Neither the viral DNA nor the expression of the putative ORF1 capsid protein of TTSuV1 or TTSuV2 was endogenously present in the five representative cell lines tested in this study.
The present study first aimed to identify potential permissive cell lines supporting TTSuV propagation. We 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, BHK-21 and MARC-145. These cell lines are known to be permissive to a wide variety of animal virus infections. In order to rule out the possibility of endogenous contamination of cultured cell lines with TTSuV1 or TTSuV2, the expression of both viral DNA and ORF1 protein was subjected to TTSuV1 or TTSuV2 real-time qPCR and IFA detection, respectively. OIE disease-free porcine serum that had been shown to have a high level of anti-TTSuV2 ORF1 antibody was also included as a control (15
). The results of the qPCR analysis showed that none of the five cell lines tested in this 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 those of the standards, and there were no specific bands corresponding to the expected PCR products (). The bands from the cell samples close in size to the standards were excised from the gel, sequenced, and found to be porcine and mammalian genomic sequences. The swine-derived trypsin and FBS products used for cell cultures were also negative (data not shown). In contrast, as expected, the commercial porcine serum was positive for TTSuV1 and TTSuV2 DNA ().
Fig 2 Detection of TTSuV1 or TTSuV2 contamination in five different cell lines (PCV1-free PK-15, 3D4/31, IPEC/J2, BHK-21, and MARC-145) and an OIE disease-free porcine serum by real-time qPCR. Fluorescence curves (A and C) and melting curves (B and D) of TTSuV1 (more ...)
To develop cell-based serological methods such as IFA or immunoperoxidase monolayer assay for TTSuV detection, we raised three specific antisera against the putative ORF1 capsid protein of TTSuV1a, TTSuV1b (Huang et al., submitted), 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 to test for susceptibility to TTSuV infection or replication by transfection with TTSuV2 DNA clones.
Construction and characterization of full-length TTSuV2 genomic DNA clones in porcine kidney PK-15 cells.
We were particularly interested in characterizing the infectivity of the TTSuV2 full-length DNA clone, since TTSuV2 has been reported to be associated with PMWS or PCVAD at a high rate of viral DNA prevalence (20
), a large viral load (1
), and a low antibody level in disease-affected pigs with an unknown mechanism (15
). We first generated two monomeric full-length TTSuV2 DNA clones, pSC-PTTV2c and pSC-TTV2-#471942, derived from prototype U.S. isolate PTTV2c-VA and German isolate TTV2-#471942, respectively (A and C) (9
). Each full-length TTSuV2 genomic DNA was inserted into cloning vector pSC-B-amp/kan, which does not contain a eukaryotic promoter. BamHI or EcoRV is the unique restriction site in the PTTV2c-VA or TTV2-#471942 genome, which was engineered at both ends of the genomic DNA to facilitate the generation of concatemers and thus to mimic the TTSuV circular DNA genome. Single digestion of the plasmid DNA of each clone with BamHI or EcoRV resulted in two different fragments of 4.3 kb and 2.8 kb. 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).
Subsequently, two copies of the full-length PTTV2c-VA genome from clone pSC-PTTV2c were ligated in tandem into the pSC-B-amp/kan vector to generate clone pSC-2PTTV2c-RR (B). Comparison of the AflII single-digestion patterns of 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 (A, right side). We utilized the same cloning strategy to produce a tandem-dimerized TTSuV2 DNA clone, pSC-2PTTV2b-RR, derived from pSC-TTV2-#471942 (D). The construct pSC-TTV2-#4719421 has two HindIII sites; one is located in the TTSuV2 genome, and the other is located in the vector. The short distance between them is 2.1 kb. When digested with HindIII alone, the construct pSC-TTV2-#4719421 produced two fragments of 2.1 and 4.9 kb, whereas the construct pSC-2PTTV2b-RR gave an additional 2.8-kb fragment representing the intact single TTSuV2 genome (A, left side), thus confirming the successful construction of the clone.
Fig 3 Identification and quality assessment of linear or circular TTSuV2 genomic DNA. (A) Comparisons of the HindIII single-digestion patterns between clones pSC-TTV2-#471942 and pSC-2PTTV2b-RR (left side) and AflII single-digestion patterns between clones (more ...)
