Note: Descriptions are shown in the official language in which they were submitted.
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INFECTIOUS GENOMIC DNA CLONE AND SEROLOGICAL PROFILE OF TORQUE
TENO SUS VIRUS 1 AND 2
REFERENCE TO RELATED APPLICATION
[0001] This patent application in a continuation-in-part of U.S. Patent
Application No.
12/861,378, which claims the benefit of U.S. Provisional Patent Application
No. 61/235,833,
filed on August 21, 2009, and U.S. Provisional Patent Application 61/316,519,
filed on March 23,
2010. This patent application also claims the benefit of U.S. Provisional
Patent Application No.
61/588,988, filed on January 20, 2012. 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
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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-
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7). TTSuV1 and FlbuV2 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-PM WS-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 IT virus,
an orphan virus,
in pigs from six different countries. Vet Microbio1104:113-7).
[00041 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. ISaito, and A.
Yoshikawa.
2002. Genomic characterization of Tr viruses (TTVs) in pigs, cats and dogs and
their relatedness
with species-specific TIVs 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
UV DNA
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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.
ISaito, 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 6RF1 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
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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).
10006]
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 postwearuing 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
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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 T1'SuV1 genotypes (la
and lb),
between two species (TTSuV1 and 2), and between porcine and human
artelloviruses. 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.
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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).
100071 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.
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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. Diliner, 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.
100081 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; Forns,
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
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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 theanti-ORF1 antibodies from one TTV or TTSuV
genotype can
cross-protect against infection withanother genotype. To our knowledge, for
human TTV or
TTSuV infection there is noinformation on this topic available to date.
Furthermore, the
antigenic diversity and relationshipof 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, ffany,
antigenic cross-reactivity between different anellovirus species/genotypes,
due to the factthat
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
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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 -lb and anti-TTSuV2. Finally, for the
first time, the
inventors assessed the antigenic relationships between two TTSuV1 genotypes
(TTSuVla and
TTSuVlb), between two species (1TSuV1 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
TTSuVla, TTSuV1b and TTSuV2, respectively.
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SUMMARY OF THE INVENTION
100101 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.
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100161 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
TifsuV1a, 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 ITsuVlb.
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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] Figure 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]
Figure 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 (104
pg; indicated by
red), five different cell lines (blue) and the porcine serum (green). For each
sample, duplicate
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determinations were made. (E) Detection of specific TTSuV1 or TTSuV2 qPCR
products
(marked by black arrowheads) by agarose gel electrophoresis.
[0025] Figure 3 illustrates identification and quality assessment of
linear or circular ITSuV2
genomic DNA. (A) Comparisons of the HindlII 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-2131TV2c-RR. (B) Quality assessment
of
concatemerized ligation products of the BamH1-digested and purified P1'1 V2c
genomic DNA.
The samples were electrophoresed in a 1% agarose gel before (linear DNA) and
after (ligation
mixture) T4 DNA ligase treatment. Linear DNA (-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] Figure 4 illustrates Immunofluorescence assay (IPA) results on PCV1-
free PK-15 cells
transfected with the ligation mixtures of linear TTSuV2 genomic DNA derived
from clones
pSC-Y1-1 V2c (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 = 200x.
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100271 Figure 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] Figure 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-
ITV2-US or
pSC-TTV2-AAA. 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><.
[00291 Figure 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 = 200x.
10030] Figure 8 illustrates expression and purification of the amino-
terminaUy truncated
TTSuVla and TTSuVlb ORF1 proteins, respectively. (A) SDS-PAGE analysis of
unpurified and
purified TTSuV1a-ORF1 products. (B) SDS-PAGE analysis of unpurified and
purified
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TTSuV1b-ORF1 products. An amino- and carboxyl-terminally double-truncated
1TSuV1b-ORF1
(lb-ctruc) of smaller product size served as the control. (C) Near-infrared
fluorescent WB
analysis of purified la- and lb-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.
100311 Figure 9 illustrates TTSuVla or TTSuVlb 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
TTSuVla WB analyses of conventional pig sera from a farm in Wisconsin.
