Immunohistochemical detection of metalloproteinase-9 (MMP-9), anti-oxidant like 1 protein (AOP-1) and synaptosomal-associated protein (SNAP-25) in the cerebella of dogs naturally infected with spontaneous canine distemper

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Toll-Like recepctors (TLRs) and retinoic acid inducible gene – I (RIG-I) activation by viral analogs in bovine endometrial cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular epidemiology of the SH (small hydrophobic) gene of human respiratory syncytial virus (HRSV), over 2 consecutive years

Human respiratory syncytial virus (HRSV) strains were isolated from nasopharyngeal aspirates collected from 965 children between 2004 and 2005, yielding 424 positive samples. We sequenced the small hydrophobic protein (SH) gene of 117 strains and compared them with other viruses identified worldwide. Phylogenetic analysis showed a low genetic variability among the isolates but allowed us to classify the viruses into different genotypes for both groups, HRSVA and HRSVB. It is also shown that the novel BA-like genotype was well segregated from the others, indicating that the mutations are not limited to the G gene. (C) 2011 Elsevier B.V. All rights reserved.

THE CARE NEEDS OF WOMEN INFECTED WITH THE HUMAN PAPILLOMA VIRUS: A COMPREHENSIVE APPROACH

This study is founded on the phenomenology of Martin Heidegger, with the objective to understand the care needs of women infected with the human papilloma virus. Participants were fourteen women who had been diagnosed with this infection. The guiding questions were: What is it like to have this diagnosis? Tell me your experience, from when you received your diagnosis until today. What has your health care been like? The questions revealed the theme - seeking care as solicitude - which showed the importance of the support of family and friends. The presence of the infection as the cause of marital conflicts and separation was another highlighted aspect. The statements showed that there was a sense of resignation after an unsuccessful attempt to find accurate and clear information in order to make assertive decisions. Health interventions for infected women must overcome the traditional models of care, including interventions for health promotion and prevention, with trained professionals who are sensitive to the subjective dimension.

Mayaro Virus Infection in Amazonia: A Multimodel Inference Approach to Risk Factor Assessment

Background: Arboviral diseases are major global public health threats. Yet, our understanding of infection risk factors is, with a few exceptions, considerably limited. A crucial shortcoming is the widespread use of analytical methods generally not suited for observational data - particularly null hypothesis-testing (NHT) and step-wise regression (SWR). Using Mayaro virus (MAYV) as a case study, here we compare information theory-based multimodel inference (MMI) with conventional analyses for arboviral infection risk factor assessment. Methodology/Principal Findings: A cross-sectional survey of anti-MAYV antibodies revealed 44% prevalence (n = 270 subjects) in a central Amazon rural settlement. NHT suggested that residents of village-like household clusters and those using closed toilet/latrines were at higher risk, while living in non-village-like areas, using bednets, and owning fowl, pigs or dogs were protective. The "minimum adequate" SWR model retained only residence area and bednet use. Using MMI, we identified relevant covariates, quantified their relative importance, and estimated effect-sizes (beta +/- SE) on which to base inference. Residence area (beta(Village) = 2.93 +/- 0.41; beta(Upland) = -0.56 +/- 0.33, beta(Riverbanks) = -2.37 +/- 0.55) and bednet use (beta = -0.95 +/- 0.28) were the most important factors, followed by crop-plot ownership (beta = 0.39 +/- 0.22) and regular use of a closed toilet/latrine (beta = 0.19 +/- 0.13); domestic animals had insignificant protective effects and were relatively unimportant. The SWR model ranked fifth among the 128 models in the final MMI set. Conclusions/Significance: Our analyses illustrate how MMI can enhance inference on infection risk factors when compared with NHT or SWR. MMI indicates that forest crop-plot workers are likely exposed to typical MAYV cycles maintained by diurnal, forest dwelling vectors; however, MAYV might also be circulating in nocturnal, domestic-peridomestic cycles in village-like areas. This suggests either a vector shift (synanthropic mosquitoes vectoring MAYV) or a habitat/habits shift (classical MAYV vectors adapting to densely populated landscapes and nocturnal biting); any such ecological/adaptive novelty could increase the likelihood of MAYV emergence in Amazonia.

