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Yellow fever (YF) is an acute and potentially fatal hemorrhagic disease caused by the Yellow Fever virus (YFV), endemic to sub-Saharan Africa and several tropical countries, including Brazil. In Brazil, the Amazon region is considered the main endemic area. YFV is maintained in a sylvatic cycle involving Neotropical primates and mosquitoes of the genera Haemagogus and Sabethes, acting as primary and secondary vectors, respectively. In March 2024, entomovirological surveillance was conducted in Santa Bárbara do Pará, Pará, Brazil. A total of 286 mosquitoes were collected, classified into 13 species across nine genera, and grouped into 33 pools. Seventeen pools were tested by RT-qPCR for Orthoflavivirus (YFV, DENV, WNV, SLEV), Alphavirus (CHIKV, MAYV), and Orthobunyavirus (OROV). YFV was detected in four Haemagogus janthinomys pools, with Ct values ranging from 22.2 to 27.9. Metagenomic sequencing confirmed the presence of YFV with assigned reads and >99% protein identity. Notably, the detection occurred without human cases or primate deaths, enabling timely vaccination of the local population. These findings confirm YFV circulation in forested areas of the Belém metropolitan region and reaffirm Hg. janthinomys as a key vector. Our study reinforces the relevance of early entomovirological surveillance and preventive strategies, such as vaccination, to mitigate yellow fever reemergence.

Viruses with encephalitogenic potential can cause neurological conditions of clinical and epidemiological importance, such as Saint Louis encephalitis virus, Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Dengue virus, Zika virus, Chikungunya virus, Mayaro virus and West Nile virus. The objective of the present study was to determine the number of arboviruses with neuroinvasive potential isolated in Brazil that corresponds to the collection of viral samples belonging to the Department of Arbovirology and Hemorrhagic Fevers, Evandro Chagas Institute (SAARB/IEC) of the Laboratory Network of National Reference for Arbovirus Diagnosis from 1954 to 2022. In the analyzed period, a total of 1,347 arbovirus samples with encephalitogenic potential were isolated from mice; 5,065 human samples were isolated exclusively by cell culture; and 676 viruses were isolated from mosquitoes. The emergence of new arboviruses may be responsible for diseases still unknown to humans, making the Amazon region a hotspot for infectious diseases due to its fauna and flora species characteristics. The detection of circulating arboviruses with the potential to cause neuroinvasive diseases is constant, which justifies the continuation of active epidemiological surveillance work that offers adequate support to the public health system regarding the virological diagnosis of circulating arboviruses in Brazil.

The rapid and disorderly urbanization in the Amazon has resulted in the insertion of forest fragments into cities, causing the circulation of arboviruses, which can involve hematophagous arthropods and free-ranging birds in the transmission cycles in urban environments. This study aimed to evaluate the circulation of arboviruses in free-ranging birds and hematophagous arthropods captured in an Environmental Protection Area in the Belem metropolitan area, Brazil. Birds were captured using mist nets, and hematophagous arthropods were collected using a human protected attraction technique and light traps. The birds’ sera were subjected to a hemagglutination inhibition test to detect antibodies against 29 arbovirus antigens. Arthropod macerates were inoculated into C6/36 and VERO cell cultures to attempt viral isolation and were tested using indirect immunofluorescence, subsequent genetic sequencing and submitted for phylogenetic analysis. Four bird sera were positive for arbovirus, and one batch of Psorophora ferox was positive for Flavivirus on viral isolation and indirect immunofluorescence. In addition, the Ilheus virus was detected in the sequencing and phylogenetic analysis. The presence of antibodies in sera from free-ranging birds and the isolation of Ilheus virus in Psorophora ferox indicate the circulation of arboviruses in forest remnants in the urban center of Belem.

Mosquitoes are recognized as the arthropod group with the greatest vectorial capacity, and the viruses they transmit constitute a significant concern in the context of global One Health. In addition, these insects act as hosts for a wide diversity of insect-specific viruses (ISVs), which exclusively infect arthropods. Expanding knowledge of ISVs is particularly relevant, given their potential influence on arbovirus replication and their role in elucidating the evolutionary processes that shape virus–vector interactions. In this study, we report the isolation and molecular analysis of three negeviruses associated with different mosquito species of the genera Culex, Coquillettidia, Mansonia, and Ochlerotatus, collected in Belém, Pará State, in the Brazilian Amazon: Loreto virus, Wallerfield virus, and a putative new species, designated Terra firme virus. Eleven pools exhibited cellular alterations consistent with cytopathic effects in invertebrate C6/36 cells but showed no evidence of replication in vertebrate Vero cells. Notably, simultaneous infections by two or three negeviruses were detected in some mosquito pools, indicating the occurrence of multiple viral infections within individual samples. Genomic analyses revealed that the isolated strains share conserved domains with previously described isolates from other countries. Phylogenetic inferences demonstrated that the investigated strains are classified within the clades Nelorpivirus and Sandewavirus. Taken together, these findings expand the currently known diversity of the negevirus group and contribute to a more comprehensive understanding of its host range and geographic distribution.

