SARS-CoV-2 Spillback to Wild Coatis in Sylvatic-Urban Hotspot, Brazil.

We tested coatis (Nasua nasua) living in an urban park near a densely populated area of Brazil and found natural SARS-CoV-2 Zeta variant infections by using quantitative reverse transcription PCR, genomic sequencing, and serologic surveillance. We recommend a One Health strategy to improve surveillance of and response to COVID-19.

Serum soluble mediators as prognostic biomarkers for morbidity, disease outcome, and late-relapsing hepatitis in yellow fever patients.

This study described a soluble mediator storm in acute Yellow Fever/YF infection along the kinetics timeline towards convalescent disease. The analyses of the YF Viral RNAnemia, chemokines, cytokines, and growth factors were performed in YF patients at acute/(D1-15) and convalescent/(D16-315) phases. Patients with acute YF infection displayed a trimodal viremia profile spreading along D3, D6, and D8-14. A massive storm of mediators was observed in acute YF. Higher levels of mediators were observed in YF with higher morbidity scores, patients under intensive care, and those progressing to death than in YF patients who progress to late-relapsing hepatitis/L-Hep. A unimodal peak of biomarkers around D4-6 with a progressive decrease towards D181-315 was observed in non-L-Hep patients, while a bimodal pattern with a second peak around D61-90 was associated with L-Hep. This study provided a comprehensive landscape of evidence that distinct immune responses drive pathogenesis, disease progression, and L-Hep in YF patients.

MEK/ERK activation plays a decisive role in Zika virus morphogenesis and release.

Brazil has experienced an increase in outbreaks caused by flaviviruses. The high incidence of dengue fever, the morbidity of Zika in children, and the high mortality of yellow fever have affected millions in recent years. Deciphering host-virus interactions is important for treating viral infections, and the mitogen-activated protein kinases (MAPK) are an interesting target because of their role in flavivirus replication. In particular, mitogen-activated protein kinase kinase (MEK), which targets extracellular-signal-regulated kinase (ERK), is necessary for dengue and yellow fever infections. In this study, we evaluated the role of the MEK/ERK pathway and the effect of the MEK inhibitor trametinib on the Asian ZIKV strain PE243 and the prototype African ZIKV strain MR766, addressing genome replication, morphogenesis, and viral release. ZIKV infection stimulated ERK phosphorylation in Vero cells at 12 and 18 hours postinfection (hpi). Trametinib showed sustained antiviral activity, inhibiting both ZIKV strains for at least four days, and electron microscopy showed probable inhibition of ZIKV morphogenesis. ZIKV PE243 can complete one cycle in Vero cells in 14 hours; genome replication was detected around 8 hpi, intracellular viral particles at 12 hpi, and extracellular progeny at 14 hpi. Treatments at 6-hour intervals showed that trametinib inhibited late stages of viral replication, and the titration of intra- or extracellular virions showed that the treatment especially affected viral morphogenesis and release. Thus, ZIKV stimulated ERK phosphorylation during viral morphogenesis and release, which correlated with trametinib inhibiting both the signaling pathway and viral replication.

Yellow Fever Molecular Diagnosis Using Urine Specimens during Acute and Convalescent Phases of the Disease.

Prior studies have demonstrated prolonged presence of yellow fever virus (YFV) RNA in saliva and urine as an alternative to serum. To investigate the presence of YFV RNA in urine, we used RT-PCR for YFV screening in 60 urine samples collected from a large cohort of naturally infected yellow fever (YF) patients during acute and convalescent phases of YF infection from recent YF outbreaks in Brazil (2017 to 2018). Fifteen urine samples from acute phase infection (up to 15 days post-symptom onset) and four urine samples from convalescent phase infection (up to 69 days post-symptom onset), were YFV PCR-positive. We genotyped YFV detected in seven urine samples (five collected during the acute phase and two collected during the YF convalescent phase). Genotyping indicated the presence of YFV South American I genotype in these samples. To our knowledge, this is the first report of wild-type YFV RNA detection in the urine this far out from symptom onset (up to 69 DPS), including YFV RNA detection during the convalescent phase of YF infection. The detection of YFV RNA in urine is an indicative of YFV infection; however, the results of RT-PCR using urine as sample should be interpreted with care, since a negative result does not exclude the possibility of YFV infection. With a possible prolonged period of detection beyond the viremic phase, the use of urine samples coupled with serological tests, epidemiologic inquiry, and clinical assessment could provide a longer diagnostic window for laboratory YF diagnosis.

