PCR (qPCR) and ELISA, The
inventors further presented the combined virological and
serological profile of TTSuV2 infection under natural or diseased
conditions using 160 porcine sera collected from different sources
(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). In the present
invention, The inventors initially aimed to assess the serological
profiles of the two TTSuV1 genotypes (TTSuV1a and TTSuV1b) in pigs,
respectively. Subsequently, the inventors aimed to compare the
virological and serological profiles of TTSuV1a and TTSuV1b with
that of TTSuV2, and to determine the degree of correlation of IgG
antibody levels between anti-TTSuV1a and -TTSuV1b and between
anti-TTSuV1a or -1b and anti-TTSuV2. Finally, for the first time,
the inventors assessed the antigenic relationships between two
TTSuV1 genotypes (TTSuV1a and TTSuV1b), between two species (TTSuV1
and TTSuV2), and between porcine and human genogroup 1
anelloviruses using ELISA and immunofluorescence assay with
antibody cross-reactions in PK-15 cells transfected with
recombinant plasmids expressing the ORF1s from TTSuV1a, TTSuV1b and
TTSuV2, respectively.
SUMMARY OF THE INVENTION
[0010] The present invention provides an infectious nucleic acid
molecule of Torque teno sus virus (TTsuV) comprising a nucleic acid
molecule encoding an infectious TTsuV which contains at least one
copy of genomic sequence having at least 85% homology to a genomic
sequence of TTsuV2.
[0011] According to one embodiment, the at least one copy of
genomic sequence having at least 95% homology to the genomic
sequence of TTsuV2.
[0012] According to another embodiment, the genomic sequence of
TTsuV2 is of genomic clone of PTTV2c-VA. In one specific example,
the genomic sequence is selected from sequences set forth in SEQ ID
NO:1.
[0013] According to a further embodiment, the genomic sequence of
TTsuV2 is of genomic clone of TTV2-#471942. In a specific example,
the genomic sequence is selected from sequences set forth in SEQ ID
NO:2.
[0014] According to an additional embodiment, the genomic sequence
of TTsuV2 comprising at least one genetic marker in intron 1. In a
specific example, the genetic marker in intron 1 is an artificially
introduced restriction site.
[0015] The present invention provides a biologically functional
plasmid or viral vector containing an infectious nucleic acid
molecule of Torque teno sus virus (TTsuV) comprising a nucleic acid
molecule encoding an infectious TTsuV which contains at least one
copy of genomic sequence having at least 85% homology to a genomic
sequence of TTsuV2.
[0016] According to one embodiment, the biologically functional
plasmid or viral vector contains more than one copy of the
infectious nucleic acid molecule.
[0017] According to one embodiment, the biologically functional
plasmid or viral vector contains tandem copies of genomic clone of
PTTV2c-VA.
[0018] The present invention provides an infectious TTsuV produced
by cells containing the infectious nucleic acid sequence of TTsuV2
is of genomic clone of PTTV2c-VA.
[0019] The present invention provides a method for diagnosing TTsuV
infection, comprising immobilizing an immunogentic fragment or a
complete protein of a polypeptide sequence of ORF1 protein of TTsuV
1 or 2, contacting a serum sample from a pig suspected of TTsuV
infection with the immobilized immunogentic fragment or complete
protein, and detecting captured antibody specific to the
immunogentic fragment.
[0020] According to one embodiment, the polypeptide sequence is
selected from the group consisting of ORF1 proteins of TTsuV
genotypes or subtypes TTsuV1a,\ or TTsuV1b.
[0021] According to another embodiment, the polypeptide sequence is
selected from the group consisting of N-terminal truncated ORF1
proteins of TTsuV genotypes or subtypes TTsuV1a, TTsuV1b or TTsuV2.
In a specific example, the polypeptide sequence is amino acid No.
317-635 of ORF1 protein of TTsuV1a. In another example, the
polypeptide sequence is amino acid No. 322-639 of ORF1 protein of
TTsuV1b.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The above-mentioned features of the invention will become
more clearly understood from the following detailed description of
the invention read together with the drawings in which:
[0023] FIG. 1 is a schematic diagrams of TTSuV2 constructs
containing full-length TTSuV2 genomic DNA. (A) pSC-PTTV2c (from the
U.S. TTSuV2 isolate PTTV2c-VA; GenBank accession no. GU456386). (B)
pSC-2PTTV2c-RR (tandem-dimerized PTTV2C-VA genomes). (C)
pSC-TTV2-#471942 (from the German TTSuV2 isolate TTV2-#471942;
GenBank accession no. GUI 88046). (D) pSC-2PTTV2b-RR
(tandem-dimerized TTV2-#471942 genomes). (E) pSC-TTV2-EU (derived
from pSC-TTV2-#471942). A HpaI site as the silent genetic marker
was introduced in this clone. (F) pSC-TTV2-US (derived from
pSC-PTTV2c). PstI and MfeI sites as the silent genetic markers were
introduced in this clone. (G) pSC-TTV2-AAA. A 104-bp deletion
mutation was introduced between the AccI and ApaI sites ranging
from the putative TATA box to the ORF1 start codon on the clone
pSC-TTV2-US. The restriction enzymes (BamHI or EcoRV) used for
plasmids constructions are shown. The plasmid backbone used for
cloning was the pSC-B-amp/kan vector (indicated by black). Grey
arrows indicate the TTSuV2 genomic copies.
[0024] FIG. 2 illustrates detection of TTSuV1 or TTSuV2
contamination in live different cell lines (PCV1-free PK-15,
3D4/31, IPEC/J2, BHK-21 and MARC-145) and an CHE diseases-free
porcine serum by real-time qPCR. Fluorescence curves (A and C) and
melting curves (B and D) of TTSuV1 (A and B) or TTSuV2 (C and D)
qPCR products are shown after 40 cycles of amplifications of the
standard template with the minimum dilution limit (10.sup.-4 pg;
indicated by red), five different cell lines (blue) and the porcine
serum (green). For each sample, duplicate determinations were made.
(E) Detection of specific TTSuV1 or TTSuV2 qPCR products (marked by
black arrowheads) by agarose gel electrophoresis.
[0025] FIG. 3 illustrates identification and quality assessment of
linear or circular TTSuV2 genomic DNA. (A) Comparisons of the
HindIII single-digestion patterns between clones pSC-TTV2-#471942
and pSC-2PTTV2b-RR (left panel) and AflII single-digestion patterns
between clones pSC-PTTV2c and pSC-2PTTV2c-RR (right panel) by
agarose gel electrophoresis. M: DNA markers. The results were
consistent to the predicted patterns of the digested fragments
(shown by black arrowheads). The 2.8-Kb fragments indicate the
intact single TTSuV2 genomic DNA from the clone pSC-2PTTV2b-RR or
pSC-2PTTV2c-RR. (B) Quality assessment of concatemerized ligation
products of the BamHl-digested and purified PTTV2c genomic DNA. The
samples were electrophoresed in a 1% agarose gel before (linear
DNA) and after (ligation mixture) T4 DNA ligase treatment. Linear
DNA (.about.2.8 Kb) and formations of the putative one-copy
(monomer), two-copy (dimer) and high-copy-number circular DNA are
indicated by arrowheads.
[0026] FIG. 4 illustrates Immunofluorescence assay (IFA) results on
PCV1-free PK-15 cells transfected with the ligation mixtures of
linear TTSuV2 genomic DNA derived from clones pSC-PTTV2c (A) or
pSC-TTV2-#471942 (C), with plasmids pSC-2PTTV2c-RR (B) or
pSC-2PTTV2b-RR (D), or with Lipofectamine LTX only (E). Cells were
stained with a rabbit anti-TTSuV2 ORF1 polyclonal antibody (Ab) and
a Texas Red-conjugated goat anti-rabbit IgG (red) at 5 days
post-transfection (the left panels), DAPI (blue) was used to stain
the cell nucleus (the middle panels). The Ab and DAPI stainings are
merged (right panels). Magnification=200.times..
[0027] FIG. 5 illustrates the putative transcription profile and
protein expression of TTSuV2 based on the PTTV2c-VA genome. (A)
Schematic diagram of three putative viral mRNAs and six viral
proteins. The TATA box, splicing sites (SD: splicing donor; SA:
splicing acceptor) and the positions of primers TTV2-448F and
TTV2-2316R were indicated at the top. The three open reading frames
(ORFs) are depicted by colored boxes. The sizes of the six ORFs and
two introns are also shown. (B) Sequencing of the RT-PCR products
amplified by primers TTV2-448F and TTV2-2316R verified the splicing
of the putative iritron 1. (C) Sequencing of the RT-PCR products
amplified by primers TTV2-448F and TTV2-2316R identified an
additional intron (intron 2). Arrows and numbers indicate the joint
site of the exons.
[0028] FIG. 6 illustrates IFA results of PCV1-free PK-15 cells
transfected with the ligation mixtures of linear TTSuV2 genomic DNA
derived from clones pSC-TTV2-EU, pSC-TTV2-US or pSC-TTV2-.DELTA.AA.
Cells were stained with an anti-TTSuV2 ORF1 antibody (Ab) and an
Alexa fluor 488-conjugated goat anti-rabbit IgC (green) at 3 days
post-transfection. DAPI (blue) was used to stain the cell nucleus.
Only merge of Ab and DAPI stainings are shown.
Magnification=200.times..
[0029] FIG. 7 illustrates transfection of nine different cell lines
with the ligation mixture of linear TTSuV2 genomic DNA derived from
the clone pSC-TTV2-US. Alexa fluor 488-conjugated antibody (Ab)
staining (green) merged with nuclear staining using DAPI (blue) are
shown. Magnification=200.times..
[0030] FIG. 8 illustrates expression and purification of the
amino-terminaUy truncated TTSuV1a and TTSuV1b ORF1 proteins,
respectively. (A) SDS-PAGE analysis of unpurified and purified
TTSuV1a-ORF1 products. (B) SDS-PAGE analysis of unpurified and
purified TTSuV1b-ORF1 products. An amino- and carboxyl-terminally
double-truncated TTSuV1b-ORF1 (lb-ctruc) of smaller product size
served as the control. (C) Near-infrared fluorescent WB analysis of
purified 1a- and 1b-GRF1 products using an anti-His-tagged mAb.
Open arrowheads indicate the truncated ORF1 protein of the expected
size whereas filled arrowheads show the presumably homodimers of
the expected proteins. M: protein markers.
[0031] FIG. 9 illustrates TTSuV1a or TTSuV1b serum WB and ELISA.
(A) WB analyses using the gnotobiotic pig serum samples from
Virginia and a commercial OIE diseases-free porcine serum as the
positive control reference serum (pos). (B) Representative results
of TTSuV1a WB analyses of conventional pig sera from a farm in
Wisconsin. Purified 1a-ORF1 protein was used as the antigen. Sera
tested negative for both TTSuV1a and TTSuV1b antibodies by WB were
pooled and used as the negative control reference serum. Open
arrowheads indicate the truncated ORF1 protein of expected size.
Only the bands in green color were considered as positive. M:
protein markers. (C) TTSuV1a or TTSuV1b ELISA results of the seven
Virginia gnotobiotic pig serum samples, positive and negative
control reference sera.
[0032] FIG. 10 illustrates serological and virological profiles of
TTSuV1 infection in 138 sera of pigs from three different herds.
(A) Distribution of TTSuV1 viremia, anti-TTSuV1a and anti-TTSuV1b
IgG among 138 serum samples. Box-and-Whisker-plots of TTSuV1a (B)
and TTSuV1b (C) serum antibody level by TTSuV1 viral DNA load. N:
Negative. The detection limit of the TTSuV1 real-time qPCR was 4
log.sub.10 copies/ml in this study.
[0033] FIG. 11 illustrates a retrospective evaluation of TTSuV1
viral loads (A), antibody levels to the ORF1 protein of TTSuV1a (B)
and TTSuV1b (C) in 10 pigs in group A from the time of their
arrival at the research facility to two months after arrival.
[0034] FIG. 12 illustrates box plots showing the comparisons of
anti-TTSuV1a (A) or anti-TTSuV1b (B) ORF1 antibody levels and
TTSuV1 (C) or PCV2 (D) viral loads between the PCVAD-affected and
-unaffected pigs.
[0035] FIG. 13 illustrates a high correlation between anti-TTSuV1a
and anti-TTSuV1b IgG in 138 serum samples. (A) Distribution of
anti-TTSuV1a, -TTSuV1b and -TTSuV2 IgG. (B) Scatter plots showing a
good linear relationship of antibody level between anti-TTSuV1a and
anti-TTSuV1b (p<0.0001).
[0036] FIG. 14 illustrates reactivity of the three purified TTSuV
ORF1 antigens: TTSuV1a (A), TTSuV1b (B) and TTSuV2 (C) with rabbit
antisera against ORF1s of TTSuV1a, TTSuV1b or TTSuV2 or with
pre-bleed rabbit serum with 2-fold serial dilutions by ELISAs. Each
antigen was tested in duplicate against each serum sample. Mean OD
values are presented.
[0037] FIG. 15 illustrates Immunofluorescence assay (IFA) results
of PCV1-free PK-15 cells transfected with the plasmids pTri-1aORF1
(A), pTri-1bORF1 (B) or pTri-2cORF1 (C) at 3 days
post-transfection. pTri-1aORF1- or pTri-1bORF1-transfected cells
were stained with the rabbit anti-TTSuV1a and -TTSuV1b ORF1
antiserum, respectively, whereas pTri-2cORF1-transfected cells were
stained with the rabbit anti-TTSuV2 ORF1 antiserum. The Alexa fluor
488-conjugated goat anti-rabbit IgG was used as the secondary Ab in
IFA (all the left panels). Ab staining merged with nuclear staining
using DAPI (blue) are shown in the right panels.
Magnification=200.times..
[0038] FIG. 16 illustrates comparison of hydrophilicity profiles of
TTSuV1a (PTTV1a-VA strain) and TTSuV1b (PTTV1b-VA strain) ORF1 and
identification of two putative common antigenic domains in ORF1 of
TTSuV1. The C-terminal region used for the expression of the
truncated 1a- or 1b-ORF1 is indicated by a box. The corresponding
alignment of amino acid (aa) sequences and aa positions of the two
domains are also shown. Favorable mismatches of the aa were
displayed as colons whereas neutral mismatches are depicted as
periods.
DETAILED DESCRIPTION OF THE INVENTION
[0039] FIG. 1 is a schematic diagrams of TTSuV2 constructs
containing full-length TTSuV2 genomic DNA. (A) pSC-PTTV2c (from the
U.S. TTSuV2 isolate PTTV2c-VA; GenBank accession no. GU456386; SEQ
ID NO:1). (B) pSC-2PTTV2c-RR (tandem-dimerized PTTV2C-VA genomes).
(C) pSC-TTV2-#471942 (from the German TTSuV2 isolate TTV2-#471942;
GenBank accession no. GUI 88046; SEQ ID NO:2). (D) pSC-2PTTV2b-RR
(tandem-dimerized TTV2-#471942 genomes). (E) pSC-TTV2-EU (derived
from pSC-TTV2-#471942). A HpaI site as the silent genetic marker
was introduced in this clone. (F) pSC-TTV2-US (derived from
pSC-PTTV2c). PstI and MfeI sites as the silent genetic markers were
introduced in this clone. (G) pSC-TTV2-AAA. A 104-bp deletion
mutation was introduced between the AccI and ApaI sites ranging
from the putative TATA box to the ORF1 start codon on the clone
pSC-TTV2-US. The restriction enzymes (BamHI or EcoRV) used for
plasmids constructions are shown. The plasmid backbone used for
cloning was the pSC-B-amp/kan vector (indicated by black). Grey
arrows indicate the TTSuV2 genomic copies.
[0040] In the present invention, the inventors describe the
construction and initial characterization of full-length DNA clones
of TTSuV2 in vitro and in vivo. The inventors provide, for the
first time, definite evidence of splicing of TTSuV2 mRNA and
expression of the putative ORF1 capsid protein by transfection of
the TTSuV2 full-length DNA clones in cultured cells. Furthermore,
rescue of TTSuV2 containing the introduced genetic markers in pigs
was confirmed by sequencing of viral DNA obtained from pigs
experimentally inoculated with the circular TTSuV2 genomic DNA.
Anellovirus is a group of single-stranded circular DNA viruses
infecting human and various other animal species. Animal models
combined with reverse genetics systems of anellovirus have not been
developed. The inventors report here the construction and initial
characterization of full-length DNA clones of a porcine
anellovirus, Torque teno sus virus 2 (TTSuV2), in vitro and in
vivo. The inventors first demonstrated that five cell lines
including PK-15 are free of TTSuV1 or TTSuV2 contamination, as
determined by real-time PCR and immunofluorescence assay (IFA)
using rabbit anti-TTSuV ORF1 sera. Recombinant plasmids harboring
monomeric or tandem-dimerized TTSuV2 genomic DNA that originated
from the United States and Germany were constructed. Circular
TTSuV2 genomic DNA with or without introduced genetic markers and
tandem-dimerized TTSuV2 plasmids were transfected into the PK-15
cells, respectively. Splicing of viral mRNAs was identified in
transfected cells. Expression of TTSuV2-specific ORF1 in cell
nuclei, especially in nucleoli, was detected by IFA. However,
evidence of productive TTSuV2 infection was not observed in 12
different cell lines including the 293TT cell line transfected with
the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2
deletion mutant did not produce ORF1 proteins, suggesting that the
observed ORF1 expression in this study is driven by TTSuV2 DNA
replication in cells. Pigs inoculated with either the
tandem-dimerized plasmids or circular DNA derived from the U.S.
strain of TTSuV2 containing genetic markers developed viremia, and
the introduced genetic markers were retained in viral DNA extracted
from the sera of infected pigs. The availability of an infectious
DNA clone of TTSuV2 will facilitate future study of porcine
anellovirus pathogenesis and biology.
[0041] Neither the viral DNA nor the expression of the putative
ORF1 capsid protein of TTSuV1 or TTSuV2 was endogenously present in
five representative cell lines tested in this study. The present
study first aimed to identify potential permissive cell lines
supporting the TTSuV propagation. The inventors selected five
commonly-used cell lines including three that are of pig origin:
PCV1-free PK-15, 3D4/31 and IPEC-J2, and two other cell lines
including BHK-21 and MARC-145. These cell lines are known to be
permissive for a wide variety of animal virus infections. In order
to rule out the possibility of endogenous contamination of TTSuV1
or TTSuV2 in cultured cell lines, both viral DNA and ORF1 protein
expression were subjected to TTSuV1 or TTSuV2 real-time qPCR and
IFA detections, respectively. An OIE diseases-free porcine serum,
which had been shown to have a high level of anti-TTSuV2 ORF1
antibody, was also included as a control (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 results obtained with the qPCR
analysis showed that none of the five cell lines tested in the
study were positive for TTSuV1 or TTSuV2 DNA, as determined by the
analyses of fluorescence curves, melting curves and agarose gel
electrophoresis, since their fluorescence curves were below the
minimum detection limit, their melting curves did not overlap with
that of the standards, and there were no detectable specific bands
corresponding to the expected PCR products (FIG. 2). In contrast,
as expected, the commercial porcine serum was positive for TTSuV1
and TTSuV2 DNA (FIG. 2).
