Reemergence of Oropouche virus between 2023 and 2024 in Brazil.

Background: Oropouche virus (OROV; species Orthobunyavirus oropoucheense) is an arthropod-borne virus that has caused outbreaks of Oropouche fever in Central and South America since the 1950s. This study investigates virological factors contributing to the reemergence of Oropouche fever in Brazil between 2023 and 2024. Methods: In this study, we combined OROV genomic, molecular, and serological data from Brazil from 1 January 2015 to 29 June 2024, along with in vitro and in vivo characterization. Molecular screening data included 93 patients with febrile illness between January 2023 and February 2024 from the Amazonas State. Genomic data comprised two genomic OROV sequences from patients. Serological data were obtained from neutralizing antibody tests comparing the prototype OROV strain BeAn 19991 and the 2024 epidemic strain. Epidemiological data included aggregated cases reported to the Brazilian Ministry of Health from 1 January 2014 to 29 June 2024. Findings: In 2024, autochthonous OROV infections were detected in previously non-endemic areas across all five Brazilian regions. Cases were reported in 19 of 27 federal units, with 83.2% (6,895 of 8,284) of infections in Northern Brazil and a nearly 200-fold increase in incidence compared to reported cases over the last decade. We detected OROV RNA in 10.8% (10 of 93) of patients with febrile illness between December 2023 and May 2024 in Amazonas. We demonstrate that the 2023-2024 epidemic was caused by a novel OROV reassortant that replicated approximately 100-fold higher titers in mammalian cells compared to the prototype strain. The 2023-2024 OROV reassortant displayed plaques earlier than the prototype, produced 1.7 times more plaques, and plaque sizes were 2.5 larger compared to the prototype. Furthermore, serum collected in 2016 from previously OROV-infected individuals showed at least a 32-fold reduction in neutralizing capacity against the reassortment strain compared to the prototype. Interpretation: These findings provide a comprehensive assessment of Oropouche fever in Brazil and contribute to a better understanding of the 2023-2024 OROV reemergence. The recent increased incidence may be related to a higher replication efficiency of a new reassortant virus that also evades previous immunity.

Phylogenetics, Epidemiology and Temporal Patterns of Dengue Virus in Araraquara, São Paulo State.

Dengue virus (DENV) is a prominent arbovirus with global spread, causing approximately 390 million infections each year. In Brazil, yearly epidemics follow a well-documented pattern of serotype replacement every three to four years on average. Araraquara, located in the state of São Paulo, has faced significant impacts from DENV epidemics since the emergence of DENV-1 in 2010. The municipality then transitioned from low to moderate endemicity in less than 10 years. Yet, there remains an insufficient understanding of virus circulation dynamics, particularly concerning DENV-1, in the region, as well as the genetic characteristics of the virus. To address this, we sequenced 37 complete or partial DENV-1 genomes sampled from 2015 to 2022 in Araraquara. Then, using also Brazilian and worldwide DENV-1 sequences we reconstructed the evolutionary history of DENV-1 in Araraquara and estimated the time to the most recent common ancestor (tMRCA) for serotype 1, for genotype V and its main lineages. Within the last ten years, there have been at least three introductions of genotype V in Araraquara, distributed in two main lineages (L Ia and L Ib, and L II). The tMRCA for the first sampled lineage (2015/2016 epidemics) was approximately 15 years ago (in 2008). Crucially, our analysis challenges existing assumptions regarding the emergence time of the DENV-1 genotypes, suggesting that genotype V might have diverged more recently than previously described. The presence of the two lineages of genotype V in the municipality might have contributed to the extended persistence of DENV-1 in the region.

SARS-CoV-2 Detection and Culture in Different Biological Specimens from Immunocompetent and Immunosuppressed COVID-19 Patients Infected with Two Different Viral Strains.