New Isolates of Pandoraviruses: Contribution to the Study of Replication Cycle Steps.

Giant viruses are complex members of the virosphere, exhibiting outstanding structural and genomic features. Among these viruses, the pandoraviruses are some of the most intriguing members, exhibiting giant particles and genomes presenting at up to 2.5 Mb, with many genes having no known function. In this work, we analyzed, by virological and microscopic methods, the replication cycle steps of three new pandoravirus isolates from samples collected in different regions of Brazil. Our data indicate that all analyzed pandoravirus isolates can deeply modify the Acanthamoeba cytoplasmic environment, recruiting mitochondria and membranes into and around the electron-lucent viral factories. We also observed that the viral factories start forming before the complete degradation of the cellular nucleus. Various patterns of pandoravirus particle morphogenesis were observed, and the assembly of the particles seemed to be started either by the apex or by the opposite side. On the basis of the counting of viral particles during the infection time course, we observed that pandoravirus particles could undergo exocytosis after their morphogenesis in a process that involved intense recruitment of membranes that wrapped the just-formed particles. The treatment of infected cells with brefeldin affected particle exocytosis in two of the three analyzed strains, indicating biological variability among isolates. Despite such particle exocytosis, the lysis of host cells also contributed to viral release. This work reinforces knowledge of and reveals important steps in the replication cycle of pandoraviruses.IMPORTANCE The emerging Pandoraviridae family is composed of some of the most complex viruses known to date. Only a few pandoravirus isolates have been described until now, and many aspects of their life cycle remain to be elucidated. A comprehensive description of the replication cycle is pivotal to a better understanding of the biology of the virus. For this report, we describe new pandoraviruses and used different methods to better characterize the steps of the replication cycle of this new group of viruses. Our results provide new information about the diversity and biology of these giant viruses.

Recent sylvatic yellow fever virus transmission in Brazil: the news from an old disease.

Yellow fever (YF) is an acute viral disease, affecting humans and non-human primates (NHP), caused by the yellow fever virus (YFV). Despite the existence of a safe vaccine, YF continues to cause morbidity and mortality in thousands of people in Africa and South America. Since 2016, massive YF outbreaks have taken place in Brazil, reaching YF-free zones, causing thousands of deaths of humans and NHP. Here we reviewed the main epidemiological aspects, new clinical findings in humans, and issues regarding YFV infection in vectors and NHP in Brazil. The 2016-2019 YF epidemics have been considered the most significant outbreaks of the last 70 years in the country, and the number of human cases was 2.8 times higher than total cases in the previous 36 years. A new YFV lineage was associated with the recent outbreaks, with persistent circulation in Southeast Brazil until 2019. Due to the high number of infected patients, it was possible to evaluate severity and death predictors and new clinical features of YF. Haemagogus janthinomys and Haemagogus leucocelaenus were considered the primary vectors during the outbreaks, and no human case suggested the occurrence of the urban transmission cycle. YFV was detected in a variety of NHP specimens presenting viscerotropic disease, similar to that described experimentally. Further studies regarding NHP sensitivity to YFV, YF pathogenesis, and the duration of the immune response in NHP could contribute to YF surveillance, control, and future strategies for NHP conservation.

Analyses of the Kroon Virus Major Capsid Gene and Its Transcript Highlight a Distinct Pattern of Gene Evolution and Splicing among Mimiviruses.