[0042] Neither the viral DNA nor the expression of the putative
ORF1 capsid protein of TTSuV1 or TTSuV2 was endogenously present in
five representative cell lines tested in this study. The present
study first aimed to identify potential permissive cell lines
supporting the TTSuV propagation. The inventors selected five
commonly-used cell lines including three that are of pig origin:
PCV1-free PK-15, 3D4/31 and IPEC-J2, and two other cell lines
including BHK-21 and MARC-145. These cell lines are known to be
permissive for a wide variety of animal virus infections. In order
to rule out the possibility of endogenous contamination of TTSuV1
or TTSuV2 in cultured cell lines, both viral DNA and ORF1 protein
expression were subjected to TTSuV1 or TTSuV2 real-time qPCR and
IFA detections, respectively. An OIE diseases-free porcine serum,
which had been shown to have a high level of anti-TTSuV2 ORF1
antibody, was also included as a control (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 results obtained with the qPCR
analysis showed that none of the five cell lines tested in the
study were positive for TTSuV1 or TTSuV2 DNA, as determined by the
analyses of fluorescence curves, melting curves and agarose gel
electrophoresis, since their fluorescence curves were below the
minimum detection limit, their melting curves did not overlap with
that of the standards, and there were no detectable specific bands
corresponding to the expected PCR products (FIG. 2). In contrast,
as expected, the commercial porcine serum was positive for TTSuV1
and TTSuV2 DNA (FIG. 2).
[0043] To develop cell-based serological methods such as IFA or
immunoperoxidase monolayer assay (IPMA) for TTSuV detection, the
inventors raised three specific antisera against the putative ORF1
capsid protein of TTSuV1a, TTSuV1b (Huang, Y. W., K. K. Harrall, B.
A. Dryman, T. Opriessnig, E. M. Vaugh, M. B. Roof, and X. J. Meng.
2012. Serological profile of Torque teno sus virus species 1
(TTSuV1) in pigs and antigenic relationships between two TTSuV1
genotypes (1a and 1b), between two species (TTSuV1 and 2), and
between porcine and human anelloviruses. J. Virol. Submitted
Manuscript) or TTSuV2 in rabbits. When the five cell lines were
stained with each of the three virus-specific antisera,
respectively, no positive fluorescence signals were detected,
indicating the absence of endogenous TTSuV1 or TTSuV2 ORF1
expression (data not shown). The IFA results were consistent with
the qPCR detection, which demonstrated that the five selected cell
lines were not contaminated with TTSuV1 or TTSuV2 and thus can be
used for testing the susceptibility of TTSuV infection or
replication by transfection with TTSuV2 DNA clones.
[0044] Construction and characterization of full-length TTSuV2 DNA
clones in porcine kidney PK-15 cells. The inventors were
particularly interested in characterizing the infectivity of TTSuV2
full-length DNA clone since TTSuV2 has been reported to be
associated with PMWS or PCVAD at a high prevalence rate of viral
DNA (Kekarainen, T., M. Sibila, and J. Segales. 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 87:833-7), a high viral load (Aramouni, M., J. Segales, M.
Sibila, G. E. Martin-Valls, D. Nieto, and T. Kekarainen. 2011.
Torque teno sus virus 1 and 2 viral loads in postweaning
multisystemic wasting syndrome (PMWS) and porcine dermatitis and
nephropathy syndrome (PDNS) affected pigs. Vet Microbiol
153:377-81) and a low antibody level in disease-affected pigs with
an unknown mechanism (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 inventors first generated two monomeric full-length
TTSuV2 DNA clones, pSC-PTTV2c and pSC-TTV2-#471942, derived from a
prototype U.S. isolate PTTV2c-VA and a German isolate TTV2-#471942,
respectively (FIGS. 1A & 1C) (Gallei, A., S. Pesch, W. S.
Esking, C. Keller, and V. F. Ohlinger. 2010. 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 143:202-12; Huang, Y. W., Y. Y. Ni, B. A.
Dryman, and X. J. Meng. 2010. Multiple infection of porcine Torque
teno virus in a single pig and characterization of the full-length
genomic sequences of four U.S. prototype PTTV strains: implication
for genotyping of PTTV. Virology 396:287-97). Each of the
full-length TTSuV2 genomic DNA was inserted into a cloning vector
pSC-B-amp/kan that does not contain a eukaryotic promoter. The
restriction site BamHI or EcoRV is the unique site on the PTTV2c-VA
or TTV2-#471942 genome, which was engineered at both ends of
genomic DNA to facilitate the generation of concatemers and thus to
mimic the TTSuV circular DNA genome. BamHI or EcoRV single
digestion of the plasmid DNA of each clone clearly resulted in two
different fragments of 4.3-Kb and 2.8-Kb in size. The 4.3-Kb
fragment represented the backbone vector whereas the 2.8-Kb
fragment represented the inserted monomeric TTSuV2 genomic DNA
(data not shown).
[0045] Subsequently, two copies of the full-length PTTV2c-VA genome
from the clone pSC-PTTV2c were ligated in tandem into the
pSC-B-amp/kan vector to generate the clone pSC-2PTTV2c-RR (FIG.
1B). Comparison of the AflII single digestion patterns between
pSC-PTTV2c and pSC-2PTTV2c-RR showed that the latter clone had an
additional 2.8-Kb fragment representing the intact single TTSuV2
genomic DNA (FIG. 3A, right panel). The inventors utilized the same
cloning strategy to produce a tandem-dimerized TTSuV2 DNA clone,
pSC-2PTTV2b-RR, derived from pSC-TTV2-#471942 (FIG. 1D). Similarly,
when digested with HindIII alone, an additional 2.8-Kb fragment
representing the intact single TTSuV2 genome was presented in this
construct, compared to its monomeric parent clone (FIG. 3A, left
panel), thus confirming the successful construction of the
clone.
[0046] Circular TTSuV2 DNA was generated by tandem ligation of the
purified linear TTSuV2 genomic DNA excised from the clone
pSC-PTTV2c or pSC-TTV2-#471942. Typical monomer, dimer and
high-copy-molecules of concatemerized TTSuV2 DNA were observed in
the ligation products (FIG. 3B). The ligation mixture from
PTTV2c-VA or TTV2-#471942 was transfected into PCV1-free PK-15
cells. IFA conducted at five days post-transfection, using the
rabbit antiserum against PTTV2c-VA ORF1, indicated that TTSuV2 ORF1
antigen was expressed in the nuclei of the transfected cells with
approximately 5% positive rate (FIGS. 4A & 4C). No fluorescent
signal was observed in mock-transfected cells stained with the same
anti-TTSuV2 serum (FIG. 4E) or in circular TTSuV2 DNA-transfected
cells stained with the anti-TTSuV1a ORF1, anti-TTSuV1b ORF1 (Huang,
Y. W., K. K. Harrall, B. A. Dryman, T. Opriessnig, E. M. Vaugh, M.
B. Roof, and X. J. Meng. 2012. Serological profile of Torque teno
sus virus species 1 (TTSuV1) in pigs and antigenic relationships
between two TTSuV1 genotypes (1a and 1b), between two species
(TTSuV1 and 2), and between porcine and human anelloviruses. J.
Virol. Submitted Manuscript) or pre-bleed rabbit serum (data not
shown). Passaging of the transfected cells for two times did not
eliminate but reduced the fluorescent signal (data not shown). When
the transfected cells were continuously passaged for up to 20
passages, no positive signal was detectable, suggesting that TTSuV2
infection did not occur (data not shown).
[0047] The inventors next tested whether direct transfection of
plasmid DNA of the tandem-dimerized clone pSC-2PTTV2c-RR or
pSC-2PTTV2b-RR into PK-15 cells resulted in the synthesis of TTSuV2
ORF1. The tandem-dimerized double-stranded DNA does not represent
genomic anellovirus DNA but might represent an infectious
replicative intermediate. IFA at 5 days post-transfection using the
same anti-TTSuV2 ORF1 antiserum confirmed that both DNA clones also
expressed ORF1 in transfected PK-15 cells (FIGS. 4B & 4D).
Again, the ORF1 was expressed in cell nuclei. However, the
fluorescent intensity and positive rate were lower than that in
circular TTSuV2 DNA-transfected cells (FIGS. 4B & 4D). The
inventors did not observe the localization of ORF1 antigen in the
cytoplasm of the transfected cells.
[0048] Experimental identification of two introns in the TTSuV2
genome. Although the transcriptional profile using cloned TTSuV
full-length genomic DNA has not been reported, we previously
speculated that TTSuV likely expresses two essential viral mRNA
transcripts, mRNA1 and mRNA2, to produce the four known ORF
counterparts of human TTV (FIG. 5A) (Huang, Y. W., Y. Y. Ni, B. A.
Dryman, and X. J. Meng. 2010. Multiple infection of porcine Torque
teno virus in a single pig and characterization of the full-length
genomic sequences of four U.S. prototype PTTV strains: implication
for genotyping of PTTV. Virology 396:287-97). The continuous mRNA1
encodes ORF1 and ORF2 whereas removal of the putative intron of
1341 nt (designated intron 1 here), corresponding to nt positions
648-1988 in PTTV2c-VA genome, generates the putative mRNA2 that
encodes two discontinuous ORFs, ORF 1/1 and ORF2/2 (Huang, Y. W.,
Y. Y. Ni, B. A. Dryman, and X. J. Meng. 2010. Multiple infection of
porcine Torque teno virus in a single pig and characterization of
the full-length genomic sequences of four U.S. prototype PTTV
strains: implication for genotyping of PTTV. Virology 396:287-97).
The inventors also speculated that more spliced mRNAs and their
encoding proteins of TTSuV may exist, as shown in human TTV
(Mueller, B., A. Maerz, K. Doberstein, T. Finsterbusch, and A.
Mankertz. 2008. Gene expression of the human Torque Teno Virus
isolate P/1C1. Virology 381:36-45; Qiu, J., L. Kakkola, F. Cheng,
C. Ye, M. Soderlund-Venermo, K. Hedman, and D. J. Pintel. 2005.
Human circovirus TT virus genotype 6 expresses six proteins
following transfection of a full-length clone, J Virol
79:6505-10).
[0049] To verify whether the splicing of the putative intron 1 in
TTSuV2 occurred, total RNA was extracted in PK-15 cells transfected
with circular PTTV2c-VA DNA followed by DNase I treatment and
RT-PCR analysis. Two PCR product bands of approximately 500 bp and
600 bp in sizes were visualized by agarose gel electrophoresis.
Sequencing of the cloned PCR fragments resulted in the
identification of two sequences. As expected, the large cDNA
fragment of 583 bp was exactly the intron 1-spliced product (FIG.
5B), whereas the small cDNA product of 492 bp contained two
splicing regions including the intron 1 and an additional 91-nt
intron, corresponding to nt positions 2103-2193 in PTTV2c-VA
genome, which was designated intron 2 in this study (FIG. 5C). The
splicing sites are conserved among all published TTSuV2 sequences
(data not shown). Therefore, in this study for the first time the
inventors experimentally demonstrated the existence of splicing of
intron 1 and the viral mRNA2 transcripts. The inventors also
identified a novel viral mRNA transcript, termed mRNA3, which
encodes two putative proteins, ORF1/1/2 and ORF2/2/3, and which
switches reading frames from 1 to 2, and 2 to 3, respectively, due
to splicing of intron 2 (FIG. 5A). The mRNA3 transcript contains at
least three exons on the TTSuV2 genome. Since the inventors failed
to determine the 5'- and 3'-ends of the viral mRNA transcripts by
rapid amplification of cDNA ends (RACE)-PCR, it is possible that
there exists an additional TTSuV2 intron in the upstream of ORF2,
as known in human TTV transcripts (Mueller, B., A. Maerz, K.
Doberstein, T. Finsterbusch, and A. Mankertz. 2008. Gene expression
of the human Torque Teno Virus isolate P/1C1. Virology 381:36-45).
However, human TTV genome does not contain a short intron
corresponding to the TTSuV intron 2 in the downstream of the large
intron (intron 1).
[0050] Nevertheless, transfection of PK-15 cells with circularized
TTSuV2 genomic DNA resulted in the synthesis of viral mRNA
transcripts and the expression of ORF1 protein, indicating that the
TTSuV2 concatemers mimicked the transcription and protein
expression from the natural circular genome of TTSuV2.
[0051] A tandem-dimerized TTSuV2 clone, pSC-2PTTV2c-RR, is
infectious when inoculated in the CD pigs. To test the infectivity
of TTSuV2 DNA clones in pigs, the inventors first performed a pilot
study with three groups of CD pigs with two pigs per group. The
pigs were inoculated with PBS buffer (pig nos. 1 and 2) in group 1,
the tandem-dimerized clone pSC-2TTV2c-RR (pig nos. 3 and 4) in
group 2, and pSC-2TTV2b-RR (pig nos. 5 and 6) in group 3,
respectively. Serum samples were collected from animals at 0, 7,
14, 21, 28, 35 and 42 days post-inoculation (DPI). Pig no. 2 died
of septicemia due to an unidentified bacterial infection shortly
after inoculation.
[0052] TTSuV2 DNA was detected in two pigs inoculated with
pSC-2TTV2c-RR beginning at 28 DPI by real-time qPCR. The viral
loads, although very low, increased weekly until 42 DPI before
necropsy at 44 DPI in both pigs. The viral loads in serum of pig
no. 3 increased from 1.93.times.10.sup.3 at DPI 28 to
5.59.times.10.sup.3 at DPI 35 and 4.36.times.10.sup.4 at DPI 42
whereas the serum viral loads in pig no. 4 elevated from
5.07.times.10.sup.3 at DPI 28 to 4.49.times.10.sup.4 at DPI 35 and
8.87.times.10.sup.4 at DPI 42. Moderate microscopic lesions in
brain (lymphoplasmacytic encephalitis mainly perivascular), liver
(lymphohistiocytic hepatitis) and kidney (lymphoplasmacytic
interstitial nephritis) were observed in pig no. 3 but not in no.
4. The remaining three pigs including pigs inoculated with the
clone pSC-2TTV2b-RR did not develop viremia throughout the study.
However, pig no. 5 had mild lymphohistiocytic multifocal hepatitis.
The results from this pilot pig experiment indicated that the clone
pSC-2PTTV2c-RR originated from a U.S. strain of TTSuV2 is
infectious.
[0053] Characterization of two TTSuV2 full-length DNA clones with
engineered genetic markers and a derived mutant clone in vitro. To
further rule out the possible contamination of other indigenous
TTSuV2 infections in the pilot animal study, it is critical to
introduce tractable genetic markers in the TTSuV2 genome so that
the cloned virus and the potential indigenous contaminating virus
in pigs can be discriminated in inoculated animals. The inventors
introduced a unique HpaI restriction site and two unique
restriction sites, PstI and MfeI, into two TTSuV2 monomeric DNA
clones pSC-TTV2-#471942 and pSC-PTTV2c to produce two new clones
pSC-TTV2-EU and pSC-TTV2-US, respectively (FIGS. 1E and 1F). The
positions of these sites, located in the intron 1, were expected to
not change the putative ORF1 capsid amino acid sequence. PK-15
cells were transfected with ligation mixtures of the linear TTSuV2
genomic DNA excised from these two marker clones, respectively. The
ORF1 expression in nuclei of the transfected cells was detected by
IFA at 3 days post-transfection, similar to the patterns of their
parental clones (FIG. 6), indicating that the clones with
introduced genetic markers are replication competent.
[0054] A mutant clone pSC-TTV2-.DELTA.AA with a 104-bp deletion (nt
positions 332-437) from the putative TATA box (nt positions
283-289; FIG. 5A) to the ORF1 (nt 528) and ORF2 (nt 445) start
codons was generated based on the clone pSC-TTV2-US (FIG. 1G). When
transfected into the PK-15 cells, the circularized DNA from this
mutant clone did not express the ORF1 antigen (FIG. 6), suggesting
that the deleted region likely contains a cis-acting element
important for viral mRNA transcription or TTSuV2 ORF1 translation.
The result of the deletion mutant clone also implied that the
observed expression of ORF1 is likely driven by the
replication-competent TTSuV2 DNA since the tandem-dimerized clone
and concatemerized ligation products from the parental PTTV2c-VA
genome were both infectious in pigs (see below).
[0055] Expression of the TTSuV2 ORF1 protein in various cell lines
transfected with the circularized TTSuV2 DNA from the clone
pSC-TTV2-US. From the in vitro transfection experiments described
above, it appeared that, although the TTSuV2 putative ORF1 capsid
protein is expressed, the PK-15 cells do not support the
cell-to-cell spread of TTSuV2 recovered from the introduced TTSuV2
DNA clones. Alternatively, it is possible that the assembly of
TTSuV2 virions in the transfected PK-15 cells may be deficient. To
search for another cell line that may be permissive for TTSuV2
infection, the inventors subsequently transfected eleven other
different cell lines with the circularized TTSuV2 DNA from the
clone pSC-TTV2-US, respectively. These cell lines included the four
cell lines (3D4/31, IPEC-J2, BHK-21 and MARC-145) that were tested
negative for TTSuV1 or TTSuV2 at both the DNA and protein levels.
The plain cells of the other seven cell lines (ST, Vero, and 293TT,
HeLa, Huh-7, HepG2 and CHO-K1) were also negative for TTSuV2 ORF1
as determined by IFA (data not shown).
[0056] After transfection, all the eleven cell lines expressed the
ORF1 protein at 3 days post-transfection (FIG. 7; the results of
BHK-21 and CHO-K1 not shown). The percentages of transfected cells
with positive IFA signals were subjectively categorized into three
levels: IPEC-J2, ST, PCV1-free PK-15, Huh-7 and HepG2 with a high
level of positive rates (>5%); 3D4/31, Vero, MARC-145 and 293TT
with a middle level of positive rates (between 2-5%); Hela, BHK-21
and CHO-K1 with a low level of positive rates (<2%). In general,
TTSuV2-specific antibody staining patterns of individual positive
cells by IFA could be divided into three different types: (i) cells
displaying dense nuclear staining; (ii) cells displaying large
nuclear inclusion staining; and (iii) cells displaying punctate
nuclear staining. The last two patterns indicated the localization
of ORF1 antigen in cell nucleoli. No cytoplasmic staining was
observed in the transfected cells.
