Torque Teno Sus Virus Species Research Articles

Epidemiology and evolutionary analysis of Torque teno sus virus

Single stranded (ss) DNA viruses are increasingly being discovered due to the ongoing development of modern technologies in exploring the virosphere. Characterized by high rates of recombination and nucleotide substitutions, it could be comparable to RNA virus ones. Torque teno sus virus (TTSuV) is a standard ssDNA virus with a high population diversity, whose evolution is still obscure, further, it is frequently found in co-infections with other viruses threatening the porcine industry and therefore share the same host and epidemiological context. Here, we implement and describe approach to integrate viral nucleotide sequence analysis, surveillance data, and a structural approach to examine the evolution of TTSuVs, we collected samples from pigs displaying respiratory signs in China and revealed a high prevalence of TTSuV1 and TTSuVk2, frequently as part of co-infections with porcine circoviruses (PCVs), especially in spleen and lung. In addition, thirty six strains sequenced were obtained to investigate their genetic diversity in China. The evolutionary history of TTSuVs were unveiled as following: At the nucleotide sequence level, TTSuVs ORF1 was confirmed to be a robust phylogenetic maker to study evolution comparably to full genomes. Additionally, extensive recombination discovered within TTSuVk2a (also 5 out of the 36 sequenced strains in this study revealed to be recombination). Then, pairwise distance, phylogenetic trees, and amino acid analysis confirmed TTSuVs species, and allowed to define circulating genotypes (TTSuV1a-1, 1a-2, 1b-1, 1b-2, 1b-3, and k2a-1, k2a-2, k2b). Selection analysis uncovered seven and six positive selected sites in TTSuV1 and TTSuVk2, respectively. At the protein structure level, mapping of sites onto the three-dimensional structure revealed that several positive selected sites locate into potential epitopes, which might related to the potential escaping from host immune response. Our result could assist future studies on swine ssDNA virus classification, surveillance and control.

Prevalence of the Novel Torque Teno Sus Virus Species k2b from Pigs in the United States and Lack of Association with Post-Weaning Multisystemic Wasting Syndrome or Mulberry Heart Disease.

The family Anelloviridae includes a number of viruses infecting humans (Torque teno viruses, TTV) and other animals including swine (Torque teno sus viruses, TTSuV). Two genetically distinct TTSuV species have been identified from swine thus far (TTSuV1 and TTSuVk2), although their definitive association with disease remains debatable. In 2012, a novel TTSuV species was identified from commercial swine serum and classified in the genus Kappatorquevirus as TTSuVk2b. The other Kappatorquevirus species, TTSuVk2a, has been associated with post-weaning multisystemic wasting syndrome (PMWS) when coinfected with porcine circovirus type 2 (PCV2). Therefore, in this study, we initially amplified a portion of TTSuVk2b ORF1 and, subsequently, assessed the molecular prevalence of the virus in pigs in the United States. A total of 127 serum and 115 tissue samples were obtained from pigs with PMWS or mulberry heart disease (MHD) in six states and tested by PCR for the presence of TTSuVk2b DNA. Approximately 27.6% of the serum and 21.7% of tissue samples tested positive for TTSuVk2b DNA, and the positive products were confirmed by sequencing. However, we did not detect a correlation between TTSuVk2b infection and PMWS or MHD. The near full-length genomic sequence of US TTSuVk2b was determined, and sequence analysis revealed that the US TTSuVk2b isolates were 95% identical to the TTSuVk2b isolate from Spain, with most of the variations clustering in ORF1. We conclude that the novel TTSuVk2b species is present in pigs in the United States and its potential association with a disease warrants further investigation.

The pathogenic role of torque teno sus virus 1 and 2 and their correlations with various viral pathogens and host immunocytes in wasting pigs

