Serological and Molecular Evidence of the Circulation of the Venezuelan Equine Encephalitis Virus Subtype IIIA in Humans, Wild Vertebrates and Mosquitos in the Brazilian Amazon.

Understanding the interaction between viruses and ecosystems in areas with or without anthropic interference can contribute to the organization of public health services, as well as prevention and disease control. An arbovirus survey was conducted at Caxiuanã National Forest, Pará, Brazil, where 632 local residents, 338 vertebrates and 15,774 pools of hematophagous arthropods were investigated. Neutralization antibodies of the Venezuelan Equine Encephalitis virus, subtype IIIA, Mucambo virus (MUCV) were detected in 57.3% and 61.5% of humans and wild vertebrates, respectively; in addition, genomic fragments of MUCV were detected in pool of Uranotaenia (Ura.) geometrica. The obtained data suggest an enzootic circulation of MUCV in the area. Understanding the circulation of endemic and neglected arboviruses, such as MUCV, represents an important health problem for the local residents and for the people living in the nearby urban centers.

Absence of Anti-RBD Antibodies in SARS-CoV-2 Infected or Naive Individuals Prior to Vaccination with CoronaVac Leads to Short Protection of Only Four Months Duration.

The COVID-19 pandemic is the biggest public health threat facing the world today. Multiple vaccines have been approved; however, the emergence of viral variants such as the recent Omicron raises the possibility of booster doses to achieve adequate protection. In Brazil, the CoronaVac (Sinovac, Beijing, China) vaccine was used; however, it is important to assess the immune response to this vaccine over time. This study aimed to monitor the anti-SARS-CoV-2 antibody responses in those immunized with CoronaVac and SARS-CoV-2 infected individuals. Samples were collected between August 2020 and August 2021. Within the vaccinated cohort, some individuals had a history of infection by SARS-CoV-2 prior to immunization, while others did not. We analyzed RBD-specific and neutralizing-antibodies. Anti-RBD antibodies were detected in both cohorts, with a peak between 45-90 days post infection or vaccination, followed by a steady decline over time. In those with a previous history of COVID-19, a higher, longer, more persistent response was observed. This trend was mirrored in the neutralization assays, where infection, followed by immunization, resulted in higher, longer lasting responses which were conditioned on the presence of levels of RBD antibodies right before the vaccination. This supports the necessity of booster doses of CoronaVac in due course to prevent serious disease.

Reporter Virus Neutralization Test Evaluation for Dengue and Zika Virus Diagnosis in Flavivirus Endemic Area.

Reporter virus neutralization test (RVNT) has been used as an alternative to the more laborious and time-demanding conventional PRNT assay for both DENV and ZIKV. However, few studies have investigated how these techniques would perform in epidemic areas with the circulation of multiple flavivirus. Here, we evaluate the performance of ZIKV and DENV Rluc RVNT and ZIKV mCh RVNT assays in comparison to the conventional PRNT assay against patient sera collected before and during ZIKV outbreak in Brazil. These samples were categorized into groups based on (1) acute and convalescent samples according to the time of disease, and (2) laboratorial diagnostic results (DENV and ZIKV RT-PCR and IgM-capture ELISA). Our results showed that DENV Rluc assay presented 100% and 78.3% sensitivity and specificity, respectively, with 93.3% accuracy, a similar performance to the traditional PRNT. ZIKV RVNT90, on the other hand, showed much better ZIKV antibody detection performance (around nine-fold higher) when compared to PRNT, with 88% clinical sensitivity. Specificity values were on average 76.8%. Even with these results, however, ZIKV RVNT90 alone was not able to reach a final diagnostic conclusion for secondary infection in human samples due to flavivirus cross reaction. As such, in regions where the flavivirus differential diagnosis represents a challenge, we suggest the establishment of a RVNT panel including other flaviviruses circulating in the region, associated with the other serological techniques such as IgM ELISA and the investigation of seroconversion, in order to help define an accurate diagnostic conclusion using serology.

Neurological disease caused by Oropouche virus in northern Brazil: should it be included in the scope of clinical neurological diseases?

We describe two neurological cases of Oropouche virus infection in northern Brazil, where the virus is endemic but neglected as a pathogen. This study reiterates the necessity of developing protocols for diagnosing infections and training medical personnel to recognize the pathogenicity of Oropouche virus in neurological infections.

Evaluation of immunoglobulin M-specific capture enzyme-linked immunosorbent assays and commercial tests for flaviviruses diagnosis by a National Reference Laboratory.

