Human pegivirus -1 (HPgV-1) RNA frequency and genotype distribution in pediatric oncology patients with febrile neutropenia.

Human Pegivirus-1, typically regarded as a commensal virus, exhibits high prevalence in humans. Its frequency and impact on oncologic pediatric patients with febrile neutropenia (FN), a frequent chemotherapy complication, remains unexplored. In this study, we assessed HPgV-1 RNA prevalence in pediatric patients experiencing FN. Blood samples were collected from 30 children, 15 presenting FN and 15 comprising a control group of either undergoing treatment or in remission. Overall, HPgV-1 RNA was detected in 23.3 % of samples (26.7 % among FN patients and 20.0 % among those under treatment or in remission). Phylogenetic analysis unveiled HPgV-1 genotype 2 predominance among these samples, the most prevalent strain circulating in Brazil. Our findings prompt crucial inquiries into the role of HPgV-1 RNA in FN: is it an incidental finding and if it can influences this clinical entity? Further investigation is imperative to elucidate HPgV-1 implications in vulnerable patients cohorts, potentially informing new approaches and understanding viral dynamics in immunocompromised populations.

Metagenomic Analysis for Diagnosis of Hemorrhagic Fever in Minas Gerais, Brazil.

Viral hemorrhagic fever poses a significant public health challenge due to its severe clinical presentation and high mortality rate. The diagnostic process is hindered by similarity of symptoms across different diseases and the broad spectrum of pathogens that can cause hemorrhagic fever. In this study, we applied viral metagenomic analysis to 43 serum samples collected by the Public Health Laboratory (Fundação Ezequiel Dias, FUNED) in Minas Gerais State, Brazil, from patients diagnosed with hemorrhagic fever who had tested negative for the standard local hemorrhagic disease testing panel. This panel includes tests for Dengue virus (DENV) IgM, Zika virus IgM, Chikungunya virus IgM, yellow fever IgM, Hantavirus IgM, Rickettsia rickettsii IgM/IgG, and Leptospira interrogans IgM, in addition to respective molecular tests for these infectious agents. The samples were grouped into 18 pools according to geographic origin and analyzed through next-generation sequencing on the NextSeq 2000 platform. Bioinformatic analysis revealed a prevalent occurrence of commensal viruses across all pools, but, notably, a significant number of reads corresponding to the DENV serotype 2 were identified in one specific pool. Further verification via real-time PCR confirmed the presence of DENV-2 RNA in an index case involving an oncology patient with hemorrhagic fever who had initially tested negative for anti-DENV IgM antibodies, thereby excluding this sample from initial molecular testing. The complete DENV-2 genome isolated from this patient was taxonomically classified within the cosmopolitan genotype that was recently introduced into Brazil. These findings highlight the critical role of considering the patient's clinical condition when deciding upon the most appropriate testing procedures. Additionally, this study showcases the potential of viral metagenomics in pinpointing the viral agents behind hemorrhagic diseases. Future research is needed to assess the practicality of incorporating metagenomics into standard viral diagnostic protocols.

Unveiling microbial worlds: exploring viral metagenomics among waste pickers at Latin America's largest dumpsite

ABSTRACT Waste pickers constitute a marginalized demographic engaged in the collection of refuse, facing considerable occupational hazards that heighten their susceptibility to contract infectious diseases. Moreover, waste pickers contend with societal stigmatization and encounter barriers to accessing healthcare services. To explore the viral profile of waste pickers potentially linked to their occupational environment, we conducted a metagenomic analysis on 120 plasma specimens sampled from individuals employed at the Cidade Estrutural dumpsite in Brasilia city, Brazil. In total, 60 blood donors served as a comparative control group. Specimens were pooled and subjected to Illumina NextSeq 2000 sequencing. Viral abundance among waste pickers revealed the presence of significant pathogens, including HIV, HCV, and Chikungunya, which were not detected in the control group. Additionally, elevated levels of anelloviruses and Human pegivirus-1 were noted, with a comparable incidence in the control group. These findings underscore the utility of metagenomics in identifying clinically relevant viral agents within underserved populations. The implications of this study extend to informing public health policies aimed at surveilling infectious diseases among individuals facing socioeconomic disparities and limited access to healthcare resources.

