Isolation of Flaviviruses and Alphaviruses with Encephalitogenic Potential Diagnosed by Evandro Chagas Institute (Pará, Brazil) in the Period of 1954-2022: Six Decades of Discoveries.

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 innate immune response in Zika virus infection.

Zika virus (ZIKV; Flaviviridae, Flavivirus) was discovered in 1947 in Uganda, Africa, from the serum of a sentinel Rhesus monkey (Macaca mulatta). It is an enveloped, positive-sense, single-stranded RNA virus, which encodes a single polyprotein that is cleaved into 10 individual proteins. In 2015, the Zika-epidemic in Brazil was marked mainly by the exponential growth of microcephaly cases and other congenital defects. With regard to host-pathogen relationships, understanding the role of the immune response in the pathogenesis ZIKV infection is challenging. The innate immune response is the first-line immunological defence, in which pathogen-associated molecular patterns are recognized by pattern-recognition receptors that trigger macrophages, dendritic cells, natural killer cells and endothelial cells to produce several mediators, which modulate viral replication and immune evasion. In this review, we have summarized current knowledge on the innate immune response against ZIKV.

Cell Death And Zika Virus: An Integrated Network Of The Mechanisms Of Cell Injury.

Zika virus (ZIKV) is an arbovirus that is transmitted by Aedes mosquitos. Its prototype was isolated in 1947 from serum of a sentinel Rhesus monkey (Macaca mulatta) in the Zika forest of Uganda. As a member of the genus Flavivirus, family Flaviviridae, ZIKV is enveloped and icosahedral and possesses a single-stranded, positive-sense RNA genome of approximately 10.7 kb. Epidemiologically, infection by ZIKV has become a global health concern in recent years because of the occurrence of epidemics, its speed of dissemination, routes of transmission, and the sequelae it can cause especially in newborns. At the neural level, there are still many gaps in our understanding of the mechanisms that induce ZIKV infection-associated microcephaly. However, some studies already demonstrated that underlying cell death is determinant to induce the congenital malformation. In this report, we reviewed the various mechanisms of cell injury involved in the immunopathogenesis of ZIKV infection and discussed its relationship with the death of neuronal and glial cells development and microcephaly.

Characterization of Triniti virus supports its reclassification in the family Peribunyaviridae.

Triniti virus (TNTV) has been isolated in Trinidad and Tobago and in Brazil. To date little is known about this virus, which is classified as an ungrouped virus within the family Togaviridae. Here, three isolates of TNTV were characterized both genetically and antigenically. The genome was shown to contain three RNA segments: small (S), medium (M) and large (L). Genome organization, protein sizes and protein motifs were similar to those of viruses in the genus Orthobunyavirus, family Peribunyaviridae. Antigenic reactivity revealed the three TNTV isolates to be closely related, but no serologic cross-reaction with other orthobunyaviruses. Morphological observation by transmission electron microscopy indicated that virus size and symmetry were compatible with those of viruses in the family Peribunyaviridae. Our serological, morphological and molecular results support the taxonomic reclassification of TNTV as a member of the genus Orthobunyavirus, family Peribunyaviridae.

Potential role of dengue virus, chikungunya virus and Zika virus in neurological diseases.

This study showed that laboratory markers of recent infection by dengue, Zika or chikungunya arboviruses were detected in the biological samples of approximately one-third of patients with encephalitis, myelitis, encephalomyelitis or Guillain-Barré syndrome, in a surveillance programme in Piauí state, Brazil, between 2015-2016. Fever and myalgia had been associated with these cases. Since in non-tropical countries most infections or parainfectious diseases associated with the nervous system are attributed to herpesviruses, enteroviruses, and Campylobacter jejuni, the present findings indicate that in tropical countries, arboviruses may now play a more important role and reinforce the need for their surveillance and systematic investigation in the tropics.

In situ inflammasome activation results in severe damage to the central nervous system in fatal Zika virus microcephaly cases.