Circular TTSuV2 DNA was generated by tandem ligation of the purified linear TTSuV2 genomic DNA excised from clone pSC-PTTV2c or pSC-TTV2-#471942. Typical monomer, dimer, and high-copy-number molecules of concatemerized TTSuV2 DNA were observed in the ligation products (B). The ligation mixture from PTTV2c-VA or TTV2-#471942 was transfected into PCV1-free PK-15 cells. IFA conducted at 5 days posttransfection using rabbit antiserum against PTTV2c-VA ORF1 indicated that the TTSuV2 ORF1 antigen was expressed in the nuclei of the transfected cells with an approximately 5% positivity rate (A and C). No fluorescent signal was observed in mock-transfected cells stained with the same anti-TTSuV2 serum (E) or in circular TTSuV2 DNA-transfected cells stained with the anti-TTSuV1a ORF1, anti-TTSuV1b ORF1 (Huang et al., submitted), or prebleed rabbit serum (data not shown). Passage of the transfected cells two times did not eliminate but did reduce the fluorescent signal (data not shown). When the transfected cells were continuously passaged for up to 20 passages, no positive signal was detectable, suggesting that TTSuV2 infection did not occur (data not shown).
Fig 4 IFA results of PCV1-free PK-15 cells transfected with the ligation mixtures of linear TTSuV2 genomic DNA derived from clone pSC-PTTV2c (A) or pSC-TTV2-#471942 (C) with plasmid pSC-2PTTV2c-RR (B) or pSC-2PTTV2b-RR (D) or with Lipofectamine LTX only (E). (more ...)
We next tested whether direct transfection of plasmid DNA of tandem-dimerized clone pSC-2PTTV2c-RR or pSC-2PTTV2b-RR into PK-15 cells resulted in the synthesis of TTSuV2 ORF1. A transfection efficiency of 50 to 60% in PK-15 cells was measured by using a green fluorescent protein (GFP) expression construct with the Lipofectamine LTX reagent (data not shown). The tandem-dimerized double-stranded DNA does not represent genomic anellovirus DNA but might represent an infectious replicative intermediate. IFA at 5 days posttransfection using the same anti-TTSuV2 ORF1 antiserum confirmed that both DNA clones also expressed ORF1 in transfected PK-15 cells (B and D). Again, ORF1 was expressed in cell nuclei. However, the fluorescence intensity and positivity rate were lower than those in circular TTSuV2 DNA-transfected cells (B and D). We did not observe localization of the ORF1 antigen in the cytoplasm of the transfected cells.
Experimental identification of two introns in the TTSuV2 genome.
Although the transcriptional profile of 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 (A) (16
). Continuous mRNA1 encodes ORF1 and ORF2, whereas removal of the putative intron of 1,341 nucleotides (nt) (designated intron 1 here), corresponding to nucleotide positions 648 to 1988 in the PTTV2c-VA genome, generates putative mRNA2, which contains two discontinuous ORFs, ORF1/1 and ORF2/2 (16
). We also speculated that more spliced mRNAs and their encoded proteins of TTSuV may exist, as shown for human TTV (30
Fig 5 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 positions (more ...)
To determine whether the splicing of putative intron 1 in TTSuV2 occurred, total RNA was extracted from PK-15 cells transfected with circular PTTV2c-VA DNA, followed by DNase I treatment and RT-PCR analysis. Two PCR product bands of approximately 500 bp and 600 bp 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 (B), whereas the small cDNA product of 492 bp contained two splicing regions, including intron 1 and an additional 91-nt intron, corresponding to nucleotide positions 2103 to 2193 in the PTTV2c-VA genome, which was designated intron 2 in this study (C). The splicing sites are conserved among all of the published TTSuV2 sequences (data not shown). Therefore, in this study, for the first time, we experimentally demonstrated the existence of splicing of intron 1 and the viral mRNA2 transcripts. We also identified a novel viral mRNA transcript, termed mRNA3, which encodes two putative proteins, ORF1/1/2 and ORF2/2/3, and which switches reading frames from 1 to 2 and 2 to 3, respectively, due to the splicing of intron 2 (A). The mRNA3 transcript contains at least three exons in the TTSuV2 genome. Since we failed to determine the 5′ and 3′ ends of the viral mRNA transcripts by rapid amplification of cDNA ends-PCR, it is possible that there exists an additional TTSuV2 intron in the region upstream of ORF2, as known in human TTV transcripts (30
). However, the human TTV genome does not contain a short intron corresponding to TTSuV intron 2 in the region downstream of the large intron (intron 1).
Nevertheless, transfection of PK-15 cells with circularized TTSuV2 genomic DNA resulted in the synthesis of viral mRNA transcripts and expression of the ORF1 protein, indicating that the TTSuV2 concatemers likely mimicked the transcription of and protein expression from the natural circular genome of TTSuV2.
A tandem-dimerized TTSuV2 clone, pSC-2PTTV2c-RR, is infectious when inoculated into CD pigs.