The inclusion of Mimiviridae members in the putative monophyletic nucleocytoplasmic large DNA virus (NCLDV) group is based on genomic and phylogenomic patterns. This shows that, along with other viral families, they share a set of genes known as core or "hallmark genes," including the gene for the major capsid protein (MCP). Although previous studies have suggested that the maturation of mimivirus MCP transcripts is dependent on splicing, there is little information about the processing of this transcript in other mimivirus isolates. Here we report the characterization of a new mimivirus isolate, called Kroon virus (KV) mimivirus. Analysis of the structure, synteny, and phylogenetic relationships of the MCP genes in many mimivirus isolates revealed a remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. In addition, sequencing of KV and Acanthamoeba polyphaga mimivirus (APMV) MCP transcripts has shown that inside the family, even related giant viruses may present different ways to process the MCP mRNA. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.IMPORTANCE Mimivirus isolates have been obtained by prospecting studies since 2003. Based on genomic and phylogenomic studies of conserved genes, these viruses have been clustered together with members of six other viral families. Although the major capsid protein (MCP) gene is an important member of the so-called "hallmark genes," there is little information about the processing and structure of this gene in many mimivirus isolates. In this work, we have analyzed the structure, synteny, and phylogenetic relationships of the MCP genes in many mimivirus isolates; these genes showed remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.

Vaccinia Virus among Domestic Dogs and Wild Coatis, Brazil, 2013-2015.

To determine their potential role as a source of human infection, we tested domestic dogs (urban) and wild coatis (wild) in Brazil for vaccinia virus. Our findings of positive neutralizing antibodies and quantitative PCR results for 35/184 dogs and 13/90 coatis highlight a potential public health risk.

High prevalence of dengue antibodies and the arginine variant of the FcγRIIa polymorphism in asymptomatic individuals in a population of Minas Gerais State, Southeast Brazil.

Dengue is the most prevalent arthropod-borne viral illness in humans worldwide. Single-nucleotide polymorphisms (SNPs) in genes involved in the immune response, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), IgG Fc receptor II-A (FcγRIIa), vitamin D receptor (VDR), and tumor necrosis factor alpha (TNF-α), were previously reported to be associated with susceptibility to dengue disease in different human populations. Therefore, due to the relevant association of host immune and genetic status with disease susceptibility/severity of dengue, this work aims to verify the frequency of anti-dengue virus antibodies and some dengue-associated risk SNPs in a population in Minas Gerais State, Southeast Brazil. A total of 1560 individuals were genotyped for polymorphisms in DC-SIGN (rs4804803), FcγRIIa (rs1801274), VDR (rs7975232), and TNF-α (rs1800629). The presence of anti-dengue antibodies (IgM and/or IgG) in these samples was also assayed. Anti-dengue antibodies were detected at an overall frequency of 16.86%, indicating a virus infection in asymptomatic individuals. The genotypic frequencies of all SNPs studied did not differ between the asymptomatic and control groups. Regarding the allelic frequencies of the four SNPs analyzed, a higher frequency was detected of the G allele of FcγRIIa/rs1801274 in the asymptomatic individuals when compared to that in the control group (p = 0.03). Therefore, the results showed a high prevalence of asymptomatic individuals in Minas Gerais State, with a potential association between the presence of the G allele of FcγRIIa/rs1801274 and protection against symptomatic disease.

Screening for Zika virus RNA in sera of suspected cases: a retrospective cross-sectional study.

BACKGROUND: Zika virus (ZIKV) became a global human health concern owing to its rapid spread worldwide and its association with congenital and neurological disorders. The current epidemiological profile of arboviruses in Brazil is characterized by widespread co-circulation of Dengue virus, Chikungunya virus, and ZIKV throughout the country. These viruses cause acute diseases frequently with overlapping symptoms, which could result in an inaccurate diagnosis based solely on clinical and epidemiological grounds. Here we conducted a screening for ZIKV RNA in serum samples from patients across Brazil with suspected ZIKV infection. METHODS: Using RT-qPCR, we investigated ZIKV RNA in 3001 serum samples. Samples were passively acquired through a private laboratory network, between December 2015 and August 2016, from 27 Brazilian Federative Units. We performed descriptive statistics on demographic variables including sex, age, and geographic location. RESULTS: ZIKV was detected in 11.4% (95%CI = 10.3-12.6%) of the sera. ZIKV RNA was detected in sera collected throughout the country, but during the analyzed period, RNA was more frequently detected in samples from the Southeast, Midwest, and North regions (3.9 to 5.8 times higher) when compared to the Northeast and South regions. CONCLUSIONS: These data reinforce the importance of laboratory diagnosis, surveillance systems, and further epidemiological studies to understand the dynamics of outbreaks and diseases associated with ZIKV and other arboviruses.