[0057] To test if some of these IFA-positive cells were susceptible
to TTSuV2 infection, supernatants collected from cell lysates of
PK-15, ST and 293TT cells transfected with circularized TTSuV2 DNA
were inoculated into all cell lines with high level positive rates
and some with middle level positive rates including the 293TT cell
line, respectively. The inoculated cells were cultured for 3 to 5
days and examined by IFA. No fluorescent signal was detected in
these cells (data not shown), indicating that none of the tested
cell lines are susceptible to productive TTSuV2 infection.
[0058] Rescue of TTSuV2 from concatamerized TTSuV2 DNA of the clone
pSC-TTV-US in CD/CD pigs. With the introduced genetic markers in
the full-length DNA clones that can be used to distinguish between
infections caused by the cloned virus and potential indigenous
contaminating virus, the inventors performed an additional study in
CD/CD pigs to further verify the in vivo infectivity of the TTSuV2
genomic DNA clones. Twelve CD/CD pigs were assigned into three
groups with four pigs each. Pigs in each group were inoculated with
PBS buffer, concatamerized "TTV2-EU DNA", and "TTV2-US DNA",
respectively. Pre-inoculation serum samples for all pigs (collected
at 30 days prior to inoculation) were tested negative for TTSuV1 or
TTSuV2 DNA by real-time qPCR. Serum samples were collected from all
animals at 0, 7, 14, 21, 28 and 35 DPI.
[0059] TTSuV2 DNA was detected in all eight inoculated pigs, but
unfortunately, it was also detected in two negative control pigs,
indicating contamination by other indigenous strains of TTSuV2 in
the research facility or the source pigs, which is not uncommon.
One pig (no. 133) inoculated with the concatamerized "TTV2-US DNA"
had a detectable viremia even at 0 DPI, whereas the other pigs
developed viremia at 14 or 21 DPI. Except for pig no. 133, the
seven TTSuV2 DNA-inoculated pigs and the two TTSuV2-positive pigs
in negative control group had an increased viral load until
necropsy, indicating active virus infection. The inventors
speculated that the source of the TTSuV2 contamination was likely
due to the 1-month waiting period between the date of
pre-inoculation serum sample testing (for which all animals were
all negative) and 0 DPI.
[0060] However, thanks to the introduced genetic markers in the
TTSuV2 DNA clones used in this study, the inventors were still able
to determine if the TTSuV2 DNA clones were infectious in pigs,
which was the main objective of our study. Since the inventors have
previously demonstrated that a single pig can be infected by
multiple strains of TTSuV2 and TTSuV1 (9, 17), then prior infection
or concurrent infection of an indigenous TTSuV2 strain should not
interfere with the infection of pigs by the TTSuV2 DNA clones the
inventors intended to test in this study. To determine if the
genetic markers of TTV2-EU or TTV2-US were present in viruses
recovered from the sera of infected pigs under the mixed TTSuV2
infection status, the inventors amplified and sequenced a 620-bp
region containing the engineered genetic markers from selected
samples at 35 DPI from both inoculated and negative control pigs.
The results showed that only the serum samples from pigs
experimentally inoculated with the concatamerized "TTV2-US DNA"
were found to have identical TTSuV2 sequences to the introduced
genetic markers PstI and MfeI, whereas serum samples from the
negative control group and from pigs inoculated with concatamerized
"TTV2-EU DNA" did not contain any introduced genetic markers (data
not shown). Therefore, this pig study further confirmed the initial
pilot pig study that the TTSuV2-US full-length DNA clone is
infectious in pigs. The results also experimentally verified, for
the first time, that pigs can be co-infected by different strains
of TTSuV2.
[0061] Little is known about the etiology and molecular biology of
anelloviruses due to the lack of a cell culture system to propagate
human TTV or TTSuV and the lack of a suitable animal model combined
with reverse genetics systems for anellovirus studies. Reports of
TTSuV DNA sequences detected in commercial porcine vaccine
products, porcine-derived human drugs and in porcine-derived
trypsin by nested PCR suggested a widespread contamination of TTSuV
(Kekearainen, T., L. Martinez-Guino, and J. Segales. 2009. Swine
torque teno virus detection in pig commercial vaccines, enzymes for
laboratory use and human drugs containing components of porcine
origin. J Gen Virol 90:648-53; Krakowka, S., S. S. Ringler, P.
Arumugam, J. McKillen, K. McIntosh, C. Hartunian, A. Hamberg, M.
Rings, G. Allan, and J. A. Ellis. 2008. Evaluation of Mycoplasma
hyopneumoniae bacterins for porcine torque teno virus DNAs. Am J
Vet Res 69:1601-7). Cell cultures may be one of the major sources
for TTSuV contamination in biological products of pig origin.
Therefore, the present study was first aimed at examining whether
five selected cell lines harbor endogenous DNA and protein antigen
of TTSuV1 or TTSuV2, and to further identify TTSuV-negative cell
lines that are potentially permissive for TTSuV propagation.
[0062] Surprisingly, none of the five cell lines tested in the
study were found to be positive for TTSuV1 or TTSuV2 DNA or ORF1
antigen (FIG. 2). Furthermore, screening of seven additional
commonly-used cell lines also yielded negative results as
determined by IFA detection, indicating that TTSuV contamination in
cell cultures is probably not as common as the inventors originally
thought. Our result was distinct from a recent study by a Brazilian
group that reported TTSuV DNA contamination in 15 out of 25 cell
lines (Teixeira, T. F., D. Dezen, S. P. Cibulski, A. P. Varela, C.
L. Holz, A. C. Franco, and P. M. Roehe. 2011. Torque teno sus virus
(TTSuV) in cell cultures and trypsin. PLoS One 6:e17501). In that
study, the five cell lines that were also used here in our study,
including PK-15, ST, BHK-21, Vero and MA-104 cells (from which the
MARC-145 cell line is derived) had been shown to have detectable
TTSuV1 and/or TTSuV2 sequences by using a one-round duplex PCR
assay (Teixeira, T. F., D. Dezen, S. P. Cibulski, A. P. Varela, C.
L. Holz, A. C. Franco, and P. M. Roehe. 2011. Torque teno sus virus
(TTSuV) in cell cultures and trypsin. PLoS One 6:e17501). It is
unclear why there is such a major discrepancy between our results
in this study and those by the Brazilian group. A reliable approach
to prove the presence of a contaminating virus in cell cultures
used in biological products is to determine its susceptibility to
virus infection, which has been exemplified by PCV1 (Beach, N. M.,
L. Cordoba, S. P. Kenney, and X. J. Meng. 2011. Productive
infection of human hepatocellular carcinoma cells by porcine
circovirus type 1. Vaccine 29:7303-6; Hattermann, K., C. Roedner,
C. Schmitt, T. Finsterbusch, T. Steinfeldt, and A. Mankertz. 2004.
Infection studies on human cell lines with porcine circovirus type
1 and porcine circovirus type 2. Xenotransplantation 11:284-94; Ma,
H., S. Shaheduzzaman, D. K. Willliams, Y. Gao, and A. S. Khan.
2011. Investigations of porcine circovirus type 1 (PCV1) in
vaccine-related and other cell lines. Vaccine 29:8429-37; Tischer,
I., H. Gelderblom, W. Vettermann, and M. A. Koch. 1982. A very
small porcine virus with circular single-stranded DNA. Nature
295:64-6). Theoretically, the possibility of TTSuV contamination in
cell cultures is very low, since anellovirus has been shown to be
extremely difficult to propagate in vitro. The present study
utilized the (i) more sensitive qPCR assay (compared to the
one-round PCR in the Teixeira et al study); (ii) the IFA; and (iii)
transfection of circular TTSuV genomic DNA into the cells as the
positive control (see below) to demonstrate the absence of TTSuV at
both the DNA and amino acids levels in 12 representative cell lines
including four of pig origin (PK-15, ST, 3D4/31 and IPEC-J2).
Therefore, based on the results from this study, the inventors
conclude that, contrary to what some may believe, there is very
little, if any, endogenous TTSuV contamination in well-established
continuous cell lineages. Instead, detection of contaminating TTSuV
DNA sequences in biological products reported by other groups may
come from the porcine-derived trypsin or serum (Kekearainen, T., L.
Martinez-Guino, and J. Segales. 2009. Swine torque teno virus
detection in pig commercial vaccines, enzymes for laboratory use
and human drugs containing components of porcine origin. J Gen
Virol 90:648-53; Teixeira, T. F., D. Dezen, S. P. Cibulski, A. P.
Varela, C. L. Holz, A. C. Franco, and P. M. Roehe. 2011. Torque
teno sus virus (TTSuV) in cell cultures and trypsin. PLoS One
6:e17501). The latter was actually confirmed in the present study
for the first time (FIG. 2).
[0063] Subsequently, the inventors demonstrated that all of these
TTSuV-free cell lines supported TTSuV2 ORF1 expression by
transfection with the circular TTSuV2 genomic DNA or the
tandem-dimerized TTSuV2 plasmids (FIG. 4, FIG. 6 and FIG. 7). The
TTSuV2 ORF1 protein was expressed in cell nuclei, especially in
nucleoli, which is consistent with the localization of human TTV
ORF1 in Huh-7 cells transfected with the circular full-length TTV
genomic DNA by immunoblotting with the ORF1-specific antibody
(Mueller, B., A. Maerz, K. Doberstein, T. Finsterbusch, and A.
Mankertz. 2008. Gene expression of the human Torque Teno Virus
isolate P/1C1. Virology 381:36-45). Most recently, it was also
reported that TTSuV1 or TTSuV2 ORF1-GFP fusion protein expressed
from the recombinant construct was accumulated in nucleoli of the
PK-15 cells (Martinez-Guino, L., M. Ballester, J. Segales, and T.
Kekarainen. 2011. Expression profile and subcellular localization
of Torque teno sus virus proteins. J Gen Virol 92:2446-57).
[0064] In addition, in this study TTSuV2-specific roRNA splicing
events were detected in transfected PK-15 cells by RT-PCR,
indicating the synthesis of viral mRNA transcripts in the
transfected cells. While the inventors experimentally demonstrated
the existence of two viral mRNAs transcripts (mRNA2 and mRNA3)
(FIG. 5), the putative mRNA 1 encoding the full-length ORF1 of
TTSuV2 was not detected (data not shown), which may suggest a lower
quantity and integrity of mRNA1 than that of mRNA2 and mRNA3. In
accordance with the result described by Martinez-Guino et al.,
splicing of the 91-nt intron 2 sequence in mRNA3 also occurred in
the post-transcription of TTSuV2 ORF1-GFP fusion gene based on
none-full-length viral clone (Martinez-Guino, L., M. Ballester, J.
Segales, and T. Kekarainen. 2011. Expression profile and
subcellular localization of Torque teno sus virus proteins. J Gen
Virol 92:2446-57).
[0065] The synthesis of viral mRNA transcripts and the subsequent
expression of the ORF1 or ORF1-related viral proteins in
transfected cells were driven by the endogenous TTSuV2 promoter.
The processes were also regulated by the unidentified cis-acting
elements, as we showed in this study that deletion of a 104-bp
sequence downstream of the TATA box completely eliminated ORF1
expression (FIG. 6). To our knowledge, this is the first
demonstration of porcine anellovirus viral mRNA and protein
expression and mutagenesis analysis based on the viral DNA
concatemers produced from circularized viral genomes or a
tandem-dimerized full-length clone.
[0066] It appeared that both PTTV2c-VA and TTV2-#471942 DNA
concatemers were replication-competent when transfected into cells
since they mimicked the natural TTSuV2 circular genome. However,
the rescue of PTTV2c-VA ("TTV2-US"), but not TTV2-#471942
("TTV2-EU"), was only demonstrated in two in vivo animal
experiments. The major sequence difference between these two TTSuV2
strains was in the GC-rich region. It has been proposed that the
GC-rich region in anelloviruses forms unique stem-loop structures,
which may play a significant role in viral replication (Miyata, H.,
H. Tsunoda, A. Kazi, A. Yamada, M. A. Khan, J. Murakami, T.
Kamahora, K. Shiraki, and S. Hino. 1999. Identification of a novel
GC-rich 113-nucleotide region to complete the circular,
single-stranded DNA genome of TT virus, the first human circovirus.
J Virol 73:3582-6; Okamoto, H., T. Nishizawa, M. Ukita, M. Taka
hash i, M. Fukuda, H. Iizuka, Y. Miyakawa, and M. Mayumi. 1999. The
entire nucleotide sequence of a TT virus isolate from the United
States (TUS01): comparison with reported isolates and phylogenetic
analysis. Virology 259:437-48). Further in-depth mutagenesis
analysis, which was not the scope of the present study, is required
to explain this discrepancy between the two clones.
[0067] The inventors also showed that, although the three cell
lines (PK-15, ST and 293TT) tested in the study supported a limited
level of TTSuV2 replication, the infection of these cells by
TTSuV2, if any, was non-productive since the supernatants of the
transfected cells did not induce a second-round infection. Most
recently, the 293TT cell line was shown to be susceptible for human
TTV propagation due to its expression of SV40 large T antigen at a
high level (5). The authors proposed that the human TTV genome
contains a conserved octanucleotide in the UTR forming a stem-loop
as the putative origin of replication. Five 4-bp motifs (CGGG and
GGGC) were found adjacent to the stem-loop, which may act as the
recognition sites for the SV40 large T antigen to facilitate TTV
replication (de Villiers, E. M., S. S. Borkosky, R. Kimmel, K.
Gunst, and J. W. Fei. 2011. The diversity of torque teno viruses:
in vitro replication leads to the formation of additional
replication-competent subviral molecules. J Virol 85:7284-95).
However, when the inventors performed a sequence alignment analysis
of the corresponding sequences among human TTV, TTSuV, Torque teno
canis virus (dog anellovirus) and Torque teno felis virus (cat
anellovirus), neither the conserved octanucleotide nor the 4-bp
motif was identified in the latter three anelloviruses (data not
shown). Therefore, the SV40 large T protein expressed in 293TT
cells likely does not provide the proposed helper effect on TTSuV
replication. Further study is needed to screen whether additional
cell lines are permissive to TTSuV2 infection.
[0068] Previous studies from our group and others have demonstrated
that, even under strictly controlled experimental conditions in
research facilities, TTSuV-negative pigs can easily acquire TTSuV
infection due to the ubiquitous nature of this virus in pigs and
environments (Gauger, P. C., K. M. Lager, A. L. Vincent, T.
Opriessnig, M. E. Kehrli, Jr., and A. K. Cheung. 2011. Postweaning
multisystemic wasting syndrome produced in gnotobiotic pigs
following exposure to various amounts of porcine circovirus type 2a
or type 2b. Vet Microbiol 153:229-39; 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). Although our second in vivo experiment
in the present study unfortunately "validated" these previous
reports, our results did demonstrate the successful rescue of
TTSuV2 in pigs inoculated with either the tandem-dimerized plasmids
or circular TTSuV2 DNA with the introduced genetic markers.
Unfortunately, due to the presence of indigenous TTSuV2 in the
CD/CD pigs from the second animal study, the inventors could not
analyze or correlate any pathological lesions in the inoculated
pigs to TTSuV infection. Therefore, a future study using the
germ-free gnotobiotic pig and the infectious DNA clone is warranted
to characterize the pathological lesions solely attributable to
TTSuV2 infection. The availability of the pig model combined with
the reverse genetics system of anellovirus described in this study
will facilitate future studies of porcine and even human
anellovirus biology and pathogenesis.
[0069] The family Anelloviridae includes human and animal Torque
teno viruses (TTV) with extensive genetic diversity. The antigenic
diversity among anelloviruses has never been assessed. Using Torque
teno sus virus (TTSuV) as a model, the inventors describe here the
first investigation on antigenic relationships among different
anelloviruses. Using the TTSuV1a or TTSuV1b ELISA based on the
respective recombinant ORF1 antigen and TTSuV1-specific real-time
PCR, the combined serological and virological profile of TTSuV1
infection in pigs was determined and compared with that of TTSuV2.
TTSuV1 is likely not associated with porcine circovirus associated
disease since both the viral loads and antibody levels were not
different between affected and unaffected pigs and since there was
no synergistic effect of concurrent PCV2/TTSuV1 infections. The
inventors did observe a higher correlation of IgG antibody levels
between anti-TTSuV1a and -TTSuV1b than between anti-TTSuV1a or -1b
and anti-TTSuV2 in these serum samples, implying potential
antigenic cross-reactivity. To confirm this, rabbit antisera
against the putative ORF1 capsid proteins of TTSuV1a, TTSuV1b or
TTSuV2 were raised and the antigenic relationships and diversity
among these TTSuVs were analyzed by ELISA. Additionally, antibody
cross-reactivity was analyzed using PK-15 cells transfected with
one of the three TTSuV ORF1 constructs. The results demonstrate
antigenic cross-reactivity between the two genotypes, TTSuV1a and
TTSuV1b, but not between the two species, TTSuV1a or 1b and TTSuV2.
In addition, an anti-genogroup 1 human TTV serum did not react with
any of the three TTSuV antigens. The results add to the knowledge
base on diversity among anelloviruses and have important
implications for diagnosis, classification and vaccine development
of TTSuVs.
[0070] Expression and purification of the N-terminally truncated
TTSuV1a and TTSuV1b ORF1 proteins. Previously the inventors had
successfully expressed a truncated TTSuV2 ORF1 protein in E. coli
(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). Using a similar
strategy, the C-terminal region of the TTSuV1a-ORF1 or TTSuV1b-ORF1
gene with a C-terminally engineered 8.times.His-tag was inserted
into the triple expression vector pTriEx1.1-Neo, resulting in two
recombinant constructs, pTri-1aORF1 and pTri-1bORF1. The inventors
also constructed an ORF1 C-terminally truncated version of 1b-ORF1
as a control, termed pTri-1bORF1-ctruc, which is 71-aa shorter than
1b-ORF1, to compare the size with that of pTri-1bORF1 in SDS-PAGE
and WB analysis.
