Rolling circle amplification assays to detect members of the family Anelloviridae from pig and human biological samples

The present study describes the optimization of Rolling Circle Amplification (RCA) assays to detect members of the family Anelloviridae from pig and human sera. Standard RCA involves the prolonged elongation of random primers bound to a circular DNA template by Phi29 DNA polymerase. The RCA products, that are tandem repeat copies of the viral complete genome, were used for the Sequence Independent Single Primer Amplification (SISPA) method, based on the use of endonuclease restriction of target sequences. These products were separated on agarose gel and different length products were ligated to specific linkers followed by PCR amplification and sequencing. With this combined approach it was possible to detect Torque teno sus virus (TTSuV) 2 in swine. Afterwards, a specific RCA technique was optimized. RCA was first carried out using short Anellovirus specific primers based on a highly conserved region among available anellovirus full-length genomes. Then, products from RCA were used as templates to amplify full-length genomes with Anellovirus specific PCR. Amplification products were run on agarose gel and full-length genomes were isolated based on the expected size. With this novel Anello-RCA/PCR approach it was possible to detect TTSuV 1 and 2 in swine and Torque teno virus (TTV) in human serum samples, which tested previously negative by conventional PCR. Based on these results, we demonstrated that combined RCA-SISPA previously set up for other species is applicable also for TTV DNA detection in swine, and Anello-RCA/PCR is a useful technique to discover members of the genus Iotatorquevirus and Alphatorquevirus. In comparison with the standard RCA combined with the SISPA approach, the Anello-RCA/PCR assay is faster and cheaper, resulting in full-length genomic sequences of Anelloviruses. Furthermore, the technique could be applicable to other species whose TTVs have not been fully characterized as yet, increasing the probability to obtain new viral species belonging to the family Anelloviridae

"Prevalenza e significato dell'infezione da TorqueTenovirus (TTV) in bambini con patologie respiratorie croniche"

TorqueTenovirus (TTV), è un piccolo virus privo di involucro esterno e con un genoma a DNA circolare a singolo filamento di polarità negativa di circa 3,8 kilobasi. Recentemente classificato nel genere Anellovirus, TTV risulta dotato di un’elevata eterogeneità genetica e, in considerazione della sua capacità di indurre viremia cronica in un’alta percentuale di soggetti sani, è da molti ritenuto un virus apatogeno. Recenti studi, condotti su neonati affetti da malattie respiratorie acute (ARD), hanno però dimostrato che TTV replica attivamente nel tratto respiratorio e che i suoi titoli correlano sia con la severità della malattia respiratoria osservata che con la percentuale di alcune popolazioni linfocitarie circolanti. Questi dati suggeriscono un ruolo del virus nelle patologie respiratorie dell’età pediatrica e una sua probabile azione immunomodulante. Per questo lavoro di tesi, un gruppo di bambini di età compresa tra 8 mesi e 16 anni affetti da patologie respiratorie croniche (bronchiectasie ed asma) ed un gruppo di bambini sani, sono stati esaminati per la presenza e i titoli di TTV. Le bronchiectasie costituiscono una grave patologia respiratoria caratterizzata da dilatazione, irreversibile e focale,dei bronchi, solitamente accompagnata da infiammazione cronica ed associata a diverse condizioni patologiche congenite e/o acquisite. L’asma è una malattia infiammatoria cronica a carico delle vie aeree, nella cui patogenesi giocano un ruolo importante numerosi tipi cellulari, fra cui mastociti, eosinofili e linfociti T. Lo scopo della tesi è stato quello di indagare il significato dell’infezione da TTV in soggetti affetti dalle suddette patologie respiratorie croniche. I risultati ottenuti hanno dimostrato che TTV è altamente prevalente nei soggetti esaminati e che i suoi titoli, nel plasma e nelle secrezioni respiratorie,erano inversamente correlati con alcuni importanti indici di funzionalità respiratoria, sia nei soggetti con asma che in quelli con bronchiectasie. In quest’ultimo gruppo di soggetti, inoltre, i titoli del virus correlavano positivamente con il grado di severità della patologia bronchiettasica, valutato mediante tomografia ad alta risoluzione. Nonostante non sia possibile attribuire a TTV un ruolo diretto nell’induzione delle malattie respiratorie studiate, i dati suggeriscono comunque un possibile contributo del virus nell’aggravare la severità di queste gravi patologie dell'infanzia