Molecular surveillance of dengue in Minas Gerais provides insights on dengue virus 1 and 4 circulation in Brazil.

Dengue, caused by any of the four types of Dengue virus (DENV) is the most important arbovirus in the world. In this study we performed a molecular surveillance of dengue during the greatest dengue outbreak that took place in Divinópolis, Minas Gerais state, Southeast Brazil, in 2013. Samples from 100 patients with clinical symptoms of dengue were studied and 26 were positive. The capsid/premembrane (CprM) and envelope gene sequences of some samples were amplified and sequenced. Molecular analyses demonstrated that two DENV-1 lineages, belonging to genotype V were introduced and co-circulated in Divinópolis. When compared to each other, those lineages presented high genetic diversity and showed unique amino acids substitutions in the envelope protein, including in domains I, II, and III. DENV-4 strains from Divinópolis clustered within genotype IIb and the most recent common ancestor was probably introduced into the city three years before the 2013 epidemic. Here we demonstrated for the first time the circulation of DENV-4 and the co-circulation of two DENV-1 lineages in Midwest region of Minas Gerais, Brazil. Moreover our analysis indicated the introduction of five DENV-1 lineages, genotype V into Brazil, in different times. J. Med. Virol. 89:966-973, 2017. © 2016 Wiley Periodicals, Inc.

Absence of vaccinia virus detection in a remote region of the Northern Amazon forests, 2005-2015.

Vaccinia virus (VACV) circulates in Brazil and other South America countries and is responsible for a zoonotic disease that usually affects dairy cattle and humans, causing economic losses and impacting animal and human health. Furthermore, it has been detected in wild areas in the Brazilian Amazon. To better understand the natural history of VACV, we investigated its circulation in wildlife from French Guiana, a remote region in the Northern Amazon forest. ELISA and plaque reduction neutralization tests were performed to detect anti-orthopoxvirus antibodies. Real-time and standard PCR targeting C11R, A56R and A26L were applied to detect VACV DNA in serum, saliva and tissue samples. No evidence of VACV infection was found in any of the samples tested. These findings provide additional information on the VACV epidemiological puzzle. The virus could nevertheless be circulating at low levels that were not detected in areas where no humans or cattle are present.

Dengue outbreaks in Divinopolis, south-eastern Brazil and the geographic and climatic distribution of Aedes albopictus and Aedes aegypti in 2011-2012.

OBJECTIVE: To entomologically monitor Aedes spp. and correlate the presence of these vectors with the recent epidemic of dengue in Divinopolis, Minas Gerais State, Brazil. METHODS: Ovitraps were installed at 44 points in the city, covering six urban areas, from May 2011 to May 2012. After collection, the eggs were incubated until hatching. In the 4th stage of development, the larvae were classified as Ae. aegypti or Ae. albopictus. RESULTS: In total, 25 633 Aedes spp. eggs were collected. February was the month with the highest incidence, with 5635 eggs collected and a hatching rate of 46.7%. Ae. aegypti eggs had the highest hatching rate, at 72.3%, whereas Ae. albopictus eggs had 27.7%. Climate and population density influenced the number of eggs found. Indicators of vector presence were positively correlated with the occurrence of dengue cases. CONCLUSION: These data reinforce the need for entomological studies, highlight the relevance of Ae. albopictus as a possible disease vector and demonstrate its adaptation. Ae. albopictus, most commonly found in forested areas, comprised a substantial proportion of the urban mosquito population.