[0071] The three recombinant proteins, 1a-ORF1, 1b-ORF1 and
1bORF1-ctruc were found to be insoluble and expressed within the
bacteria as inclusion bodies. Purification of the crude lysates
from 1a-ORF1 products with a nickel-affinity column resulted in
visualization of two bands of .about.40 KDa (white arrowheads) and
.about.70 KDa (black arrowheads), as analyzed by Coomassie blue
staining (FIG. 8A). The .about.40 KDa band is the expected product
of the truncated 1a-ORF1 protein, whereas the .about.70 KDa
polypeptide is an unknown product but should be derived from the
former since it also reacted with an anti-His-tagged Mab (see
below). Expression of 1b-ORF1 or 1bORF1-ctruc showed a smear in the
crude lysates (FIG. 8B). After purification, two bands of .about.40
KDa and .about.70 KDa, similar to 1a-ORF1, were also identified in
the purified 1b sample, whereas only a .about.30 KDa polypeptide
(white arrowheads) was detected in the purified 1b-ctruc sample
(FIG. 8B). The bands of .about.40 KDa and .about.30 KDa were
consistent with the expected sizes of 1b-ORF1 and 1bORF1-ctruc
protein products, respectively. All the identified polypeptides in
the purified products were detected by WB using the anti-His-tagged
Mab (FIG. 8C). The results indicated that both the truncated
1a-ORF1 and 1b-ORF1 proteins were successfully expressed in E. coli
and thus can be used as antigens for TTSuV1a and TTSuV1b antibody
detection in porcine sera.
[0072] Development of TTSuV1a- and TTSuV1b-based serum WB and
indirect ELISAs. In order to identify reference positive and
negative sera as controls, a total of 100 serum samples from
different sources including those from the gnotobiotic pigs were
collected. Samples were screened for anti-TTSuV1a or anti-TTSuV1b
IgG seropositivity by serum WB analysis using the purified 1a-ORF1
or 1b-ORF1 as the antigens, respectively. A TTSuV2-seropositive and
TTSuV1/TTSuV2-DNA positive porcine serum (Huang, Y. W., A. R.
Patterson, T. Opriessnig, B. A. Dryman, A. Gallei, K. K. Harrall,
E. M. Vaughn, M. B. Roof, and X. J. Meng. 2012. Rescue of a porcine
anellovirus (Torque teno sus virus 2) from cloned genomic DNA in
pigs. J. Virol. Submitted Manuscript) showed reactivity with the
1a-ORF1 and the 1b-ORF1 antigen, as the .about.40 KDa band was
presented in the WB analysis (FIG. 9A; two rightmost lanes).
Therefore, this serum was considered to be TTSuV1a- and
TTSuV1b-seropositive and thus was used as a reference positive
control for the ELISAs. All the seven Virginia and 12 Iowa
gnotobiotic pigs had no detectable TTSuV1a and TTSuV1b antibodies
(FIG. 9A). Except for a few serum samples from conventional pigs
from a Wisconsin swine farm (FIG. 9B; the two lanes on the left),
the remaining samples were tested positive for both TTSuV1a and
TTSuV1b antibodies by the WB analysis. The dual-negative serum
samples from Wisconsin conventional pigs were pooled and used as a
negative control reference serum.
[0073] With the available positive and negative control reference
sera, TTSuV1a- and TTSuV1b-based ELISAs were subsequently developed
and standardized, respectively. The concentrations of the purified
1a-ORF1 or 1b-ORF1 antigen, porcine sera and IgG conjugate were
determined by a checkerboard titration assay to ensure low
background signal and to give the highest difference of OD.sub.450
values between the positive and negative controls. WB-negative
gnotobiotic porcine sera showed very low OD values (<0.1)
compared to the negative control reference serum (FIG. 9C),
suggesting that these pig sera should not serve as a negative
control reference for detection of porcine field samples in the
ELISA test.
[0074] Association of TTSuV1 viral DNA loads and anti-TTSuV1a and
anti-TTSuV1b IgG antibody levels. A total of 160 serum samples were
collected and evaluated for the prevalence and viral DNA load of
TTSuV1 by real-time qPCR and for seroprevalence and antibody levels
(represented by S/N values) of anti-TTSuV1a and anti-TTSuV1b IgG by
the ELISAs. Among the 160 samples, 138 sera in groups A to C were
collected from three herds under field conditions whereas the
remaining 22 sera in groups D (gnotobiotic pigs) and E were
collected from pigs raised and housed under strictly controlled
experimental conditions in research facilities.
[0075] None of the 12 TTSuV1a/TTSuV1b-seronegative gnotobiotic pigs
in group D had a detectable viremia. In group E pigs, only one pig
was viremic whereas six were seropositive for TTSuV1a and among
them, one pig was also seropositive for TTSuV1b.
[0076] In groups A and C, 44 of 138 pigs were viremic (31.9%)
whereas 128 were TTSuV1a-seropositive (92.8%) and 121 were
TTSuV1b-seropositive (87.7%) (FIG. 10A). The incidence of TTSuV1
viremia was much lower than the TTSuV1a or 1b seropositive rate,
suggesting previous clearance of the virus by neutralizing
antibodies during the post-TTSuV1 infection convalescent period.
Similar to the previously obtained results for TTSuV2 (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), pigs with
undetectable TTSuV1 viral DNA load were more likely to have lower
levels of TTSuV1a and TTSuV1b antibody titers than pigs with TTSuV1
viral DNA load at the levels of 10.sup.4 to 10.sup.6 copies/ml
(p<0.05) in these three groups (FIGS. 10B & 10C).
[0077] All three markers of TTSuV1 infection, TTSuV1 DNA and
TTSuV1a/1b antibodies, were found in 40 serum samples. Notably, the
number of pigs that were TTSuV1a/TTSuV1b-dually seropositive but
viral DNA-negative (77 samples) was higher than that of pigs with
TTSuV1a- or TTSuV1b-seropositivity only (FIG. 10A). In addition,
the total number of porcine sera with both antibodies was 117
(40+77) among the 138 serum samples, implying that (i) co-infection
rates of pigs with TTSuV1a and TTSuV1b are high, which was
expected; and (ii) a certain degree of cross-reactivity may exist
between anti-TTSuV1a and anti-TTSuV1b IgG antibodies.
[0078] The inventors had previously demonstrated that, over a
two-month period, the 10 group-A pigs had decreasing TTSuV2 viral
loads that were associated with elevated anti-TTSuV2 ORF1 IgG
antibody levels (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). Whether
an analogous situation for TTSuV1 in these ten pigs existed was
subsequently analyzed in this study, by comparing the TTSuV1 viral
DNA loads and the anti-TTSuV1a or anti-TTSuV1b antibody levels in
sera from the time of their arrival until two months later. Five of
ten pigs were TTSuV1 DNA negative during the two months, and in
four pigs (ID#4314, 4316, 4319 and 4321) the viral DNA loads
decreased after two months, including in 3 pigs (ID#4314, 4319 and
4321) with no detectable TTSuV1 DNA (FIG. 11A). In contrast, both
the anti-TTSuV1a and anti-TTSuV1b antibody titers increased in all
10 pigs (FIGS. 11B & 11C). These results were consistent with
those of the TTSuV2 study.
[0079] TTSuV1 is likely not associated with PCVAD. The inventors
had previously found that PCVAD-affected pigs had a significantly
lower level of TTSuV2 antibody than PCVAD-unaffected pigs in group
B (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). However,
determination of the levels of anti-TTSuV1a and anti-TTSuV1b IgG
antibodies in these serum samples did not reveal a difference
between the PCVAD-affected and -unaffected pigs (FIGS. 12A &
12B). In addition, there was no statistically significant
difference of TTSuV1 viral loads between the PCVAD-affected and
-unaffected pigs (FIG. 12C). In contrast, PCV2 viral load was
significantly higher (p<0.05) in PCVAD-affected pigs compared to
PCVAD-unaffected pigs (FIG. 12D).
[0080] The inventors further analyzed whether there existed a PCV2
and TTSuV1 synergistic effect associated with PCVAD. Serum viral
DNA prevalence rates (viremia) of PCVAD-affected pigs were as
follows: 50% (16/32) for PCV2 and TTSuV1, 56% (14/25) for PCV2
only, 0% (0/1) for TTSuV1 only, and 0% (0/2) for no detectable
virus. These proportions were not significantly different
(p=0.4339). The above results suggested that TTSuV1 is likely not
associated with PCVAD.
[0081] Comparison and correlations of seroprevalence and antibody
levels among anti-TTSuV 1a, anti-TTSuV1b and anti-TTSuV2. Mixed
infections of TTSuV1 and TTSuV2 are common in pigs, as determined
by the presence of viral DNA of both TTSuV1 and TTSuV2 in the same
pig using PCR (Gallei, A., S. Pesch, W. S. Esking, C. Keller, and
V. F. Ohlinger. 2010. 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
143:202-12; 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; 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; Huang, Y.
W., Y. Y. Ni, B. A. Dryman, and X. J. Meng. 2010. Multiple
infection of porcine Torque teno virus in a single pig and
characterization of the full-length genomic sequences of four U.S.
prototype PTTV strains: implication for genotyping of PTTV.
Virology 396:289-97). In this study, the inventors provided the
serological evidence to support this conclusion by analyzing the
seroprevalence distribution of anti-TTSuV1a, -TTSuV1b and -TTSuV2
IgG in the 138 serum samples in groups A-C. As shown in FIG. 6A, 82
of 138 serum samples were triple-seropositive, indicating that
these pigs had been infected by TTSuV1 (TTSuV1a and/or TTSuV1b) and
TTSuV2.
[0082] The distribution of dual seropositive samples was
significantly different. A total of 117 (82+30+5) porcine sera were
dually-seropositive for both anti-TTSuV1a and anti-TTSuV1b, which
was consistent with the number calculated in FIG. 10A. In contrast,
dual seropositivity to anti-TTSuV1a and anti-TTSuV2, or to
anti-TTSuV1b and anti-TTSuV2, each occurred in only one sample
(FIG. 13A).
[0083] Furthermore, correlations of antibody levels between
anti-TTSuV1a and anti-TTSuV 1b, between anti-TTSuV1a and
anti-TTSuV2, and between anti-TTSuV1b and anti-TTSuV2 were assessed
in the 138 serum samples by using Spearman's correlation
coefficient. A good linear relationship was observed between the
anti-TTSuV1a and anti-TTSuV1b (FIG. 13B; Spearman's rank
correlation coefficient=0.91, p<0.0001). When all the 160
samples were included, a better agreement was obtained (Spearman's
rank correlation coefficient=0.93, p<0.0001). A lesser degree of
correlation between anti-TTSuV1a and anti-TTSuV2 or between
anti-TTSuV1b and anti-TTSuV2 was found when compared to that
between anti-TTSuV1a and anti-TTSuV1b (data not shown). The results
further revealed an association of seroprevalence and antibody
levels between anti-TTSuV1a and anti-TTSuV1b, and thus it is
logical to hypothesize that there exists an antigenic
cross-reactivity between the two TTSuV1a and TTSuV1b genotypes.
[0084] Analysis of antigenic relationships among TTSuV1a, TTSuV1b
and TTSuV2 by ELISA. Three antisera against the truncated
recombinant ORF1s of TTSuV1a, TTSuV1b or TTSuV2 were raised by
immunization of rabbits with the respective purified recombinant
antigen. Cross-immunoreactivity studies were initially performed to
assess whether one of these antigens could cross-react with
antisera against the other two antigens in an ELISA format. The
pre-bleed rabbit serum was used as the negative control. As
expected, each of three TTSuV antigens reacted with its
corresponding homologous antiserum but not with the pre-bleed
negative control serum (OD values<0.1) that were serially
diluted from 1:200 to 1:1600 (FIG. 14A-14C).
[0085] The TTSuV2 antigen did not appear to cross-react with
TTSuV1a or TTSuV1b antiserum even at 1:200 dilution since the OD
value was relatively low (FIG. 14C). In contrast, the TTSuV1b
antigen did cross-react with the anti-TTSuV1a serum (as shown at
1:200 and 1:400 dilutions, both OD values>0.5) but not with the
anti-TTSuV2 serum (FIG. 14B) whereas the TTSuV1a antigen likely
cross-reacted with the anti-TTSuV1b serum (at 1:200 dilution) but
not with the anti-TTSuV2 serum (FIG. 14A). The ELISA results
strongly supported our hypothesis that there is an antigenic
cross-reactivity between the two TTSuV1a and TTSuV1b genotypes but
not between the two species TTSuV1a or 1b and TTSuV2.
[0086] Demonstration of antigenic relationships among TTSuV1a,
TTSuV1b and TTSuV2, and between TTSuVs and a genogroup 1 human TTV
by IFA. In order to definitely analyze the antigenic
cross-reactivity among these viruses, an antibody cross-reactivity
experiment was performed by using IFA staining. PK-15 cells were
transfected with three plasmid constructs, pTri-1aORF1, pTri-1bORF1
and pTri-2cORF1, which harbor the truncated ORF1 capsid genes from
TTSuV1a, TTSuV1b and TTSuV2, respectively. Three days
post-transfection, cells were stained with anti-TTSuV1a,
anti-TTSuV1b, anti-TTSuV2 and pre-bleed serum, respectively. As
shown in FIG. 15, cells transfected with pTri-1aORF1 (FIG. 15A) or
pTri-1bORF1 (FIG. 15B) stained positive with both anti-TTSuV1a and
anti-TTSuV1b but not with the anti-TTSuV2 or the pre-bleed serum
(data not shown), whereas cells transfected with pTri-2cORF1 only
reacted with anti-TTSuV2 serum (FIG. 15C). Each TTSuV1 antiserum
reacted stronger with its own homologous antigen than the
heterologous antigen based on comparison of the positive cell
numbers and fluorescence intensity (FIGS. 15A and 15B). The
truncated ORF1s were expressed in both nuclei and cytoplasm of the
transfected cells (FIG. 15), which was different from what we found
in cells transfected with full-length TTSuV DNA clones (15),
probably due to the lack of most of the putative nuclear
localization signals (NLS) located at the N-terminal part of the
ORF1 in the truncated genes (computer analysis; data not shown).
Table 1 summarizes the results of the cross-reactive immunostaining
study. In addition, when transfected cells were each stained with
an anti-human genogroup 1 TTV ORF1 antiserum (AK47; raised in
rabbits), no fluorescent signal was detected. Mock-transfected
cells did not stain with any of the five antisera (Table 1). The
IFA result further confirmed the presence of antigenic
cross-reactivity between TTSuV1a and TTSuV1b as shown by the ELISA
but not between the TTSuV1a or 1b and TTSuV2. The results also
revealed that there was no antigenic cross-reactivity between
genogroup 1 human TTV and porcine anelloviruses.
[0087] Identification of two putative antigenic sites on the ORF1
shared by TTSuV1a and TTSuV1b by sequence analyses. The full-length
ORF1 proteins between TTSuV1 and TTSuV2 shared only 22.4-25.8%
amino acid (aa) sequence identity with no significantly conserved
regions identified (14). The ORF1 proteins of the two TTSuV species
share only 19.1-21.0% aa sequence identity with that of the human
genogroup 1 TTV isolate P/1C1 (GenBank accession no. AF298585). The
high ORF1 sequence divergences between TTSuV1 and TTSuV2 and
between porcine and human anelloviruses likely account for the
absence of antigenic cross-reactivity observed in this study.
[0088] However, the aa sequence identity of ORF1 between the two
TTSuV1a and TTSuV1b genotypes (six isolates available in GenBank)
ranged between 49.4-52.4%. The inventors have previously found that
conserved sites exist in the ORF1 of different TTSuV1 stains except
for the four proposed variable regions (30.0-37.5% aa identity)
(Huang, Y. W., Y. Y. Ni, B. A. Dryman, and X. J. Meng. 2010.
Multiple infection of porcine Torque teno virus in a single pig and
characterization of the full-length genomic sequences of four U.S.
prototype PTTV strains: implication for genotyping of PTTV.
Virology 396:289-97). In order to identify the common antigenic
sites on the ORF1 between the genotypes TTSuV1a and TTSuV1b, the
inventors performed a comparative analysis of hydrophilicity
profiles of the ORF1 aa sequences between PTTV1a-VA and PTTV1b-VA.
Two conserved hydrophilic regions located at the middle and
C-terminal regions were identified (FIG. 16). The C-terminal
antigenic domain appeared to be more antigenic than the domain in
the middle region. Alignment of the two putative antigenic regions
among all published TTSuV1 sequences revealed a high degree of
sequence conservation (data not shown).
[0089] The immunology of anellovirus is poorly understood (20).
Detection of specific adaptive immune responses can provide
insights into anellovirus epidemiology. By analogy to the chicken
anemia virus (CAV), another single-stranded circular DNA virus, the
ORF1 product of anelloviruses is believed to function as the
putative capsid protein and thus represents the major viral antigen
(Crowther, R. A., J. A. Berriman, W. L. Curran, G. M. Allan, and D.
Todd. 2003. Comparison of the structures of three circoviruses:
chicken anemia virus, porcine circovirus type 2, and beak and
feather disease virus. J Virol 77:13036-41; Maggi, F., and M.
Bendinelli. 2009. Immunobiology of the Torque teno viruses and
other anelloviruses. Curr Top Microbiol Immunol 331:65-90).
[0090] Detection of human TTV IgG antibodies in human populations
based on the human TTV ORF1 as the antigen has been reported
(Maggi, F., and M. Bendinelli. 2009. Immunobiology of the Torque
teno viruses and other anelloviruses. Curr Top Microbiol Immunol
331:65-90). Handa et al reported a 38% prevalence of human TTV
antibody among 100 American blood donors when using the N-terminal
part (aa 1-411) containing the arginine-rich region of ORF1 of a
human genotype 1b TTV isolate as the antigen (Handa, A., B.
Dickstein, N. S. Young, and K. E. Brown. 2000. Prevalence of the
newly described human circovirus, TTV, in United States blood
donors. Transfusion 40:245-51). In contrast, antibody reactivity in
humans to the N-terminus of ORF1 (ORF1-N) of a human TTV genotype 6
was not detected by a Finish group. After removal of the
arginine-rich region (aa 1-62), the arginine-deleted constructs
(ORF1AArg and ORF1-NAArg) as well as the C-terminal portion
(ORF1-C; aa 344-737) were expressed, 48% human TTV IgG prevalence
was detected in sera of 21 healthy Finnish adults using the three
products as the antigens (Kakkola, L., H. Bonden, L. Hedman, N.
Kivi, S. Moisala, J. Julin, J. Yla-Liedenpohja, S. Miettinen, K.