The pathogenic role of torque teno sus virus (TTSuV) in swine is controversial among different studies. The present study intended to evaluate the potential pathogenicity of TTSuV based on its correlations with the histopathological changes, various common concurrently infected viral pathogens including porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PPV), as well as changes in the distribution and population of host immunocytes such as B lymphocytes, T lymphocytes, and macrophages by using the superficial inguinal lymph nodes (siLNs) of wasting pigs. A tissue microarray consisting of 270 available siLNs collected from 262 clinically wasting and 8 healthy pigs, respectively, were used for the detection of TTSuV1, TTSuV2, PCV2, PRRSV, and PPV by either in situ hybridization (ISH) or immunohistochemical (IHC) staining, and for the detection of various subsets of immunocytes by IHC staining with monoclonal antibodies to CD3, CD79a, and lysozyme. The slides were then subject to digital scanning followed by a semi-quantitative positive pixel evaluation for further statistical analysis. Although a high prevalence of TTSuV1 and/or TTSuV2 infection was noted in both wasting and healthy pigs, the wasting pigs had a significantly higher intensity in both TTSuV1 and TTSuV2 ISH-positive signals than healthy ones did. In the wasting pigs, a significant positive correlation in the tissue viral load was noted between TTSuV1 and TTSuV2 and between TTSuV2 and PCV2, but not between TTSuV1 and PCV2. Conversely, a significant negative correlation in the tissue viral load was revealed between TTSuV2, but not TTSuV1, and PRRSV. The tissue viral load of TTSuV1 was significantly correlated with B cell hyperplasia, while the tissue viral load of TTSuV2 was significantly correlated with increased macrophage population. The ISH positivity of TTSuV2 was significantly correlated with lymphoid depletion and granulomatous inflammation, which are the characteristic histopathological findings in postweaning multisystemic wasting syndrome-affected pigs. These findings suggest that both TTSuV species may have the potential involving the development of porcine circovirus-associated lymphoid lesions via alternating the host immune system.

High prevalence of co-infection with multiple Torque teno sus virus species in Italian pig herds.

Torque teno viruses (TTVs) are a large group of vertebrate-infecting small viruses with circular single-stranded DNA, classified in the Anelloviridae family. In swine, two genetically distinct species, Torque teno sus virus 1a (TTSuV1a) and 1b (TTSuV1b) are currently grouped into the genus Iotatorquevirus. More recently, a novel Torque teno sus virus species, named Torque teno sus virus k2b (TTSuVk2b), has been included with Torque teno sus virus k2a (TTSuVk2a) into the genus Kappatorquevirus. In the present study, TTSuV1 (TTSuV1a and TTSuV1b), TTSuVk2a and TTSuVk2b prevalence was evaluated in 721 serum samples of healthy pigs from Sardinian farms, insular Italy. This is the largest study to date on the presence of TTSuV in healthy pigs in Italy. The global prevalence of infection was 83.2% (600/721), being 62.3% (449/721), 60.6% (437/721), and 11.5% (83/721) the prevalence of TTSuV1, TTSuVk2a and TTSuVk2b, respectively. The rate of co-infection with two and/or three species was also calculated, and data show that co-infections were significantly more frequent than infections with single species, and that TTSuV1+TTSuVk2a double infection was the prevalent combination (35.4%). Quantitative results obtained using species-specific real time-qPCR evidenced the highest mean levels of viremia in the TTSuV1 subgroup, and the lowest in the TTSuVk2b subgroup. Interestingly, multiple infections with distinct TTSuV species seemed to significantly affect the DNA load and specifically, data highlighted that double infection with TTSuVk2a increased the viral titers of TTSuV1, likewise the co-infection with TTSuVk2b increased the titers of TTSuVk2a.

Discovery of a novel Torque teno sus virus species: genetic characterization, epidemiological assessment and disease association

The study describes a novel Torque teno sus virus (TTSuV) species, provisionally named Torque teno sus virus k2b (TTSuVk2b), originally found in commercial pig sera by applying the rolling-circle amplification technique. Full-length sequences of TTSuVk2b were obtained, annotated and used in the phylogenetic analyses, which revealed that TTSuVk2b is a novel Anellovirus species within the genus Kappatorquevirus of the family Anelloviridae. Quantitative PCR techniques were developed to determine total TTSuV DNA quantities as well as the prevalence and viral DNA quantities of TTSuV1, TTSuVk2a and TTSuVk2b. The mean total TTSuV load in seven commercial sera was determined at 6.3 log(10) DNA copies ml(-1) of serum, with TTSuVk2b loads being the lowest at 4.5 log(10) DNA copies ml(-1) of serum. Subsequently, prevalence and loads of TTSuVs were determined in pig sera from 17 countries. TTSuVk2b prevalence ranged from 0 to 100 % with viral loads from 3.3 to 4.6 log(10) copies ml(-1) of sera. TTSuVk2a, so far the only species in the genus Kappatorquevirus, has been linked to an economically important swine disease, namely post-weaning multisystemic wasting syndrome (PMWS). Considering the grouping of TTSuVk2b in the same genus as TTSuVk2a, TTSuVk2b prevalence and viral DNA load were determined in PMWS-affected animals and healthy counterparts. This revealed that TTSuVk2a and TTSuVk2b are not only genetically related, but also that their viral loads in serum are elevated in PMWS animals compared with those of healthy pen mates. In summary, the present work describes a novel TTSuV species including its genetic characterization, epidemiological assessment and potential disease association.