First genome sequence of St. Louis encephalitis virus (SLEV) isolated from a human in Brazil.

St. Louis encephalitis virus (SLEV), a member of the family Flaviviridae, genus Flavivirus, is a causative agent of encephalitis in the Americas. In Brazil, sporadic cases of SLEV infection have been reported since 1953, but the first outbreak of SLEV in Brazil was identified only in 2007, concomitant with an outbreak of dengue virus (DENV) serotype 3. This finding, along with other reports, indicates that SLEV circulation in Brazil is largely unknown, and there may be epidemiological implications of the co-circulation of SLEV, DENV and other flaviviruses in Brazil. Here, we describe the first complete genome sequence of an SLEV strain isolated from a human patient in Brazil, strain BeH 355964. Phylogenetic analysis was performed to determine the genotype of BeH 355964 using the full-length genome and envelope (E) gene sequences separately. Both analyses showed that BeH 355964 could be classified as genotype V. Although the number of single gene sequences available is greater (such as for the E gene), the phylogenetic tree based on the complete genome sequence was better supported and provided further information about the virus.

Evaluation of humoral immune response after yellow fever infection: an observational study on patients from the 2017-2018 sylvatic outbreak in Brazil.

Between 2016 and 2018, Brazil experienced major sylvatic yellow fever (YF) outbreaks that caused hundreds of casualties, with Minas Gerais (MG) being the most affected state. These outbreaks provided a unique opportunity to assess the immune response triggered by the wild-type (WT) yellow fever virus (YFV) in humans. The plaque reduction neutralization test (PRNT) is currently the standard method to assess the humoral immune response to YFV by measuring neutralizing antibodies (nAbs). The present study aimed to evaluate the humoral immune response of patients from the 2017-2018 sylvatic YF outbreak in MG with different disease outcomes by using PRNTs with a WT YFV strain, isolated from the 2017-2018 outbreak, and a vaccine YFV strain. Samples from naturally infected YF patients were tested, in comparison with healthy vaccinees. Results showed that both groups presented different levels of nAb against the WT and vaccine strains, and the levels of neutralization against the strains varied homotypically and heterotypically. Results based on the geometric mean titers (GMTs) suggest that the humoral immune response after a natural infection of YFV can reach higher levels than that induced by vaccination (GMT of patients against WT YFV compared to GMT of vaccinees, P < 0.0001). These findings suggest that the humoral immune responses triggered by the vaccine and WT strains of YFV are different, possibly due to genetic and antigenic differences between these viruses. Therefore, current means of assessing the immune response in naturally infected YF individuals and immunological surveillance methods in areas with intense viral circulation may need to be updated.IMPORTANCEYellow fever is a deadly febrile disease caused by the YFV. Despite the existence of effective vaccines, this disease still represents a public health concern worldwide. Much is known about the immune response against the vaccine strains of the YFV, but recent studies have shown that it differs from that induced by WT strains. The extent of this difference and the mechanisms behind it are still unclear. Thus, studies aimed to better understand the immune response against this virus are relevant and necessary. The present study evaluated levels of neutralizing antibodies of yellow fever patients from recent outbreaks in Brazil, in comparison with healthy vaccinees, using plaque reduction neutralization tests with WT and vaccine YFV strains. Results showed that the humoral immune response in naturally infected patients was higher than that induced by vaccination, thus providing new insights into the immune response triggered against these viruses.

YELLOW ALERT: Persistent Yellow Fever Virus Circulation among Non-Human Primates in Urban Areas of Minas Gerais State, Brazil (2021-2023).