Introduction-The dynamics of SARS-CoV-2 shedding and replication in humans remain incompletely understood. Methods-We analyzed SARS-CoV-2 shedding from multiple sites in individuals with an acute COVID-19 infection by weekly sampling for five weeks in 98 immunocompetent and 25 immunosuppressed individuals. Samples and culture supernatants were tested via RT-PCR for SARS-CoV-2 to determine viral clearance rates and in vitro replication. Results-A total of 2447 clinical specimens were evaluated, including 557 nasopharyngeal swabs, 527 saliva samples, 464 urine specimens, 437 anal swabs and 462 blood samples. The SARS-CoV-2 genome sequences at each site were classified as belonging to the B.1.128 (ancestral strain) or Gamma lineage. SARS-CoV-2 detection was highest in nasopharyngeal swabs regardless of the virus strain involved or the immune status of infected individuals. The duration of viral shedding varied between clinical specimens and individual patients. Prolonged shedding of potentially infectious virus varied from 10 days up to 191 days, and primarily occurred in immunosuppressed individuals. Virus was isolated in culture from 18 nasal swab or saliva samples collected 10 or more days after onset of disease. Conclusions-Our findings indicate that persistent SARS-CoV-2 shedding may occur in both competent or immunosuppressed individuals, at multiple clinical sites and in a minority of subjects is capable of in vitro replication.

Understanding Sabiá virus infections (Brazilian mammarenavirus).

BACKGROUND: Only two naturally occurring human Sabiá virus (SABV) infections have been reported, and those occurred over 20 years ago. METHODS: We diagnosed two new cases of SABV infection using metagenomics in patients thought to have severe yellow fever and described new features of histopathological findings. RESULTS: We characterized clinical manifestations, histopathology and analyzed possible nosocomial transmission. Patients presented with hepatitis, bleeding, neurological alterations and died. We traced twenty-nine hospital contacts and evaluated them clinically and by RT-PCR and neutralizing antibodies. Autopsies uncovered unique features on electron microscopy, such as hepatocyte "pinewood knot" lesions. Although previous reports with similar New-World arenavirus had nosocomial transmission, our data did not find any case in contact tracing. CONCLUSIONS: Although an apparent by rare, Brazilian mammarenavirus infection is an etiology for acute hemorrhagic fever syndrome. The two fatal cases had peculiar histopathological findings not previously described. The virological diagnosis was possible only by contemporary techniques such as metagenomic assays. We found no subsequent infections when we used serological and molecular tests to evaluate close contacts.

Sulfated ß-glucan from Agaricus subrufescens inhibits flavivirus infection and nonstructural protein 1-mediated pathogenesis.

Despite substantial morbidity and mortality, no therapeutic agents exist for treatment of dengue or Zika, and the currently available dengue vaccine is only recommended for dengue virus (DENV)-immune individuals. Thus, development of therapeutic and/or preventive drugs is urgently needed. DENV and Zika virus (ZIKV) nonstructural protein 1 (NS1) can directly trigger endothelial barrier dysfunction and induce inflammatory responses, contributing to vascular leak in vivo. Here we evaluated the efficacy of the (1-6,1-3)-ß-D-glucan isolated from Agaricus subrufescens fruiting bodies (FR) and its sulfated derivative (FR-S) against DENV-2 and ZIKV infection and NS1-mediated pathogenesis. FR-S, but not FR, significantly inhibited DENV-2 and ZIKV replication in human monocytic cells (EC50 = 36.5 and 188.7 µg/mL, respectively) when added simultaneously with viral infection. No inhibitory effect was observed when FR or FR-S were added post-infection, suggesting inhibition of viral entry as a mechanism of action. In an in vitro model of endothelial permeability using human pulmonary microvascular endothelial cells (HPMECs), FR and FR-S (0.12 µg/mL) inhibited DENV-2 NS1- and ZIKV NS1-induced hyperpermeability by 50% and 100%, respectively, as measured by Trans-Endothelial Electrical Resistance. Treatment with 0.25 µg/mL of FR and FR-S inhibited DENV-2 NS1 binding to HPMECs. Further, FR-S significantly reduced intradermal hyperpermeability induced by DENV-2 NS1 in C57BL/6 mice and protected against DENV-induced morbidity and mortality in a murine model of dengue vascular leak syndrome. Thus, we demonstrate efficacy of FR-S against DENV and ZIKV infection and NS1-induced endothelial permeability in vitro and in vivo. These findings encourage further exploration of FR-S and other glycan candidates for flavivirus treatment alone or in combination with compounds with different mechanisms of action.

