Identification of Viruses in Molossus Bats from the Brazilian Amazon: A Descriptive Metagenomic Analysis.

Bats are widely distributed in Brazil, including the Amazon region, and their association with viral pathogens is well-known. This work aimed to evaluate the metavirome in samples of Molossus sp. bats captured in the Brazilian Amazon from 2019 to 2021. Lung samples from 58 bats were divided into 13 pools for RNA isolation and sequencing followed by bioinformatic analysis. The Retroviridae family showed the highest abundance of viral reads. Although no complete genome could be recovered, the Paramyxoviridae and Dicistroviridae families showed the formation of contigs with satisfactory identity and size characteristics for further analysis. One contig of the Paramyxoviridae family was characterized as belonging to the genus Morbillivirus, being grouped most closely phylogenetically to Porcine morbillivirus. The contig related to the Dicistroviridae family was identified within the Cripavirus genus, with 94%, 91%, and 42% amino acid identity with Culex dicistrovirus 2, Rhopalosiphum padi, and Aphid lethal paralysis, respectively. The presence of viruses in bats needs constant updating since the study was able to identify viral sequences related to families or genera still poorly described in the literature in association with bats.

Phylogenetic analysis of rabies surveillance samples from north and northeast Brazil.

Viruses of the Lyssavirus genus are classified into several genotypes (GT1 to GT7), of which only GT1 (classic rabies virus-RABV) has a cosmopolitan distribution and circulates in Brazil. GT1 is subdivided into several antigenic variants (AgV) maintained in independent cycles with a narrow host range and distinct geographic distributions, namely, AgV1 and AgV2 found in dogs, AgV3 in the vampire bats Desmodus rotundus, and AgV4 and AgV6 in bats non-hematophagous Tadarida brasiliensis and Lasiurus cinereus, a common variant of marmoset (Callithrix jacchus), and crab-eating fox (Cerdocyon thous). In this study, we performed phylogenetic analysis to identify at the antigenic variant level; six RABV genomes derived from the Rabies Surveillance in the north and northeast regions of Brazil. The analysis resulted in the formation of 11 monophyletic clusters, each corresponding to a particular variant, with high bootstrap support values. The samples were positioned inside the AgV3, AgV6, and Callithrix variant clades. This is the first report of the AgV6 variant found in northern Brazil, which provides valuable information for rabies surveillance in the country. The possibility of viral spillover has been much debated, as it deals with the risk of shifting transmission from a primary to a secondary host. However, more genomic surveillance studies should be performed, with a greater number and diversity of samples to better understand the transmission dynamics of each variant to detect changes in its geographic distribution and spillover events.

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.

Zika Virus Infection Damages the Testes in Pubertal Common Squirrel Monkeys (Saimiri collinsi).

During the Zika virus (ZIKV) outbreak and after evidence of its sexual transmission was obtained, concerns arose about the impact of the adverse effects of ZIKV infection on human fertility. In this study, we evaluated the clinical-laboratory aspects and testicular histopathological patterns of pubertal squirrel monkeys (Saimiri collinsi) infected with ZIKV, analyzing the effects at different stages of infection. The susceptibility of S. collinsi to ZIKV infection was confirmed by laboratory tests, which detected viremia (mean 1.63 × 106 RNA copies/µL) and IgM antibody induction. Reduced fecal testosterone levels, severe testicular atrophy and prolonged orchitis were observed throughout the experiment by ultrasound. At 21 dpi, testicular damage associated with ZIKV was confirmed by histopathological and immunohistochemical (IHC) analyses. Tubular retraction, the degeneration and necrosis of somatic and germ cells in the seminiferous tubules, the proliferation of interstitial cells and an inflammatory infiltrate were observed. ZIKV antigen was identified in the same cells where tissue injuries were observed. In conclusion, squirrel monkeys were found to be susceptible to the Asian variant of ZIKV, and this model enabled the identification of multifocal lesions in the seminiferous tubules of the infected group evaluated. These findings may suggest an impact of ZIKV infection on male fertility.

Arboviruses in Free-Ranging Birds and Hematophagous Arthropods (Diptera, Nematocera) from Forest Remnants and Urbanized Areas of an Environmental Protection Area in the Amazon Biome.

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.

Challenges of Rabies Surveillance in the Eastern Amazon: The Need of a One Health Approach to Predict Rabies Spillover.