Purified la-ORF1
protein was used as the antigen. Sera tested negative for both TTSuVla and
TTSuVlb
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) TTSuVla or TTSuVlb ELISA
results of
the seven Virginia gnotobiotic pig serum samples, positive and negative
control reference sera.
100321 Figure 10 illustrates serological and virological profiles of TTSuV1
infection in 138
sera of pigs from three different herds. (A) Distribution of TTSuV1 viremia,
anti-TTSuVla and
anti-TTSuVlb IgG among 138 serum samples. Box-and-Whisker-plots of TTSuVla (B)
and
TTSuVlb (C) serum antibody level by TTSuV1 viral DNA load. N: Negative. The
detection
limit of the TTSuV1 real-time qPCR was 4 logio copies/ ml in this study.
100331 Figure 11 illustrates a retrospective evaluation of TTSuV1 viral
loads (A), antibody
levels to the ORF1 protein of TTSuVla (B) and TTSuVlb (C) in 10 pigs in group
A from the time
of their arrival at the research facility to two months after arrival.
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[0034] Figure 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] Figure 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-ITSuV1b (p<0.0001).
[0036] Figure 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.
100371 Figure 15 illustrates Immunofluorescence assay (WA) results of
PCV1-free PK-15
cells transfected with the plasmids pTri-1a0RF1 (A), pTri-1bORF1 (B) or pTri-
2cORF1 (C) at 3
days post-transfection. pTri-1a0RF1- 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 = 200x.
[0038] Figure 16 illustrates comparison of hydrophilicity profiles of
TTSuV1a (PTTV1a-VA
strain) and TTSuVlb (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
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truncated la- or lb-ORFI 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.
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DETAILED DESCRIPTION OF THE INVENTION
100391 Figure 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-ampikan 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 T1'SuV2 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
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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 ITSuV1 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 1TSuV2 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.
10041] 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
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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
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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, TISuVlb (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 (1 __________________________________________
1SuV1) in pigs and antigenic relationships between two
22
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TTSuV1 genotypes (la and lb), 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
23
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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 (Fig. 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 PITV2c-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).
100451 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
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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 HindlII
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 (Fig. 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-
TTSuVla 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 (la 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
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positive signal was detectable, suggesting that TTSuV2 infection did not occur
(data not
shown).
100471 The inventors next tested whether direct transfection of plasmid
DNA of the
tandem-dimerized clone pSC-2PTTV2c-RR or pSC-2PTTV2b-RR into P1(45 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 (Fig. 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 (Fig. 4B St 4D). The inventors
did not observe the
localization of ORF1 antigen in the cytoplasm of the transfected cells.
100481 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
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porcine Torque teno virus in a single pig and characterization of the full-
length genomic
sequences of four U.S. prototype PDT 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 prduct 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,
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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 T1'SuV2 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
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before necropsy at 44 DPI in both pigs. The viral loads in serum of pig no. 3
increased from
1.93x103 at DPI 28 to 5.59x103 at DPI 35 and 4.36x104 at DPI 42 whereas the
serum viral loads
in pig no. 4 elevated from 5.07x103 at DPI 28 to 4.49x104 at DPI 35 and
8.87x104 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-PTIV2c to produce two
new
clones pSC-TTV2-EU and pSC-TTV2-US, respectively (Fig. 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
29
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parental clones (Fig. 6), indicating that the clones with introduced genetic
markers are
replication competent.
[0054] A mutant clone pSC-TTV2-AAA 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
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plain cells of the other seven cell lines (ST, Vero, and 293TT, HeLa, Huh-7,
HepG2 and CHO-K1)
were also negative for IlbuV2 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 2931T 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
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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
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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 Pst1 and Mfel, 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
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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
34
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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
#10932903 v1
CA 02809714 2013-03-15
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 ITV
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 mRNAI than that of mRNA2 and mRNA3. In accordance
with the
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CA 02809714 2013-03-15
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.
37
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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
38
#10932903 v1
CA 02809714 2013-03-15
replication. Further study is needed to screen whether additional cell lines
are permissive to
TTSuV2 infection.
100681 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
Microbio1153:229-39;
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.
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[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
TTSuVlb 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 -lb 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 lb 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.