Torquetenovirus in saliva: A potential biomarker for SARS-CoV-2 infection?

Torquetenovirus (TTV) is present in biological fluids from healthy individuals and measurement of its titer is used to assess immune status in individuals with chronic infections and after transplants. We assessed if the titer of TTV in saliva varied with the presence of SARS-CoV-2 in the nasopharynx and could be a marker of COVID-19 status. Saliva from 91 individuals positive for SARS-CoV-2 in nasal-oropharyngeal samples, and from 126 individuals who were SARS-CoV-2-negative, all with mild respiratory symptoms, were analyzed. Both groups were similar in age, gender, symptom duration and time after symptom initiation when saliva was collected. Titers of TTV and SARS-CoV-2 were assessed by gene amplification. Loss of smell (p = 0.0001) and fever (p = 0.0186) were more prevalent in SARS-CoV-2-positive individuals, while sore throat (p = 0.0001), fatigue (p = 0.0037) and diarrhea (p = 0.0475) were more frequent in the SARS-CoV-2 negative group. The saliva TTV and nasal-oropharyngeal SARS-CoV-2 titers were correlated (p = 0.0085). The TTV level decreased as symptoms resolved in the SARS-CoV-2 infected group (p = 0.0285) but remained unchanged in the SARS-CoV-2 negative controls. In SARS-CoV-2 positive subjects who provided 2-4 saliva samples and in which TTV was initially present, the TTV titer always decreased over time as symptoms resolved. We propose that sequential TTV measurement in saliva is potentially useful to assess the likelihood of symptom resolution in SARS-CoV-2-positive individuals and to predict prognosis.

Prolonged presence of replication-competent SARS-CoV-2 in mildly symptomatic individuals: A report of two cases.

It has been estimated that individuals with COVID-19 can shed replication-competent virus up to a maximum of 20 days after initiation of symptoms. The majority of studies that addressed this situation involved hospitalized individuals and those with severe disease. Studies to address the possible presence of SARS-CoV-2 during the different phases of COVID-19 disease in mildly infected individuals, and utilization of viral culture techniques to identify replication-competent viruses, have been limited. This report describes two patients with mild forms of the disease who shed replication-competent virus for 24 and 37 days, respectively, after symptom onset.

Identification of Polyomaviruses in Skin Cancers.

BACKGROUND: Polyomaviruses (PyVs) were initially described in animals. They have also been detected in humans with some evidence that could play a role in skin carcinogenesis. OBJECTIVES: This study aimed to verify the presence of PyVs in different skin tumour samples and to make clinical correlations with patients' epidemiological data from Clinics Hospital of Medical School of University of São Paulo, Brazil. METHODS: This is a cross-sectional study. A random selection was performed of 120 patients with histopathological exams of different cutaneous neoplasms equally divided into 6 groups and 20 patients with normal skin. The available skin specimens were analysed with 2 different techniques of PCR (conventional and real time) for detection of PyV DNA. Concomitantly, retrospective analysis of the respective medical records for the collection of epidemiological data was done. Analyses suitable for categorical data were used to compare the proportion of patients in each group. RESULTS: PyV DNA was found in 25.69% of the samples: 15% in basal cell carcinoma group, 15% in squamous cell carcinoma, 28.57% in melanoma, 15% in dermatofibrosarcoma protuberans, 13.33% in Kaposi sarcoma, 65% in Merkel cell carcinoma (MCC), and none in normal skin. Merkel cell PyV detection was statistically significant in MCC patients (p value <0.01), but no correlations were found between PyVs and others skin tumours. CONCLUSION: This study demonstrated the presence of PyVs in different skin tumours; however, no association of any PyVs found in any skin tumour with epidemiological data could be shown. Further studies are still needed to elucidate the mechanisms of PyVs in skin carcinogenesis.