Viral metagenomics unveils MW (Malawi) polyomavirus infection in Brazilian pediatric patients with acute respiratory disease.

Viral metagenomics has been extensively applied for the identification of emerging or poorly characterized viruses. In this study, we applied metagenomics for the identification of viral infections among pediatric patients with acute respiratory disease, but who tested negative for SARS-CoV-2. Twelve pools composed of eight nasopharyngeal specimens were submitted to viral metagenomics. Surprisingly, in two of the pools, we identified reads belonging to the poorly characterized Malawi polyomavirus (MWPyV). Then, the samples composing the positive pools were individually tested using quantitative polymerase chain reaction for identification of the MWPyV index cases. MWPyV-positive samples were also submitted to respiratory virus panel testing due to the metagenomic identification of different clinically important viruses. Of note, MWPyV-positive samples tested also positive for respiratory syncytial virus types A and B. In this study, we retrieved two complete MWPyV genome sequences from the index samples that were submitted to phylogenetic inference to investigate their viral origin. Our study represents the first molecular and genomic characterization of MWPyV obtained from pediatric patients in South America. The detection of MWPyV in acutely infected infants suggests that this virus might participate (coparticipate) in cases of respiratory symptoms. Nevertheless, future studies based on testing of a larger number of clinical samples and MWPyV complete genomes appear to be necessary to elucidate if this emerging polyomavirus might be clinically important.

Merkel cell polyomavirus (MCPyV) DNA prevalence in Brazilian blood donors.

Merkel cell polyomavirus (MCPyV) is an oncogenic virus that has been etiologically linked to Merkel cell carcinoma. Low levels of MCPyV DNA have been detected in blood donors with unclear impact on transfusion. The prevalence of MCPyV DNA in Brazilian blood donors is unclear. Therefore, the objective of this study was to evaluate the MCPyV DNA prevalence among Brazilian blood donors. We examined the presence of MCPyV DNA by real-time PCR (qPCR) in a total of 450 serum samples obtained from blood donors from three Brazilian regions (North, Central-West and South). The overall estimated MCPyV DNA prevalence was 1.1% (CI = 95%, 0.16-2.06%). Divided by region, in North Brazil (city of Macapa, state of Amapá) and South Brazil (city of Santa Maria, state of Rio Grande do Sul), the MCPyV prevalence was the same: 1.33% (CI = 95%, range 0.0-3.14%). In Central-West Brazil (city of Brasilia), the MCPyV prevalence was 0.6% (CI = 95%, 0.0-1.96%). All MCPyV positive samples showed a high cycle threshold (median Ct = 35.5), most probably related to the low viral load. More studies are necessary to unveil the impact of this oncogenic virus on transfusion medicine and if such exists, especially in regards of its infectivity and transmission potential.

Rapid and Sensitive Qualitative Duoplex Real-Time PCR Method for Discriminatory and Confirmatory Diagnosis of HTLV-1 and HTLV-2 Infections: Brazilian Multicentric Study.