Oropouche fever is caused by the Oropouche virus (OROV; Bunyaviridae, Orthobunyavirus), one of the most frequent arboviruses that infect humans in the Brazilian Amazon. This year, an OROV outbreak was identified in Brazil, and its vertical transmission was reported, which was associated with fetal death and microcephaly. We describe the clinical manifestations identified in three cases of congenital OROV infection with confirmed serology (OROV-IgM) in the mother-newborn binomial. One of the newborns died, and post-mortem molecular analysis using real-time RT-qPCR identified the OROV genome in several tissues. All three newborns were born in the Amazon region in Brazil, and the mothers reported fever, rash, headache, myalgia, and/or retro-orbital pain during pregnancy. The newborns presented with severe microcephaly secondary to brain damage and arthrogryposis, suggestive of an embryo/fetal disruptive process at birth. Brain and spinal images identified overlapping sutures, cerebral atrophy, brain cysts, thinning of the spinal cord, corpus callosum, and posterior fossa abnormalities. Fundoscopic findings included macular chorioretinal scars, focal pigment mottling, and vascular attenuation. The clinical presentation of vertical OROV infection resembled congenital Zika syndrome to some extent but presents some distinctive features on brain imaging and in several aspects of its neurological presentation. A recognizable syndrome with severe brain damage, neurological alterations, arthrogryposis, and fundoscopic abnormalities can be associated with in utero OROV infection.

Background The emergence and re-emergence of infectious diseases are a cause for worldwide concern. The introduction of Zika and Chikungunya diseases in the Americas has exposed unforeseen medical and logistical challenges for public health systems. Moreover, the lack of preventive measures and vaccination against known and emerging mosquito-transmitted pathogens, and the occurrence of unanticipated clinical complications, has had an enormous social and economic impact on the affected populations. In this study, we aimed to measure the seroprevalence of endemic and emerging viral pathogens in military personnel stationed in Manaus, Amazonas state. Methods We measured the seropositivity of antibodies against 19 endemic and emerging viruses in a healthy military personnel group using a hemagglutination inhibition assay (HIA). Results Overall, DENV positivity was 60.4%, and 30.9% of the individuals reacted against ZIKV. Also, 46.6%, 54.7%, 51.3% and 48.7% individuals reacted against West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Ilheus virus (ILHV) and Rocio virus (ROCV), respectively. Individuals with high DENV HIA titer reacted more frequently with ZIKV or WNV compared to those with low HIA titers. Observed cross-reactivity between Flaviviruses varied depending on the virus serogroup. Additionally, 0.6% and 0.3% individuals were seropositive for Oropouche virus (OROV) and Catu virus (CATUV) from the family Peribunyaviridae, respectively. All samples were negative for Eastern Equine Encephalitis virus (EEEV), Western Equine Encephalomyelitis virus (WEEV), Mayaro virus (MAYV), Mucambo virus (MUCV) and CHIKV from the family Togaviridae . Conclusions A high proportion of individuals in our high-risk population (~ 60%) lacked antibodies against major endemic and emerging viruses, which makes them susceptible for further infections. Military personnel serving in the Amazon region could serve as sentinels to strengthen global infectious disease surveillance, particularly in remote areas. Graphical abstract