Zika and Dengue viruses present considerable immunological cross-reactivity, resulting in a troublesome serodiagnosis due to occurrence of false positive results. Due to Brazil's wide variety of circulating flaviviruses we aimed to access the use of in house serological tests adapted by National Reference Laboratory for Arboviruses in Brazil and evaluate commercial tests available. We evaluated in house IgM ELISAs for the individual detection of anti-ZIKV, -DENV, and -YFV IgM, against a panel of samples positive for dengue, zika, yellow fever, Rocio, Ilheus, Saint Louis encephalitis, West Nile and chikungunya. We also evaluated two commercial kits for dengue and zika IgM detection recommended by the Brazilian Ministry of Health in 2015. The sensitivity and specificity for the in house ZIKV IgM ELISA was 60.0 % and 88.6 % and for the in house DENV IgM ELISA was 100 % and 82.2 %, respectively. The in house YFV IgM ELISA presented 100 % for both sensitivity and specificity. The Novagnost Zika Virus IgM test presented a sensitivity of 47.3 % and specificity of 85.3 % and the Serion ELISA classic Dengue Virus IgM, 92.8 % and 58.9 %, respectively. Overall, both in house ELISAs for ZIKV and DENV adapted and evaluated here, presented better performances than the commercial kits tested.

Zika Virus Epidemic in Brazil. II. Post-Mortem Analyses of Neonates with Microcephaly, Stillbirths, and Miscarriage.

INTRODUCTION: The recent Zika virus(ZIKV) epidemic in Brazil was characterized by a range of different clinical presentations, particularly microcephaly, Guillain-Barré syndrome, and death. In this context, we determined the causal relationship between fatal microcephaly cases and ZIKV infection. METHODS: Twelve fatal cases of neonates, whose mothers were infected with ZIKV during pregnancy, were examined; cases included nine neonatal deaths due to microcephaly, one miscarriage, and two stillbirths. Tissue samples were obtained from all cases at necropsy and were submitted for virological investigation (RT-qPCR and virus isolation) and/or histopathology (hematoxylin and eosin staining) and immunohistochemical assay for the detection of ZIKV antigens. RESULTS: ZIKV antigens and/or ZIKV RNA were detected in tissue samples of all 12 cases examined. ZIKV was recovered in one case. Results of the virological and immunohistochemical analyses, as well as the anatomic abnormalities and histopathologic changes observed at necropsy on the 12 fatal cases, are presented. CONCLUSIONS: Data from these 12 cases provide strong evidence of the causal relationship between ZIKV and congenital disease in fetuses of women who were infected with the virus during pregnancy.

Correlation between Apoptosis and in Situ Immune Response in Fatal Cases of Microcephaly Caused by Zika Virus.

Zika virus (ZIKV) is a single-stranded positive-sense RNA flavivirus that possesses a genome approximately 10.7 Kb in length. Although pro-inflammatory and anti-inflammatory cytokines and apoptotic markers belonging to the extrinsic and intrinsic pathways are suggested to be involved in fatal cases of ZIKV-induced microcephaly, their exact roles and associations are unclear. To address this, brain tissue samples were collected from 10 individuals, five of whom were diagnosed as ZIKV positive with microcephaly and a further five were flavivirus-negative controls that died because of other causes. Examination of material from the fatal cases of microcephaly revealed lesions in the cerebral cortex, edema, vascular proliferation, neuronal necrosis, gliosis, neuronophagy, calcifications, apoptosis, and neuron loss. The expression of various apoptosis markers in the neural parenchyma, including FasL, FAS, BAX, BCL2, and caspase 3 differed between ZIKV-positive cases and controls. Further investigation of type 1 and 2 helper T-cell cytokines confirmed a greater anti-inflammatory response in fatal ZIKV-associated microcephaly cases. Finally, an analysis of the linear correlation between tumor necrosis factor-α, IL-1ß, IL-4, IL-10, transforming growth factor-ß, and IL-33 expression and various apoptotic markers suggested that the immune response may be associated with the apoptotic phenomenon observed in ZIKV-induced microcephaly.

In situ immune response and mechanisms of cell damage in central nervous system of fatal cases microcephaly by Zika virus.