Zika virus (ZIKV) has caused substantial concern worldwide owing to its association with severe birth defects, such as microcephaly and other congenital malformations. Inflammasomes, i.e., multi-protein complexes that induce inflammation and pyroptosis, are predicted to contribute to the immune response to this flavivirus. Accordingly, in this study, the in situ inflammasome response was evaluated in fatal cases of ZIKV-linked microcephaly. Brain tissue samples were collected from eight babies, including four ZIKV-positive microcephalic neonates who died after birth and four flavivirus-negative neonatal controls who died of other causes and whose central nervous system (CNS) architecture was preserved. In the ZIKV-positive newborn/stillbirth babies, the major histopathological alterations included atrophy of the cortical layer, a predominance of mononuclear cell infiltration in the Virchow-Robin space, neuronal necrosis, vacuolization and neuronal degeneration, neuronophagy, and gliosis. An immunohistochemical analysis of tissues in the neural parenchyma showed significantly higher expression of the receptors NLRP1, NLRP3, and AIM2, cytokines IL-1ß, IL-18, and IL-33, and enzymes caspase 1, iNOS, and arginase 1 in ZIKV-positive microcephaly cases than in flavivirus-negative controls. These results suggest that inflammasome activation can aggravate the neuroinflammatory response and consequently increase CNS damage in neonates with fetal neural ZIKV infection and microcephaly.

Encephalitis associated with Zika virus infection and reactivation of the varicella-zoster virus in a Brazilian child.

We report a case of encephalitis associated with Zika virus infection and reactivation of varicella-zoster virus in the central nervous system of a Brazilian child. This case raises the possibility that reactivation of the latent varicella-zoster virus may be a mechanism of neurological impairment induced by acquired Zika virus infection.

Encephalitis associated with Zika virus infection and reactivation of the varicella-zoster virus in a Brazilian child

Abstract We report a case of encephalitis associated with Zika virus infection and reactivation of varicella-zoster virus in the central nervous system of a Brazilian child. This case raises the possibility that reactivation of the latent varicella-zoster virus may be a mechanism of neurological impairment induced by acquired Zika virus infection.

Potential role of dengue virus, chikungunya virus and Zika virus in neurological diseases

This study showed that laboratory markers of recent infection by dengue, Zika or chikungunya arboviruses were detected in the biological samples of approximately one-third of patients with encephalitis, myelitis, encephalomyelitis or Guillain-Barré syndrome, in a surveillance programme in Piauí state, Brazil, between 2015-2016. Fever and myalgia had been associated with these cases. Since in non-tropical countries most infections or parainfectious diseases associated with the nervous system are attributed to herpesviruses, enteroviruses, and Campylobacter jejuni, the present findings indicate that in tropical countries, arboviruses may now play a more important role and reinforce the need for their surveillance and systematic investigation in the tropics.

Gene Polymorphisms and Serum Levels of Pro- and Anti-Inflammatory Markers in Dengue Viral Infections.

Pro- and anti-inflammatory markers (tumor necrosis factor [TNF]-α, TNF-ß, interferon [IFN]-γ, interleukin [IL]-6, IL-8, IL-10, and C-reactive protein [CRP]) were investigated in 80 patients infected with dengue viruses, 100 patients presenting with febrile illness but negative for dengue, and 99 healthy subjects. Immunoenzyme methods were used for quantitative assays in the plasma. Polymorphisms of TNF-α, TNF-ß, IL-6, IL-8, and IL-10 genes were assessed by polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific oligonucleotide (ASO)-PCR for the IFN-γ. The highest mean serum levels of TNF-α, IFN-γ, IL-8, and CRP were observed in dengue-positive individuals. TNF-ß, IL-6, and IL-10 levels were significantly higher in the dengue-negative individuals. No cytokine expression pattern was evidenced according to virus serotype. Genotypic frequency distributions were statistically significant for the polymorphisms of TNF-α and IFN-γ among positive, negative, and control dengue groups and IFN-γ among groups DENV-1, DENV-2, DENV-3, and controls. Modulation of cytokine expression and polymorphisms is a complex matter and needs further explanation considering the ethnic origins of the Brazilian population.

Zika virus in the Americas: Early epidemiological and genetic findings.

Brazil has experienced an unprecedented epidemic of Zika virus (ZIKV), with ~30,000 cases reported to date. ZIKV was first detected in Brazil in May 2015, and cases of microcephaly potentially associated with ZIKV infection were identified in November 2015. We performed next-generation sequencing to generate seven Brazilian ZIKV genomes sampled from four self-limited cases, one blood donor, one fatal adult case, and one newborn with microcephaly and congenital malformations. Results of phylogenetic and molecular clock analyses show a single introduction of ZIKV into the Americas, which we estimated to have occurred between May and December 2013, more than 12 months before the detection of ZIKV in Brazil. The estimated date of origin coincides with an increase in air passengers to Brazil from ZIKV-endemic areas, as well as with reported outbreaks in the Pacific Islands. ZIKV genomes from Brazil are phylogenetically interspersed with those from other South American and Caribbean countries. Mapping mutations onto existing structural models revealed the context of viral amino acid changes present in the outbreak lineage; however, no shared amino acid changes were found among the three currently available virus genomes from microcephaly cases. Municipality-level incidence data indicate that reports of suspected microcephaly in Brazil best correlate with ZIKV incidence around week 17 of pregnancy, although this correlation does not demonstrate causation. Our genetic description and analysis of ZIKV isolates in Brazil provide a baseline for future studies of the evolution and molecular epidemiology of this emerging virus in the Americas.