Whole transcriptome analysis of multiple Sclerosis patients reveals active inflammatory profile in relapsing patients and downregulation of neurological repair pathways in secondary progressive cases

BACKGROUND: Multiple sclerosis (MS) is an inflammatory autoimmune neurologic disease that causes progressive destruction of myelin sheath and axons. Affecting more than 2 million people worldwide, MS may presents distinct clinical courses. However, information regarding key gene expression and genic pathways related to each clinicalform is still limited. OBJECTIVE: To assess the whole transcriptome of blood leukocytes from patients with remittent-recurrent (RRMS) and secondary-progressive (SPMS) forms to explore the gene expression profile of each form. METHODS: Total RNA was obtained and sequenced in Illumina HiSeq platform. Reads were aligned to human genome (GRCh38/hg38), BAM files were mapped and differential expression was obtained with DeSeq2. Up or downregulated pathways were obtained through Ingenuity IPA. Pro-inflammatory cytokines levels were also assessed. Results: The transcriptome was generated for nine patients (6 SPMS and 3 RRMS) and 5 healthy controls. A total of 731 and 435 differentially expressed genes were identified in SPMS and RRMS, respectively. RERE, IRS2, SIPA1L1, TANC2 and PLAGL1 were upregulated in both forms, whereas PAD2 and PAD4 were upregulated in RRMS and downregulated in SPMS. Inflammatory and neuronal repair pathways were upregulated in RRMS, which was also observed in cytokine analysis. Conversely, SPMS patients presented IL-8, IL-1, Neurothrophin and Neuregulin pathways down regulated. CONCLUSIONS: Overall, the transcriptome of RRMS and SPMS clearly indicated distinct inflammatory profiles, where RRMS presented marked pro-inflammatory profile but SPMS did not. SPMS individuals also presented a decrease on expression of neuronal repair pathways

Detection of Reticuloendotheliosis Virus in Muscovy Ducks, Wild Turkeys, and Chickens in Brazil.

Reticuloendotheliosis viruses (REVs) are known to cause immunosuppressive and oncogenic disease that affects numerous avian species. Reticuloendotheliosis viruses are present worldwide and recently have been reported in South America with cases of infected commercial flocks in Argentina. We surveyed for the presence of REV in birds from a state in the northern region of Brazil using real-time PCR. We report here the presence of REV in Brazil, detected in Muscovy Ducks (Cairina moschata), Wild Turkeys (Meleagris gallopavo), and chickens (Gallus gallus) at a relatively high prevalence (16.8%). Phylogenetic analysis indicated a close relationship of these strains to variants in the US. This study provides evidence of REV in the Amazon biome and provides a baseline for future surveillance of the virus in the region and throughout Brazil.

Performance of at-home self-collected saliva and nasal-oropharyngeal swabs in the surveillance of COVID-19.

Background: SARS-CoV-2 quickly spreads in the worldwide population, imposing social restrictions to control the infection, being the massive testing another essential strategy to break the chain of transmission. Aim: To compare the performance of at-home self-collected samples - saliva and combined nasal-oropharyngeal swabs (NOP) - for SARS-CoV-2 detection in a telemedicine platform for COVID-19 surveillance. Material and methods: We analyzed 201 patients who met the criteria of suspected COVID-19. NOP sampling was combined (nostrils and oropharynx) and saliva collected using a cotton pad device. Detection of SARS-COV-2 was performed by using the Altona RealStar® SARS-CoV-2 RT-PCR Kit 1.0.  Results: There was an overall significant agreement (κ coefficient value of 0.58) between saliva and NOP. Considering results in either sample, 70 patients positive for SARS-CoV-2 were identified, with 52/70 being positive in NOP and 55/70 in saliva. This corresponds to sensitivities of 74.2% (95% CI; 63.7% to 83.1%) for NOP and 78.6% (95% CI; 67.6% to 86.6%) for saliva. Conclusion: Our data show the feasibility of using at-home self-collected samples (especially saliva), as an adequate alternative for SARS-CoV-2 detection. This new approach of testing can be useful to develop strategies for COVID-19 surveillance and for guiding public health decisions.