Human T cell lymphotropic virus (HTLV) is the caustive agent of two main conditions i. e., the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and the adult T-cell leukemia/lymphoma (ATLL). HTLV diagnosis is based on serological and molecular approaches; however, an accurate and validated method is still needed. The objective of this study was to establish a rapid and sensitive molecular test to confirm and discriminate HTLV 1/2 types. The test validation was performed as a multicentric study involving HTLV confirmation centers throughout Brazil. Proviral DNA was extracted from whole blood and the amplification was performed using in-house designed primer and probe sets targeting the pol genomic region. An internal control to validate the extraction and amplification was also included. The limit of detection (LoD) of the assay was four copies/reaction for HTLV-1 and 10.9 copies/reaction for HTLV-2. The diagnostic sensitivity of the platform was 94.6% for HTLV-1, 78.6% for HTLV-2, and the specificity was 100% for both viruses. Cross-reactions of the test with human viruses including HAV, HBV, HCV, HIV-1/2, and parvovirus B19 were not observed. During the multicentric validation, the test was used to screen a total of 692 blood samples obtained from previously confirmed HTLV-positive individuals. From these, 91.1% tested positive being concordant with the previously obtained results. In conclusion, our duoplex-RT-PCR-HTLV1 /2 presented adequate efficiency for HTLV-1/2 differentiation showing high sensitivity and specificity. Therefore, it can be a suitable tool for confirmation of suspected and inconclusive HTLV cases, prenatal and pre-transplant diagnosis, in Brazil and in other countries HTLV-endemic countries.

Monitoring of HTLV-1-associated diseases by proviral load quantification using multiplex real-time PCR.

Proviral load (PVL) is one of the determining factors for the pathogenesis and clinical progression of the human T-lymphotropic virus type I (HTLV-1) infection. In the present study, we optimized a sensitive multiplex real-time PCR for the simultaneous detection and quantification of HTLV-1 proviral load and beta-globin gene as endogenous control. The values obtained for HTLV-1 PVL were used to monitor the clinical evolution in HTLV-1-infected individuals. A vector containing cloned DNA targets of the real-time PCR for the beta-globin gene and the HTLV-1pol region was constructed. For the reaction validation, we compared the amplification efficiency of the constructed vector and MT-2 cell line containing HTLV-1. The analytical sensitivity of the reaction was evaluated by the application of a standard curve with a high order of magnitude. PVL assay was evaluated on DNA samples of HTLV-1 seropositive individuals. The construct showed adequate amplification for the beta-globin and HTLV-1 pol genes when evaluated as multiplex real-time PCR (slope = 3.23/3.26, Y-intercept = 40.18/40.73, correlation coefficient r2 = 0.99/0.99, and efficiency = 103.98/102.78, respectively). The quantification of PVL using the MT-2 cell line was equivalent to the data obtained using the plasmidial curve (2.5 copies per cell). In HTLV-1-associatedmyelopathy/tropical spastic paraparesis patients, PVL was significantly higher (21315 ± 2154 copies/105 PBMC) compared to asymptomatic individuals (1253 ± 691 copies/105 PBMC). The obtained results indicate that the optimized HTLV-1 PVL assay using plasmidial curve can be applied for monitoring and follow-up of the progression of HTLV-1 disease. The use of a unique reference plasmid for both HTLV-1 and endogenous gene allows a robust and effective quantification of HTLV-1 PVL. In addition, the developed multiplex real-time PCR assay was efficient to be used as a tool to monitor HTLV-1-infected individuals.

Microbiome and oral squamous cell carcinoma: a possible interplay on iron metabolism and its impact on tumor microenvironment.

There is increasing evidence showing positive association between changes in oral microbiome and the occurrence of oral squamous cell carcinoma (OSCC). Alcohol- and nicotine-related products can induce microbial changes but are still unknown if these changes are related to cancerous lesion sites. In an attempt to understand how these changes can influence the OSCC development and maintenance, the aim of this study was to investigate the oral microbiome linked with OSCC as well as to identify functional signatures and associate them with healthy or precancerous and cancerous sites. Our group used data of oral microbiomes available in public repositories. The analysis included data of oral microbiomes from electronic cigarette users, alcohol consumers, and precancerous and OSCC samples. An R-based pipeline was used for taxonomic and functional prediction analysis. The Streptococcus spp. genus was the main class identified in the healthy group. Haemophilus spp. predominated in precancerous lesions. OSCC samples revealed a higher relative abundance compared with the other groups, represented by an increased proportion of Fusobacterium spp., Prevotella spp., Haemophilus spp., and Campylobacter spp. Venn diagram analysis showed 52 genera exclusive of OSCC samples. Both precancerous and OSCC samples seemed to present a specific associated functional pattern. They were menaquinone-dependent protoporphyrinogen oxidase pattern enhanced in the former and both 3',5'-cyclic-nucleotide phosphodiesterase (purine metabolism) and iron(III) transport system ATP-binding protein enhanced in the latter. We conclude that although precancerous and OSCC samples present some differences on microbial profile, both microbiomes act as "iron chelators-like" potentially contributing to tumor growth.