Zika virus (ZIKV) has recently caused a pandemic disease, and many cases of ZIKV infection in pregnant women resulted in abortion, stillbirth, deaths and congenital defects including microcephaly, which now has been proposed as ZIKV congenital syndrome. This study aimed to investigate the in situ immune response profile and mechanisms of neuronal cell damage in fatal Zika microcephaly cases. Brain tissue samples were collected from 15 cases, including 10 microcephalic ZIKV-positive neonates with fatal outcome and five neonatal control flavivirus-negative neonates that died due to other causes, but with preserved central nervous system (CNS) architecture. In microcephaly cases, the histopathological features of the tissue samples were characterized in three CNS areas (meninges, perivascular space, and parenchyma). The changes found were mainly calcification, necrosis, neuronophagy, gliosis, microglial nodules, and inflammatory infiltration of mononuclear cells. The in situ immune response against ZIKV in the CNS of newborns is complex. Despite the predominant expression of Th2 cytokines, other cytokines such as Th1, Th17, Treg, Th9, and Th22 are involved to a lesser extent, but are still likely to participate in the immunopathogenic mechanisms of neural disease in fatal cases of microcephaly caused by ZIKV.

Zika virus epidemic in Brazil. I. Fatal disease in adults: Clinical and laboratorial aspects.

BACKGROUND: Zika virus (ZIKV) was first detected in Brazil in May 2015 and the country experienced an explosive epidemic. However, recent studies indicate that the introduction of ZIKV occurred in late 2013. Cases of microcephaly and deaths associated with ZIKV infection were identified in Brazil in November, 2015. OBJECTIVES: To determine the etiology of three fatal adult cases. STUDY DESIGN: Here we report three fatal adult cases of ZIKV disease. ZIKV infection in these patients was confirmed by cells culture and/or real-time reverse transcriptase polymerase chain reaction (RT-qPCR) and by antigen detection using immunohistochemical assay. Samples of brain and other selected organs taken at autopsy from three patients were also analyzed by histopathological and immunohistological examination. RESULTS: The first patient, a 36-year-old man with lupus and receiving prednisone therapy, developed a fulminant ZIKV infection. At autopsy, RT-qPCR of blood and tissues was positive for ZIKV RNA, and the virus was cultured from an organ homogenate. The second patient, a previously healthy female, 16 years of age, presented classic symptoms of Zika fever, but later developed severe thrombocytopenia, anemia and hemorrhagic manifestations and died. A blood sample taken on the seventh day of her illness was positive RT-PCR for ZIKV RNA and research in the serum was positive for antinuclear factor fine speckled (1/640), suggesting Evans syndrome (hemolytic anemia an autoimmune disorder with immune thrombocytopenic purpura) secondary to ZIKV infection. The third patient was a 20-year-old woman hospitalized with fever, pneumonia and hemorrhages, who died on 13days after admission. Histopathological changes were observed in all viscera examined. ZIKV antigens were detected by immunohistochemistry in viscera specimens of patients 1 and 3. These three cases demonstrate other potential complications of ZIKV infection, in addition to microcephaly and Guillain-Barre syndrome (GBS), and they suggest that individuals with immune suppression and/or autoimmune disorders may be at higher risk of developing severe disease, if infected with ZIKV.

West Nile Virus Encephalitis: The First Human Case Recorded in Brazil.

A Brazilian ranch worker with encephalitis and flaccid paralysis was evaluated in the regional Acute Encephalitis Syndromic Surveillance Program. This was the first Brazilian patient who met the Centers for Disease Control and Prevention (CDC) confirmation criteria for West Nile virus disease. Owing to the overlapping of neurological manifestations attributable to several viral infections of the central nervous system, this report exemplifies the importance of human acute encephalitis surveillance. The syndromic approach to human encephalitis cases may enable early detection of the introduction of unusual virus or endemic occurrence of potentially alarming diseases within a region.

Nearly complete genome sequence of curionopolis virus, a culicoides-related rhabdovirus isolated in the brazilian Amazon region.

We report here the first nearly complete genome sequence related to curionopolis virus (CURV), that of strain AR440009, isolated from a pool of Culicoides sp. midges in Serra Norte, Pará State, northern Brazil. All genes showed similarities to those belonging to members of the family Rhabdoviridae.

New genome sequences of gamboa viruses (family bunyaviridae, genus orthobunyavirus) isolated in panama and Argentina.

We describe here the nearly complete open reading frame (ORF) of five Gamboa virus strains isolated in Panama and Argentina. The viruses with complete ORF showed the regular genome organization observed in other orthobunyaviruses with exception to the presence of NSs protein. All predicted proteins showed homology with viruses belonging to members of the family Bunyaviridae.

Genetic and biological characterization of selected Changuinola viruses (Reoviridae, Orbivirus) from Brazil.