Brazil has been promoting essential improvements in health indicators by implementing free-access health programs, which successfully reduced the prevalence of neglected zoonosis in urban areas, such as rabies. Despite constant efforts from the authorities to monitor and control the disease, sylvatic rabies is a current issue in Amazon's communities. The inequalities among Amazon areas challenge the expansion of high-tech services and limit the implementation of active laboratory surveillance to effectively avoid outbreaks in human and non-human hosts, which also reproduces a panorama of vulnerability in risk communities. Because rabies is a preventable disease, the prevalence in the particular context of the Amazon area highlights the failure of surveillance strategies to predict spillovers and indicates the need to adapt the public policies to a "One Health" approach. Therefore, this work assesses the distribution of free care resources and facilities among Pará's regions in the oriental Amazon; and discusses the challenges of implanting One Health in the particular context of the territory. We indicate a much-needed strengthening of the sylvatic and urban surveillance networks to achieve the "Zero by 30" goal, which is inextricable from multilateral efforts to combat the progressive biome's degradation.

Immunological impact of tetrahydrobiopterin on the central nervous system in a murine model of rabies virus infection.

Currently, the Milwaukee protocol presents healing results in human beings affected by the rabies virus. However, there are many points to clarify on the action of drugs and the immune mechanism involved in the evolution of the disease. One of the drugs used is biopterin, which is an important cofactor for nitric oxide, important for preventing vasospasm. Thus, we describe the effect of biopterin on some inflammatory factors in a rabies virus infection developed in an animal model. The immunological mediators studied in animals infected with rabies virus submitted to doses of sapropterin were Anti-RABV, IL-6, IL-2, IL-17a, INF-gamma and Anti-iNOS. It is suggested that the medication in the context of a RABV infection already installed, had the effect of modulating the inflammatory mechanisms mainly linked to the permeability of the blood-brain barrier and the migration of cytotoxic cells.

Prenatal disorders and congenital Zika syndrome in squirrel monkeys.

During the Zika virus (ZIKV) outbreak in Brazil (2015-2016), the clinical manifestations associated with its infection were complex and included miscarriage and congenital malformations, not previously described. In this study, we evaluated the prenatal conditions of pregnant female squirrel monkeys (Saimiri collinsi) infected during different gestational thirds (GTs) and assessed all clinical aspects, diagnostic imaging, viremia and the immune response. In our study, 75% of the infected animals in the 1st GT group had significant clinical manifestations, such as miscarriage and prolonged viremia associated with a late immune response. Consequently, their neonates showed fetal neuropathology, such as cerebral hemorrhage, lissencephaly or malformations of the brain grooves, ventriculomegaly, and craniofacial malformations. Thus, our study demonstrated the relevance of pregnant squirrel monkeys as a model for the study of ZIKV infection in neonates due to the broad clinical manifestations presented, including the typical congenital Zika syndrome manifestations described in humans.

Serological evidence of Eastern equine encephalitis circulation in equids in Pará state, Brazil.

Serum samples from 89 equids were analyzed (75 horses, 9 donkeys, and 5 mules) from the municipality of Viseu, Pará state, Brazil. Samples were collected in November 2014 and August 2015. The antibody prevalence against the following alphaviruses was estimated: Eastern equine encephalitis virus, Western equine encephalitis virus, Mucambo virus, and Mayaro virus. Seroprevalence was determined by the hemagglutination inhibition (HI) technique. Sera that exhibited HI antibodies with heterotypic reactions for the analyzed viruses were subjected to the 90% plaque reduction neutralization test (PRNT90). The HI prevalence of monotypic reactions to EEEV was 7.9%, and that of WEEV was 1.1%, as confirmed by PRNT90. Viral isolation attempts were negative for all tested blood samples. Our results suggest the circulation of equine encephalitis complex viruses. Future studies should evaluate the possible involvement of arthropod hosts and residents in the viral transmission in the study area.

Foram analisadas amostras de soros de 89 equídeos (75 equinos, 9 asininos e 5 muares) oriundos do município de Viseu, estado do Pará. As amostras foram coletadas no período de novembro de 2014 e agosto de 2015. Foram estimadas as prevalências de anticorpos contra os seguintes Alfavírus: vírus da Encefalite equina leste (EEEV), vírus da Encefalite equina oeste (WEEV), vírus Mucambo (MUCV), and vírus Mayaro (MAYV). A soroprevalência foi determinada por técnicas de Inibição da Hemaglutinação (IH), porém os soros que apresentaram anticorpos IH com reações heterotípicas para os vírus analisados foram submetidos ao PRNT90. A prevalência de reações monotípicas por IH para EEEV foi de 7, 9% e para WEEV foi de 1,1%, confirmadas por PRNT90. A tentativa de isolamento viral foi negativa em todas as amostras de sangue testadas. Nossos resultados sugerem a circulação de vírus do complexo das encefalites equinas. Estudos futuros devem ser realizados para avaliar possível envolvimento de hospedeiros artrópodes e residentes na área de abrangência do estudo.