Variable sources of Bk virus in renal allograft recipients.

BK virus is the causative agent of polyomavirus-associated nephropathy, a major cause of kidney transplant failure affecting 1%-10% of recipients. Previous studies that investigated the viral source on the kidney recipient pointed that the donor is implicated in the origin of human polyomavirus BK (BKPyV) infection in recipients, but giving the low genetic variability of BKPyV this subject is still controversial. The aim of this study was to determine if BKPyV replicating in kidney recipients after transplantation is always originated from the donor. Urine and blood samples from 68 pairs of living donors and kidney recipients who underwent renal transplantation from August 2010-September 2011 were screened for BKPyV by real time polymerase chain reaction. Only three recipients presented viremia. When both donors and recipients were BKPyV positive, a larger fragment of VP1 region was obtained and sequenced to determine the level of similarity between them. A phylogenetic tree was built for the 12 pairs of sequences obtained from urine and high level of similarity among all sequences was observed, indicating that homology inferences for donor and recipient viruses must be cautiously interpreted. However, a close inspection on the donor-recipient pairs sequences revealed that 3 of 12 pairs presented considerably different viruses and 4 of 12 presented mixed infection, indicating that the source of BKPyV infection is not exclusively derived from the donor. We report that about 60% of the renal recipients shed BKPyV genetically distinct from the donor, confronting the accepted concept that the donor is the main source of recipients' infection.

The first observation of the association of Merkel cell polyomavirus and Merkel cell carcinoma in Brazil.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare but aggressive primary cutaneous carcinoma with high mortality rates. The present study intends to delineate the epidemiological profile of patients with MCC seen at the Clinics Hospital of the Medical School at the University of São Paulo, Brazil, and its association with Merkel cell polyomavirus (MCPyV). METHODS: This is a retrospective study. A search was performed in the hospital's medical index for all cases of MCC from January 1994 to December 2012. Among patients with MCC, the available tumoral skin specimens were analyzed with two different techniques of polymerase chain reaction (PCR) (conventional and real-time) for detection of MCPyV DNA. Additionally, paraffin-embedded samples of patients with non-MCC skin cancers were also analyzed. Analyses suitable for categorical data (i.e., x² of Fisher) were used to compare the proportion of patients in each group. RESULTS: Nineteen patients with MCC and 20 patients with non-MCC skin cancers entered the study. All MCC samples available (13) tested positive for the presence of MCPyV DNA; however, in the non-MCC skin cancer samples, the MCPyV DNA was detected in 4 of 20 samples (20%). MCPyV DNA detection rate was higher in patients with MCC than in the other group, and its analysis was statistically significant (P < 0.01). CONCLUSIONS: This study demonstrates the association of MCPyV in Brazilian patients with MCC. However, further studies are necessary to determine the exact involvement of MCPyV in MCC pathogenesis and to define the significance of viral DNA detection in non-MCC skin cancers.

A non-functional galanin receptor-2 in a multiple sclerosis patient.