Molecular evolution pattern of Merkel cell polyomavirus identified by viral metagenomics in plasma of high-risk blood donors from the Brazilian Amazon.

Merkel cell polyomavirus (MCPyV) is a common human skin pathogen, shows high seroprevalence and is considered the etiologic agent of Merkel cell carcinoma. However, studies which detect MCPyV DNA in blood products may reveal the importance of this virus for the transfusion medicine. In this study we analyzed by viral metagenomics 36 plasma samples obtained from blood donors positive for the common blood transmitted infections from the city of Macapá (Brazilian Amazon). The generated raw data were were analyzed through a specific bioinformatics pipeline aimed at discovery of emerging viruses. The genomes of interest were analyzed phylogeographically and phylogenetically. MCPyV complete genome was recovered from one HBV-positive pool with high coverage (~ 223×) indicating acute viremia or reactivated infection. Interestingly, the phylogeographic position of the identified strain suggests its ancestry compared to MCPyV isolate from Colombian Amazon which hypothesizes that viral dissemination in the Amazon may have originated from Brazil. In conclusion, this study brings information for the genetic relationships of MCPyV isolated from blood donors from the Brazilian Amazon and demonstrates the possible phylogeographic behavior of our strain in relation to the other findings. We also demonstrated a strong evidence of viremic MCPyV phase in blood donations, however, more studies are necessary in order to understand the MCPyV impact on transfusion therapy.

Viral metagenomics performed in patients with acute febrile syndrome during Toxoplasma gondii outbreak in south Brazil

ABSTRACT Toxoplasmosis is a zoonotic infection caused by the protozoan parasite Toxoplasma gondii. The infection is widely disseminated in the human population and is usually benign or asymptomatic. Systemic T. gondii infection presents risks for pregnant women and AIDS patients. Although rare, T. gondii can cause outbreaks in urban centers. The origin of these outbreaks is not completely understood but probably results from introduction of zoonotic T. gondii strains in the population. During such outbreaks other pathogens which mimic T. gondii acute febrile syndrome may also circulate; therefore, detailed investigation of the outbreak is of extreme importance. In this study we performed viral metagenomics next-generation sequencing (mNGS) in patient samples obtained during T. gondii outbreak in Santa Maria city, South Brazil. Specific bioinformatics pipelines specialized in virus discovery were applied in order to identify co-circulating vial agents. Epstein Barr virus and Parvovirus B19 contigs were assembled and these viruses can cause symptoms similar to toxoplasmosis. In conclusion, our findings show the importance of Metagenomics next generation sequencing (mNGS) use to help characterize the outbreak more completely and in the management of the affected patients.

Low human parvovirus B19 (B19V) DNA prevalence in blood donors from Central-West Brazil.

Parvovirus B19 (B19V) transmission may occur through blood transfusion as a result of asymptomatic viral persistence in blood donors. Our study evaluated the prevalence and viral load of B19V in blood donors from Brasilia, Federal District, Central-West Brazil. B19V DNA detection and quantification were performed in 477 blood donors. The positive samples were also tested for anti-B19V IgG and haemoderivative recipients were investigated for adverse effects following transfusion. B19V DNA prevalence was 0.21  % (n=1/477). The positive B19V DNA sample was also anti-B19 IgG-positive (probably persistent infection). The viral load was low and no adverse effects following blood transfusion were registered in the recipients. This study demonstrated that the B19V DNA prevalence in blood donors from Central-West Brazil is low. Nevertheless, the mere presence of B19V DNA in blood donors strengthens the need for viral molecular screening, especially in haemoderivatives that that will go to susceptible recipients.