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CA 02809714 2013-03-15
[0070] Expression and purification of the N-terminally truncated TTSuVla
and TTSuVlb
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 TTSuVlb-ORF1 gene with a C-terminally engineered 8 X 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
lb-ORF1 as a control, termed pTri-1bORF1-ctruc, which is 71-aa shorter than lb-
ORF1, to
compare the size with that of pTri-1bORF1 in SDS-PAGE and WB analysis.
[0071] The three recombinant proteins, la-ORF1, lb-ORF1 and lbORF1-ctruc
were found to
be insoluble and expressed within the bacteria as inclusion bodies.
Purification of the crude
lysates from la-ORF1 products with a nickel-affinity column resulted in
visualization of two
bands of -40 KDa (white arrowheads) and -70 KDa (black arrowheads), as
analyzed by
Coomassie blue staining (Fig. 8A). The -40 KDa band is the expected product of
the truncated
la-ORF1 protein, whereas the -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 lb-ORF1 or 1bORF1-ctruc showed a smear in the crude lysates
(Fig. 8B). After
purification, two bands of -40 KDa and -70 KDa, similar to la-ORF1, were also
identified in the
purified lb sample, whereas only a -30 KDa polypeptide (white arrowheads) was
detected in
41
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the purified lb-ctruc sample (Fig. 8B). The bands of ¨40 KDa and ¨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-I-us-tagged
Mab (Fig. 8C). The results indicated that both the truncated la-ORF1 and lb-
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 la-ORF1 or 1b-ORF1 as the antigens, respectively. A TTSuV2-
seropositive and
TTSuVl/TTSuV2-DNA positive porcine serum (Huang, Y. W., A. R. Patterson, T.
Opriessnig, B.
A. Drymari, 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 la-ORF1 and the 1b-ORF1
antigen, as the
¨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
42
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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
TTSuVlb-based ELISAs were subsequently developed and standardized,
respectively. The
concentrations of the purified la-ORF1 or lb-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 0D450 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-TTSuVla 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
43
*10932903 vi
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TTSuV1 viremia was much lower than the TTSuVla or lb 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 TTSuVla and TTSuVlb
antibody titers
than pigs with TTSuV1 viral DNA load at the levels of 104 to 106 copies/m1 (p
<0.05) in these
three groups (Fig. 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
TTSuVla/TTSuVlb-
dually seropositive but viral DNA-negative (77 samples) was higher than that
of pigs with
TTSuVla- or TTSuVlb-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 TTSuVla and TTSuVlb are high, which was expected;
and (ii) a
certain degree of cross-reactivity may exist between anti-TTSuVla and anti-
TTSuVlb 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
44
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putative ORF1 capsid protein of Torque teno sus virus 2 (TTSuV2) and
development of Western
blot and ELISA serodiag-nostic 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-TTSuVla and anti-TTSuVlb antibody titers
increased in all 10
pigs (Fig. 1113 & 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 (Fig. 12A & 1213). 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).
#10932903 v1
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[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-
TTSuV1a, anti-TTSuVlb 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 flbuV2 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
46
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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 Figure 6A, 82 of 138 serum
samples were
triple-seropositive, indicating that these pigs had been infected by TTSuV1
(TTSuV1a and/or
TTSuVlb) 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-TTSuVla and
anti-TTSuVlb,
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-TTSuVla
and anti-
ITSuV1b, 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-
TTSuVlb (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
47
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hypothesize that there exists an antigenic cross-reactivity between the two
TTSuVla and
TTSuVlb genotypes.
[0084] Analysis of antigenic relationships among TTSuVla, TTSuVlb and
TTSuV2 by
ELISA. Three antisera against the truncated recombinant ORFls of TTSuVla,
TTSuVlb or
TTSuV2 were raised by immunization of rabbits with the respective purified
recombinant
antigen. Cross-immunoreativity 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 1lbuV
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 TTSuVla or
TTSuVlb
antiserum even at 1:200 dilution since the OD value was relatively low (Fig.
14C). In contrast,
the TTSuVlb antigen did cross-react with the anti-TTSuVla 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
ITSuVla antigen likely cross-reacted with the anti-TTSuVlb 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 TTSuVla and
TTSuVlb genotypes
but not between the two species TTSuVla or lb and TTSuV2.