Kantola, K. Hedman, and M. Soderlund-Venermo. 2008. Expression of
all six human Torque teno virus (TTV) proteins in bacteria and in
insect cells, and analysis of their IgG responses. Virology
382:182-9). Two other groups also utilized similar strategies
targeting the C-terminal region to successfully express human TTV
ORF1. Muller et al demonstrated that an ORF1-specific antiserum
against the C-terminal part of ORF1 (aa 402-733) of the human TTV
isolate P/1C1 generated in a rabbit was able to detect ORF1
expression in cell culture (21), whereas a French group reported
the detection of anti-human TTV ORF1 IgG antibodies in 69 of 70
French subjects including 30 blood donors, 30 cryptogenic hepatitis
patients and 10 healthy children using an ORF1 C-terminus-based WB
analysis (Ott, C., L. Duret, I. Chemin, C. Trepo, B. Mandrand, and
F. Komurian-Pradel. 2000. Use of a TT virus ORF1 recombinant
protein to detect anti-TT virus antibodies in human sera. J Gen
Virol 81:2949-58). Most recently, our group successfully used the
C-terminal fragment of the ORF1 protein of a U.S. strain of TTSuV2
as the antigen to detect TTSuV2-specific IgG antibodies in pig sera
by ELISA (13). Together with the present study for serological
detections of the two porcine TTV species-1 genotypes TTSuV1a and
TTSuV1b, the obtained data suggest that the C-terminal portion of
ORF1 of anelloviruses is an appropriate target for the development
of serodiagnostic assays.
[0091] Indeed, based on the CAV virion structure determined by
cryo-electron microscopic images, the C-terminal half portion of
the ORF1 is proposed to form the outer part of the capsid that is
exposed to the virion surface whereas the basic N-terminal part of
the CAV ORF1 is proposed to be inside the capsid to bind the viral
DNA, and the middle part of the ORF1 is proposed to form the inner
shell of the capsid (Crowther, R. A., J. A. Berriman, W. L. Curran,
G. M. Allan, and D. Todd. 2003. Comparison of the structures of
three circoviruses: chicken anemia virus, porcine circovirus type
2, and beak and feather disease virus. J Virol 77:13036-41). The
ORF1 polypeptide of anellovirus has been suggested to be organized
in the same way as that of CAV (Crowther, R. A., J. A. Berriman, W.
L. Curran, G. M. Allan, and D. Todd. 2003. Comparison of the
structures of three circoviruses: chicken anemia virus, porcine
circovirus type 2, and beak and feather disease virus. J Virol
77:13036-41). This proposed structure is consistent with the
computer analysis of the ORF1 hydrophilicity profiles of TTSuV1
(FIG. 16) and TTSuV2 (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). In either case, there are two conserved major
hydrophilic regions located at the middle and C-terminal regions
that span the C-terminal half portion of the ORF1.
[0092] Reliability and specificity of the established ELISAs for
differential TTSuVs antibody detections were guaranteed by
screening of the positive and negative reference sera through a
serum WB. It was further demonstrated by triple seronegativity of
TTSuV1a, TTSuV1b and TTSuV2 in gnotobiotic pigs of group D (FIG.
9). A high seropositive rate of TTSuV1a (92.8%) or TTSuV1b (87.7%)
was revealed in the 138 groups A-C pigs (FIG. 10A), which was
higher than that of TTSuV2 (.about.60%) (13), indicating a wider
spread of actual TTSuV1 infection or the presence of
long-persisting anti-TTSuV1 ORF1 antibodies in these pigs
regardless of a low incidence of TTSuV1 viremia. Accordingly, these
results, for the first time, provided serological evidence
supporting multiple infections of TTSuV1a, TTSuV1b and TTSuV2 in
the same pigs. To our knowledge, this is also the first study
demonstrating multiple anellovirus infections in the same animals
by using serological diagnosis in addition to the PCR assay.
Therefore, the subsequent question raised was to determine the
specificity of seropositivity and cross-antigenic reactivity among
different TTSuV species and genotypes.
[0093] In this study, the inventors demonstrated by investigating
four different aspects that indeed there exists antigenic
cross-reactivity between the two TTSuV1a and TTSuV1b genotypes but
not between the two TTSuV species (TTSuV1a or 1b and TTSuV2).
First, when compared to the serum samples with single TTSuV1a- or
TTSuV1b-seropositivity, the numbers of serum samples with
TTSuV1a/1b-dual seropositivity was much higher (FIG. 10A), likely
implying a certain degree of cross-antigenic reactivity between
TTSuV1a and TTSuV1b antibodies. Secondly, the number of serum
samples with dual TTSuV1a and TTSuV1b seropositivity was
significantly higher than that of dual seropositivity to TTSuV1a
and TTSuV2, or to TTSuV1b and TTSuV2 (FIG. 13A). In addition, a
high correlation of antibody levels between anti-TTSuV1a and
anti-TTSuV1b as assessed by Spearman's correlation coefficient was
observed (FIG. 13B). These analyses were conducted under the
background of multiple TTSuV infections in field samples, which led
us to propose a logical hypothesis regarding the presence of an
antigenic cross-reactivity between TTSuV1a and TTSuV1b. Thirdly,
this hypothesis was experimentally confirmed by analysis of the
antigenic relationships among TTSuV1a, TTSuV1b and TTSuV2 through
antigen-specific ELISAs (FIG. 14), and antibody cross-reactivity
studies in PK-15 cells transfected with the three TTSuV ORF1
constructs, respectively (FIG. 15 and Table 1). Finally, sequence
comparison of ORF1 of the TTSuV also supported the observed
epidemiologic and experimental data in this study: while there was
no significant sequence homology of TTSuV1a or 1b ORF1 with that of
TTSuV2, the inventors identified two putative antigenic sites on
the ORF1 that are shared by TTSuV1a and TTSuV1b (FIG. 16).
[0094] In addition, in this study the inventors also demonstrated
the absence of antigenic cross-reactivity between TTSuVs and a
human genogroup 1 TTV by IFA. Taken together, the results from this
study have important implications in predicting the antigenic
cross-reactivity among different anelloviruses based on the ORF1 aa
sequence homology. Currently, anelloviruses are classified into
nine genera according to the infected host species (human/ape,
tamarin, douroucouli, tupaia, pig, dog and cat), nucleotide
sequence identity and the genome size of primate anelloviruses
(TTV, TTMV and TTMDV) (Biagini, P., M. Bendinelli, S. Hino, L.
Kakkola, A. Mankertz, C. Niel, H. Okamoto, S. Raidal, C. G. Teo,
and D. Todd. 2011. Anelloviridae, p. 331-341. In A. M. Q. King, M.
J. Adams, E. B. Carstens, and E. J. Lefkowitz (ed.), Virus
Taxonomy, 9th Report of the ICTV. Elsevier Academic Press, London).
The ORF1 of the TTSuV (Genus Iotatorquevirus) share 15.6-22.3% aa
sequence identity with the other eight genera based on multiple
sequence alignment (data not shown), which is similar to that
between TTSuVs and the human genogroup 1 TTV (19.1-21.0%).
Therefore, it is reasonable to deduce that porcine anellovirus is
not antigenically cross-reactive with other anelloviruses in other
animal species. The ORF1 aa sequence homologues among the nine
genera range from 15.0% to 27.3% (data not shown), thus implying
that antigenic diversity between different genera does exist.
[0095] The two TTSuV species (TTSuV1 and TTSuV2) do not share
antigenicity in the ORF1 antigen since they only had 22.4-25.8% aa
sequence identity, whereas the two TTSuV1 genotypes (TTSuV1a and
1b) were antigenically related and cross-reactive due to their
higher aa sequence homology (49.4-52.4%). It is possible that the
antigenic relationship of different anelloviruses in the same genus
may depend on a threshold or a range of aa sequence homology. The
available data using TTSuV as a model will provide insights into
similar research of antigenic diversity on human anelloviruses
(TTV, TTMV and TTMDV) in the future.
[0096] The present study on TTSuV1 together with our previous study
on TTSuV2 (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) also
revealed a broader picture of the nature of mixed TTSuVs infections
under natural or clinically disease conditions by assessing the
serological and virological profiles. It is not surprising to see
in this study that several features of TTSuV1 infection were
consistent with that of TTSuV2 (FIG. 3 & FIG. 11). More
importantly, the inventors provided new evidence to support the
current opinion that TTSuV1 is likely not associated with PCVAD (1,
18, 23), by demonstrating that both viral loads and antibody levels
were not significant different between PCVAD-affected and
-unaffected pigs (FIG. 12), and that there was no significant
PCV2/TTSuV1 synergic effect. It is not known whether the presence
of ORF1 antibody is protective against homologous TTSuV infection.
However, since antibodies to TTSuV1 or TTSuV2 ORF1 do not
cross-react with the heterologous TTSuV antigen, it appears that
TTSuV1 infection and the consequent humoral immune response do not
interfere with TTSuV2 infection. Therefore, this may make the
development of a single vaccine against the two recognized TTSuV
species difficult. Together, the results from the present study
have important implications in understanding the diversity of
anellovirus, and in diagnosis and vaccine development of
TTSuVs.
[0097] Vaccines of the infectious viral and infectious molecular
DNA clones, and methods of using them, are also included within the
scope of the present invention. Inoculated pigs are protected from
viral infection and associated diseases caused by TTV2 infection or
co-infection. The novel method protects pigs in need of protection
against viral infection by administering to the pig an
immunologically effective amount of a vaccine according to the
invention, such as, for example, a vaccine comprising an
immunogenic amount of the infectious TTsuV DNA, a plasmid or viral
vector containing the infectious DNA clone of TTsuV, the
recombinant TTsuV DNA, the polypeptide expression products, the
bacteria-expressed or baculovirus-expressed purified recombinant
ORF1 capsid protein, etc. Other antigens such as PRRSV, PPV, other
infectious swine agents and immune stimulants may be given
concurrently to the pig to provide a broad spectrum of protection
against viral infections.
[0098] The vaccines comprise, for example, the infectious viral and
molecular DNA clones, the cloned TTsuV infectious DNA genome in
suitable plasmids or vectors such as, for example, the pSC-B
vector, an avirulent, live virus, an inactivated virus, expressed
recombinant capsid subunit vaccine, etc. in combination with a
nontoxic, physiologically acceptable carrier and, optionally, one
or more adjuvants. The vaccine may also comprise the infectious
TTsuV2 molecular DNA clone described herein. The infectious TTsuV
DNA, the plasmid DNA containing the infectious viral genome and the
live virus are preferred with the live virus being most preferred.
The avirulent, live viral vaccine of the present invention provides
an advantage over traditional viral vaccines that use either
attenuated, live viruses which run the risk of reverting back to
the virulent state or killed cell culture propagated whole virus
which may not induce sufficient antibody immune response for
protection against the viral disease.
[0099] Vaccines and methods of using them are also included within
the scope of the present invention. Inoculated mammalian species
are protected from serious viral infection, may also provide
protection for disease related to co-infection of TTsuV, such as
porcine dermatitis and nephropathy syndrome (PDNS), postweaning
multisystemic wasting syndrome (PMWS), and other related illness.
The vaccines comprise, for example, an inactivated or attenuated
TTsuV virus, a nontoxic, physiologically acceptable carrier and,
optionally, one or more adjuvants.
[0100] The adjuvant, which may be administered in conjunction with
the vaccine of the present invention, is a substance that increases
the immunological response of the pig to the vaccine. The adjuvant
may be administered at the same time and at the same site as the
vaccine, or at a different time, for example, as a booster.
Adjuvants also may advantageously be administered to the pig in a
manner or at a site different from the manner or site in which the
vaccine is administered. Suitable adjuvants include, but are not
limited to, aluminum hydroxide (alum), immunostimulating complexes
(ISCOMS), non-ionic block polymers or copolymers, cytokines (like
IL-1, IL-2, IL-7, IFN-.alpha., IFN-.beta., IFN-.gamma., etc.),
saponins, monophosphoryl lipid A (MLA), muramyl dipeptides (MDP)
and the like. Other suitable adjuvants include, for example,
aluminum potassium sulfate, heat-labile or heat-stable enterotoxin
isolated from Escherichia coli, cholera toxin or the B subunit
thereof, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's
incomplete or complete adjuvant, etc. Toxin-based adjuvants, such
as diphtheria toxin, tetanus toxin and pertussis toxin may be
inactivated prior to use, for example, by treatment with
formaldehyde.
[0101] The vaccines may further contain additional antigens to
promote the immunological activity of the infectious TTsuV DNA
clones such as, for example, porcine reproductive and respiratory
syndrome virus (PRRSV), porcine parvovirus (PPV), other infectious
swine agents and immune stimulants.
[0102] The new vaccines of this invention are not restricted to any
particular type or method of preparation. The cloned viral vaccines
include, but are not limited to, infectious DNA vaccines (i.e.,
using plasmids, vectors or other conventional carriers to directly
inject DNA into 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 art.
[0103] As a further benefit, the preferred live virus of the
present invention provides a genetically stable vaccine that is
easier to make, store and deliver than other types of attenuated
vaccines.
[0104] Another preferred vaccine of the present invention utilizes
suitable plasmids for delivering the nonpathogenic DNA clone to
pigs. In contrast to the traditional vaccine that uses live or
killed cell culture propagated whole virus, this invention provides
for the direct inoculation of pigs with the plasmid DNA containing
the infectious viral genome.
[0105] Additional genetically engineered vaccines, which are
desirable in the present invention, are produced by techniques
known in the art. Such techniques involve, but are not limited to,
further manipulation of recombinant DNA, modification of or
substitutions to the amino acid sequences of the recombinant
proteins and the like.
[0106] Genetically engineered vaccines based on recombinant DNA
technology are made, for instance, by identifying alternative
portions of the viral gene encoding proteins responsible for
inducing a stronger immune or protective response in pigs (e.g.,
proteins derived from ORF1, ORF1/1, ORF2, ORF2/2, etc.). Such
identified genes or immuno-dominant fragments can be cloned into
standard protein expression vectors, such as the baculovirus
vector, and used to infect appropriate host cells (see, for
example, O'Reilly et al., "Baculovirus Expression Vectors: A Lab
Manual," Freeman & Co., 1992). The host cells are cultured,
thus expressing the desired vaccine proteins, which can be purified
to the desired extent and formulated into a suitable vaccine
product. The recombinant subunit vaccines are based on
bacteria-expressed (FIG. 10, FIG. 15) or baculovirus-expressed ORF1
capsid proteins of TTsuV1a, PTTsuV1b and TTsuV2.
[0107] If the clones retain any undesirable natural abilities of
causing disease, it is also possible to pinpoint the nucleotide
sequences in the viral genome responsible for any residual
virulence, and genetically engineer the virus avirulent through,
for example, site-directed mutagenesis. Site-directed mutagenesis
is able to add, delete or change one or more nucleotides (see, for
instance, Zoller et al., DNA 3:479-488, 1984). An oligonucleotide
is synthesized containing the desired mutation and annealed to a
portion of single stranded viral DNA. The hybrid molecule, which
results from that procedure, is employed to transform bacteria.
Then double-stranded DNA, which is isolated containing the
appropriate mutation, is used to produce full-length DNA by
ligation to a restriction fragment of the latter that is
subsequently transfected into a suitable cell culture. Ligation of
the genome into the suitable vector for transfer may be
accomplished through any standard technique known to those of
ordinary skill in the art. Transfection of the vector into host
cells for the production of viral progeny may be done using any of
the conventional methods such as calcium-phosphate or DEAE-dextran
mediated transfection, electroporation, protoplast fusion and other
well-known techniques (e.g., Sambrook et al., "Molecular Cloning: A
Laboratory Manual," Cold Spring Harbor Laboratory Press, 1989). The
cloned virus then exhibits the desired mutation. Alternatively, two
oligonucleotides can be synthesized which contain the appropriate
mutation. These may be annealed to form double-stranded DNA that
can be inserted in the viral DNA to produce full-length DNA.
[0108] An immunologically effective amount of the vaccines of the
present invention is administered to a pig in need of protection
against viral infection. The immunologically effective amount or
the immunogenic amount that inoculates the pig can be easily
determined or readily titrated by routine testing. An effective
amount is one in which a sufficient immunological response to the
vaccine is attained to protect the pig exposed to the TTsuV virus.
Preferably, the pig is protected to an extent in which one to all
of the adverse physiological symptoms or effects of the viral
disease are significantly reduced, ameliorated or totally
prevented.
[0109] The vaccine can be administered in a single dose or in
repeated doses. Dosages may range, for example, from about 1
microgram to about 1,000 micrograms of the plasmid DNA containing
the infectious chimeric DNA genome (dependent upon the
concentration of the immuno-active component of the vaccine),
preferably 100 to 200 micrograms of the TTsuV DNA clone, but should
not contain an amount of virus-based antigen sufficient to result
in an adverse reaction or physiological symptoms of viral
infection. Methods are known in the art for determining or
titrating suitable dosages of active antigenic agent to find
minimal effective dosages based on the weight of the pig,
concentration of the antigen and other typical factors. Preferably,
the infectious viral DNA clone is used as a vaccine, or a live
infectious virus can be generated in vitro and then the live virus
is used as a vaccine. In that case, from about 50 to about 10,000
of the 50% tissue culture infective dose (TCID 50) of live virus,
for example, can be given to a pig.
[0110] The new vaccines of this invention are not restricted to any
particular type or method of preparation. The vaccines include, but
are not limited to, modified live vaccines, inactivated vaccines,
subunit vaccines, attenuated vaccines, genetically engineered
vaccines, etc.
[0111] The advantages of live vaccines are that all possible immune
responses are activated in the recipient of the vaccine, including
systemic, local, humoral and cell-mediated immune responses. The
disadvantages of live virus vaccines, which may outweigh the
advantages, lie in the potential for contamination with live
adventitious viral agents or the risk that the virus may revert to
virulence in the field.
[0112] To prepare inactivated virus vaccines, for instance, the
virus propagation and virus production can occur in cultured
porcine cell lines such as, without limitation PK-15 cells. Serial
virus inactivation is then optimized by protocols generally known
to those of ordinary skill in the art or, preferably, by the
methods described herein.
[0113] Inactivated virus vaccines may be prepared by treating the
TTsuV with inactivating agents such as formalin or hydrophobic
solvents, acids, etc., by irradiation with ultraviolet light or
X-rays, by heating, etc. Inactivation is conducted in a manner
understood in the art. For example, in chemical inactivation, a
suitable virus sample or serum sample containing the virus is
treated for a sufficient length of time with a sufficient amount or
concentration of inactivating agent at a sufficiently high (or low,
depending on the inactivating, agent) temperature or pH to
inactivate the virus. Inactivation by heating is conducted at a
temperature and for a length of time sufficient to inactivate the
virus. Inactivation by irradiation is conducted using a wavelength
of light or other energy source for a length of time sufficient to
inactivate the virus. The virus is considered inactivated if it is
unable to infect a cell susceptible to infection.
[0114] The preparation of subunit vaccines typically differs from
the preparation of a modified live vaccine or an inactivated
vaccine. Prior to preparation of a subunit vaccine, the protective
or antigenic components of the vaccine must be identified. In the
present invention, antigenic components of TTsuV were identified as
the ORF1 capsid proteins of TTsuV1a, TTsuV1b and TTsuV2, which were
expressed and purified in Escherichia coli (E. coli) in this
invention, and other expression system, such as baculovirus
expression system, for use as subunit recombinant capsid vaccines.