Serological Profile of Torque Teno Sus Virus Species 1 (TTSuV1) in Pigs and Antigenic Relationships between Two TTSuV1 Genotypes (1a and 1b), between Two Species (TTSuV1 and -2), and between Porcine and Human Anelloviruses

The family Anelloviridae includes human and animal torque teno viruses (TTVs) with extensive genetic diversity. The antigenic diversity among anelloviruses has never been assessed. Using torque teno sus virus (TTSuV) as a model, we describe here the first investigation of the antigenic relationships among different anelloviruses. Using a TTSuV genotype 1a (TTSuV1a) or TTSuV1b enzyme-linked immunosorbent assay (ELISA) based on the respective putative ORF1 capsid 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 (PCVAD), because both the viral loads and antibody levels were not different between affected and unaffected pigs and because there was no synergistic effect of concurrent PCV2/TTSuV1 infections. We did observe a higher correlation of IgG antibody levels between anti-TTSuV1a and -TTSuV1b than between anti-TTSuV1a or -1b and anti-TTSuV2 antibodies in these sera, implying potential antigenic cross-reactivity. To confirm this, rabbit antisera against the putative capsid proteins of TTSuV1a, TTSuV1b, or TTSuV2 were generated, and the antigenic relationships among these TTSuVs were analyzed by an ELISA and by an immunofluorescence assay (IFA) using PK-15 cells transfected with one of the three TTSuV ORF1 constructs. The results demonstrate antigenic cross-reactivity between the two genotypes TTSuV1a and TTSuV1b but not between the two species TTSuV1a or -1b and TTSuV2. Furthermore, an anti-genogroup 1 human TTV antiserum did not react with any of the three TTSuV antigens. These results have important implications for an understanding of the diversity of anelloviruses as well as for the classification and vaccine development of TTSuVs.

The prevalence of Torque teno sus virus (TTSuV) is common and increases with the age of growing pigs in the United States

Infection with the Torque teno sus virus (TTSuV) is believed to be common yet limited information is available on the epidemiology of TTSuV. The objectives of this study were to develop novel and improve existing diagnostic methods for TTSuV infection and to investigate the prevalence of TTSuV species 1 (TTSuV1) and 2 (TTSuV2) in the USA. Three hundred and four blood or fetal thoracic fluid samples were collected from pigs on 40 US farms in 12 States. Samples were collected from fetuses and in pre-suckle neonates (n=73), suckling pigs (1-20 days of age; n=27), nursery pigs (21-55 days of age; n=60), finisher pigs (8-25 weeks of age; n=90) and adults (>25 weeks of age; n=54). Samples were tested by a new quantitative differential real-time PCR for TTSuV1 and TTSuV2 DNA and by ELISA for detection of anti-TTSuV2-antibodies. The prevalence of TTSuV1 DNA ranged from 8.2% (fetuses and neonates) to 81% (finisher pigs) and the prevalence of TTSuV2 DNA ranged from 3.7% (suckling pigs) to 67% (finisher pigs). Evidence of fetal TTSuV infection was minimal. Mixed infection of TTSuV1 and TTSuV2 was seen in 6.7% of the nursery pigs, 52.2% of the finisher pigs, and 22.2% of the mature pigs. The prevalence of TTSuV1 was higher than that of TTSuV2. Anti-TTSuV2 antibodies were not detected in the fetuses and neonates and the seroprevalence of TTSuV2 was between 3.8% and 100% in growing pigs. The results of this study indicate that vertical transmission may not be a main route of TTSuV transmission in pigs in the USA.

