Isolation and sequencing of Orthoflavivirus ilheusense from mosquitoes collected in the Brazilian cerrado, West-Central region, Brazil.

The Orthoflavivirus ilheusense (ILHV) is an arbovirus that was first isolated in Brazil in 1944 during an epidemiologic investigation of yellow fever. Is a member of the Flaviviridae family and it belongs to the antigenic complex of the Ntaya virus group. Psorophora ferox is the primary vector of ILHV and this study presents the isolation and phylogenetic analysis of ILHV in a pool of Ps. ferox collected in the state of Goiás in 2021. Viral isolation tests were performed on Vero cells and C6/36 clones. The indirect immunofluorescence test (IFI) was used to confirm the positivity of the sample. The positive sample underwent RT-qPCR, sequencing, and phylogenetic analysis. This is the first report of ILHV circulation in this municipality and presented close relationship between this isolate and another ILHV isolate collected in the city of Belém (PA).

Evaluation of Multiple RNA Extraction Protocols for Chikungunya Virus Screening in Aedes aegypti Mosquitoes.

Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the virus's diagnosis and surveillance. Most molecular biology methodologies use nucleic acid extraction as the first step and require quality RNA for their execution. In this context, four RNA extraction protocols were evaluated in Ae. aegypti experimentally infected with CHIKV. Six pools were tested in triplicates (n = 18), each containing 1, 5, 10, 20, 30, or 40 mosquitoes per pool (72 tests). Four commercial kits were compared: QIAamp®, Maxwell®, PureLink®, and PureLink® with TRIzol®. The QIAamp® and PureLink® with TRIzol® kits had greater sensitivity. Two negative correlations were observed: as the number of mosquitoes per pool increases, the Ct value decreases, with a higher viral load. Significant differences were found when comparing the purity and concentration of RNA. The QIAamp® protocol performed better when it came to lower Ct values and higher RNA purity and concentration. These results may provide help in CHIKV entomovirological surveillance planning.

Genomic characterization of a novel Hepatovirus identified in Maranhão state, Brazil.

Bats are efficient reservoirs of a number of viruses with zoonotic potential, and are involved directly in the transmission cycle of many zoonoses. In the present study, which is part of a larger project that is documenting the viromes of the bat species found in the Mid-North states of Maranhão and Piauí, we analyzed 16 pooled samples obtained from four species of bat of the genus Artibeus-Artibeus obscurus, Artibeus cinereus, Artibeus lituratus and Artibeus planirostris. We describe and identify a Hepatovirus, denominated Hepatovirus H isolate sotense, which was found in a pool of internal organs (liver and lungs) extracted from a specimen of A. planirostris, a frugivorous bat, collected in the Cerrado biome of Maranhão state. This material was analyzed using new generation sequencing, which produced a contig of 7390 nucleotides and presented a degree of identity with a number of existing Hepatovirus sequences available for bats (amino acid identity of 61.5% with Bat hepatovirus C of Miniopterus cf. manavi, 66.6% with Bat hepatovirus G of Coleura afra, 67.4% with Hepatovirus G2 of Rhinolophus landeri, and 75.3% with Hepatovirus H2 of Rhinolophus landeri). The analysis of the functional domains of this contig confirmed a pattern consistent with the characteristics of the genus Hepatovirus (Picornaviridae). In the phylogenetic tree with several other Hepatovirus species, this genome also grouped in a monophyletic clade with Hepatovirus H (HepV-H1; HepV-H2, and HepV-H3) albeit on an external branch, which suggests that it may be a distinct genotype within this species. This is the first isolate of Hepatovirus H identified in bats from South America, and represents an important discovery, given that most studies of viruses associated with bats in the state of Maranhão have focused on the family Rhabdoviridae.

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.

A novel hantavirus identified in bats (Carollia perspicillata) in Brazil.

Bats play an essential role in maintaining ecosystems. Their unique characteristics increase the likelihood of interactions with various species, making them a potential source for the emergence and spread of infectious diseases. Hantaviruses are continuously expanding their range of hosts. This study presents the identification of a partial genome associated with Hantavirus in samples collected from neotropical bats. We conducted a metagenomic study using samples from Carollia perspicillata in Maranhão, Brazil. Tissue fragments were used for RNA extraction and subsequent sequencing. The resulting data was subjected to bioinformatic analysis. A sequence showing an identity of 72.86% with the L gene in the reference genome was obtained. The phylogenetic analysis revealed the study sequence, denoted as Buritiense, clustering within the Mobatvirus clade. The intragroup analysis showed a broader dispersion and were markedly asymmetric. This observation suggests the possibility that Buritiense could potentially represent a new species within the bat-borne hantaviruses, but further analyses are needed to provide additional insights if bats plays a role as reservoirs and the potential for transmission to human populations.