Multiple Sclerosis (MS) is an inflammatory neurodegenerative disease that affects approximately 2.5 million people globally. Even though the etiology of MS remains unknown, it is accepted that it involves a combination of genetic alterations and environmental factors. Here, after performing whole exome sequencing, we found a MS patient harboring a rare and homozygous single nucleotide variant (SNV; rs61745847) of the G-protein coupled receptor (GPCR) galanin-receptor 2 (GALR2) that alters an important amino acid in the TM6 molecular toggle switch region (W249L). Nuclear magnetic resonance imaging showed that the hypothalamus (an area rich in GALR2) of this patient exhibited an important volumetric reduction leading to an enlarged third ventricle. Ex vivo experiments with patient-derived blood cells (AKT phosphorylation), as well as studies in recombinant cell lines expressing the human GALR2 (calcium mobilization and NFAT mediated gene transcription), showed that galanin (GAL) was unable to stimulate cell signaling in cells expressing the variant GALR2 allele. Live cell confocal microscopy showed that the GALR2 mutant receptor was primarily localized to intracellular endosomes. We conclude that the W249L SNV is likely to abrogate GAL-mediated signaling through GALR2 due to the spontaneous internalization of this receptor in this patient. Although this homozygous SNV was rare in our MS cohort (1:262 cases), our findings raise the potential importance of impaired neuroregenerative pathways in the pathogenesis of MS, warrant future studies into the relevance of the GAL/GALR2 axis in MS and further suggest the activation of GALR2 as a potential therapeutic route for this disease.

Characterization of clinical predictors of naturally occurring NS3/NS4A protease polymorphism in genotype 1 hepatitis C virus mono and HIV co-infected patients.

Spontaneously occurring resistance may impair the success of protease inhibitors based regimens in HCV treatment. This study aimed to evaluate associations between amino acid substitutions in NS3/NS4A domain and clinical features of 247 HCV mono or HCV/HIV co-infected patients. Fourteen samples (5.7%) harbored at least one resistance-associated substitution (RAS). The following RASs were detected in NS3 region: T54S (6-2.4%), V55A (7-2.8%), and Q80R (2-0.8%). S122G occurred in 86.9% of HCV genotype 1b samples with either natural polymorphisms or RASs. Advanced liver fibrosis and HIV co-infection were not related to NS3/NS4A amino acid substitutions.

Polyomavirus detection in multiple sclerosis patients under natalizumab therapy: Profile and frequency of urinary shedding

Patients undergoing Natalizumab (NTZ) therapy are at risk of progressive multifocal leukoencephalopathy (PML). Besides John Cunningham virus (JCV), BK polyomavirus might represent an additional concern for such patients since it can also infect CNS cells. Currently, data regarding the presence of anti-JCV antibodies added to previous immunosuppressive therapy and prolonged NTZ therapy has been used to classify patients at risk of developing PML. Here, we investigated the profile shedding of JCV and BKV in multiple sclerosis (MS) patients during treatment with NTZ. Serial blood and urine samples from 97 MS patients receiving either NTZ or ß-interferon were investigated for polyomavirus shedding. While all blood samples tested negative, 36% of the patients shed polyomavirus in the urine in at least one time point. From these, 21.7%, 9.3%, and 5.1% shed JCV, BKV, and both polyomavirus, respectively. No difference was observed between the rates of urinary shedding of patients treated with NTZ (38.9%) and patients treated with other drugs (34.5%), also no PML event was diagnosed during the follow-up. Therefore, urinary shedding might not be interfered by therapy condition. In our study, we also observed 14/27 (52%) of anti-JCV antibodies prevalence, and nearly half of them (42%) did not present any event of urinary shedding during the follow-up

Persistence of Zika Virus in Breast Milk after Infection in Late Stage of Pregnancy.

We detected Zika virus in breast milk of a woman in Brazil infected with the virus during the 36th week of pregnancy. Virus was detected 33 days after onset of signs and symptoms and 9 days after delivery. No abnormalities were found during fetal assessment or after birth of the infant.

Expression of human endogenous retrovirus K and W in babies.

Here we determined the relative expression of HERV-K and W proviruses in HIV infected and non-infected mothers as well as their respective babies up to 1 year-old. HIV-infected mothers, their babies and uninfected control groups presented expression of both HERV-K and HERV-W with relatively high frequency. While the level of HERV-K expression was similar among groups, the level of HERV-W expression in HIV-infected mothers was four-fold higher than the uninfected mothers from the control group (p < 0.01). HERV-W was down regulated in HIV-exposed babies in comparison to non-exposed babies. To our knowledge, this is the first report of HERV transcriptional activity in babies from 0-1 year-old.