Short Communication: Human Bone Marrow Stromal Cells Exhibit Immunosuppressive Effects on Human T Lymphotropic Virus Type 1 T Lymphocyte from Infected Individuals.

Human multipotent mesenchymal stromal cells (MSCs) display immunoregulatory functions that can modulate innate and adaptive cellular immune responses. The suppressive and immunomodulatory activities of MSCs occur through the action of soluble factors that are constitutively produced and released by these cells or, alternatively, after MSC induction by stimuli of inflammatory microenvironments. However, to date the contribution of MSCs in the inflammatory microenvironment resulting from viral infection is unknown. In our study, we evaluated the MSC immunosuppressive effect on human T lymphotropic virus type 1 (HTLV-1) infected T lymphocytes. To evaluate if MSC immunoregulation can influence the proliferation of HTLV-1 infected T lymphocytes, we compared the proliferation of lymphocytes obtained from HTLV-1 infected and healthy individuals cocultured in the presence of MSCs. It was observed that the lymphoproliferative inhibition by MSCs on infected lymphocytes was similar compared to the cells obtained from healthy individuals. In addition, this suppressive effect was related to a significant increase of indoleamine-2,3-dioxygenase and prostaglandin E2 gene expression (p ≤ .05). Furthermore, the HTLV-1 pol gene was less expressed after coculturing with MSCs, suggesting that the MSC immunoregulation can have effective suppression on HTLV-1 infected T cells. In conclusion, this study suggests that MSCs could be involved in the immunomodulation of the HTLV-1 infected T lymphocytes.

Seroprevalence of Chikungunya virus in blood donors from Northern and Southeastern Brazil

ABSTRACT Background: Chikungunya virus, an arbovirus that belongs to the Alphavirus genus of the Togaviridae family, causes a febrile illness accompanied by rash and arthralgia. It is estimated that during outbreaks, the prevalence of Chikungunya virus RNA in viremic blood donations varies between 0.4 and 2.1%; therefore, this virus may be transmitted by transfusion. In Brazil, Chikungunya virus has been claimed to cause extensive outbreaks, however, the seroprevalence of anti-Chikungunya virus IgG among Brazilian blood donors is unknown. Methods: Eight hundred and ninety-seven blood samples were collected from volunteer blood donors in two distant localities long after the Chikungunya virus first appeared in Brazil. In 2015, 442 samples were collected from the Hemotherapy Service of Macapá, Amapá in the northern Brazilian Amazon. To evaluate the dissemination course of the virus in Brazil, in 2016, 455 blood samples were collected from the southeastern region (Blood Center of Ribeirão Preto, Ribeirão Preto, São Paulo). All samples were tested for the presence of anti-Chikungunya virus IgG and viral RNA. Results: One sample (0.2%) obtained from the Hemotherapy Center of Macapá tested positive for anti-Chikungunya virus IgG and no sample from the Blood Center of Ribeirão Preto was seroreactive to anti-Chikungunya virus IgG. All blood donations were Chikungunya virus RNA negative. Conclusions: This study, performed during 2015-2016, indicates that the transfusion risk of Chikungunya virus in this period was low. However, due to the constant advance of this virus in Brazil, further studies during outbreaks are needed to evaluate the presence of Chikungunya virus RNA in blood donations and the respective transfusion-transmission risk.

TAX-mRNA-Carrying Exosomes from Human T Cell Lymphotropic Virus Type 1-Infected Cells Can Induce Interferon-Gamma Production In Vitro.