The genus Orbivirus of the family Reoviridae comprises 22 virus species including the Changuinola virus (CGLV) serogroup. The complete genome sequences of 13 CGLV serotypes isolated between 1961 and 1988 from distinct geographical areas of the Brazilian Amazon region were obtained. All viral sequences were obtained from single-passaged CGLV strains grown in Vero cells. CGLVs are the only orbiviruses known to be transmitted by phlebotomine sandflies. Ultrastructure and molecular analysis by electron microscopy and gel electrophoresis, respectively, revealed viral particles with typical orbivirus size and morphology, as well as the presence of a segmented genome with 10 segments. Full-length nucleotide sequencing of each of the ten RNA segments of the 13 CGLV serotypes provided basic information regarding the genome organization, encoded proteins and genetic traits. Segment 2 (encoding VP2) of the CGLV is uncommonly larger in comparison to those found in other orbiviruses and shows varying sizes even among different CGLV serotypes. Phylogenetic analysis support previous serological findings, which indicate that CGLV constitutes a separate serogroup within the genus Orbivirus. In addition, six out of 13 analysed CGLV serotypes showed reassortment of their genome segments.

Levantamento soroepidemiológico para arbovírus em macaco-prego-galego (Cebus flavius) de vida livre no estado da Paraíba e em macaco-prego (Cebus libidinosus) de cativeiro do nordeste do Brasil / Epidemiologic survey for arbovirus in galician capuchin monkeys (Cebus flavius) free living in Paraíba and captive capuchin monkey (Cebus libidinosus) from northeast Brazil

Este estudo descreve a primeira investigação de anticorpos para arbovírus em primatas não humanos do Novo Mundo no nordeste brasileiro. No período de março de 2008 a setembro de 2010 foram colhidos soros sanguíneos de 31 macacos-prego-galegos (Cebus flavius) de vida livre na Paraíba e de 100 macacos-prego (Cebus libidinosus) em cativeiro nos estados de Alagoas, Paraíba, Pernambuco, Piauí e Rio Grande do Norte. Para a pesquisa de anticorpos utilizou-se o teste de inibição da hemaglutinação (IH), usando antígenos de 19 diferentes tipos de arbovírus, pertencentes aos gêneros Flavivirus,Alphavirus e Bunyavirus. As amostras de soro foram testadas nas diluições de 1:20 a 1:1280. Dentre as amostras examinadas, todas as de C. flavius foram negativas e 46 por cento das de C. libidinosus em cativeiro apresentaram anticorpos para arbovírus. Foram detectados anticorpos para nove (9/19) arbovírus. Foram observadas 17 reações heterotípicas, para dois ou mais vírus, do gênero Flavivirus, e 15 para o gênero Alphavirus, com títulos variando de 1:20 a 1:1280. Quinze amostras apresentaram reação monotípica para ILHV (n=4), MAYV (n=6), SLEV (n=1), ROCV (n=2), OROV (n=1) e MUCV (n=1). Estes resultados sugerem que houve intensa circulação de arbovírus na população estudada de macacos-prego em cativeiro.

This paper describes the first investigation of arbovirus antibodies on New World non-human primates from Northeast Brazil. From March 2008 to September 2010 blood serum samples were collected from 31 wild blond capuchin monkeys (Cebus flavius) from Paraíba and 100 captive capuchin monkeys from Alagoas, Paraíba, Pernambuco, Piauí and Rio Grande do Norte. The haemagglutination-inhibition test (HI) was employed for 19 arbovirus of the Flavivirus,Alphavirus and Bunyavirus genus. Serum samples were tested from 1:20 to 1:1280 dilutions. Among the primates tested all C. flavius were negative and 46 percent C. libidinosus presented antibodies to arbovirus. Antibodies were detected for nine arbovirus (9/19). Seventeen heterotypic reactions were observed for at least two Or Flavirus and 15 for Alphavirus, at titers varying between 1:20 to 1:1280. Fifteen samples presented monotypic reaction for ILHV (n=4), MAYV (n=6), SLEV (n=1), ROCV (n=2), OROV (n=1) and MUCV (n=1). These results suggest that there was an intense arbovirus circulation in the studied population of captive capuchin monkeys.

Seroprevalence of flaviviruses antibodies in water buffaloes (Bubalus bubalis) in Brazilian Amazon.

BACKGROUND: The state of Pará encompasses 26% of Brazilian Amazon where an enormous diversity of arboviruses has been found. This study aimed to assess the prevalence and distribution of hemagglutination-inhibition antibodies against antigens of six Flavivirus (yellow fever virus, Ilheus virus, Saint Louis encephalitis virus, Cacipacore virus, Bussuquara virus and Rocio virus) in water buffaloes in Pará state, Brazil. The prevalence of antibodies in these farm animals is important to determine the circulating arboviruses. FINDINGS: All investigated arboviruses were detected in the species studied and our results indicate that water buffaloes are susceptible to Flavivirus infection. Furthermore, there is solid evidence of active circulation of these viruses in the Brazilian Amazon. CONCLUSIONS: Water buffaloes showed higher prevalence of heterotypic antibody reactions and we hypothesized that they can serve as sentinels to detect the movement of such arboviruses in the Brazilian Amazon.