[0086] Demonstration of antigenic relationships among TTSuVla, TTSuVlb and
TTSuV2,
and between TTSuVs and a genogroup 1 human TTV by [FA. In order to definitely
analyze the
antigenic cross-reactivity among these viruses, an antibody cross-reactivity
experiment was
48
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performed by using IFA staining. PK-15 cells were transfected with three
plasmid constructs,
pTri-1a0RF1, pTri-1bORF1 and pTri-2cORF1, which harbor the truncated ORF1
capsid genes
from TTSuVla, TTSuVlb and ITSuV2, respectively. Three days post-transfection,
cells were
stained with anti-TTSuVla, anti-TTSuV1b, anti-TTSuV2 and pre-bleed serum,
respectively. As
shown in Fig. 15, cells transfected with pTri-1a0RF1 (Fig. 15A) or pTri-1bORF1
(Fig. 15B)
stained positive with both anti-TTSuVla and anti-TT'SuV1b 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 (Fig. 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 ITV 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
TTSuVla and TTSuVlb as shown by the ELISA but not between the TTSuVla or lb
and
49
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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 TTSuVla 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 TISuV1b
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
#10932903 v1
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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).
100901 Detection of human TTV IgG antibodies in human populations based
on the human
Try ORF1 as the antigen has been reported (Maggi, F., and M. Bendirtelli.
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 lb 'ITV
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 arginirte-deleted constructs (ORF1AArg and ORF1-NAArg)
as well as the
51
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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 ITV 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 1TSuV2-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
ITSuV1a 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
52
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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 TTSuVla,
TTSuV1b and
TTSuV2 in gnotobiotic pigs of group D (Fig. 9). A high seropositive rate of 1-
lbuVla (92.8%) or
TTSuV1b (87.7%) was revealed in the 138 groups A-C pigs (Fig. 10A), which was
higher than
that of TTSuV2 (-60%) (13), indicating a wider spread of actual TTSuV1
infection or the
53
*10932903 v1
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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 TTSuV1 a, TTSuVlb 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 TTSuVla and
TTSuVlb
genotypes but not between the two TTSuV species (TTSuVla or lb and TTSuV2).
First, when
compared to the serum samples with single TTSuVla- or TTSuVlb-seropositivity,
the numbers
of serum samples with TTSuVla/lb-dual seropositivity was much higher (Fig.
10A), likely
implying a certain degree of cross-antigenic reactivity between TTSuVla and
ITSuVlb
antibodies. Secondly, the number of serum samples with dual TTSuVla and
TTSuVlb
seropositivity was significantly higher than that of dual seropositivity to
TTSuVla and TTSuV2,
or to TTSuVlb and TTSuV2 (Fig. 13A). Inaddition, a high correlation of
antibody levels
between anti-TTSuVla and anti-TTSuVlb 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 TTSuVla and TTSuVlb. Thirdly, this
hypothesis was
experimentally confirmed by analysis of the antigenic relationships among
TTSuVla, TTSuVlb
and TTSuV2 through antigen-specific ELISAs (Fig. 14), and antibody cross-
reactivity studies in
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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 TTSuVla or lb ORF1 with that of TTSuV2, the inventors identified
two putative
antigenic sites on the ORF1 that are shared by ITSuVla and TTSuVlb (Fig. 16).
100941 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 (Try, 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.
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[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 (TTSuVla and lb) 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.
100961 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 serodiag-nostic
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
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cross-react with the heterologous TTSuV antigen, it appears that ITSuV1
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 TTSIN
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,
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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.
100991 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 T'TsuV,
such as porcine
dermatitis and nephropathy syndrome (PDNS), postweanirtg multisystemic wasting
syndrome
(PMWS), and other related illness. The vaccines comprise, for example, an
inactivated or
attenuated TTsttV virus, a nontoxic, physiologically acceptable carrier and,
optionally, one or
more adjuvants.
1001001 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-a, IFN-13, IFN-y, etc.), saponins,
monophosphoryl lipid A
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(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.