Hantavirus Pulmonary Syndrome is an, often fatal, emerging zoonotic disease in the Americas caused by hantaviruses (family: Hantaviridae). In Brazil, hantavirus routine diagnosis is based on serology (IgM-ELISA) while RT-PCR is often used to confirm acute infection. A Semi-nested RT-PCR and an internally controlled RT-qPCR assays were developed for detection and quantification of four hantaviruses strains circulating in the Brazilian Amazon: Anajatuba (ANAJV) and Castelo dos Sonhos (CASV) strains of Andes virus (ANDV) species; and Rio Mamoré (RIOMV) and Laguna Negra (LNV) strains of LNV species. A consensus region in the N gene of these hantaviruses was used to design the primer sets and a hydrolysis probe. In vitro transcribed RNA was diluted in standards with known concentration. MS2 bacteriophage RNA was detected together with hantavirus RNA as an exogenous control in a duplex reaction. RT-qPCR efficiency was around 100% and the limit of detection was 0.9 copies/μL of RNA for RT-qPCR and 10 copies/μL of RNA for Semi-nested RT-PCR. There was no amplification of either negative samples or samples positive to other pathogens. To assess the protocol for clinical sensitivity, specificity and general accuracy values, both assays were used to test two groups of samples: one comprising patients with disease (n = 50) and other containing samples from healthy individuals (n = 50), according to IgM-ELISA results. A third group of samples (n = 27) infected with other pathogens were tested for specificity analysis. RT-qPCR was more sensitive than semi-nested RT-PCR, being able to detect three samples undetected by conventional RT-PCR. RT-qPCR clinical sensitivity, specificity and general accuracy values were 92.5%, 100% and 97.63%, respectively. Thus, the assays developed in this study were able to detect the four Brazilian Amazon hantaviruses with good specificity and sensitivity, and may become powerful tools in diagnostic, surveillance and research applications of these and possibly other hantaviruses.

West Nile virus is a flavivirus transmitted by mosquitoes, mainly of the genus Culex. In Brazil, serological studies have already indicated the circulation of the virus since 2003, with the first human case detected in 2014. The objective of the present paper is to report the first isolation of WNV in a Culex (Melanoconion) mosquito. Arthropods were collected by protected human attraction and CDC light bait, and taxonomically identified and analyzed by viral isolation, complement fixation and genomic sequencing tests. WNV was isolated from samples of Culex (Melanoconion) mosquitoes, and the sequencing analysis demonstrated that the isolated strain belonged to lineage 1a. The finding of the present study presents the first evidence of the isolation and genome sequencing of WNV in arthropods in Brazil.

Background Zika virus (ZIKV) is a flavivirus that has gained global attention due to its association with congenital microcephaly and neuroinflammatory responses. Markers of endothelial activation and purinergic signaling have been identified in the context of ZIKV neuropathogenesis, although the underlying mechanisms remain poorly understood. Methods Brain tissue samples from fatal cases of ZIKV‐induced microcephaly were analyzed using immunohistochemistry to detect endothelial activation markers (E‐selectin, P‐selectin, ICAM‐1, and VCAM‐1) and purinergic receptors (P2X4, P2X7, and P2Y2). Quantitative analysis measured the expression patterns of these molecules and assessed their contribution to neuroinflammation and blood–brain barrier disruption. Results ZIKV‐positive cases exhibited significant endothelial activation, with increased expression of adhesion molecules mediating leukocyte recruitment. Purinergic receptor upregulation suggested a role in excitotoxicity and neuroinflammatory exacerbation. Statistical analysis revealed a marked difference in marker expression between ZIKV‐infected cases and controls (p < 0.0001). Conclusion The interaction between endothelial activation and purinergic signaling may be associated with the vascular dysfunction and neuronal damage observed in ZIKV‐associated microcephaly. Understanding these associations could contribute to the development of targeted interventions for Zika congenital syndrome.

Macrophages in the kidney play a pathogenic role in inflammation and fibrosis. Our study aimed to understand the polarisation of the M1 and M2 phenotypic profiles of macrophages in injured kidney tissue retrieved from fatal cases of yellow fever virus (YFV). A total of 11 renal tissue biopsies obtained from patients who died of yellow fever (YF) were analysed. To detect antibodies that promote the classical and alternative pathways of macrophage activation, immunohistochemical analysis was performed to detect CD163, CD68, inducible nitric oxide synthase (iNOS), arginase 1, interleukin (IL)-4, IL-10, interferon (IFN)-γ, IFN-β, tumour necrosis factor (TNF)-α, IL-13, and transforming growth factor (TGF)-β. There was a difference in the marker expression between fatal cases of YFV and control samples, with increased expression in the cortical region of the renal parenchyma. The immunoexpression of CD68 and CD163 receptors suggests the presence of activated macrophages migrating to infectious foci. The rise in IL-10, IL-4, and IL-13 indicated their potential role in the inactivation of the inflammatory macrophage response and phenotypic modulation of M2 macrophages. The altered expression of IFN-γ and IFN-β demonstrates the importance of the innate immune response in combating microorganisms. Our findings indicate that the polarisation of M1 and M2 macrophages plays a vital role in the renal immune response to YFV.