Detection of the Epstein-Barr virus in blood and bone marrow mononuclear cells of patients with aggressive B-cell non-Hodgkin's lymphoma is not associated with prognosis

The Epstein-Barr virus (EBV) is associated with a large spectrum of lymphoproliferative diseases. Traditional methods of EBV detection include the immunohistochemical identification of viral proteins and DNA probes to the viral genome in tumoral tissue. The present study explored the detection of the EBV genome, using the BALF5 gene, in the bone marrow or blood mononuclear cells of patients with diffuse large B-cell lymphomas (DLBCL) and related its presence to the clinical variables and risk factors. The results show that EBV detection in 21.5% of patients is not associated with age, gender, staging, B symptoms, international prognostic index scores or any analytical parameters, including lactate dehydrogenase (LDH) or beta-2 microglobulin (B2M). The majority of patients were treated with R-CHOP-like (rituximab. cyclophosphamide, doxorubicin, vincristine and prednisolone or an equivalent combination) and some with CHOP-like chemotherapy. Response rates [complete response (CR) + partial response (PR)] were not significantly different between EBV-negative and -positive cases, with 93.2 and 88.9%, respectively. The survival rate was also similar in the two groups, with 5-year overall survival (OS) rates of 64.3 and 76.7%, respectively. However, when analyzing the treatment groups separately there was a trend in EBV-positive patients for a worse prognosis in patients treated with CHOP-like regimens that was not identified in patients treated with R-CHOP-like regimens. We conclude that EBV detection in the bone marrow and blood mononuclear cells of DLBC patients has the same frequency of EBV detection on tumoral lymphoma tissue but is not associated with the risk factors, response rate and survival in patients treated mainly with immunochemotherapy plus rituximab. These results also suggest that the addition of rituximab to chemotherapy improves the prognosis associated with EBV detection in DLBCL.

Prospective surveillance study of acute respiratory infections, influenza-like illness and seasonal influenza vaccine in a cohort of juvenile idiopathic arthritis patients

Background Acute respiratory infections (ARI) are frequent in children and complications can occur in patients with chronic diseases. We evaluated the frequency and impact of ARI and influenza-like illness (ILI) episodes on disease activity, and the immunogenicity and safety of influenza vaccine in a cohort of juvenile idiopathic arthritis (JIA) patients. Methods Surveillance of respiratory viruses was conducted in JIA patients during ARI season (March to August) in two consecutive years: 2007 (61 patients) and 2008 (63 patients). Patients with ARI or ILI had respiratory samples collected for virus detection by real time PCR. In 2008, 44 patients were immunized with influenza vaccine. JIA activity index (ACRPed30) was assessed during both surveillance periods. Influenza hemagglutination inhibition antibody titers were measured before and 30-40 days after vaccination. Results During the study period 105 ARI episodes were reported and 26.6% of them were ILI. Of 33 samples collected, 60% were positive for at least one virus. Influenza and rhinovirus were the most frequently detected, in 30% of the samples. Of the 50 JIA flares observed, 20% were temporally associated to ARI. Influenza seroprotection rates were higher than 70% (91-100%) for all strains, and seroconversion rates exceeded 40% (74-93%). In general, response to influenza vaccine was not influenced by therapy or disease activity, but patients using anti-TNF alpha drugs presented lower seroconversion to H1N1 strain. No significant differences were found in ACRPed30 after vaccination and no patient reported ILI for 6 months after vaccination. Conclusion ARI episodes are relatively frequent in JIA patients and may have a role triggering JIA flares. Trivalent split influenza vaccine seems to be immunogenic and safe in JIA patients.

Molecular basis oh herpes simplex virus entry into the cell and retargeting of the viral tropism for the design of oncolytic herpesviruses