Chikungunya risk for Brazil.

This study aimed to show, based on the literature on the subject, the potential for dispersal and establishment of the chikungunya virus in Brazil. The chikungunya virus, a Togaviridae member of the genusAlphavirus, reached the Americas in 2013 and, the following year, more than a million cases were reported. In Brazil, indigenous transmission was registered in Amapa and Bahia States, even during the period of low rainfall, exposing the whole country to the risk of virus spreading. Brazil is historically infested by Ae. aegypti and Ae. albopictus, also dengue vectors. Chikungunya may spread, and it is important to take measures to prevent the virus from becoming endemic in the country. Adequate care for patients with chikungunya fever requires training general practitioners, rheumatologists, nurses, and experts in laboratory diagnosis. Up to November 2014, more than 1,000 cases of the virus were reported in Brazil. There is a need for experimental studies in animal models to understand the dynamics of infection and the pathogenesis as well as to identify pathophysiological mechanisms that may contribute to identifying effective drugs against the virus. Clinical trials are needed to identify the causal relationship between the virus and serious injuries observed in different organs and joints. In the absence of vaccines or effective drugs against the virus, currently the only way to prevent the disease is vector control, which will also reduce the number of cases of dengue fever.

Emergence and potential for spread of Chikungunya virus in Brazil.

BACKGROUND: In December 2013, an outbreak of Chikungunya virus (CHIKV) caused by the Asian genotype was notified in the Caribbean. The outbreak has since spread to 38 regions in the Americas. By September 2014, the first autochthonous CHIKV infections were confirmed in Oiapoque, North Brazil, and in Feira de Santana, Northeast Brazil. METHODS: We compiled epidemiological and clinical data on suspected CHIKV cases in Brazil and polymerase-chain-reaction-based diagnostic was conducted on 68 serum samples from patients with symptom onset between April and September 2014. Two imported and four autochthonous cases were selected for virus propagation, RNA isolation, full-length genome sequencing, and phylogenetic analysis. We then followed CDC/PAHO guidelines to estimate the risk of establishment of CHIKV in Brazilian municipalities. RESULTS: We detected 41 CHIKV importations and 27 autochthonous cases in Brazil. Epidemiological and phylogenetic analyses indicated local transmission of the Asian CHIKV genotype in Oiapoque. Unexpectedly, we also discovered that the ECSA genotype is circulating in Feira de Santana. The presumed index case of the ECSA genotype was an individual who had recently returned from Angola and developed symptoms in Feira de Santana. We estimate that, if CHIKV becomes established in Brazil, transmission could occur in 94% of municipalities in the country and provide maps of the risk of importation of each strain of CHIKV in Brazil. CONCLUSIONS: The etiological strains associated with the early-phase CHIKV outbreaks in Brazil belong to the Asian and ECSA genotypes. Continued surveillance and vector mitigation strategies are needed to reduce the future public health impact of CHIKV in the Americas.

Chikungunya risk for Brazil / Risco do chikungunya para o Brasil

This study aimed to show, based on the literature on the subject, the potential for dispersal and establishment of the chikungunya virus in Brazil. The chikungunya virus, a Togaviridae member of the genusAlphavirus, reached the Americas in 2013 and, the following year, more than a million cases were reported. In Brazil, indigenous transmission was registered in Amapa and Bahia States, even during the period of low rainfall, exposing the whole country to the risk of virus spreading. Brazil is historically infested by Ae. aegypti and Ae. albopictus, also dengue vectors. Chikungunya may spread, and it is important to take measures to prevent the virus from becoming endemic in the country. Adequate care for patients with chikungunya fever requires training general practitioners, rheumatologists, nurses, and experts in laboratory diagnosis. Up to November 2014, more than 1,000 cases of the virus were reported in Brazil. There is a need for experimental studies in animal models to understand the dynamics of infection and the pathogenesis as well as to identify pathophysiological mechanisms that may contribute to identifying effective drugs against the virus. Clinical trials are needed to identify the causal relationship between the virus and serious injuries observed in different organs and joints. In the absence of vaccines or effective drugs against the virus, currently the only way to prevent the disease is vector control, which will also reduce the number of cases of dengue fever.