Experimental infection of golden hamsters with Guama virus (Peribunyaviridae, Orthobunyavirus).

The Guama virus (GMAV) is a member of Peribunyaviridae family, Orthobunyavirus genus. Several strains of the virus were isolated in South and Central Americas from several hosts, such as humans, wild animals, including nonhuman primates, wild rodents and mosquitoes as well as mice used as sentinels. The virus is able to cause febrile disease in humans. Here we describe for the first time pathologic and biochemical findings in golden hamsters (Mesocricetus auratus) infected with the prototype GMAV. Blood and organs of infected and control animals were collected every 24 h after infection from the 1st to the 7th day post infection (dpi) and at 21 dpi when experiment was ended. The tissues were processed for histopathology and immunohistochemistry. The blood and serum were used to determine viremia and biochemical markers plus to detect anti-GMAV antibodies. The viremia was early detected already on the 1st dpi and it was no longer detected on the 3rd dpi. Total anti-GMAV antibodies were detected from the 6th dpi. Hepatic markers as ALT of infected animals were increased and showed statistically significant difference in comparison with control animals, indicating damage of the liver; indeed the liver was the most affected organ, but other organs presented lesions and positive GMAV immunostaining as brain, lung, liver, spleen, and kidney. Our findings indicate that golden hamsters are a good animal model for experimental infection of the GMAV.

Metagenomic Analysis of Samples from Three Bat Species Collected in the Amazon Rain Forest.

We report here the sequencing of five microbiome samples collected from different bat species in the Amazon rain forest. All contigs matching virus sequences were assigned to members of the Retroviridae family, while the bacterial contigs matched several bacterial species mostly belonging to the Proteobacteria phylum.

Complete Endogenous Retrovirus Genome Sequence from a Brazilian Vampire Bat (Desmodus rotundus).

The strain Desmodus rotundus endogenous retrovirus (DrERV) QR09 was obtained from a bat tissue sample collected from Desmodus rotundus in the Brazilian rain forest. The complete genome was sequenced using the next-generation sequencing strategy. The full-length genome of DrERV QR09 is 8,256 nucleotides in length and showed high similarity with other DrERVs.

First isolation of West Nile virus in Brazil.

BACKGROUND: Serological evidence of West Nile virus (WNV) infection has been reported in different regions of Brazil from equine and human hosts but the virus had never been isolated in the country. OBJECTIVES: We sought to identify the viral etiology of equine encephalitis in Espírito Santo state. METHODS: We performed viral culture in C6/36 cells, molecular detection of WNV genome, histopathology and immunohistochemistry from horse cerebral tissue. We also carried out sequencing, phylogenetic analysis and molecular clock. FINDINGS: Histopathologic analysis from horse cerebral tissue showed injury related to encephalitis and WNV infection was confirmed by immunohistochemistry. The virus was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) from brain tissue and subsequently isolated in C6/36 cells. WNV full-length genome was sequenced showing the isolated strain belongs to lineage 1a. The molecular clock indicated that Brazilian WNV strain share the same common ancestor that were circulating in US during 2002-2005. MAIN CONCLUSIONS: Here we report the first isolation of WNV in Brazil from a horse with neurologic disease, which was clustered into lineage 1a with others US WNV strains isolated in beginning of 2000's decade.

First isolation of West Nile virus in Brazil

BACKGROUND Serological evidence of West Nile virus (WNV) infection has been reported in different regions of Brazil from equine and human hosts but the virus had never been isolated in the country. OBJECTIVES We sought to identify the viral etiology of equine encephalitis in Espírito Santo state. METHODS We performed viral culture in C6/36 cells, molecular detection of WNV genome, histopathology and immunohistochemistry from horse cerebral tissue. We also carried out sequencing, phylogenetic analysis and molecular clock. FINDINGS Histopathologic analysis from horse cerebral tissue showed injury related to encephalitis and WNV infection was confirmed by immunohistochemistry. The virus was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) from brain tissue and subsequently isolated in C6/36 cells. WNV full-length genome was sequenced showing the isolated strain belongs to lineage 1a. The molecular clock indicated that Brazilian WNV strain share the same common ancestor that were circulating in US during 2002-2005. MAIN CONCLUSIONS Here we report the first isolation of WNV in Brazil from a horse with neurologic disease, which was clustered into lineage 1a with others US WNV strains isolated in beginning of 2000's decade.