1001011 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.
1001021 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.
1001031 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.
1001041 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
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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.
[00105] 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.
[00106] 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 TrsuV1a, PTTsuV1b
and TTsuV2.
[00107] 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
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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.
[00108] 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.
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[00109] 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.
[00110] 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.
[00111] 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.
[00112] To prepare inactivated virus vaccines, for instance, the virus
propagation and virus
production can occur in cultured porcine cell lines such as, without
limitation P1(45 cells. Serial
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virus inactivation is then optimized by protocols generally known to those of
ordinary skill in
the art or, preferably, by the methods described herein.
[00113] 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.
[001141 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,
TTsuVlb and TTsuV2, which were expressed and purified in Escherichia coil (E.
colt) 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
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form virus substructures or identifiable parts or units of such substructures;
oligoglycosides,
glycolipids or gl.ycoproteins 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.
[00115] 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, Mani.atis et at,
"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.
[00116] 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
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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.
[00117] 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.
[00118] 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 CsC1 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.
[00119] 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
ORB, ORF2, ORF111, and ORF2/2, etc.). Such identified genes or immuno-dominant
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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., "Bacu.lovirus
Expression Vectors: A Lab Manual," Freeman Sz 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.
[00120] 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.
[00121] 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-domin.ant proteins of the virus. The
transfer vector
includes, for example, linearized baculovirus DNA and a plasmid containing the
desired
polyn.ucleotides. The host cell line may be co-transfected with the linearized
baculovirus DNA
and a plasmid in order to make a recombinant baculovirus.
[00122] 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.
1001231 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
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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.
1001241 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 immu.no-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.
[00125] 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 ITV. 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,
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transdermally, parenterally, etc. The parenteral route of administration
includes, but is not
limited to, intramuscular, intravenous, intraperitoneal and subcutaneous
routes.
[00126] 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.
[00127] 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 he prepared using a colloid mill. Emulsions, for example, may be
prepared using
a homogenizer.
[00128] 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
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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.
[00129] 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
C. to about 28
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.
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EXAMPLES
Example 1. Cell lines and cell cultures.
1001301 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, NC) (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
ovarycell 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 GlutaMAXTm-I (Invitrogen,
Carlsbad, CA)
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-
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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 jig/m1
hygromycin B and
antibiotics. All cells were grown at 37 C with 5% CO2.
Example 2. Analysis of TTSuV1 or TTSuV2 contamination in cultured cells by
real-time
quantitative PCR (qPCR).
[00131] 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. Haifa11, 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
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cell line using the QIAamp DNA mini kit (Qiagen) and was subsequently
subjected to TTSuV1
or TTSuV2 qPCR detection in a 25-41 PCR system using SensiMix SYBR &
Fluorescein kit
(Quantace Ltd) as described previously (Huang, Y. W., B. A. Dryman, K. K.
Harm11, 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 quantdtation 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.
[001321 The inventors have previously expressed and purified a recombinant
truncated
ORF1 protein of TTSuV2 (PTTV2c-VA strain) (Huang, Y. W., K. K. Harrall, B. A.
Dryman, N. M.
Beach, S. P. Kenney, T. Opriessnig, E. M. Vaughn, M. B. Roof, and X. J. Meng.
2011. Expression
of the putative ORF1 capsid protein of Torque teno sus virus 2 (TTSuV2) and
development of
Western blot and ELISA serodiagnostic assays: correlation between TTSuV2 viral
load and IgG
antibody level in pigs. Virus Res 158:79-88). The purified protein products
were used to
immunize two New Zealand white rabbits as a custom antibody production service
at Rockland
Immunochemicals (Gilbertsville, PA). Serum samples from both rabbits were
collected before
immunization (pre-bleed) and at 45 days post-immunization.
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Example 4. Construction of full-length genomic DNA clones of TTSuV2.
[00133] 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
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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.
[00134] 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 (G1TAAC; 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-AAA, 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
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AccI and Apal 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.
[00135] The PCV1-free PK-15 cells were seeded at 2x105 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 (-2 ug)
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).