o objetivo deste estudo foi mostrar, com base na literatura sobre o tema, o potencial de dispersão e estabelecimento do vírus chikungunya no brasil. O vírus chikungunya, um Togaviridae do gênero Alphavirus, atingiu as Américas em 2013 e, no ano seguinte, mais de um milhão de casos foram notificados. No Brasil, foi registrada transmissão autóctone no Amapá e Bahia, mesmo durante o período de baixo índice pluviométrico, expondo ao risco de propagação do vírus todo o território nacional. Historicamente, o Brasil é infestado por Ae. Aegypti e Ae. Albopictus, também vetores do dengue. É possível que o chikungunya se dissemine, sendo importante que medidas sejam tomadas para evitar que o vírus se torne endêmico no País. A adequada assistência a pacientes com febre de chikungunya requer treinamento de clínicos, reumatologistas, enfermeiros e especialistas em diagnóstico laboratorial. Até novembro de 2014 foram notificados no Brasil mais de 1.000 casos da virose. Estudos experimentais em modelos animais devem ser realizados para conhecer a dinâmica da infecção e a patogenia, bem como identificar mecanismos fisiopatológicos que possam contribuir para identificar drogas com efeito sobre o vírus. São necessários ensaios clínicos para identificar a relação causal do vírus com sérias lesões observadas em diferentes órgãos e nas articulações. Na ausência de vacinas ou drogas efetivas contra o vírus, a única forma de prevenir a doença é atualmente o controle vetorial, o que também reduzirá o número de casos de dengue.

Callithrix penicillata: a feasible experimental model for dengue virus infection.

Although the murine models have the feasibility to reproduce some signs of dengue Virus (DENV) infection, the use of isogenic hosts with polarized immune response patterns does not reproduce the particularities of human disease. Our goal was to investigate the kinetics of peripheral blood biomarkers in immunocompetent Callithrix penicillata non-human primates subcutaneously infected with DENV-3. The viral load of infected animals was determinated by quantitative real time PCR. Measurements of DENV-3/IgM were performed, and several parameters were assessed by hemogram: red blood cells count, hemoglobin, hematocrit, white blood cells count, neutrophils, monocytes, lymphocytes, and platelets count. The coagulogram was performed by prothrombin time (PT), and activated partial thromboplastin time (APTT) assays. The renal function was monitored by urea and creatinine, and the liver function by the aspartate (AST), and alanine (ALT) aminotransferases. Also, the level of the cytokines IL-6, TNF-α, IL-2, IFN-γ, IL-4 and IL-5 was quantified during the experimental study. Data analysis was performed considering relevant differences when baseline fold changes were found outside from 0.75 to 1.5 range. Our data demonstrated that infected animals presented relevant signs of dengue disease, including peaks of viremia at 5 days-post-infection (dpi), peaks of anti-DENV-3 IgM at 15 dpi and hemaglutination inhibition assay (HIA) from 15 to at 60 dpi. Despite early monocytosis, slight neutrophilia and lymphocytosis, animals developed persistent leucopenia starting at 4 dpi. Anemia episodes were steady at 3-4 dpi. Patent thrombocytopenia was observed from 1 to 15 dpi with sporadic decrease of APTT. A substantial increase of ALT and AST was observed with higher peak at 4 dpi. Moreover, early increases of TNF-alpha and IFN-gamma besides late increase of IFN-gamma were observed. The analysis of biomarkers network pointed out two relevant strong axes during early stages of dengue fever, a protective axes TNF-alpha/Lymphocytes/Platelets, and a pathological IL-2/IL-6/Viremia/Monocyte/PT bond. Later on, the biomarker network highlighted the interaction IFN-gamma/PLT/DENV-3(IgM;HAI)/PT, and the involvement of type-2 cytokines (IL-4; IL-5). Our findings demonstrated that C. penicillata is a feasible experimental model for dengue virus infection, which could be useful to pathogenesis studies, discovery of novel antiviral drugs as well as to evaluate vaccine candidates against DENV.