Herpes simplex virus 1 (HSV-1) infects oral epitelial cells, then spreads to the nerve endings and estabilishes latency in sensory ganglia, from where it may, or may not reactivate. Diseases caused by virus reactivation include mild diseases such as muco-cutaneous lesions, and more severe, and even life-threatening encephalitis, or systemic infections affecting diverse organs. Herpes simplex virus represents the most comprehensive example of virus receptor interaction in Herpesviridae family, and the prototype virus encoding multipartite entry genes. In fact, it encodes 11-12 glycoproteins and a number of additional membrane proteins: five of these proteins play key roles in virus entry into subsceptible cells. Thus, glycoprotein B (gB) and glycoprotein C (gC) interact with heparan sulfate proteoglycan to enable initial attachment to cell surfaces. In the next step, in the entry cascade, gD binds a specific surface receptor such as nectin1 or HVEM. The interaction of glycoprotein D with the receptor alters the conformation of gD to enable the activation of gB, glycoprotein H, and glycoprotein L, a trio of glycoproteins that execute the fusion of the viral envelope with the plasma membrane. In this thesis, I described two distinct projects: I. The retargeting of viral tropism for the design of oncolytic Herpesviruses: • capable of infecting cells through the human epitelial growth factor receptor 2 (HER2), overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis; • detargeted from its natural receptors, HVEM and nectin1. To this end, we inserted a ligand to HER2 in gD. Because HER2 has no natural ligand, the selected ligand was a single chain antibody (scFv) derived from MAb4D5 (monoclonal antibody to HER2), herein designated scHER2. All recombinant viruses were targeted to HER2 receptor, but only two viruses (R-LM113 and R-LM249) were completely detargeted from HVEM and nectin1. To engineer R-LM113, we removed a large portion at the N-terminus of gD (from aa 6 to aa 38) and inserted scHER2 sequence plus 9-aa serine-glycine flexible linker at position 39. On the other hand, to engineer R-LM249, we replaced the Ig-folded core of gD (from aa 61 to aa 218) with scHER2 flanked by Ser-Gly linkers. In summary, these results provide evidence that: i. gD can tolerate an insert almost as big as gD itself; ii. the Ig-like domain of gD can be removed; iii. the large portion at the N-terminus of gD (from aa 6 to aa 38) can be removed without loss of key function; iv. R-LM113 and R-LM249 recombinants are ready to be assayed in animal models of mammary and ovary tumour. This finding and the avaibility of a large number of scFv greatly increase the collection of potential receptors to which HSV can be redirected. II. The production and purification of recombinant truncated form of the heterodimer gHgL. We cloned a stable insect cell line expressing a soluble form of gH in complex with gL under the control of a metalloprotein inducible promoter and purified the heterodimer by means of ONE-STrEP-tag system by IBA. With respect to biological function, the purified heterodimer is capable: • of reacting to antibodies that recognize conformation dependent epitopes and neutralize virion infectivity; • of binding a variety cells at cell surface. No doubt, the availability of biological active purified gHgL heterodimer, in sufficient quantities, will speed up the efforts to solve its crystal structure and makes it feasible to identify more clearly whether gHgL has a cellular partner, and what is the role of this interaction on virus entry.

Untersuchungen zur posttranslationalen präS-Translokation des großen Hüllproteins des Hepatitis-B-Virus

Untersuchungen zur posttranslationalen präS-Translokation des großen Hüllproteins des Hepatitis-B-Virus. Das große (L) Hüllprotin des Hepatitis-B-Virus (HBV) besitzt die ungewöhnliche Eigenschaft, mittels partieller, posttranslationaler Translokation seiner präS-Domäne durch intrazelluläre Membranen zwei unterschiedliche Transmembrantopologieen auszubilden. Unter Berücksichtigung der Hypothese eines HBV-spezifischen Transmembrankanals, der sich möglicherweise während der Virusmorphogenese bilden und die präS-Translokation ermöglichen könnte, wurden Parameter untersucht, welche die L-Topologie beeinflussen. Dazu wurden Wildtyp-L-Proteine und L-Mutanten in Säugerzellen synthetisiert und deren Topologie mittels Proteaseschutzversuchen untersucht. Ich konnte zeigen, daß alle Faktoren, für die angenommen wurde, daß sie für die Ausbildung einer HBV-spezifischen Pore und die damit verbundene präS-Reorientierung wichtig seien, entbehrlich sind. Im einzelnen konnte nachgewiesen werden, daß die posttranslationale präS-Translokation weder die Helferfunktion der HBV S und M Proteine, noch die kovalente Dimerausbildung der Hüllproteine benötigt. Weiterhin ergaben die Untersuchungen, daß keine der amphipathischen Transmembrandomänen des L-Proteins an der präS-Reorientierung beteiligt ist. Vielmehr wurde die hydrophobe Transmembrandomäne 2 (TM2) als ausreichend und essentiell für diesen Prozeß identifiziert. Zellfraktionierungsstudien ergaben weiterhin, daß die präS-Reorientierung und damit die duale Topologie des L-Proteins innerhalb des Endoplasmatischen Retikulums (ER) herbeigeführt wird. Letztlich konnte eine Interaktion des L-Proteins mit zellulären Chaperonen (Hsc70, Hsp40, BiP) gezeigt werden, was eine Beteiligung dieser Proteine am Translokationsprozeß nahelegt.