Delivery Mode Shapes the Composition of the Lower Airways Microbiota in Newborns

Radical alterations in the human microbiota composition are well-known to be associated with many pathological conditions. If these aberrations are established at the time of birth, the risk of developing correlated pathologies throughout life is significantly increased. For this reason, all newborns should begin their lives with a proper microbiota in each body district. The present study aimed at demonstrating a correlation between the mode of delivery and the development of a well-balanced microbiota in the lower airways of newborns. 44 pregnant women were enrolled in this study. Microbiological comparative analysis was carried out on tracheobronchial secretions of babies born through vaginal delivery (VD) or caesarean section (CS). All samples showed the presence of bacterial DNA, regardless of the mode of delivery. No viable cultivable bacteria were isolated from the CS samples. On the contrary, VD allowed colonization of the lower airways by alive cultivable bacteria. The identification of bacterial species revealed that Lactobacillus spp. and Bacteroides vulgatus were the most common microorganisms in the lower airways of vaginally-delivered newborns. Data obtained from quantitative PCRs showed a significantly higher total bacterial load, as well as Firmicutes and Lactobacillus spp. amount, in VD samples than CS ones, while no statistically significant difference was found in Torque Teno Virus (TTV) load between samples. Taken together, our findings confirm the hypothesis that passage through the maternal vaginal canal determines more beneficial colonization of the lower airways in newborns

The Grizzly, January 27, 2011

Bonner Leaders Host Hunger Banquet • UC Alumnus Discusses Alternative Careers in Science • Students Find Internships While Studying Abroad • Berman Museum Opens Two New Exhibitions • MLK Celebrations • Student Experiences Australian Flood • Students Travel to Spain for New Experiences • Internship Profile: Ananda Holton • Diversity Through Monologues • Opinion: Scheduling Classes is Too Difficult; Tragedy in Arizona: The Work of a Madman; Snapshots of London From my Time Studying Abroad • Swimming Training Trip to Florida Proves Rewardinghttps://digitalcommons.ursinus.edu/grizzlynews/1827/thumbnail.jp

The Grizzly, March 24, 2011

Phi Kappa Sigma Wins Airband 2011 • Music Department Visits Ireland During Spring Break • Students Give Back Through Philadelphia Reads Program • Former U.S. Poet Laureate Robert Pinsky Speaks • Playing Favorites with Bobbi Block • UC Community Comes Together to Break Ground for New Stadium • Planned Parenthood No Longer Able to Provide Services • Jamaica Welcomes Bonner Leaders for Spring Break • Junior Jumpstart • Diversity through Film • Theater Review: Playing Favorites • Opinions: Japan Equipped to Rebuild after Natural Disaster; Facing Segregation in the 21st Century Through Campus Programs • Women\u27s Lacrosse Looks for Return Trip to Playoffshttps://digitalcommons.ursinus.edu/grizzlynews/1832/thumbnail.jp

Studio longitudinale della viremia di Torque Teno Virus (TTV) nel trapianto di organo solido

