Detection of torque teno sus virus 1 and 2 in porcine tissues by in situ hybridization using multi-strained pooled probes
Introduction
Torque teno virus (TTV) is a group of novel non-enveloped DNA viruses with a circular, single-stranded, negative sense DNA genome. Depending on the host species, the size of the genome of TTV ranges from 2.8 to 3.9 kb with low nucleotide homology (Biagini, 2009). In pigs, two major groups, recently categorized as two different species and named as torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2), have been characterized (Aramouni et al., 2011). Similar to porcine parvovirus (PPV) and porcine reproductive and respiratory virus (PRRSV), TTSuV has been regarded as a common co-factor in porcine circovirus-associated diseases (PCVAD) (Opriessnig and Halbur, 2012). Nevertheless, the pathogenicity of TTSuV1 and TTSuV2 has been in a controversy for a period of time. TTSuV2 has been extrapolated to have a higher pathogenic potential since its viral load was significantly higher in PMWS-affected pigs than in healthy ones, while the viral load of TTSuV1 was similar in both groups (Aramouni et al., 2011). On the other hand, inoculation of TTSuV1-containing tissue homogenates prior to PCV2 or combined with PRRSV may contribute to PMWS expression or experimental reproduction of porcine dermatitis and nephritis syndrome (PDNS), respectively, in gnotobiotic piglets (Ellis et al., 2008, Krakowka et al., 2008). Aside from this, no significant histopathological changes were revealed in TTSuV2-inoculated specific pathogen-free piglets (Mei et al., 2011). To date, a direct positive correlation between TTSuV and disease has not yet been well established.
Clarification of target cell is the first step to understand the pathogenesis of viral infection, which remains unclear for TTV. The present knowledge regarding the infection of TTSuV is primarily derived from the polymerase chain reaction (PCR)-based results (Kekarainen et al., 2006, Nieto et al., 2011). Despite this molecular technique could be used for the evaluation of the prevalence as well as the quantification of viral load of TTSuV1 and TTSuV2, the information regarding the correlation between viral infection and histopathological changes cannot be revealed. In situ hybridization (ISH) is a measurement to display viral nucleic acids directly in tissues, providing a way to observe the virus-infected target cells, and to connect the viral detection with the histopathological changes simultaneously. To our knowledge, so far only two reports showed some data regarding the tissue distribution of TTSuV by using ISH with oligonucleotide probe. Krakowka and Ellis (2008) reported that scattered weak signals of TTSuV1 genome were detected in the bone marrow of TTSuV1-inoculated gnotobiotic pigs, while Martin-Valls et al. (2008) demonstrated a small to moderate amount of TTSuV2 signal in the lymphoid tissues of PMWS-affected pigs. Although the copy number of TTSuV genome detected by real-time PCR could reach above 108/μg of total DNA from 25 mg tissue in some naturally infected pigs (Nieto et al., 2013), to our experience, the presence of apparent tissue signals of TTSuV genome has not yet been well demonstrated by conventional ISH procedure.
Considering the extensive divergence of TTSuV DNA sequences, ranging from 15% to 30%, among various strains (Cortey et al., 2011, Huang et al., 2010) and the fact of more than one genotype or subtype of the same species of TTSuV occurring in the same host (Huang et al., 2010), the aims of this study were to establish a highly effective ISH for the detection of TTSuV1 and TTSuV2 in the tissue by using multi-strained pooled probe, rather than single-strained probe, to perform TTSuV screening with the high throughput tissue microarray (TMA) composed of 278 porcine superficial inguinal lymph nodes (siLNs), and to further evaluate the tissue tropism and potential target cells of TTSuV.
Section snippets
Experimental design
Tissues of siLNs from 50 wasting pigs were subjected to TTSuV detection by PCR. The amplicons were subcloned and sequenced. Seven different strains of TTSuV1 and TTSuV2 were selected for probe synthesis by using DIG-labeled PCR program. The probes of the 7 selected strains from each species of TTSuV were then mixed in an equal ratio to form the pooled probe. Single-strained PCR-derived DIG-labeled probes as well as DIG-labeled oligonucleotide were also designed and used for comparisons with the
Synthesis of PCR-derived DIG-labeled probes
Within the selected 50 post-weaning pigs displaying wasting clinically, the PCR results of the siLNs showed that 18 pigs were TTSuV1-positive (36%) and 21 pigs were TTSuV2-positive (42%), in which 11 pigs were double-positive (22%). Following cloning and sequencing, seven diversified strains of each TTSuV1 and TTSuV2 were randomly selected. The percentage identity (PI) of the sequences between the TTSuV1 standard strains (NCBI Genbank; accession number: GU188045, AY823990) and the TTSuV1
Discussion
In the present study, a modified ISH by means of multi-strained pooled probe for the detection of TTSuV1 and TTSuV2 in tissues was established to overcome the challenge of apparent genetic diversity of TTSuV (Cortey et al., 2011, Huang et al., 2010) and to reduce the bias caused by sequential specificity of single-strained probe (Anderson et al., 2003). The results of the multi-strained pooled probe-based ISH demonstrated a significantly enhanced sensitivity in TTSuV detection due to the
Acknowledgements
This research was supported in part by grants NSC 101-2313-B-002-024 and NSC 102-2313-B-002-028-MY3 from the Ministry of Science and Technology of the Republic of China (Taiwan).
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