Such protective or antigenic components include certain amino acid
segments or fragments of the viral capsid proteins which raise a
particularly strong protective or immunological response in pigs;
single or multiple viral capsid proteins themselves, oligomers
thereof, and higher-order associations of the viral capsid proteins
which form virus substructures or identifiable parts or units of
such substructures; oligoglycosides, glycolipids or glycoproteins
present on or near the surface of the virus or in viral
substructures such as the lipoproteins or lipid groups associated
with the virus, etc. Preferably, the ORF1 protein is employed as
the antigenic component of the subunit vaccine. Other proteins may
also be used such as those encoded by the nucleotide sequence in
the ORF2, ORF1/1, and ORF2/2 gene. These immunogenic components are
readily identified by methods known in the art. Once identified,
the protective or antigenic portions of the virus (i.e., the
"subunit") are subsequently purified and/or cloned by procedures
known in the art. The subunit vaccine provides an advantage over
other vaccines based on the live virus since the subunit, such as
highly purified subunits of the virus, is less toxic than the whole
virus.
[0115] If the subunit vaccine is produced through recombinant
genetic techniques, expression of the cloned subunit such as the
ORF1, ORF2, ORF1/1, and ORF2/2 genes, for example, may be expressed
by the method provided above, and may also be optimized by methods
known to those in the art (see, for example, Maniatis et al
"Molecular Cloning: A Laboratory Manual," Cold Spring Harbor
Laboratory, Cold Spring Harbor, Mass. (1989)). On the other hand,
if the subunit being employed represents an intact structural
feature of the virus, such as an entire capsid protein, the
procedure for its isolation from the virus must then be optimized.
In either case, after optimization of the inactivation protocol,
the subunit purification protocol may be optimized prior to
manufacture.
[0116] To prepare attenuated vaccines, the live, pathogenic virus
is first attenuated (rendered nonpathogenic or harmless) by methods
known in the art or, preferably, as described herein. For instance,
attenuated viruses may be prepared by the technique of the present
invention which involves the novel serial passage through
embryonated pig eggs. Attenuated viruses can be found in nature and
may have naturally-occurring gene deletions or, alternatively, the
pathogenic viruses can be attenuated by making gene deletions or
producing gene mutations. The attenuated and inactivated virus
vaccines comprise the preferred vaccines of the present
invention.
[0117] Genetically engineered vaccines, which are also desirable in
the present invention, are produced by techniques known in the art.
Such techniques involve, but are not limited to, the use of RNA,
recombinant DNA, recombinant proteins, live viruses and the
like.
[0118] For instance, after purification, the wild-type virus may be
isolated from suitable clinical, biological samples such as serum,
fecal, saliva, semen and tissue samples by methods known in the
art, preferably by the method taught herein using infected pigs or
infected suitable cell lines. The 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. The cDNA of viral genome is
cloned into a suitable host by methods known in the art (see
Maniatis et al., id.), and the virus genome is then analyzed to
determine essential regions of the genome for producing antigenic
portions of the virus. Thereafter, the procedure is generally the
same as that for the modified live vaccine, an inactivated vaccine
or a subunit vaccine.
[0119] Genetically engineered vaccines based on recombinant DNA
technology are made, for instance, by identifying the portion of
the viral gene which encodes for proteins responsible for inducing
a stronger immune or protective response in pigs (e.g., proteins
derived from ORF1, ORF2, ORF1/1, and ORF2/2, etc.). Such identified
genes or immuno-dominant fragments can be cloned into standard
protein expression vectors, such as the baculovirus vector, and
used to infect appropriate host cells (see, for example, O'Reilly
et al., "Baculovirus Expression Vectors: A Lab Manual," Freeman Co.
(1992)). The host cells are cultured, thus expressing the desired
vaccine proteins, which can be purified to the desired extent and
formulated into a suitable vaccine product.
[0120] Genetically engineered proteins, useful in vaccines, for
instance, may be expressed in insect cells, yeast cells or
mammalian cells. The genetically engineered proteins, which may be
purified or isolated by conventional methods, can be directly
inoculated into a porcine or mammalian species to confer protection
against TTsuV.
[0121] An insect cell line (like sf9, sf21, or HIGH-FIVE) can be
transformed with a transfer vector containing polynucleic acids
obtained from the virus or copied from the viral genome which
encodes one or more of the immuno-dominant proteins of the virus.
The transfer vector includes, for example, linearized baculovirus
DNA and a plasmid containing the desired polynucleotides. The host
cell line may be co-transfected with the linearized baculovirus DNA
and a plasmid in order to make a recombinant baculovirus.
[0122] Alternatively, DNA from the isolated TTsuV which encode one
or more capsid proteins can be inserted into live vectors, such as
a poxvirus or an adenovirus and used as a vaccine.
[0123] An immunologically effective amount of the vaccine of the
present invention is administered to an porcine or mammalian
species in need of protection against said infection or syndrome.
The "immunologically effective amount" can be easily determined or
readily titrated by routine testing. An effective amount is one in
which a sufficient immunological response to the vaccine is
attained to protect the pig or other mammal exposed to the TTsuV
virus, or TTsuV co-infection, which may cause porcine dermatitis
and nephropathy syndrome (PDNS), postweaning multisystemic wasting
syndrome (PMWS) or related illness. Preferably, the pig or other
mammalian species is protected to an extent in which one to all of
the adverse physiological symptoms or effects of the viral disease
are found to be significantly reduced, ameliorated or totally
prevented.
[0124] The vaccine can be administered in a single dose or in
repeated doses. Dosages may contain, for example, from 1 to 1,000
micrograms of virus-based antigen (dependent upon the concentration
of the immuno-active component of the vaccine), but should not
contain an amount of virus-based antigen sufficient to result in an
adverse reaction or physiological symptoms of viral infection.
Methods are known in the art for determining or titrating suitable
dosages of active antigenic agent based on the weight of the bird
or mammal, concentration of the antigen and other typical
factors.
[0125] The vaccine can be administered to pigs. Also, the vaccine
can be given to humans such as pig farmers who are at high risk of
being infected by the viral agent. It is contemplated that a
vaccine based on the TTsuV can be designed to provide broad
protection against both porcine and human TTV. In other words, the
vaccine based on the TTsuV can be preferentially designed to
protect against human TTV infection through the so-called
"Jennerian approach" (i.e., cowpox virus vaccine can be used
against human smallpox by Edward Jenner). Desirably, the vaccine is
administered directly to a porcine or other mammalian species not
yet exposed to the TTV virus. The vaccine can conveniently be
administered orally, intrabuccally, intranasally, transdermally,
parenterally, etc. The parenteral route of administration includes,
but is not limited to, intramuscular, intravenous, intraperitoneal
and subcutaneous routes.
[0126] When administered as a liquid, the present vaccine may be
prepared in the form of an aqueous solution, a syrup, an elixir, a
tincture and the like. Such formulations are known in the art and
are typically prepared by dissolution of the antigen and other
typical additives in the appropriate carrier or solvent systems.
Suitable carriers or solvents include, but are not limited to,
water, saline, ethanol, ethylene glycol, glycerol, etc. Typical
additives are, for example, certified dyes, flavors, sweeteners and
antimicrobial preservatives such as thimerosal (sodium
ethylmercurithiosalicylate). Such solutions may be stabilized, for
example, by addition of partially hydrolyzed gelatin, sorbitol or
cell culture medium, and may be buffered by conventional methods
using reagents known in the art, such as sodium hydrogen phosphate,
sodium dihydrogen phosphate, potassium hydrogen phosphate,
potassium dihydrogen phosphate, a mixture thereof, and the
like.
[0127] Liquid formulations also may include suspensions and
emulsions which contain suspending or emulsifying agents in
combination with other standard co-formulants. These types of
liquid formulations may be prepared by conventional methods.
Suspensions, for example, may be prepared using a colloid mill.
Emulsions, for example, may be prepared using a homogenizer.
[0128] Parenteral formulations, designed for injection into body
fluid systems, require proper isotonicity and pH buffering to the
corresponding levels of mammalian body fluids. Isotonicity can be
appropriately adjusted with sodium chloride and other salts as
needed. Suitable solvents, such as ethanol or propylene glycol, can
be used to increase the solubility of the ingredients in the
formulation and the stability of the liquid preparation. Further
additives which can be employed in the present vaccine include, but
are not limited to, dextrose, conventional antioxidants and
conventional chelating agents such as ethylenediamine tetraacetic
acid (EDTA). Parenteral dosage forms must also be sterilized prior
to use.
[0129] The following examples demonstrate certain aspects of the
present invention. However, it is to be understood that these
examples are for illustration only and do not purport to be wholly
definitive as to conditions and scope of this invention. It should
be appreciated that when typical reaction conditions (e.g.,
temperature, reaction times, etc.) have been given, the conditions
both above and below the specified ranges can also be used, though
generally less conveniently. The examples are conducted at room
temperature (about 23.degree. C. to about 28.degree. C.) and at
atmospheric pressure. All parts and percents referred to herein are
on a weight basis and all temperatures are expressed in degrees
centigrade unless otherwise specified.
EXAMPLES
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 Carolina State University, Raleigh, N.C.) (Schierack, P., M.
Nordhoff, M. Pollmann, K. D. Weyrauch, S. Amasheh, U. Lodemann, J.
Jores, B. Tachu, S. Kleta, A. Blikslager, K. Tedin, and L. H.
Wieler. 2006. Characterization of a porcine intestinal epithelial
cell line for in vitro studies of microbial pathogenesis in swine.
Histochem Cell Biol 125:293-305), and a hamster ovary cell line
CHO-K1 (ATCC CCL-61, passage 12) were each cultured in Dulbecco's
modified Eagle's medium (DMEM) and nutrient mixture F-12 (Ham)
(1:1) with GlutaMAX.TM.-I (Invitrogen, Carlsbad, Calif.)
supplemented with 5% FBS and antibiotics. A monkey kidney cell line
subclone MARC-145 (passage 42) derived from MA-104 (ATCC CRL-2378),
a human cervical cancer cell line HeLa (ATCC CCL-2, passage 10),
two human hepatocellular carcinoma cell lines Huh-7 (subclone 10-3;
a gift from Dr. Suzanne U. Emerson at NIAID, NIH) (Emerson, S. U.,
H. Nguyen, J. Graff, D. A. Stephany, A. Brockington, and R. H.
Purcell. 2004. In vitro replication of hepatitis E virus (HEV)
genomes and of an HEV replicon expressing green fluorescent
protein. J Virol 78:4838-46) and HepG2 (ATCC CRL-10741, passage 7)
were each grown in DMEM supplemented with 10% fetal bovine serum
(FBS) and antibiotics. A human 293 cell line, 293TT, engineered to
stably express high levels of SV40 large T antigen (a gift from Dr.
John T. Schiller, Laboratory of Cellular Oncology, National Cancer
Institute, Bethesda, Md.) (Buck, C. B., D. V. Pastrana, D. R. Lowy,
and J. T. Schiller. 2004. Efficient intracellular assembly of
papillomaviral vectors. J Virol 78:751-7), was cultured in DMEM-10
medium (DMEM with 10% inactivated FBS, 1% non-essential amino acids
and 1% GlutaMAX-I) supplemented with 400 .mu.g/ml hygromycin B and
antibiotics. All cells were grown at 37.degree. C. with 5%
CO.sub.2.
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, 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). A TTSuV1 or TTSuV2 standard template and a porcine
serum sample from a commercial company used in cell culture, which
is supposed to be OIE (The World Organization for Animal Health)
diseases-free, were included as controls. All samples were run in
duplicate on the same plate.
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.
Example 4
Construction of Full-Length Genomic DNA Clones of TTSuV2
[0133] Two PCR fragments (E and F) covering the full-length genome
of the U.S. strain of TTSuV2 isolate PTTV2c-VA (GenBank accession
no. GU456386; SEQ ID NO:1) were re-amplified from the constructs
reported previously (Huang, Y. W., Y. Y. Ni, B. A. Dryman, and X.
J. Meng. 2010. Multiple infection of porcine Torque teno virus in a
single pig and characterization of the full-length genomic
sequences of four U.S. prototype PTTV strains: implication for
genotyping of PTTV. Virology 396:287-97), which were subsequently
assembled into a full-length genomic DNA by overlapping PCR using
the Herculase II Fusion DNA Polymerase (Stratagene) in the vector
pSC-B-amp/kan (Stratagene). The monomeric TTSuV2 DNA fragment was
flanked by a BamHI restriction site at both ends. The resulting
construct was designated pSC-PTTV2c (FIG. 1A). The full-length
PTTV2c genome was excised from the clone pSC-PTTV2c using BamHI
digestion, purified and ligated head-to-tail to form concatemers.
Two-copy concatemers were cloned into the BamHI-pre-digested
pSC-B-amp/kan vector to produce a tandem-dimerized TTSuV2 DNA
clone, pSC-2PTTV2c-RR (FIG. 1B). Similarly, two plasmids harboring
monomeric and tandem-dimerized TTSuV2 genomic DNA originated from
German TTSuV2 isolate TTV2-#471942 (GenBank accession no. GU188046;
SEQ ID NO:2) (Gallei, A., S. Pesch, W. S. Esking, C. Keller, and V.
F. Ohlinger. 2010. 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
143:202-12) were constructed with the EcoRV site on the same vector
backbone, respectively. Since the TTV2-#471942 strain was
classified into the TTSuV2 subtype 2b together with the U.S.
isolate PTTV2b-VA based upon phylogenetic analysis (data not
shown), the inventors designated these two clones pSC-TTV2-#471942
(FIG. 1C) and pSC-2PTTV2b-RR (FIG. 1D), respectively.
Example 5
Introduction of Genetic Markers into the Two TTSuV2 Monomeric DNA
Clones and Construction of a TTSuV2 Deletion Mutant
[0134] An HpaI restriction enzyme site was engineered into the
putative spliced region (intron) of TTSuV2 genome in the clone
pSC-TTV2-#471942 for introducing a genetic marker to discriminate
between the cloned virus and the potential indigenous viruses in
the subsequent animal study. To create the unique HpaI site
(GTTAAC; mutations are underlined; SEQ ID NO:3), three point
mutations, C to T, C to A and T to A at nucleotide (nt) positions
1817, 1819 and 1820 corresponding to the TTV2-#471942 genome were
generated by a fusion PCR technique using two pairs of primers
containing the desired mutations. The fusion PCR product replaced
the corresponding region on the clone pSC-TTV2-#471942 by using the
cloning site KpnI at both ends. The mutations did not change the
putative ORF1 capsid amino acid sequence. The resulting full-length
DNA clone was named pSC-TTV2-EU (FIG. 1E). Using the same strategy,
two unique restriction sites, PstI (CTGCAG; SEQ ID NO:4) and MfeI
(CAATTG; SEQ ID NO:5), were introduced into the putative intron of
the PTTV2c-VA genome in the pSC-PTTV2c clone (FIG. 1F). The new
clone, designed pSC-TTV2-US, contained three silent mutations at nt
positions 1613 (A to T), 1784 (T to C) and 1787 (C to T)
corresponding to the PTTV2c-VA genome. A mutant clone
pSC-TTV2-.DELTA.AA, with a 104-bp deletion (nt positions 332-437)
from the putative TATA box to the ORF1/ORF2 start codon on the
clone pSC-TTV2-US, was also generated by removing the short
deletion fragment with double-digestion with the AccI and ApaI
enzymes followed by formation of two blunt ends with a Klenow
enzyme and self-ligation (FIG. 1G). All mutagenesis were confirmed
by DNA sequencing.
Example 6
In Vitro Transfection of TTSuV DNA Clones
[0135] The PCV1-free PK-15 cells were seeded at 2.times.10.sup.5
cells per well onto a 6-well plate and grown until 60%-70%
confluency before transfection. Two micrograms of the
tandem-dimerized clones pSC-2PTTV2b-RR and pSC-2PTTV2c-RR were
directly transfected into the cells, respectively, using
Lipofectamine LTX (Invitrogen) according to the manufacturer's
protocol. For monomeric clones pSC-PTTV2c, pSC-TTV2-#471942,
pSC-TTV2-EU, pSC TTV2-US and pSC-TTV2-AAA, the respective genomic
fragment was excised by BamHI or EcoRV enzyme, gel-purified, and
re-ligated with the T4 DNA ligase overnight. The ligation mixtures
(.about.2 .mu.g) were used for transfection using Lipofectamine
LTX, respectively. Cells were cultured for 3 to 5 days, and then
subjected to an immunofluorescence assay (IFA) to detect the
expression of ORF1. Alternatively, transfected cells were passaged
into new 6-well plates and were cultured for 3 days before
detection of ORF1 expression by IFA. Transfection of the other 11
cell lines and IFA detection were similar.
Example 7
Immunofluorescence Assay (IFA)
[0136] Transfected or passaged cells on 6-well plates were washed
times with PBS and fixed with acetone. Five hundred microliters of
the anti-TTSuV2 ORF1 antiserum at a 1:500 dilution in PBS, was
added to the cells for each well and incubated for 1 hour at room
temperature. Cells were washed 3 times with PBS and 500 .mu.l Texas
Red- or Alexa Fluor 488-conjugated goat anti-rabbit IgG
(Invitrogen) at a 1:300 dilution was subsequently added. After
incubation for 1 hour at room temperature, the cells were washed
with PBS, stained with 500 .mu.l DAPI (KPL, Inc.) at a 1:1000
dilution and visualized under a fluorescence microscope.
Example 8
RT-PCR
[0137] Total RNA was extracted from PCV1-free PK-15 cells
transfected with circular TTSuV2 DNA using the RNeasy mini kit
(Qiagen) followed by an RNase-free DNase I treatment. The cDNA
synthesis was performed using SuperScript II reverse transcriptase
(Invitrogen) with oligo-dT as the reverse primer. PCR was performed
in a 50-.mu.L reaction with the Advantage 2 PCR kit (Clontech)
using primers TTV2-448F (5'-GAAGAAAGATGGCTGACGGTAGCGTACT-3'; SEQ ID
NO:6) and TTV2-2316R (5'-AGGTGCTTGAGGAGTCGTCGCTTG-3'; SEQ ID NO:7).
The PCR products were gel-purified, cloned into a pCR2.1 vector
(Invitrogen) by TA cloning strategy and sequenced.
Example 9
In Vivo Transfection of Colostrum Deprived (CD) Pigs with the
Tandem-Dimerized TTSuV2 Clones
[0138] It has been previously demonstrated that the infectivity of
infectious DNA clones for viruses with a circular genome can be
tested by direct inoculation of dimerized full-length genomic DNA
into animals (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). Therefore, in this study, a pilot animal study was
initially conducted to determine the infectivity of the two
tandem-dimerized TTSuV2 clones pSC-2TTV2c-RR and pSC-2TTV2b-RR.