Changuinola Virus Serogroup, New Genomes within the Genus Orbivirus (Family Reoviridae) Isolated in the Brazilian Amazon Region.

We report here the first complete genome sequence of a Changuinola virus (CGLV) serotype Irituia virus (BE AN 28873) isolated from a wild rodent (Oryzomys goeldi) in the municipality of Ipixuna, State of Pará, northern Brazil. All genome segments showed similarity with those belonging to members of the genus Orbivirus, family Reoviridae.

Circulation of hantaviruses in the influence area of the Cuiabá-Santarém Highway.

We describe evidence of circulation of hantaviruses in the influence area of the Santarém-Cuiabá Highway (BR-163) in the Brazilian Amazon through the prevalence of specific antibodies against hantaviruses in inhabitants living in four municipalities of this area: Novo Progresso (2.16%) and Trairão (4.37%), in state of Pará (PA), and Gua-rantã do Norte (4.74%) and Marcelândia (9.43%), in state of Mato Grosso. We also demonstrate the ongoing association between Castelo dos Sonhos virus (CASV) and hantavirus pulmonary syndrome (HPS) cases in the Castelo dos Sonhos district (municipality of Altamira, PA) and the first report of CASV in the municipalities of Novo Progresso and Guarantã do Norte. The results of this work highlight the risk for a possible increase in the number of HPS cases and the emergence of new hantavirus lineages associated with deforestation in this Amazonian area after the conclusion of paving works on BR-163 Highway.

Circulation of hantaviruses in the influence area of the Cuiabá-Santarém Highway

We describe evidence of circulation of hantaviruses in the influence area of the Santarém-Cuiabá Highway (BR-163) in the Brazilian Amazon through the prevalence of specific antibodies against hantaviruses in inhabitants living in four municipalities of this area: Novo Progresso (2.16 percent) and Trairão (4.37 percent), in state of Pará (PA), and Gua-rantã do Norte (4.74 percent) and Marcelândia (9.43 percent), in state of Mato Grosso. We also demonstrate the ongoing association between Castelo dos Sonhos virus (CASV) and hantavirus pulmonary syndrome (HPS) cases in the Castelo dos Sonhos district (municipality of Altamira, PA) and the first report of CASV in the municipalities of Novo Progresso and Guarantã do Norte. The results of this work highlight the risk for a possible increase in the number of HPS cases and the emergence of new hantavirus lineages associated with deforestation in this Amazonian area after the conclusion of paving works on BR-163 Highway.

Mayaro fever virus, Brazilian Amazon.

In February 2008, a Mayaro fever virus (MAYV) outbreak occurred in a settlement in Santa Barbara municipality, northern Brazil. Patients had rash, fever, and severe arthralgia lasting up to 7 days. Immunoglobulin M against MAYV was detected by ELISA in 36 persons; 3 MAYV isolates sequenced were characterized as genotype D.

Oropouche fever epidemic in Northern Brazil: epidemiology and molecular characterization of isolates.

BACKGROUND: Oropouche fever virus is an important arbovirus associated with febrile disease that re-emerged in 2006 in several municipalities of Pará State, Bragantina region, Amazon, Brazil, 26 years after the last epidemic. OBJECTIVE: To investigate an Oropouche fever outbreak in this region. STUDY DESIGN: A serologic survey and prospective study of acute febrile cases were performed in Magalhães Barata (urban and rural areas) and Maracanã (rural area) municipalities. Serology (IgM-ELISA and hemagglutination-inhibition [HI]), virus isolation, RT-PCR and real-time-PCR were used to confirm Oropouche virus (OROV) as responsible for the febrile outbreaks. RESULTS: Real-time-PCR showed high titers of OROV in acute-phase serum samples from febrile patients. From 113 of 119 acutely febrile patients with paired serum samples, OROV infections was confirmed by serologic conversion (n=76) or high titers (n=37) for both HI and IgM-ELISA. Patients had a febrile disease characterized by headache, chills, dizziness, photophobia, myalgia, nausea, and vomiting. Females and children under 15 years of age were most affected. Nucleotide sequencing of six OROV isolates identified that genotype II was associated with the human disease epidemic. CONCLUSIONS: Oropouche fever, which has re-emerged in the Bragantina region in eastern Amazon 26 years after the last epidemic, is caused by genotype II, a lineage previously found only in Peru and western Brazil.