To test the infectivity of TTSuV2 DNA clones in pigs, we first performed a pilot study with three groups of CD pigs with two pigs per group. The pigs were inoculated with PBS buffer (pigs 1 and 2) in group 1, the tandem-dimerized clone pSC-2TTV2c-RR (pigs 3 and 4) in group 2, and pSC-2TTV2b-RR (pigs 5 and 6) in group 3, respectively. Serum samples were collected from the animals at 0, 7, 14, 21, 28, 35, and 42 dpi. Pig no. 2 died of septicemia due to an unidentified bacterial infection shortly after inoculation.
TTSuV2 DNA was detected by real-time qPCR beginning at 28 dpi in two pigs inoculated with pSC-2TTV2c-RR. The viral loads of both pigs, although very light, increased weekly until 42 dpi before necropsy at 44 dpi. The viral load in the serum of pig no. 3 increased from 1.93 × 103 copies/ml at 28 dpi to 5.59 × 103 copies/ml at 35 dpi and 4.36 × 104 copies/ml at 42 dpi, whereas the serum viral loads in pig no. 4 rose from 5.07 × 103 copies/ml at 28 dpi to 4.49 × 104 copies/ml at 35 dpi and 8.87 × 104 copies/ml at 42 dpi. Moderate microscopic lesions in the brain (lymphoplasmacytic encephalitis, mainly perivascular), liver (lymphohistiocytic hepatitis), and kidney (lymphoplasmacytic interstitial nephritis) were observed in pig 3 but not in pig 4. The remaining three pigs, including pigs inoculated with clone pSC-2TTV2b-RR, did not develop viremia during this study. However, pig no. 5 had mild lymphohistiocytic multifocal hepatitis. The results of this pilot pig experiment indicated that clone pSC-2PTTV2c-RR, from a U.S. strain of TTSuV2, is infectious.
Characterization of two TTSuV2 full-length genomic DNA clones with engineered genetic markers and a derived mutant clone in vitro.
To further rule out possible contamination with other indigenous TTSuV2 strains in the pilot animal study, it is critical to introduce tractable genetic markers into the TTSuV2 genome so that the cloned virus and the potential indigenous contaminating virus in pigs can be discriminated in inoculated animals. We introduced a unique HpaI restriction site and two unique restriction sites, PstI and MfeI, into two TTSuV2 monomeric DNA clones, pSC-TTV2-#471942 and pSC-PTTV2c, to produce two new clones, pSC-TTV2-EU and pSC-TTV2-US, respectively (E and F). The positions of these sites, located in intron 1, were expected not to 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. ORF1 expression in the nuclei of transfected cells was detected by IFA at 3 days posttransfection, similar to the patterns of their parental clones (), indicating that the clones with introduced genetic markers were replication competent.
Fig 6 IFA results of PCV1-free PK-15 cells transfected with the ligation mixtures of linear TTSuV2 genomic DNA derived from clone pSC-TTV2-EU, pSC-TTV2-US, or pSC-TTV2-ΔAA. Cells were stained with an anti-TTSuV2 ORF1 antibody and Alexa Fluor 488-conjugated (more ...)
Mutant clone pSC-TTV2-ΔAA, with a 104-bp deletion (nucleotide positions 332 to 437) from the putative TATA box (nucleotide positions 283 to 289; A) to the ORF1 (nt 528) and ORF2 (nt 445) start codons, was generated based on clone pSC-TTV2-US (G). When transfected into the PK-15 cells, the circularized DNA from this mutant clone did not express the ORF1 antigen (), suggesting that the deleted region likely contains a cis-acting element important for viral mRNA transcription or TTSuV2 ORF1 translation. The result obtained with the deletion mutant clone also implied that the observed expression of ORF1 is likely driven by 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).
Expression of the TTSuV2 ORF1 protein in various cell lines transfected with the circularized TTSuV2 DNA from 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 is deficient. To search for another cell line that may be permissive for TTSuV2 infection, we subsequently transfected 11 other different cell lines with the circularized TTSuV2 DNA from clone pSC-TTV2-US, respectively. These cell lines included the four cell lines (3D4/31, IPEC-J2, BHK-21, and MARC-145) that were negative for TTSuV1 or TTSuV2 at both the DNA and protein levels. Plain cells of the other seven lines (ST, Vero, 293TT, HeLa, Huh-7, HepG2, and CHO-K1) were also negative for TTSuV2 ORF1, as determined by IFA (data not shown).