Briefly, six, 26-day old, CD pigs that were seronegative and viral
DNA-negative for TTSuV1 and TTSuV2 were assigned into three groups
of two each. Each group of pigs was housed separately and
maintained under conditions that met all requirements of the
Institutional Animal Care and Use Committee. The pigs in each group
were injected by using a combination of intra-lymphoid (superficial
inguinal lymph nodes) and intramuscular routes with the plasmid DNA
of the full-length TTSuV2 clones. The two pigs (nos. 1 and 2) in
group 1 were each given 1 ml of PBS buffer and used as the negative
control. The two pigs (nos. 3 and 4) in group 2 were each injected
with 200 .mu.g of the pSC-2TTV2c-RR plasmid DNA whereas the
remaining two pigs (nos. 5 and 6) in group 3 were each inoculated
with 200 .mu.g of the pSC-2TTV2b-RR clone.
[0139] Pigs were monitored daily for evidence of TTSuV2 infection
for a total of 44 days. All pigs were necropsied at 44 days
post-inoculation. Serum samples were collected from all pigs prior
to inoculation and weekly thereafter until termination of the
study. The samples were tested for the presence of TTSuV DNA and
quantified for viral loads by a singleplex TTSuV2-specific
real-time qPCR (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). Samples of tissues including brain, lung, lymph nodes,
liver, kidney, thymus, spleen, small intestines, large intestines,
heart, tonsil, bone marrow were collected during necropsies and
processed for microscopic examination. The tissues were examined in
fashion blinded to the treatment status of the pigs and given a
subjective score for severity of tissue lesions ranged from 0
(normal) to 3 (severe) (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; Halbur, P. G., P. S. Paul, M. L. Frey, J. Landgraf, K.
Eernisse, X. J. Meng, M. A. Lum, J. J. Andrews, and J. A. Rathje.
1995. Comparison of the pathogenicity of two US porcine
reproductive and respiratory syndrome virus isolates with that of
the Lelystad virus. Vet Pathol 32:648-60).
Example 10
In Vivo Transfection of Cesarean Derived, Colostrum Deprived
(CD/CD) Pigs with the Circularized TTSuV2 Genomic DNA Containing
Genetic Markers
[0140] To further verify the results from the initial pilot pig
study, the inventors introduced tractable genetic markers into the
full-length DNA clones and conducted another CD/CD pig study.
Approximately 600 .mu.g of circular or concatamerized TTSuV2
genomic DNA derived from the clone pSC-TTV2-EU or pSC-TTV2-US was
generated by ligation of the linearized TTSuV2 genomic DNA. To
determine the infectivity of the full-length DNA clones, the
inventors inoculated four, 40-day-old, CD/CD pigs (nos. 129, 135,
139 and 140 in group 1) each with 150 .mu.g of concatamerized
"TTV2-EU DNA" by a combination of both the intra-lymph node route
and intramuscular route. Another four CD/CD pigs (nos. 133, 137,
138 and 141) in group 2, which were housed in a separate room, were
each similarly inoculated with 150 .mu.g of concatamerized "TTV2-US
DNA". The remaining four CD/CD pigs (nos. 127, 132, 136 and 142) in
group 3 were each injected with 1.5 ml of PBS buffer and served as
negative controls. All pigs were monitored for evidence of TTSuV2
infection for a total of 35 days, at which time they were
necropsied. Viremia was tested by a TTSuV2 real-time qPCR (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). A TTSuV2 genomic region of 620 bp
containing the engineered genetic markers in TTV2-EU or TTV2-US was
amplified from the sera of inoculated pigs by PCR using primers
TTV2-tagF (5'-TGACACAGGA/CGTAGGAAATGCAGT-3'; SEQ ID NO:8) and
TTV2-tagR (5'-TGAAGTATTTAGGGTCATTTGTAGCA-3'; SEQ ID NO:9) from
selected serum samples of pigs with viremia. The PCR products were
gel-purified and cloned into a pCR2.1 vector by using the TA
cloning strategy. The white bacterial clones on the
X-gal-containing agar plates were picked up for subsequent DNA
extraction and sequencing.
Example 11
Sources of Porcine Sera
[0141] Porcine sera used in this study were described previously
(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). Briefly, serum
samples for serum Western blot (WB) analysis were collected from 20
conventional adult boars with no clinical symptoms from a Virginia
pig farm, seven gnotobiotic pigs from Virginia (nos. 4 to 7, 224,
229 and 230; kindly provided by Drs. Lijuan Yuan and Guohua Li from
Virginia Tech) and 12 from Iowa (group D), five cesarean-derived,
colostrum-deprived (CD/CD) pigs and approximately 50 conventional
piglets from a Wisconsin pig farm. A TTSuV2-seropositive porcine
serum, which was manufactured in New Zealand and free of all known
OIE (The World Organization for Animal Health) notifiable diseases,
was also used in this study.
[0142] One hundred and sixty porcine serum samples were used for
assessing the virological and serological profiles of TTSuV1a and
TTSuV1b infection and were divided into five groups (A to E) as
described previously (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): (i) Twenty group-A samples were from 10
specific-pathogen-free (SPF) pigs (60-80 days old at arrival) free
of known pathogens and were collected at arrival in the facility
and two months after arrival; (ii) Sixty group-B samples were
collected from 105 days old pigs in a farm with an outbreak of
porcine circovirus associated disease (PCVAD): 30 were from
clinically affected pigs and 30 were clinically unaffected pigs;
(iii) Fifty-eight group-C samples were collected from 28 days old
pigs with unknown disease status: 28 were clinically affected and
30 were clinically unaffected; (iv) Twelve group-D samples were
from 14-42 days old gnotobiotic pigs located in Iowa; (v) Ten
group-E sera were from 21-30 days old SPF pigs used for an
experimental PCV2 infection study.
Example 12
Construction of the TTSuV1a- and TTSuV1b-ORF1 Expression
Plasmids
[0143] The C-terminal part of the ORF1 of two TTSuV1 strains,
PTTV1a-VA (GenBank accession no. GU456383; SEQ ID NO:10) and
PTTV1b-VA (GenBank accession no. GU456384; SEQ ID NO:11) was
amplified, respectively, from the available PCR fragments reported
previously. The amplicon was expected to encode a truncated
PTTV1a-VA ORF1 protein of 319 aa (positions 317-635 corresponding
to PTTV1a-VA) or a truncated PTTV1b-VA ORF1 protein of 318 aa
(positions 322-639 corresponding to PTTV1b-VA). An additional
methionine was introduced at the N-terminus of each amplified
fragments. Two ORF1 expression plasmids, designated pTri-1aORF1 and
pTri-1bORF1, were each constructed by cloning the respective PCR
product into a bacterial/insect/mammalian-triple expression vector
pTriEx1.1-Neo (Novagen) between the NcoI and XhoI restriction sites
to generate two C-terminally 8.times.His-tagged fusion proteins.
The recombinant plasmids were confirmed by DNA sequencing. The
TTSuV2 ORF1 expression construct, pTri-2cORF1, had been described
previously (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).
Example 13
Expression and Purification of the Recombinant TTSuV1a- and
TTSuV1b-ORF1 Proteins
[0144] The two plasmids were each transformed into Rosetta 2 (DE3)
pLacI competent cells (Novagen). The bacteria were grown in 100-ml
of Overnight Express TB Media (Novagen) for 16-18 hours at
37.degree. C. and then the bacterial culture was harvested by
centrifugation at 3,400 rpm for 15 minutes at 4.degree. C. The
resulting bacterial pellet was treated with BugBuster and rLysozyme
according to the manufacture's protocol (Novagen). Benzonase
Nuclease (Novagen) was added to degrade DNA and RNA. The resulting
inclusion bodies were lysed in 6 M guanidine hydrochloride, 0.1 M
sodium phosphate, 0.01 M Tris-Chloride, and 0.01 M imidazole with a
pH value of 8.0. The lysate supernatants were collected by
centrifugation and were used for His-tagged protein purification by
a Ni-NTA His Bind Resin 50% (Novagen) under denaturing condition
with 8 M urea. Proteins were dialyzed as described previously (13).
The recombinant His-tagged TTSuV1a- or TTSuV1b ORF1 proteins used
as the antigen for ELISA and rabbit immunization were quantified
using a NanoDrop spectrophotometry and frozen at -80.degree. C.
until use.
Example 14
Generation of Anti-ORF1 Antisera of TTSuV1a and TTSuV1b in
Rabbits
[0145] The two ORF1 proteins of TTSuV1a and TTSuV1b expressed in E.
coli were purified and used to immunize two New Zealand white
rabbits, respectively, at a custom antibody production service at
Rockland Immunochemicals (Gilbertsville, Pa.). Antisera were
harvested at 50 days post-immunization.
Example 15
SDS-PAGE, Anti-His-Tagged WB and Serum WB Analysis
[0146] The unpurified or purified recombinant TTSuV1 ORF1 proteins
were resolved on a 4-12% Bis-Tris Polyacrylamide Gel (Invitrogen)
by electrophoresis and were subsequently transferred onto a
polyvinylidene difluoride (PVDF) membrane. Proteins were detected
on the PVDF membrane using an anti-6.times.His-tagged Mab at a
1:1000 dilution at 4.degree. C., followed by incubation with an
IRDye 800CW conjugated goat anti-rabbit IgG (LI-COR Biosciences) at
a 1:10,000 dilution at room temperature. After three washing steps
using Tris buffered saline/0.05% Tween 20 (TBS-T; Sigma), the
membrane was analyzed using the Odyssey Infrared Imaging System
(LI-COR Biosciences).
[0147] For serum WB analysis, the purified TTSuV1a- or TTSuV1b-ORF1
proteins were incubated with individual porcine sera at a 1:200
dilution and with IRDye 800CW conjugated rabbit F(ab').sub.2
anti-swine IgG (Rockland Immunochemicals, Inc.) at a 1:10,000
dilution at room temperature. The membrane was then analyzed using
the Odyssey Infrared Imaging System.
Example 16
Indirect ELISAs
[0148] TTSuV1a- and TTSuV1b-based ELISAs were developed. The
optimal concentration of the antigens and the optimal dilutions of
sera and HRP conjugates were determined by checkerboard titrations.
Similar to the TTSuV2-based ELISA reported previously, the optimal
amount of the ORF1 antigen of TTSuV1a or TTSuV1b was 68 ng per
well. The optimal ELISA results were obtained by using a 1:100
dilution of serum samples and a 1:4000 dilution of IgG
conjugates.
[0149] The ELISA was initiated by diluting the purified ORF1
proteins in carbonate coating buffer (pH=9.6) that was used for
coating 96-well ELISA plates (Greiner Bio-One) with 100 .mu.l/well.
After incubation at 37.degree. C. for 2 hours, each well was washed
3 times with 300 .mu.l of Tris-buffered saline-Tween 20 solution
(TBS-T) and blocked with protein-free blocking buffer (Pierce) at a
volume of 300 .mu.l for 1 hour at 37.degree. C. One hundred .mu.l
of each diluted serum sample was transferred to the corresponding
well on the ELISA plates and incubated at 37.degree. C. for 2
hours. After washing the wells three times with 300 .mu.l of TBS-T
buffer, the diluted HRP-conjugated rabbit anti-swine IgG (Rockland)
was added to each well in a volume of 100 .mu.l and the plate was
incubated at 37.degree. C. for 1 hour. A volume of 100 .mu.l of
Sure Blue Reserve 1-Component (KPL) was added to each well and
incubated for 10 minutes at room temperature. The reaction was
stopped by adding 100 .mu.l/well of 1 N HCL. The plates were then
read at 450 nm using a spectrophotometer. All serum samples were
run in duplicates. Positive and negative controls run in
quadruplicates were included on each plate. In general, the mean OD
value of the negative control was less than 0.5 whereas the mean OD
value of the positive control was greater than 1.5. The ELISA value
was calculated as the S/N value that was expressed as a ratio of
the mean OD value of a sample to the mean OD value of the negative
control (n=4). A subjective cut-off S/N value of 1.2 was used to
distinguish between positive and negative samples.
Example 17
Real-Time qPCR Assay for Quantitation of TTSuV1
[0150] A SYBR green-based TTSuV1-specific real-time quantitative
PCR (qPCR) developed recently in our laboratory was used to measure
the total TTSuV1 viral loads (both TTSuV1a and TTSuV1b) in the five
groups of pig sera as described previously (12). The minimal
detection limit was 1.0.times.10.sup.4 copies per ml in this study.
The TTSuV1 qPCR assay does not cross-amplify TTSuV2 DNA (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). Quantitation of TTSuV2 and PCV2 viral
loads in group-B sera had been reported previously (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).
Example 18
Statistical Analyses
[0151] Data were analyzed using SAS software (version 9.2; SAS
Institute Inc., Cary, N.C.) and GraphPad Prism software (version
5.0; San Diego, Calif.), respectively. Antibody levels (represented
by S/N values) were compared between categories of log.sub.10 viral
titers using the Kruskal-Wallis test followed by Dunn's procedure.
For each group that contained clinically affected and non-affected
pigs (groups B and C), log.sub.10 virus titers were compared
between pigs with and without clinical signs using a Wilcoxon
2-sample test. Antibody levels were compared between pigs with and
without disease using a 2-sample t-test. Using a cutoff point of
1.2, the proportion of pigs with antibodies was compared between
affected and unaffected pigs using a Fisher's exact test.
[0152] Correlations between S/N values for TTSuV1a and S/N values
for TTSuV1b, and between S/N values for TTSuV1a or TTSuV1b
(separately) and TTSuV2 were assessed using Spearman's correlation
coefficient. The correlations were separately generated for a
combination of 3 groups (group-A to group-C).
[0153] To assess the synergistic effects between PCV2 and TTSuV1 on
disease prevalence, the pigs in group B were categorized as
follows: pigs positive for both PCV2 and TTSuV1, pigs only positive
for PCV2, pigs only positive for TTSuV1, and pigs with neither PCV2
nor TTV1. Subsequently, the proportions of affected pigs were
compared between the groups using Fisher's exact test. Statistical
significance was set to alpha=0.05.
Example 19
Transfection of PK-15 Cell with TTSuV Expression Constructs
[0154] PK-15 cells were seeded onto a 6-well plate and grown until
70%-80% confluency before transfection. Two micrograms of each of
the three constructs pTri-1aORF1, pTri-1bORF1 and pTri-2cORF1,
mixed with 10 .mu.l of Lipofectamine LTX (Invitrogen), were
transfected into the cells, respectively. Cells were cultured for 3
days and were subjected to IFA to detect the ORF1 expression.
Example 20
Immunofluorescence Assay (IFA)
[0155] Five rabbit antisera were used for IFA staining, including
anti-TTSuV1a, anti-TTSuV1b, anti-TTSuV2, pre-bleed rabbit negative
control serum, and rabbit anti-human genogroup-1 TTV ORF1 antiserum
(AK47; a generous gift from Dr. Annette Mankertz at the Robert
Koch-Institute, Berlin, Germany) (21). Transfected cells were fixed
with acetone. Five hundred microliters of each of the five
antisera, at a 1:500 dilution in PBS, was added on top of the cells
in each well and incubated for 1 hour at room temperature. After
three washing steps with PBS, the cells were incubated with 500
.mu.l Alexa Fluor 488-labeled goat anti-rabbit IgG (Invitrogen) at
a 1:200 dilution for 1 hour incubation at room temperature. Cells
were stained with 500 .mu.l DAPI (KPL, Inc.) at a 1:1000 dilution
and visualized under a fluorescence microscope.