Yellow fever virus (YFV) is the agent of yellow fever (YF), which affects both humans and non-human primates (NHP). Neotropical NHP are highly susceptible to YFV and considered sentinels for YFV circulation. Brazil faced a significant YF outbreak in 2017-2018, with over 2000 human cases and 2000 epizootics cases, mainly in the State of Minas Gerais, Brazil. This study aimed to investigate whether YFV circulation persisted in NHP after the human outbreak had subsided. To this end, NHP carcass samples collected in Minas Gerais from 2021 to 2023 were screened for YFV. RNA was extracted from tissue fragments and used in RT-qPCR targeting the YFV 5'UTR. Liver and lung samples from 166 animals were tested, and the detection of the ß-actin mRNA was used to ensure adequacy of RNA isolation. YFV RNA was detected in the liver of 18 NHP carcasses collected mainly from urban areas in 2021 and 2022. YFV positive NHP were mostly represented by Callithrix, from 5 out of the 12 grouped municipalities (mesoregions) in Minas Gerais state. These findings reveal the continued YFV circulation in NHP in urban areas of Minas Gerais during 2021 and 2022, with the attendant risk of re-establishing the urban YFV cycle.

Neuro-COVID-19 With or Without the Multisystem Inflammatory Syndrome (MIS-C): A Single-Center Study : COVID-19: Neurologic Manifestations in Children.

This study evaluates the range of neurological manifestation in children with COVID-19 (neuro-COVID-19) both with and without the multisystem inflammatory syndrome (MIS-C) and the persistence of symptoms after hospital discharge. The study was conducted as a prospective study of children and adolescents under 18 years of age who were admitted to a children's hospital for infectious diseases from January 2021 to January 2022. The children had no previous neurological or psychiatric disorders. Out of the 3021 patients evaluated, 232 were confirmed to have COVID-19 and 21 of these patients (9%) showed neurological manifestations associated with the virus. Of these 21 patients, 14 developed MIS-C, and 7 had neurological manifestations unrelated to MIS-C. There was no statistical difference regarding the neurological manifestations during hospitalization and outcomes between patients with neuro-COVID-19 who had or did not have MIS-C, except for seizures that occurred more frequently in patients with neuro-COVID-19 without MIS-C (p-value = 0.0263). One patient died, and 5 patients still had neurological or psychiatric manifestations at discharge, which persisted for up to 7 months. The study highlights that SARS-CoV-2 infection can affect the central and peripheral nervous system, particularly in children and adolescents with MIS-C, and that it is crucial to be vigilant for long-term adverse outcomes, as the neurological and psychiatric effects of COVID-19 in children are emerging during an important stage of brain development.

Ecological drivers of sustained enzootic yellow fever virus transmission in Brazil, 2017-2021.

Beginning December 2016, sylvatic yellow fever (YF) outbreaks spread into southeastern Brazil, and Minas Gerais state experienced two sylvatic YF waves (2017 and 2018). Following these massive YF waves, we screened 187 free-living non-human primate (NHPs) carcasses collected throughout the state between January 2019 and June 2021 for YF virus (YFV) using RTqPCR. One sample belonging to a Callithrix, collected in June 2020, was positive for YFV. The viral strain belonged to the same lineage associated with 2017-2018 outbreaks, showing the continued enzootic circulation of YFV in the state. Next, using data from 781 NHPs carcasses collected in 2017-18, we used generalized additive mixed models (GAMMs) to identify the spatiotemporal and host-level drivers of YFV infection and intensity (an estimation of genomic viral load in the liver of infected NHP). Our GAMMs explained 65% and 68% of variation in virus infection and intensity, respectively, and uncovered strong temporal and spatial patterns for YFV infection and intensity. NHP infection was higher in the eastern part of Minas Gerais state, where 2017-2018 outbreaks affecting humans and NHPs were concentrated. The odds of YFV infection were significantly lower in NHPs from urban areas than from urban-rural or rural areas, while infection intensity was significantly lower in NHPs from urban areas or the urban-rural interface relative to rural areas. Both YFV infection and intensity were higher during the warm/rainy season compared to the cold/dry season. The higher YFV intensity in NHPs in warm/rainy periods could be a result of higher exposure to vectors and/or higher virus titers in vectors during this time resulting in the delivery of a higher virus dose and higher viral replication levels within NHPs. Further studies are needed to better test this hypothesis and further compare the dynamics of YFV enzootic cycles between different seasons.