Viral Interference between the Insect-Specific Virus Brejeira and the Saint Louis Encephalitis Virus In Vitro.

The Saint Louis encephalitis virus (SLEV) is an encephalitogenic arbovirus (Flaviviridae family) that has a wide geographical distribution in the western hemisphere, especially in the Americas. The negevirus Brejeira (BREV) was isolated for the first time in Brazil in 2005. This study aimed to verify the existence of a possible interfering effect of BREV on the course of SLEV infection and vice versa. We used clone C6/36 cells. Three combinations of MOIs were used (SLEV 0.1 × BREV 1; SLEV 1 × BREV 0.1; SLEV 1 × BREV 1) in the kinetics of up to 7 days and then the techniques of indirect immunofluorescence (IFA), a plaque assay on Vero cells, and RT-PCR were performed. Our results showed that the cytopathic effect (CPE) caused by BREV was more pronounced than the CPE caused by SLEV. Results of IFA, the plaque assay, and RT-PCR showed the suppression of SLEV replication in the co-infection condition in all the MOI combinations used. The SLEV suppression was dose-dependent. Therefore, the ISV Brejeira can suppress SLEV replication in Aedes albopictus cells, but SLEV does not negatively interfere with BREV replication.

Detection of a Multiple Circulation Event of Dengue Virus 2 Strains in the Northern Region of Brazil.

Dengue virus serotype 2 (DENV-2) is responsible for dengue epidemics on a global scale and is associated with severe cases of the disease. This study conducted a phylogenetic investigation of DENV-2 isolates from 2017 to 2021 originating from the northern states of Brazil. A total of 32 samples from DENV-2 isolates were analyzed, including 12 from Acre, 19 from Roraima, and one from Tocantins. Only one lineage of the Asian-American genotype and one lineage of the cosmopolitan genotype were observed: Lineage 1, Asian-American genotype (connection to Puerto Rico); Lineage 5, cosmopolitan genotype (connection to Peru). Our results provide important data regarding the study of DENV genotypes and lineage distribution and open up possibilities for probable introduction and dissemination routes.

Virome analysis of Desmodus rotundus tissue samples from the Amazon region.

BACKGROUND: Bats are renowned for harboring a high viral diversity, their characteristics contribute to emerging infectious diseases. However, environmental and anthropic factors also play a significant role in the emergence of zoonotic viruses. Metagenomic is an important tool for investigating the virome of bats and discovering new viruses. RESULTS: Twenty-four families of virus were detected in lung samples by sequencing and bioinfomatic analysis, the largest amount of reads was focused on the Retroviridae and contigs assembled to Desmodus rotundus endogenous retrovirus, which was feasible to acquire complete sequences. The reads were also abundant for phages. CONCLUSION: This lung virome of D. rotundus contributes valuable information regarding the viral diversity found in bats, which is useful for understanding the drivers of viral cycles and their ecology in this species. The identification and taxonomic categorization of viruses hosted by bats carry epidemiological significance due to the potential for viral adaptation to other animals and humans, which can have severe repercussions for public health. Furthermore, the characterization of endogenized viruses helps to understanding the host genome and the evolution of the species.

Sequencing and Analysis of the Mitochondrial Genome of Aedes aegypti (Diptera: Culicidae) from the Brazilian Amazon Region.

Aedes aegypti is a mosquito native to the African continent, which is now widespread in the tropical and subtropical regions of the world. In many regions, it represents a major challenge to public health, given its role in the cycle of transmission of important arboviruses, such as Dengue, Zika, and Chikungunya. Considering the epidemiological importance of Ae. aegypti, the present study sequenced the partial mitochondrial genome of a sample collected in the municipality of Balsas, in the Brazilian state of Maranhão, followed by High Throughput Sequencing and phylogenetic analyses. The mitochondrial sequence obtained here was 15,863 bp long, and contained 37 functional subunits (thirteen PCGs, twenty-two tRNAs and two rRNAs) in addition to a partial final portion rich in A+T. The data obtained here contribute to the enrichment of our knowledge of the taxonomy and evolutionary biology of this prominent disease vector. These findings represent an important advancement in the understanding of the characteristics of the populations of northeastern Brazil and provide valuable insights into the taxonomy and evolutionary biology of this prominent disease vector.

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.

Dengue virus serotype 2 genotype III evolution during the 2019 outbreak in Mato Grosso, Midwestern Brazil.