Virus a RNA e a DNA: importanza dell’ampliamento dello spettro d’ospite nell’evoluzione virale

L’ampliamento dello spettro d’ospite è strettamente connesso al processo evolutivo a cui i virus sono assoggettati e rappresenta una notevole sfida alla loro capacità di adattarsi. L’attitudine a superare le barriere di specie è conseguente alla costante e relativamente rapida evoluzione che caratterizza i virus; allo stesso tempo, la forza selettiva esercitata dal nuovo ospite rappresenterà un ulteriore stimolo per le capacità adattative del virus. Ad oggi, i meccanismi genetici ed evolutivi responsabili del salto di specie virale, cioè la trasmissione di un virus da un ospite tradizionale ad uno precedentemente resistente all’infezione, sono parzialmente sconosciuti. Nel seguente lavoro verranno presentati gli studi effettuati sulle dinamiche evolutive caratterizzanti virus a RNA e a DNA in cui si sono osservate variazioni dello spettro d’ospite. Gli studi hanno riguardato i coronavirus, con particolare riferimento al ruolo svolto dai pipistrelli nell’evoluzione dei coronavirus SARS-correlati, e l’importanza del gatto nell’evoluzione dei parvovirus dei carnivori. Nella prima sezione saranno mostrate le correlazioni genetiche dei coronavirus identificati in Italia nei pipistrelli appartenenti alla specie Rhinolophus ferrumequinum con i ceppi europei e del resto del mondo, allo scopo di chiarire l’origine evolutiva dei coronavirus dei pipistrelli correlati al virus della SARS (Bat-SARS-like CoV) europei, gli eventi migratori che hanno caratterizzato la loro diffusione nel continente e le potenziali ripercussioni sulla salute pubblica. Nella seconda sezione saranno evidenziate le caratteristiche molecolari dei ceppi di parvovirus circolanti nella popolazione felina, valutandone la diversità di sequenza e la complessità genetica, allo scopo di ottenere importanti informazioni in merito all’evoluzione del virus e alle interazioni tra il parvovirus e l’ospite.

Functional characterization of modified vaccinia virus Ankara-encoded anti-apoptotic proteins

Candidate vaccines based on the highly attenuated orthopoxvirus strain MVA are tested against various infectious and cancer diseases and, more profound, vaccines based on wildtype and recombinant viruses have been found safe and immunogenic in clinical trials. Compared to conventional vaccine strains, MVA lacks many functional genes for potentially important regulators of virus-host interactions. However, some gene functions responsible for counteraction of cellular antiviral pathways are still conserved in the genome of MVA and the inhibition of apoptosis seems to be one important mechanism, the virus is still able to interact with.rnrnVaccinia viruses encode several proteins which prevent the induction of virus-induced apoptosis. The vaccinia virus anti-apoptotic protein F1 was shown to counteract the activation of the mitochondrial pathway of apoptosis in a highly effective manner. Another vaccinia virus protein, N1, like F1 shows structural and functional similarity to members of the cellular anti-apoptotic bcl-2 family and was also shown to inhibit apoptosis. The vaccinia virus early protein E3 inhibits programmed cell death by binding to and sequestration of dsRNA molecules, normally inducing cellular antiviral pathways also driving the induction of apoptosis. All three anti-apoptotic genes were functionally analyzed during this work.rn

Role of αvβ3 – Integrin and TLR2 in the innate response to Herpes Simplex Virus infection and Delivery of retargeted oncolytic Herpes Simplex via carrier cells

In the first part of my thesis I studied the mechanism of initiation of the innate response to HSV-1. Innate immune response is the first line of defense set up by the cell to counteract pathogens infection and it is elicited by the activation of a number of membrane or intracellular receptors and sensors, collectively indicated as PRRs, Patter Recognition Receptors. We reported that the HSV pathogen-associated molecular patterns (PAMP) that activate Toll-like receptor 2 (TLR2) and lead to the initiation of innate response are the virion glycoproteins gH/gL and gB, which constitute the conserved fusion core apparatus across the Herpesvirus. Specifically gH/gL is sufficient to initiate a signaling cascade which leads to NF-κB activation. Then, by gain and loss-of-function approaches, we found that αvβ3-integrin is a sensor of and plays a crucial role in the innate defense against HSV-1. We showed that αvβ3-integrin signals through a pathway that concurs with TLR2, affects activation/induction of interferons type 1, NF-κB, and a polarized set of cytokines and receptors. Thus, we demonstrated that gH/gL is sufficient to induce IFN1 and NF-κB via this pathway. From these data, we proposed that αvβ3-integrin is considered a class of non-TLR pattern recognition receptors. In the second part of my thesis I studied the capacity of human mesenchymal stromal cells isolated by fetal membranes (FM-hMSCs) to be used as carrier cells for the delivery of retargeted R-LM249 virus. The use of systemically administrated carrier cells to deliver oncolytic viruses to tumoral targets is a promising strategy in oncolytic virotherapy. We observed that FM-hMSCs can be infected by R-LM249 and we optimized the infection condition; then we demonstrate that stromal cells sustain the replication of retargeted R-LM249 and spread it to target tumoral cells. From these preliminary data FM-hMSCs resulted suitable to be used as carrier cells