Histopathological investigation in porcine infected with torque teno sus virus type 2 by inoculation

BackgroundPorcine torque teno sus virus (TTSuV) is a small icosahedral and non-enveloped virus which contains a single-stranded (ssDNA), circular and negative DNA genome and infects mainly vertebrates and is currently classified into the 'floating' genus Anellovirus of Circoviridae with two species. Viral DNA of both porcine TTSuV species has a high prevalence in both healthy and diseased pigs worldwide and multiple infections of TTSuV with distinct genotypes or subtypes of the same species has been documented in the United States, Europe and Asia. However, there exists no information about histopathological lesions caused by infection with porcine TTSuV2.MethodsPorcine liver tissue homogenate with 1 ml of 6.91 × 107genomic copies viral loads of porcine TTSuV2 that had positive result for torque teno sus virus type 2 and negative result for torque teno sus virus type 1 and porcine pseudorabies virus type 2 were used to inoculate specific pathogen-free piglets by intramuscular route and humanely killed at 3,7,10,14,17,21 and 24 days post inoculation (dpi), the control pigs were injected intramuscularly with 1 ml of sterile DMEM and humanely killed the end of the study for histopathological examination routinely processed, respectively.ResultsAll porcine TTSuV2 inoculated piglets were clinic asymptomatic but developed myocardial fibroklasts and endocardium, interstitial pneumonia, membranous glomerular nephropathy, and modest inflammatory cells infiltration in portal areas in the liver, foci of hemorrhage in some pancreas islet, a tiny amount red blood cells in venule of muscularis mucosae and outer longitudinal muscle, rarely red blood cells in the microvasculation and infiltration of inflammatory cells (lymphocytes and eosinophils) of tonsil and hilar lymph nodes, infiltration of inflammatory lymphocytes and necrosis or degeneration and focal gliosis of lymphocytes in the paracortical zone after inoculation with porcine TTSuV2-containing tissue homogenate.ConclusionsAnalysis of these presentations revealed that porcine TTSuV2 was readily transmitted to TTSuV-negative swine and that infection was associated with characteristic pathologic changes in specific pathogen-free piglets inoculated with porcine TTSuV2. Those results indicated no markedly histopathological changes happened in those parenchymatous organs, especially the digestive system and immune system when the specific pathogen-free pigs were infected with porcine TTSuV2, hence, to some extent, it was not remarkable pathological agent for domestic pigs at least. So, porcine TTSuV2 could be an unrecognized pathogenic viral infectious etiology of swine. This study indicated a directly related description of lesions responsible for TTSuV2 infection in swine.

A multiple SYBR Green I-based real-time PCR system for the simultaneous detection of porcine circovirus type 2, porcine parvovirus, pseudorabies virus and Torque teno sus virus 1 and 2 in pigs

Multiple viral infections are common in pigs under intensive production conditions. All five of the viruses included in this study are associated with multifactorial diseases that cause significant economic losses in swine farming worldwide. The development is described of a novel multiple real-time PCR system based on the use of SYBR Green I that allows the simultaneous detection and differentiation of porcine circovirus 2 (PCV-2), porcine parvovirus (PPV), pseudorabies virus (PRV) and Torque teno sus virus species 1 and 2 (TTSuV1 and TTSuV2) in pigs. The method was able to distinguish between all five viral agents, and tests of other DNA viruses proved the specificity of the system. The multiple real-time PCR system was sensitive, as the limits of detection ranged from 3.65×10(3) to 5.04×10(3) copies of DNA template per reaction. The coefficients of variation were low for both intra-assay and inter-assay variability. In addition, the results of the multiple real-time PCR system tests were 100% consistent with previous results based on specific PCR assay testing of field samples. This method could be a useful tool for epidemiological studies and disease management.

Expression profile and subcellular localization of Torque teno sus virus proteins

In the present study, the expression, generation and subcellular localization of Torque teno sus virus (TTSuV) proteins were characterized into two genetically distinct TTSuV species (TTSuV1 and TTSuV2). Following transfection of three TTSuV1 and TTSuV2 full-length ORF (ORF1, ORF2 and ORF3) expression constructs into porcine kidney cells, alternative splice variants encoding new TTSuV protein isoforms were identified for the first time. Proteins encoded from ORF1 and ORF3 were localized in the nucleoli of porcine kidney cells and that of ORF2 in the cytoplasm and nucleus excluding the nucleoli. The subcellular localization of the different protein isoforms was not only similar between distinct TTSuV species but also to the ones described in human Torque teno virus (TTV). Results of the present in vitro study were not based on full-length viral clones but suggested that alternative splicing strategy to generate TTSuV protein isoforms probably occurs in vivo. Obtained data provide new information on molecular biology of TTSuV and anelloviruses, which until now has been solely based on results obtained from human TTV.