TABLE-US-00001 TABLE 1 Reactivity of anti-TTSuV1a, anti-TTSuV1b,
anti-TTSuV2, pre- bleed rabbit and anti human TTV (AK47) sera in
PCV1-free PK-15 cells transfected with plasmids encoding truncated
ORF1s from TTSuV1a, TTSuV1b and TTSuV2, respectively, as determined
by IFA. The intensity of the fluorescent signal was determined
visually and expressed ranging from - to ++. Ab Pre-bleed Trans-
Anti- Anti- Anti- rabbit Anti-human fection TTSuV1a TTSuV1b TTSuV2
serum TTV (AK47) pTri- ++ + - - - 1aORF1 pTri- + ++ - - - 1bORF1
pTri- - - ++ - - 2cORF1 Mock - - - - -
Sequence CWU 1
1
1112803DNATorque teno sus virus 1tcatgacagg gttcaccgga agggctgcaa
aattacagct aaaaccacaa gtctaacaca 60ataaaccaca aagtattaca ggaaactgca
ataaatttag aaatagatta cacataacca 120ccaaaccaca ggaaacctac
acataaccac caaaccacag gaaacataac caccaaacca 180caggaaactg
tgcaaaaaag gggaaataaa ttctattggc tgggcctgaa gtcctcatta
240gaataataaa agaaccaatc agaagaactt cctcttttag agtatataag
taagtgcgca 300gacgaatggc tgagtttatg ccgctggtgg tagacacgaa
cagagctgag tgtctaaccg 360cctgggcggg tgccggagct cctgagagcg
gagtcaaggg gcttatcggg caggcggtaa 420tccagcggaa ccgggccccc
ctcgatggaa gaaagatggc tgacggtagc gtactgcgcc 480cacggattat
tctgcggatg taaagacccg aaaaaacacc ttgaaaaatg ccttacagac
540gctatcgcag acgccgaagg agaccgacac ggagatggag gcaccggagg
tggagacgct 600tctttcgata tcggtatcga cgcgctcctc gccgccgccg
cacaaaggta aggagacgga 660ggagaaaagc tccggtcata caatggttcc
ctcctagccg gaggacctgc ctcatagagg 720gcttctggcc gttgagctac
ggacactggt tccgtacctg tctccctatg agaaggctga 780acggactcat
tttcacgggt ggcggttgtg actggacaca gtggagttta caaaacttat
840accatgaaaa acttaactgg agaaatatat ggacagcttc taatgttggc
atggaatttg 900ctagattttt aagaggaaaa ttttacttct tcagacaccc
ctggagaagc tatattatta 960cttgggacca agacattcct tgcaaacctt
taccatacca aaacttacat ccactactta 1020tgctattaaa aaaacaacat
aaacttgtac tatctcaaaa agactgtaat ccaaacagaa 1080gacaaaaacc
agtaacttta aaaataagac ctccaccaaa attaacatca cagtggagat
1140taagcagaga actagcaaaa atgccacttg tcagactagg agtcagtcta
atagacctct 1200cagaaccatg gttagaaggc tggggaaatg ctttttacag
cgtactggga tatgaagcta 1260gtaaacactc agggagatgg tcaaactgga
cacaaataaa atacttctgg atatatgaca 1320caggagtagg aaatgcagtt
tatgtcattt tattaaaaca agaggtggat gataatccag 1380gggcaatggc
aacaaaattt gtaactggac caggacaaca cccagatgcc atagacagga
1440tcgaacaaat aaatgaagga tggccttact ggcttttctt ttacggacag
tcagaacaag 1500acataaaaaa attagcacac gatcaagaaa tagcaaggga
atatgcaaac aatccaaaat 1560ctaaaaaatt aaaaatagga gtgataggat
gggctagcag taactttaca acagcaggca 1620gctcacaaaa tcaaacacca
caaacaccag aagccataca aggaggatac gtagcatatg 1680caggctcaaa
aatacaagga gcaggagcaa ttacaaactt atacacagat gcatggccgg
1740gagaccaaaa ttggccacct ctaaatagag aacaaacaaa ctttaactgg
ggcttaagag 1800gactctgtat aatgagagat aatatgaaac tgggagctca
agaactagat gatgaatgta 1860caatgctcac actttttgga ccttttgtgg
aaaaagcaaa cacagctttt gctacaaatg 1920accctaaata cttcagacca
gaactcaaag actataacat agtaatgaaa tatgccttta 1980aatttcagtg
gggaggccac ggaaccgaaa gattcaaaac aaccatcgga gatcccagca
2040ccataccatg tccctttgaa cccggggaac ggtaccacca cggggtacaa
gaccccgcca 2100aggtacaaaa cacagtcctc aacccttggg actatgactg
tgacgggatt gttagaacag 2160atactctcaa aagacttctc gaactcccca
cagagacgga ggagacggag aaggcgtacc 2220cactccttgg acaaaaaaca
gagaaagagc cattatcaga ctccgacgaa gagagcgtta 2280tctcaagcac
gagcagtgga tcctctcaag aagaagagac gcagagaaga agacagcaca
2340agccaagcaa gcgacgactc ctcaagcacc tccagcgggt ggtaaagaga
atgaagacac 2400tgtgatagat aaatatagaa acctagcaga cccctcactc
aatgtcacag gacacatgga 2460aaaattcatg caactgcaca tacaaaacgt
acaagaaata agagctaaaa atgctaaaaa 2520atccctcaat aaactttact
tttctgatta ataccggcct cctgtgtcca atctattttt 2580cctacacccc
ttcaaaatgg cgggcgggac acaaaatggc ggaggaaact aagggggggg
2640caagcccccc cccgggggtt gagggggggt ttccccccct ccccccggtg
cagggggcgg 2700agcccccgca ccccccctgc gggggctccg ccccctgcac
ccccgggagg gggggaaacc 2760ccccctcaac cccccgcggg gggcaagccc
ccctgcaccc ccc 280322802DNATorque teno sus virus 2tcatgacagg
gttcaccgga agggctgcaa aattacagct aaaaccacaa gtctaacaca 60ataaaccaca
aagtattaca ggaaactgca ataaatttag aaataaatta cacataacca
120ccaaaccaca ggaaactgtg caaaaaagag gaaataaatt ctattggctc
ggtctaaagt 180cctcattaga ataataaaag aaccaatcag aagaacttcc
tcttttagag tatataagta 240agtgcgcaga cgaatggctg agtttatgcc
gctggtggta gacacgaaca gagctgagtg 300tctaaccgcc tgggcgggtg
ccggagctcc agagagcgga gtcaaggggc ctatcgggca 360ggcggtaatc
cagcggaacc gggcccccac cctccatgga agaaagatgg ctgacggtag
420cgtactgcgc ccacggatta ttctgcggct gtaaaaaccc aaaaaaacat
ctagaaaaat 480gccttacaga cgctatcgca gacgccggag gagacccacc
agaagatgga ggcaccggag 540gtggagacgc tactttcgat atcggtatcg
acgcgctcct cgccgccgcc gcgcaaaggt 600aaggagacgg aggaggaaag
ctccggtcat acaatggaac cctcctagcc ggaggacctg 660cctcatagag
ggcttctggc cgttgagcta cggacactgg ttccgtacct gtctccctat
720gagaaggcta aacggactta tattcacggg tggcggttgt gactggactc
aatggagttt 780acaaaacttg ttccatgaaa aacttaactg gaggaatata
tggacagctt ctaacgtagg 840catggaattt gctagatttt taagaggaaa
attctacttc ttcagacatc cctggagaag 900ctacatagta acatgggatc
aggacattcc ttgtaaacct ctcccatatc aaaacttgca 960acctctatta
atgctactca aaaaacagca taaattagta ctctcacaaa aagactgtaa
1020cccgagcaga aaacagaaac cggtcacatt aaaattcaga cctccaccaa
aattaacatc 1080acaatggaga ctaagtagag aactctctaa aatacctctg
attagactag gaataagcct 1140tatagacctc tcagaaccat ggctagaagg
gtggggaaat gcattttaca gtgtcctcgg 1200atatgaagct tctaaacact
ccgggagatg gtcaaactgg acacaaatga aatatttttg 1260gatatatgac
acaggcgtag gaaatgcagt ctacgttatt ttactaaaaa aagatgtaga
1320tgacaatccc ggagatatgg ctacaaaatt tgtaacagga caagggcaac
acccagacgc 1380tatagatcat atagaaatgg tcaatgaagg gtggccttac
tggctatttt tttatggaca 1440atcagaacaa gacattaaaa aactagcaca
cgaccaagac atagctagag aatatgccag 1500agacccaaaa tccaaaaaac
taaaaatagg agtcataggg tgggcgagta gcaactatac 1560aacagcaggc
agtaatcaaa acaccacagc acaaacacca gaagcaatac agggaggata
1620tgtggcctat gcaggttcaa gaataccagg cgcaggatca atcaccaatt
tatttcaaat 1680gggatggcca ggagatcaaa actggccccc cacaaaccaa
gaacaaacca attttaactg 1740gggactcaga ggactctgcg tactcagaga
taatatgaaa ttaggagcac aagaactaga 1800cgatgaatgt acaatgctct
cactttttgg gccttttgtt gaaaaagcaa acacagcctt 1860tgctacaaat
gaccctaaat acttcagacc agaactcaaa gactataatg tagtaatgaa
1920atatgccttt aaatttcagt ggggaggcca cggaaccgaa agattcaaaa
caaccattgg 1980agatcccagc accataccat gtcccttcga acccggggaa
cggtaccacc acggggtaca 2040agaccccgcc aaggtacaaa acacagtgct
caacccttgg gactatgact gtgacgggat 2100tgttagaaca gatactctca
aaagacttct cgaactcccc acagagacgg aggagacgga 2160gaaggcgtac
ccactccttg gacaaaaaac agagaaagag ccattatcag actccgacga
2220agagagcgtt atctcaagca cgagcagtgg atcgtctcaa gaagaagaga
cgcagagaag 2280aagacagcac aagccaagca agcgacgact cctcaagcac
ctccagcggg tggtaaagag 2340aatgaaaaca ctgtaataga taaatataga
aacctagcag acccctcact caatgtcact 2400ggacacatgg aaaaattcat
gcagctacac atacaaaacg tacaagaaat aagagctaaa 2460aatgctaaaa
aatccctcaa taaactttac ttttctgatt aatagcggcc tcctgtgtcc
2520aatctatttt tcctacaccc attcaaaatg gcgggcggga cacaaaatgg
cggagggact 2580aagggggggg caagcccccc ccacccccca tgcgggggct
ccgccccctg cacccccttg 2640ctaagtcaca aaatggcggc gcggctagga
cacaaaatgg cggcgtagcg ggggggggga 2700cccccccgca ccccccctgc
ggggctccgc cccctgcacc cccgggaggg ggggaaaccc 2760cccctcaacc
ccccgcgggg ggcaagcccc cctgcacccc cc 2802310DNAArtificial
Sequencesythetically synthesized sequence 3nngttaacnn
10410DNAArtificial Sequencesynthetically synthesized sequence
4nnctgcagnn 10510DNAArtificial Sequencesynthetically synthesized
sequence 5nncaattgnn 10628DNATorque teno sus virus 6gaagaaagat
ggctgacggt agcgtact 28724DNATorque teno sus virus 7aggtgcttga
ggagtcgtcg cttg 24825DNATorque teno sus virus 8tgacacagga
cgtaggaaat gcagt 25926DNATorque teno sus virus 9tgaagtattt
agggtcattt gtagca 26102878DNATorque teno sus virus 10tacacttccg
ggttcaggag gctcaatttg gctagcttcg ctcgcaccac gtttgctgcc 60aggcggacct
gattgaagac tgaaaaccgt taaattcaaa tttgaaaatg gcgggcaaaa
120tggcggaagg ggggcggagt ttatgcaaat taatttatgc aaagtaggag
gagctcgatt 180ttaatttatg caaagtagga ggagtcaatt ctgattggtc
gggagctcaa gtcctcattt 240gcatagggtg taaccaatca gatttaaggc
gttcccccaa aagtgaatat aagtaagcgc 300agttccgaat ggctgagttt
atgccgccag cggtagacag aactgtctag cgactgggcg 360ggtgccggag
gatccctgat ccggagtcaa ggggcctatc gggcaggagc agctgagcgg
420agggcctaca tgaaggagaa agactactgg gaggaagcct ggctgaccag
ctgtacatct 480atacacgacc accactgcaa ctgcggtagc tggagagacc
acctgtggac gctatgcgct 540ttagacgacg cagatttggc cgccgccgca
gatattatag aaagagaaga ggcggatgga 600ggagaagatt tcggattcgt
agacggagac cctggagacg ctggcgggta aggagatggc 660ggcgttccgt
cttccgtaga gggggacgta gagcgcgccc ctaccgcatt agcgcttgga
720accctaaggt tctcagaaac tgccgcatca cgggatggtg gccagttata
cagtgtatgg 780acgggatgga gtggataaaa tacaagccta tggacttaag
agtcgaggca aactggatat 840tcaataaaca ggacagtaaa atagagacag
aacagatggg atacctgatg cagtatggag 900gagggtggtc aagcggagta
atcagcttag agggactatt caatgaaaac agactgtgga 960gaaatatatg
gtcaaaaagc aatgacggga tggacttggt cagatacttt ggctgtagaa
1020ttagactata tccaacagag aatcaggact acttgttctg gtatgacaca
gaatttgacg 1080aacagcaaag gagaatgcta gatgaataca cacaacctag
tgtgatgctg caggctaaaa 1140actcgcgtct aatagtgtgt aaacagaaga
tgccaattag acgcagagta aaaagtattt 1200ttataccgcc gcctgcacag
ttaacaactc agtggaaatt tcaacaggaa ctatgtcagt 1260ttccactgtt
taactgggcc tgtatctgca tagacatgga cacgccgttc gactacaacg
1320gcgcatggcg aaatgcctgg tggctaatga gaaggctgca aaacggaaac
atggagtaca 1380tagaaagatg gggcagaata ccaatgacag gagacacaga
actaccacca gcagacgact 1440tcaaggcagg aggggtgaac aaaaacttca
aaccgacagg tattcaaaga atatacccga 1500tagtagcggt atgccttgta
gaagggaaca aaagagtagt caaatgggcc acagtacaca 1560atggtcccat
agacagatgg agaaaaaaac agacaggaac tttaaagctc tctaacctga
1620gaggcctagt actgagagta tgctcagaga gtgaaacata ctataagtgg
acaggatcag 1680aatttacagg ggcatttcaa caagactggt ggccagtagg
cggaacagaa tacccgcttt 1740gtaccattaa aatggaccca gaatatgaaa
accctacagt agaggtatgg tcctggaaag 1800caaatatacc gacatcaggg
actcttaaag actacttcgg actgagtaca gggcaacagt 1860ggaaagacac
tgactttgcg aggctgcaac tacctagaag cagccacaat gtggactttg
1920gacataaagc tagatttggg ccattttgcg ttaaaaagcc tccagtagag
ttcagagata 1980cagccccaaa cccactaaat atatgggtaa aatacacgtt
ctattttcag ttcggcggca 2040tgtaccagcc tcccaccgga atccaagatc
cctgcacttc taacccgacc tatcctgtca 2100gaatggtcgg agcagttaca
caccccaaat acgccgggca aggcggaatc acgacccaaa 2160ttggagatca
aggtatcacc gctgcctcta tccgtgccat cagtgcagct ccaccagata
2220cctacacgca gtcggcgttc ctcaaagccc cggaaaccga gaaagaagag
gaaagagaga 2280gtgagaccag tttcacgagt gccgaaagct cttctgaggg
agatggatcg tctgatgacc 2340aagcagagag acgcgctgcc agaaagcgag
tcatcaagtt acttctcaag cgactcgctg 2400acagacccgt ggacaacaag
cgacgacgat tttcagagtg accctgaccc cctcaccaat 2460aaacgcaaaa
aacgcttgca attctaactc tgtctctgtg acttcattgg gggggtccgg
2520gggggcttgc ccccccgtta gttgggttct cgcactcccg cctgccaagt
gaaactcggg 2580gaggagtgag tgcgggacat cccgtgtaat ggctacataa
ctacccggct ttgcttcgac 2640agtggccgtg gctcgaccct cacacaacac
tgcagatagg gggcgcaatt gggatcgtta 2700gaaaactatg gccgagcatg
gggggggctc cgcccccccc aacccccccg gtgggggggc 2760caaggccccc
cctacacccc cccatggggg gctgccgccc cccaaacccc ccgcgtcgga
2820tggggggggc tgcgcccccc ccaaaccccc cttgcccggg gctgtgcccc ggaccccc
2878112875DNATorque teno sus virus 11tacacttccg ggttcaggag
gctcaatttg gctcgcttcg ctcgcaccac gtttgctgcc 60aagcggacct gattgaagac
tgacaaccgt tacattcaaa tttgaaaatg gcgcccaaac 120atggcggcgg
ggggcggagt ttatgcaaat taatttatgc aaagtaggag gagctccatt
180ttaatttatg caaagtagga ggagtcaatt ctgattggtc gggagctcaa
gtcctcattt 240gcatagggtg taaccaatca gatttaaggc gttcccatta
aagcgaatat aagtaagtga 300ggttccgaat ggctgagttt atgccgccag
cggtagacag aactgtctag cgactgggcg 360ggtgccggag gatccctgat
ccggagtcaa ggggcctatc gggcaggagc agctgagcgg 420agggcctatg
ccggaacact gggaggaagc ctggttggaa gctaccaagg gctggcacga
480tctcgactgc cgctgcggta actggcagga ccacctatgg ctcctactcg
ccgatggaga 540cgccgctttg gccgccgccg tagacgctat agaaagagac
gctatgggtg gagaagacgt 600tactaccgct acagaccgcg ttactatagg
agacgatggc tggtaaggag aaggcggcgt 660tccgtctacc gtagaggtgg
acgtagagcg cgcccctacc gaataagtgc ttttaaccca 720aaagtaatgc
ggagggtggt gattagaggt tggtggccaa tattacagtg tctaaaagga
780caggaatcac taagatatag accactgcag tgggacactg aaaaacagtg
gagagtaaag 840aaagactatg aggacaacta cggctacttg gtgcagtacg
gaggaggttg ggggagtggt 900gaagtgacat tggagggatt atatcaggaa
cacttactct ggagaaactc ttggtcaaag 960ggaaatgatg gcatggacct
agtgagatac tttggctgca tagtatacct gtacccactg 1020caggaccaag
actactggtt ttggtgggat acagacttta aagaactata cgcagagagc
1080atcaaagaat actcccagcc aagtgttatg atgatggcca aacgcactag
actagtaata 1140gctagagaca gagcaccaca cagaagaaga gtaagaaaaa
ttttcatacc cccgccaagc 1200agagacacca cacaatggca atttcagaca
gacttctgca aaaggccact attcacatgg 1260gcggcaggat taatagacat
gcagaaacca tttgatgcaa acggagcgtt tagaaacgcc 1320tggtggctag
aaacaaggaa tgaccaggga gaaatgaaat acattgaact atggggaagg
1380gtgccaccac agggtgacac agaactgcca aaacagagtg agtttaagaa
gggagataat 1440aaccctaact ataacataac ggaaggacat gaaaaaaata
tttacccaat aatcatatac 1500gttgaccaga aagaccagaa aacaagaaaa
aaatactgtg tatgctacaa caaaacttta 1560aatagatgga gaaaagccca
ggcgagtaca ttagcaatag gagatcttca aggactagta 1620ctgcgtcagc
ttatgaatca ggagatgaca tactactgga aatcgggaga gttttcctca
1680ccattcctgc aaagatggaa aggaactagg ctaataacca tagacgcaag
aaaggcagac 1740acagaaaacc caaaagtaag ttcgtgggaa tgggggcaaa
actggaacac aagcggaaca 1800gtgctacagg aggtattcaa catttcactg
aacaacactc aaataagaca ggatgacttt 1860gcaaaattga cactgccaaa
gtcaccacat gacatagact ttggacatca cagcagattt 1920ggaccattct
gtgttaaaaa cgaaccacta gaattccaac tactgcctcc aacaccaact
1980aacctatggt ttcagtacaa atttctcttt cagtttggcg gtgaatacca
gccaccaaca 2040ggtatccgcg atccctgcat tgatacacca gcctatcctg
tgccgcagtc aggaagtgtt 2100acacacccca aattcgccgg aaagggcgga
atgctcacgg aaacagaccg ttggggtatc 2160actgctgcct cttccagaac
cctcagtgca gatacaccca ccgaagcagc gcaaagtgca 2220cttctcagag
gggacgcgga aaagaaagga gaggaaaccg aggaaaccgc gtcatcgtcc
2280agtatcacga gtgccgaaag ctctactgag ggagatggat cgtctgatga
tgaagagaca 2340atcagacgca gaaggaggac ctggaagcga ctcagacgga
tggtcagaca gcagcttgac 2400cgacgaatgg accacaagcg acagcgactt
cattgatacc cccataagag aaagatgcct 2460caataaaaaa caaaaaaaac
gctaaacagt gtccgcctat tagtgggggg gtccgggggg 2520gcttgccccc
ccgtaagcgg ggttaccgca ctaactccct gccaagtgaa actcggggac
2580gagtgagtgc gggacatccc gtgtaatggc tacataacta cccggctttg
cttcgacagt 2640ggccgtggct cgaccctcgc acaacactgc aggtaggggg
cgcaattggg atcgttagaa 2700aactatggcc gagcatgggg ggggctccgc
cccccccaac ccccccggtg ggggggccaa 2760ggccctccct acaccccccc
atggggggct gccgcccccc aaaccccccg cgtcggatgg 2820ggggggctgc
gcccccccca aacccccctt gcccggggct gtgccccgga ccccc 2875
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