After transfection, all of the 11 cell lines expressed the ORF1 protein at 3 days posttransfection (; BHK-21 and CHO-K1 results not shown). The percentages of transfected cells with positive IFA signals were subjectively categorized into the following three levels: IPEC-J2, ST, PCV1-free PK-15, Huh-7, and HepG2 with a high positivity rate (>5%); 3D4/31, Vero, MARC-145, and 293TT with a medium positivity rate (2 to 5%); and HeLa, BHK-21, and CHO-K1 with a low positivity rate (<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 transfected cells.
Fig 7 Transfection of nine different cell lines with the ligation mixture of linear TTSuV2 genomic DNA derived from clone pSC-TTV2-US. Images of Alexa Fluor 488-conjugated antibody staining (green) merged with nuclear staining with DAPI (blue) are shown. Magnification, (more ...)
To test if some of these IFA-positive cells were susceptible to TTSuV2 infection, supernatants collected from lysates of PK-15, ST, and 293TT cells transfected with circularized TTSuV2 DNA were inoculated into all of the cell lines with a high rate of positivity and some with a medium rate of positivity, including the 293TT cell line, respectively. The inoculated cells were cultured for 3 to 5 days and examined by IFA. No fluorescent signal was detected in these cells (data not shown), indicating that none of the cell lines tested are susceptible to TTSuV2 infection.
Rescue of TTSuV2 from concatemerized TTSuV2 genomic DNA of clone pSC-TTV2-US in CD/CD pigs.
With the introduced genetic markers in the full-length DNA clones that can be used to distinguish between infections caused by the cloned virus and potential indigenous contaminating virus, we performed an additional study with CD/CD pigs to further verify the in vivo infectivity of the TTSuV2 genomic DNA clones. Twelve CD/CD pigs were assigned to three groups of four pigs each. Pigs in each group were inoculated with PBS buffer, concatemerized TTV2-EU DNA, and TTV2-US DNA, respectively. Preinoculation serum samples from all of the pigs (collected 30 days prior to inoculation) were negative for TTSuV1 or TTSuV2 DNA by real-time qPCR. Serum samples were collected from all of the animals at 0, 7, 14, 21, 28, and 35 dpi.
TTSuV2 DNA was detected in all eight inoculated pigs, but unfortunately, it was also detected in two negative-control pigs (no. 136 and 142), indicating contamination with other strains of TTSuV2 indigenous in the research facility or the source pigs, which is not uncommon (see Table S1 in the supplemental material). One pig (no. 133) inoculated with the concatemerized TTSuV2-US DNA had detectable viremia even at 0 dpi, whereas the other pigs developed viremia at 14 or 21 dpi (see Table S1 in the supplemental material). Except for pig no. 133, the seven TTSuV2 DNA-inoculated pigs and the two TTSuV2-positive pigs in the negative-control group had increased viral loads until necropsy, indicating active virus infection (see Table S1 in the supplemental material). We speculated that the source of the TTSuV2 contamination was likely due to the 1-month waiting period between the date of preinoculation serum sample testing (by which all of the animals were negative) and 0 dpi. Since total DNA was extracted from these samples without DNase I pretreatment, to rule out the possibility of the presence of inoculated free DNA in the sera from pig no. 133, serum samples from this pig and from pigs no. 136 and 142 in the negative-control group with positive PCR results were subjected to DNase I treatment or left treated before DNA extraction followed by qPCR. The result was consistent with Table S1 in the supplemental material, showing that there was no significant viral load difference between DNase I-treated and untreated samples (see Table S2 in the supplemental material).
However, thanks to the introduced genetic markers in the TTSuV2 DNA clones used in this study, we were still able to determine if the TTSuV2 DNA clones were infectious in pigs, which was the main objective of our study. Since we have previously demonstrated that a single pig can be infected with multiple strains of TTSuV2 and TTSuV1 (9
), prior infection or concurrent infection with an indigenous TTSuV2 strain should not interfere with the infection of pigs with the TTSuV2 DNA clones we intended to test in this study. To determine if the genetic markers of TTSuV2-EU or TTSuV2-US were present in viruses recovered from the sera of infected pigs with the mixed TTSuV2 infection status, we 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 concatemerized TTSuV2-US DNA had TTSuV2 sequences identical to the introduced genetic markers PstI and MfeI, whereas serum samples from the negative-control group and from pigs inoculated with concatemerized TTV2-EU DNA did not contain any introduced genetic markers (data not shown). Therefore, this pig study further confirmed the initial pilot pig study in which the TTSuV2-US full-length DNA clone was infectious in pigs. The results also experimentally verified, for the first time, that pigs can be coinfected with different strains of TTSuV2.