RIASSUNTO Torque Teno Virus (TTV) è il prototipo di un vasto gruppo di virus caratterizzati da un genoma a singolo filamento di DNA circolare e polarità negativa. Sebbene di dimensioni ridotte (ca. 3,8 kilobasi, kb), il genoma virale codifica per almeno quattro Open-Reading Frames (ORFs) e presenta un tratto ricco in G-C. La regione genomica non tradotta (UnTranslatedRegion, UTR) contiene le sequenze regolatorie coinvolte nella replicazione, risultando una regione estremamente conservata tra gli isolati di TTV noti. Le regioni tradotte, in particolare la regione codificante per la proteina capsidica, presentano un elevato grado di variabilità genetica. Secondo la recente classificazione tassonomica da parte del Comitato Internazionale di Tassonomia dei Virus (International Committee on Taxonomy of Viruses , ICTV), gli isolati di TTV noti sono raggruppati in 29 specie diverse appartenenti al genere Alphatorquevirus all’interno della famiglia Anelloviridae. TTV fu identificato per la prima volta nel 1997 nel siero di un paziente giapponese affetto da acuta epatite post-trapianto ad eziologia sconosciuta e suscitò sin da subito un notevole interesse clinico. L'elevata prevalenza del virus nella popolazione generale mondiale ha condotto a considerare TTV come virus “orfano” di patologia. Le problematiche riscontrate nelle prove sperimentali in vitro di isolamento colturale del virus hanno reso difficoltoso lo studio dei meccanismi di interazione virus-ospite. Ad oggi l’unico metodo che permette di rilevare la presenza di TTV nell’uomo è la diagnosi del DNA virale con saggi molecolari in molteplici matrici biologiche. Con l'avvento e successiva ottimizzazione delle metodiche di Next Generation Sequencing (NGS), TTV ha suscitato ulteriormente uno spiccato interesse a tal punto che, attualmente, viene considerato il principale componente del viroma umano. TTV induce viremia cronica in oltre l’80% della popolazione globale ed ha la capacità di infettare diversi distretti dell’ospite: l’alta prevalenza, associata alla persistenza, hanno suggerito una possibile correlazione tra TTV e sistema immunitario, ipotesi che ha condotto a studi ulteriori atti a sostenere la possibilità di utilizzare TTV come marcatore per il monitoraggio dello status immunologico dell'ospite. Si ritiene tale considerazione molto utile soprattutto nell'ambito del trapianto d'organo solido, in cui la terapia immunosoppressiva deve essere somministrata in modo mirato in quanto se da un lato evita il rischio di rigetto dell’organo trapiantato, dall’altro abbattendo il sistema immunitario rende l’ospite più suscettibile a infezioni. Già da molti lavori presenti in letteratura emerge la possibilità di considerare TTV come possibile marker di funzionalità immunitaria: il monitoraggio dello status immunologico del paziente costituisce un’importante possibilità per evitare o quanto meno ridurre la possibilità di complicanze post-trapianto. Lo studio del “comportamento” di TTV nel singolo paziente potrebbe esser utile ai fini di una terapia individualizzata. Lo scopo di questo lavoro di tesi, è quello di studiare l’andamento della viremia di TTV e delle relative specie in pazienti trapiantati di fegato nel periodo post-operatorio mediante l’utilizzo di metodi biomolecolari qualitativi e quantitativi ottimizzati in modo specie-specifico. ABSTRACT Torque Teno Virus (TTV) is the prototype of a large group of viruses characterized by a single-stranded circular DNA genome and negative polarity. Although small in size (about 3.8 kilobases, kb), the viral genome encodes at least four Open-Reading Frames (ORFs) and has a G-C-rich trait. The UnTranslated genomic Region (UTR) contains regulatory sequences involved in replication, resulting in an extremely conserved region among known TTV isolates. The translated regions, particularly the coding region for the capsid protein, show a high degree of genetic variability. According to the International Committee on Taxonomy of Viruses (ICTV), TTV isolates are grouped into 29 different species belonging to the genus Alphatorquevirus within the Anelloviridae family. TTV was first identified in 1997 in a Japanese patient suffering from acute post-transplant hepatitis of unknown etiology and immediately aroused considerable clinical interest. The high prevalence of the virus in the general world population has led to consider TTV as an "orphan" virus of pathology. The problems encountered in the in vitro experiments of virus culture isolation made it difficult to study the mechanisms of virus-host interaction. To date, the only method able to detect the presence of TTV in multiple biological matrices is the diagnosis of viral DNA with molecular assays. With the advent of Next Generation Sequencing (NGS) methods and the discovery of new TTV isolates, the virus has further aroused considerable interest to such an extent that it is currently considered the main component of the human virome. TTV induces chronic viraemia in over 80% of the global population and can infect different host districts. The high prevalence and persistence of the virus, have suggested the existence of a possible correlation between TTV and the immune system, a hypothesis that led to further studies to support the possibility of using TTV as a marker for monitoring the host's immunological status. This consideration seems very useful especially in the context of solid organ transplantation, in which immunosuppressive therapy must be administered in a targeted way since on the one hand it avoids the risk of rejection of the transplanted organ, on the other side by breaking down the system immune system makes the host more susceptible to infections. The idea of considering TTV as a possible marker of immune function has already emerged from many studies: monitoring the patient's immunological status is an important possibility to avoid or at least reduce the possibility of post-transplant complications. The study of the kinetics of TTV in a single patient could be useful for individualized therapy. The aim of this experimental work, based on an important project concerning the "Microbiome and Transplantation” is to study the trend of TTV viraemia and related TTV species in liver transplant patients by optimized species-specific biomolecular methods

Global impact of Torque teno virus infection in wild and domesticated animals

Infection with Torque teno viruses (TTVs) is not restricted to humans. Different domestic and wild animal species are naturally infected with species-specific TTVs worldwide. Due to the global spread of the infection, it is likely that essentially all animals are naturally infected with species-specific TTVs, and that co-evolution of TTVs with their hosts probably occurred. Although TTVs are potentially related to many diseases, the evidence of the widespread infection in healthy human and nonhuman hosts raised doubts about their pathogenic potential. Nonetheless, their role as superimposed agents of other diseases or as triggers for impairment of immune surveillance is currently under debate. The possible contribution of animal TT viruses to interspecies transmission and their role as zoonotic agents are currently topics of discussion