Caratterizzazione genomica di virus influenzali suini in Italia mediante next generation sequencing

I sottotipi H1N1, H1N2 e H3N2 di influenza A virus sono largamente diffusi nella popolazione suina di tutto il mondo. Nel presente lavoro è stato sviluppato un protocollo di sequenziamento di c.d. nuova generazione, su piattaforma Ion Torrent PGM, idoneo per l’analisi di tutti i virus influenzali suini (SIV). Per valutare l’evoluzione molecolare dei SIV italiani, sono stati sequenziati ed analizzati mediante analisi genomica e filogenetica un totale di sessantadue ceppi di SIV appartenenti ai sottotipi H1N1, H1N2 e H3N2, isolati in Italia dal 1998 al 2014. Sono stati evidenziati in sei campioni due fenomeni di riassortimento: tutti i SIV H1N2 esaminati presentavano una neuraminidasi di derivazione umana, diversa da quella dei SIV H1N2 circolanti in Europa, inoltre l’emoagglutinina (HA) di due isolati H1N2 era originata dal riassortimento con un SIV H1N1 avian-like. L’analisi molecolare dell’HA ha permesso di rivelare un’inserzione di due amminoacidi in quattro SIV H1N1 pandemici e una delezione di due aminoacidi in quattro SIV H1N2, entrambe a livello del sito di legame con il recettore cellulare. E’ stata inoltre evidenziata un’elevata omologia di un SIV H1N1 con ceppi europei isolati negli anni ’80, suggerendo la possibile origine vaccinale di questo virus. E’ stato possibile, in aggiunta, applicare il nuovo protocollo sviluppato per sequenziare un virus influenzale aviare altamente patogeno trasmesso all’uomo, direttamente da campione biologico. La diversità genetica nei SIV esaminati in questo studio conferma l’importanza di un continuo monitoraggio della costellazione genomica dei virus influenzali nella popolazione suina.

Monocyte differentiation toward regulatory dendritic cells is not affected by respiratory syncytial virus-induced inflammatory mediators

Airway epithelial cells were shown to drive the differentiation of monocytes into dendritic cells (DCs) with a suppressive phenotype. In this study, we investigated the impact of virus-induced inflammatory mediator production on the development of DCs. Monocyte differentiation into functional DCs, as reflected by the expression of CD11c, CD123, BDCA-4, and DC-SIGN and the capacity to activate T cells, was similar for respiratory syncytial virus (RSV)-infected and mock-infected BEAS-2B and A549 cells. RSV-conditioned culture media resulted in a partially mature DC phenotype, but failed to up-regulate CD80, CD83, CD86, and CCR7, and failed to release proinflammatory mediators upon Toll-like receptor (TLR) triggering. Nevertheless, these DCs were able to maintain an antiviral response by the release of Type I IFN. Collectively, these data indicate that the airway epithelium maintains an important suppressive DC phenotype under the inflammatory conditions induced by infection with RSV.

Subcellular Localization of the Non-Structural Proteins 3C and 3CD of the Honeybee Virus Deformed Wing Virus

Apis mellifera L., the European honeybee, is a crucial pollinator of many important agricultural crops in the United States. Recently, honeybee colonies have been affected by Colony Collapse Disorder (CCD), a disorder in which the colony fails due to the disappearance of a key functional group of worker bees. Though no direct causalrelationship has been confirmed, hives that experience CCD have been shown to have a high incidence of Deformed Wing Virus (DWV), a common honeybee virus. While the genome sequence and gene-order of DWV has been analyzed fairly recently, few other studies have been performed to understand the molecular characterization of the virus.Since little is known about where DWV proteins localize in infected host cells, the objective of this project was to determine the subcellular localization of two of the important non-structural proteins that are encoded in the DWV genome. This project focused on the protein 3C, an autocatalytic protease which cleaves itself from a longer polyprotein and helps to cut all of the other proteins apart from one another so that they can become functional, and 3D, the RNA-dependent RNA polymerase (RdRp) which is critical for replication of the virus because it copies the viral genome. By tagging nested constructs containing these two proteins and tracking where they localized in living cells, this study aimed to better understand the replication of DWV and to elicit possible targetsfor further research on how to control the virus. Since DWV is a picorna-like virus, distantly related to human viruses such as polio, and picornavirus non-structural proteins aggregate at cellular membranes during viral replication, the major hypothesis was that the 3C and 3CD proteins would localize at cellular organelle membranes as well. Using confocal microscopy, both proteins were found to localize in the cytoplasm, but the 3CDprotein was found to be mostly diffuse cytoplasmic, and the 3C protein was found to localize more specifically on membranous structures just outside of the nucleus.