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.

Rabies Virus in Bats, State of Pará, Brazil, 2005-2011.

Rabies is an acute, progressive zoonotic viral infection that in general produces a fatal outcome. This disease is responsible for deaths in humans and animals worldwide and, because it can affect all mammals, is considered one of the most important viral infections for public health. This study aimed to determine the prevalence of rabies in bats of different species found in municipalities of the state of Pará from 2005 to 2011. The rabies virus was detected in 12 (0.39%) bats in a total of 3100 analyzed, including hematophagous, frugivorous, and insectivorous bats. Of these, eleven were characterized as AgV3, which is characteristic of the hematophagous bat Desmodus rotundus (E. Geoffroy 1810); one insectivorous animal showed a different profile compatible with the Eptesicus pattern and may therefore be a new antigenic variant. This study identified the need for greater intensification of epidemiological surveillance in municipalities lacking rabies surveillance (silent areas); studies of rabies virus in bats with different alimentary habits, studies investigating the prevalence of AgV3, and prophylactic measures in areas where humans may be infected are also needed.

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.

Prevalence of antibodies to equine alphaviruses in the State of Pará, Brazil / Prevalência de anticorpos para alphavirus em equinos do estado do Pará, Brasil

The State of Pará comprises 26% of Brazilian Amazon region, where a large diversity of arboviruses has been described. This study sought to assess the prevalence and distribution of hemagglutination inhibition (HI) antibodies against antigens of four alphaviruses (Togaviridae: Alphavirus ) from the species: Eastern equine encephalitis (EEEV), Western equine encephalitis (WEEV), Mayaro virus (MAYV), and Mucambo virus (MUCV) in 753 serum samples of horses in Pará State, Brazil. All investigated arboviruses were detected and indicate that horses are susceptible to these alphaviruses, and show evidences of their active circulation in farm animals in the Brazilian Amazon.(AU)

O estado do Pará corresponde a 26% da Amazônia brasileira, onde uma grande diversidade de arbovírus foi descrita. Este estudo procurou avaliar a prevalência e a distribuição de anticorpos inibidores da hemaglutinação (IH) contra antígenos de quatro alfavirus (Togaviridae: Alphavirus ), das espécies: Vírus da encefalite equina do leste (EEEV), Vírus da encefalite equina do oeste (WEEV), Vírus mayaro (MAYV) e Vírus mucambo (MUCV), de 753 amostras de soro de equinos no estado do Pará, Brasil. Todos os arbovirus pesquisados foram detectados, indicando que os equinos são suscetíveis a esses Alphavirus e mostrando evidências de sua circulação ativa em animais de fazenda na Amazônia brasileira.(AU)

Diagnosis of arboviruses using indirect sandwich IgG ELISA in horses from the Brazilian Amazon.

BACKGROUND: The Amazon as a whole is the largest reservoir of arboviruses worldwide, while the Brazilian Amazon hosts the largest variety of arboviruses isolated to date. In this study, the results of an indirect sandwich IgG ELISA, standardized for 19 arbovirustypes circulating among horses in Brazilian Amazon, were compared to results of the hemagglutination inhibition test. A screening test assessed the conditional probability distribution and a Pearson linear correlation test determined the correlation strength among the absorbance values recorded for viruses from the same family. FINDINGS: Sensitivity varied between 40.85 and 100%; the specificity was low and ranged from 39.71 to 67.0%; and the accuracy varied between 41 and 65.2%. The test developed in this study yielded a large number of serological cross-reactions. CONCLUSIONS: The test can be employed to detect IgG antibodies within one arbovirus family; however, the hemagglutination test or other more specific techniques, such as the serum neutralization test in mice or the plaque-reduction neutralization test, are essential complementary methods for positive cases.

Diagnosis of arboviruses using indirect sandwich IgG ELISA in horses from the Brazilian Amazon

The Amazon as a whole is the largest reservoir of arboviruses worldwide, while the Brazilian Amazon hosts the largest variety of arboviruses isolated to date. In this study, the results of an indirect sandwich IgG ELISA, standardized for 19 arbovirustypes circulating among horses in Brazilian Amazon, were compared to results of the hemagglutination inhibition test. A screening test assessed the conditional probability distribution and a Pearson linear correlation test determined the correlation strength among the absorbance values recorded for viruses from the same family.