[00136] 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
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hour at room temperature. Cells were washed 3 times with PBS and 500 pi 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 I DAPI (KPL, Inc.) at a 1:1000 dilution and visualized under a
fluorescence
microscope.
Example 8. RT-PCR.
[00137] 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- L
reaction with
the Advantage 2 PCR kit (Clontech) using primers 1TV2-448F (5'-
GAAGAAAGATGGCTGACGGTAGCGTACT-3'; SEQ ID NO:6) and TTV2-2316R (5'-
AGGTGC1"1GAGGAGTCGTCGC1-1G-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 trartsfection of colostrum deprived (CD) pigs with the
tandem-dimerized
TT SuV2 clones.
[00138] 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
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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 inta-
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 gg of the pSC-2TTV2c-RR plasmid DNA whereas the remaining
two pigs (nos.
and 6) in group 3 were each inoculated with 200 gg of the pSC-2TTV2b-RR clone.
1001391 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
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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.
1001401 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 j_tg 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
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inventors inoculated four, 40-day-old, CD/CD pigs (nos. 129, 135, 139 and 140
in group 1) each
with 150 jig 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 jig 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 quantitaiion 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
1TV2-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.
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[00141] 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 adultboars
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
fromVirginia
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.
[00142] 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 (1lbuV2) 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
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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 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.
[00143] 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 PTIV1a-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-1a0RF1 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 Ncol and XhoI restriction sites to generate two C-
terminally 8 x 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.
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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.
[00144] 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 C and then the bacterial culture was harvested by
centrifugation at 3,400 rpm
for 15 minutes at 4 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 8Ø 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 C until
use.
Example 14. Generation of anti-ORF1 antisera of TTSuV1a and TTSuV1b in
rabbits.
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[00145] 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 WE analysis.
[00146] 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-6xHis-tagged Mab at a 1:1000 dilution at 4 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).
[00147] For serum WB analysis, the purified TTSuV1a- or ITSuV1b-ORF1 proteins
were
incubated with individual porcine sera at a 1:200 dilution and with IRDye
800CW conjugated
rabbit F(ab1)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.
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[00148] 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 TTSuVlb 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.
[00149] 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
1.11/well. After incubation at 37 C for 2 hours, each well was washed 3 times
with 300 Al of Tris-
buffered saline-Tween 20 solution (TBS-T) and blocked with protein-free
blocking buffer
(Pierce) at a volume of 300 1 for 1 hour at 37 C. One hundred 1 of each
diluted serum sample
was transferred to the corresponding well on the ELISA plates and incubated at
37 C for 2
hours.
After washing the wells three times with 300 I of TBS-T buffer, the diluted
HRP-conjugated
rabbit anti-swine IgG (Rockland) was added to each well in a volume of 100 Al
and the plate
was
incubated at 37 C for 1 hour. A volume of 100 I 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 1/ well of 1 N HCL. The plates were then read at 450 nm using a
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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.
[00150] 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
TTSuVlb) in the five groups of pig sera as described previously (12). The
minimal detection
limit was 1.0X104 copies per ml in this study. The TTSuV1 qPCR assay does not
cross-amplify
ITSuV2 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
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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.
[00151] Data were analyzed using SAS software (version 9.2; SAS Institute
Inc., Cary, NC)
and GraphPad Prism software (version 5.0; San Diego, CA), respectively.
Antibody levels
(represented by S/N values) were compared between categories of logio 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), logio 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.
[00152] 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).
[00153] 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 ITV1.
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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.
[00154] 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-
1a0RF1, pTri-1bORF1
and pTri-2cORF1, mixed with 10 I 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).
[00155] 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 I Alexa Fluor 488-labeled goat
anti-rabbit IgG
(Invitrogen) at a 1:200 dilution for 1 hour incubation at room temperature.
Cells were stained
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with 500 pl DAPI (KPL, Inc.) at a 1:1000 dilution and visualized under a
fluorescence
microscope.
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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
++.
Pm-blood Anti-human
ti -T-1MA '' la Anti -TisSif.' I b Anti -T TSu V2
rabbit scrum TIN ( A K47)
pl LaORE I -"^r*
n- tb0 RYI 1.4-+
ti - 2cORFI 4+
h44-,Kk
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