Estudo experimental sobre a patogenicidade do Vírus Ilhéus em hamsters dourados (Mesocricetus auratus) / Pathogenesis of the Ilheus virus in golden hamsters (Mesocricetus auratus)

Visando investigar a patogenicidade do Flavivirus Ilhéus (VILH) foi inoculada, via intraperitoneal, 9,8 DL de suspensão 50 viral em hamsters dourados jovens (Mesocricetus auratus) e, diariamente, soros e vísceras (cérebro, fígado, coração, baço, rins e pulmões) de animais infectados e de controles não-infectados foram obtidos sob anestesia. Durante o experimento foi determinado o título viral do VILH em soros e vísceras infectados, em camundongos recém-nascidos. Ademais, a detecção de antígeno e os níveis de anticorpos por testes de fixação do complemento e inibição da hemaglutinação foram realizados nos soros. Exame histopatológico por HE e a detecção de antígenos virais por Imunohistoquímica (IHQ) foram realizados nos tecidos dos animais. A dose inoculada ocasionou a morte dos animais por encefalite no sétimo dia pós inoculação. Todos os órgãos estudados apresentaram alterações teciduais detectáveis por histopatologia. Volumosa presença de antígeno viral foi detectada por IHQ no cérebro, e, em menor quantidade, no fígado, baço e rins; porém, nestes órgãos, a presença de antígeno viral foi transitória e de leve intensidade, o que corroborou com os títulos virais obtidos nesses órgãos. Não foram encontrados antígenos virais em coração e pulmões, sugerindo que os títulos (DL ) 50 observados nesses órgãos, durante a titulação em camundongos, decorreram da presença do VILH na corrente sanguínea (viremia). Os achados deste estudo reforçam o importante e conhecido neurotropismo do VILH.

The pathogenesis of the Ilheus flavivirus (Flaviviridae) was investigated in golden hamsters (Mesocricetus auratus) using an inoculum of 9.8 LD50 via intraperitoneal (IP). For ten days, two infected and one control animals were anesthetized, and blood and viscera fragments (brain, liver, heart, lung, spleen and kidneys) were collected on a daily basis for determination of viral titers in newborn mice and antigens/antibody by complement fixation and hemagglutination inhibition tests. Additionally, the pathology of animal tissues was studied by the the hematoxylin and eosin method, viral antigens were detected by immunochemistry, and all collected viscera showed histopathological changes. Large amounts of ILHV antigens were detected by immunohistochemistry in the brain, and in lower quantities in the liver, spleen and kidneys, corroborating with newborn viral titers in them. This inoculum resulted in a fatal outcome of all infected animals seven days after experimentation. Viral antigens were not found in the heart and lungs, suggesting that the viral titers obtained were caused by viremia and not by viral damage. The information in this study confirms the neurotropism and neuropathogenicity of ILHV.

A febre do Oropouche: uma revisão dos aspectos epidemiológicos e moleculares na Amazônia brasileira / Oropouche fever: an overview of the epidemiological and molecular aspects in the brazilian Amazon region

O vírus Oropouche (VORO Bunyaviridae, Orthobunyavirus) é um dos mais importantes arbovírus que infectam humanos na Amazônia Brasileira, sendo o agente causador da febre do Oropouche. Entre os anos de 1961 e 2006, um grande número de epidemias foi registrado em diferentes centros urbanos do estado do Pará (Belém, Santa Isabel, Castanhal, Santarém, Oriximiná, Serra Pelada, zona Bragantina - Igarapé Açu, Maracanã e Magalhães Barata), do Amazonas (Manaus e Barcelos), Acre (Xapuri), Amapá (Mazagão), Maranhão (Porto Franco), Tocantins (Tocantinópolis) e Rondônia (Ariquemes e Oro Preto D'Oeste). Estudos moleculares têm demonstrado a presença de pelo menos três linhagens distintas do VORO na Amazônia Brasileira (genótipos I, II e III), sendo os genótipos I e II os mais frequentemente encontrados em regiões da Amazônia ocidental e oriental, respectivamente. O genótipo III do VORO, previamente encontrado somente no Paraná, foi recentemente descrito na região Sudeste do Brasil. A associação de dados epidemiológicos e moleculares vêm contribuindo substancialmente para a caracterização das cepas do VORO isoladas durante epidemias, no período de pelo menos quatro décadas, bem como permitindo um melhor entendimento a respeito da epidemiologia molecular do VORO no que tange à emergência de novas linhagens genéticas e à dinâmica evolutiva desse arbovírus nas Américas e principalmente na Amazônia Brasileira. Este trabalho tem por objetivo apresentar uma revisão dos aspectos epidemiológicos e moleculares do VORO enfatizando sua distribuição, a dinâmica das epidemias ocorridas entre 1961 e 2006, bem como a dispersão de diferentes genótipos no Brasil.

Oropouche virus isolation, southeast Brazil.

An Oropouche virus strain was isolated from a novel host (Callithrix sp.) in Arinos, Minas Gerais State, southeastern Brazil. The virus was identified by complement fixation test and confirmed by reverse transcription-polymerase chain reaction. Phylogenetic analysis identified this strain as a genotype III isolate previously recognized only in Panama.