Human T cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma. The development of HAM/TSP, a chronic neuroinflammatory disease, is correlated to complex interaction between the host immune response and the infecting virus. Tax expression plays an important role in HAM/TSP pathogenesis by activating various cellular genes, including the cytokines interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Exosomes have emerged as an important factor of cell-to-cell communication contributing to diverse cellular processes, including immune modulation. Considering the potential role of exosomes in modulating the immune response and inflammation, the main objective of this study was to examine if HTLV-1-infected cells produce exosomes carrying viral proteins or inflammatory molecules, which can participate in the chronic inflammation that is observed in patients with HAM/TSP. Exosomes were isolated from HTLV-1-infected cell line, evaluated for the tax mRNA presence, and tested for the ability to activate peripheral mononuclear cells (PBMC) in inducing an inflammatory immune response. We observed that the proinflammatory cytokines, IFN-γ and TNF-α, were upregulated in T cells after treatment of the PBMC with Tax-carrying exosomes compared to the negative control. Interleukin-4, Granzyme B, and Perforin did not show alterations. Taken together, these results suggest that exosomes carrying tax-mRNA isolated from HTLV-1-infected cells might induce the production of proinflammatory cytokines and activate T helper (Th)1, and not Th2-immune response. If this finding is further confirmed, this study may have impact on investigations on the pathogenesis of HAM-TSP and the inflammatory response involved in this disease.

Detection of HTLV-1 proviral DNA in BM mononuclear cells and cultured mesenchymal stromal cells isolated from patients with HTLV-1 infection.

The bone marrow (BM) biology during HTLV-1 infection is obscure. In this study, we investigated BM mononuclear cells and mesenchymal stromal cells (MSC) from HTLV-1 asymptomatic and symptomatic individuals. An infiltration of CD4+ T-cell lymphocytes in the BM of HTLV-1-infected individuals was observed when compared to healthy controls. The provirus detection in the BM CD4+ T cells confirmed the presence of integrated HTLV DNA. In regard to MSC, we observed that the number of fibroblast progenitor cells was lower in HTLV-1 infected individuals than in healthy controls. Isolated HTLV-1 infected BM-MSC demonstrated surface expression markers and in vitro differentiation potential similar to uninfected individuals. The presence of HTLV-1 proviral DNA in the BM-MSC of HTLV-1-infected patients was demonstrated but no p19 antigen was detected in supernatant from cultured MSC. We suppose that HTLV-1 infects human MSC probably by cell-to-cell contact from the infected CD4+ T-lymphocytes infiltrated into the bone marrow.

No evidence of xenotropic murine leukemia virus-related virus infection in Brazilian multiply transfused patients with sickle cell disease and beta-thalassemia major.

Although xenotropic murine leukemia virus-related virus (XMRV) has been regarded as a laboratory contaminant, it remains one of the most controversial viruses. The objective of the study was to determine if XMRV is present in 44 patients with beta-thalassemia major, 48 with sickle cell disease, and 89 volunteer blood donors. After RNA/ DNA extraction from plasma/buffy coat the samples were screened for XMRV sequences by conserved nested GAG primers. None of the RNA samples showed a positive result. Surprisingly, four DNA samples obtained from blood donors were positive for XMRV provirus. The subsequent phylogenetic analysis revealed that these sequences are identical to the positive control (murine leukemia retrovirus) and are probably consistent with laboratory contamination. XMRV infection (provirus and viral RNA) was absent in multiply transfused patients and volunteer blood donors. The positive result obtained from some blood donors probably reflects laboratory contamination. We believe that XMRV does not pose risk to blood transfusion.

Molecular and clinical evaluation of the acute human parvovirus B19 infection: comparison of two cases in children with sickle cell disease and discussion of the literature

Human parvovirus B19 is a well-known cause of severe conditions in patients with sickle cell disease, but the molecular mechanisms of the infection are insufficiently understood. The different clinical outcome of the acute parvovirus B19 infection in two pediatric patients with sickle cell disease has been examined. One of them developed life-threatening condition requiring emergency transfusions, while the other had asymptomatic infection, diagnosed occasionally. Both cases had high viral load and identical subgenotype, indicating that the viral molecular characteristics play a minimal role in the infection outcome.