DENV-2 was the main responsible for a 70% increase in dengue incidence in Brazil during 2019. That year, our metagenomic study by Illumina NextSeq on serum samples from acute febrile patients (n = 92) with suspected arbovirus infection, sampled in 22 cities of the state of Mato Grosso (MT), in the middle west of Brazil, revealed eight complete genomes and two near-complete sequences of DENV-2 genotype III, one Human parvovirus B19 genotype I (5,391 nt) and one Coxsackievirus A6 lineage D (4,514 nt). These DENV-2 sequences share the aminoacidic identities of BR4 lineage on E protein domains I, II and III, and were included in a clade with sequences of the same lineage circulating in the southeast of Brazil in the same year. Nevertheless, 11/34 non-synonymous mutations are unique to three strains inthis study, distributed in the E (n = 6), NS3 (n = 2) and NS5 (n = 3) proteins. Other 14 aa changes on C (n = 1), E (n = 3), NS1 (n = 2), NS2A (n = 1) and NS5 (n = 7) were first reported in a genotype III lineage, having been already reported only in other DENV-2 genotypes. All 10 sequences have mutations in the NS5 protein (14 different aa changes). Nine E protein aa changes found in two sequences, six of which are unique, are in the ectodomain; where the E:M272T change is on the hinge of the E protein at domain II, in a region critical for the anchoring to the host cell receptor. The NS5:G81R mutation, in the methyltransferase domain, was found in one strain of this study. Altogether, these data points to an important evolution of DENV-2 genotype III lineage BR4 during this outbreak in 2019 in MT. Genomic surveillance is essential to detect virus etiology and evolution, possibly related to immune evasion and viral fitness changes leading to future novel outbreaks.

Shotgun Metagenomic Sequencing Reveals Virome Composition of Mosquitoes from a Transition Ecosystem of North-Northeast Brazil.

A wide diversity of pathogenic mosquito-borne viruses circulate in the Brazilian Amazon, and the intense deforestation can contribute to the spread of these viruses. In this context, this study aimed to investigate the viral diversity in mosquitoes of the genera Aedes, Culex, Haemagogus, and Sabethes from a transition area between the Amazon, Cerrado, and Caatinga biomes in Brazil. Metagenomic high-throughput sequencing was used to characterize the virome of 20 mosquito pools. A total of 15 virus-like genomes were identified, comprising species genomically close to insect-specific viruses of the families Iflaviridae, Metaviridae, Lispiviridae, Rhabdoviridae, Xinmoviridae, and Parvoviridae and species of plant viruses of the families Solemoviridae, Virgaviridae, and Partitiviridae. However, sequences of viruses associated with human and animal diseases were not detected. Most of the recovered genomes were divergent from those previously described. These findings reveal that there are a large number of unknown viruses to be explored in the middle-north of Brazil.

Emergence of a New Strain of DENV-2 in South America: Introduction of the Cosmopolitan Genotype through the Brazilian-Peruvian Border.

Dengue virus 2 (DENV-2) seriously contributes to dengue-related mortality. It includes five nonsylvatic genotypes, with cosmopolitan being the most widespread with a significant contribution to the total number of DENV-2 cases globally. In South America, the cosmopolitan genotype was first recorded in 2019 in Madre de Dios, Peru, and then in Goiás (Midwest Brazil) in November 2021. In this study, we tested 163 human serum samples from Acre (Northern Brazil) collected during a DENV outbreak between 2020 and 2021 for all DENV genotypes by RT-qPCR. Of the 163 samples, 139 were positive for DENV-2, and 5 were positive for DENV-1. Five DENV-2-positive samples from early 2021 were sequenced, and the sequences clustered with the three other DENV-2 cosmopolitan genotype sequences already recorded on the continent. These results create a geographical link, suggesting the possible route of introduction of the DENV-2 cosmopolitan genotype into Brazil through the border with Peru, from which it may have dispersed to Midwest Brazil.

The Importance of Entomo-Virological Investigation of Yellow Fever Virus to Strengthen Surveillance in Brazil.

The largest outbreak of sylvatic yellow fever virus (YFV) in eight decades was recorded in Brazil between 2016-2018. Besides human and NHP surveillance, the entomo-virological approach is considered as a complementary tool. For this study, a total of 2904 mosquitoes of the Aedes, Haemagogus and Sabethes genera were collected from six Brazilian states (Bahia, Goiás, Mato Grosso, Minas Gerais, Pará, and Tocantins) and grouped into 246 pools, which were tested for YFV using RT-qPCR. We detected 20 positive pools from Minas Gerais, 5 from Goiás, and 1 from Bahia, including 12 of Hg. janthinomys and 5 of Ae. albopictus. This is the first description of natural YFV infection in this species and warns of the likelihood of urban YFV re-emergence with Ae. albopictus as a potential bridge vector. Three YFV sequences from Hg. janthinomys from Goiás and one from Minas Gerais, as well as one from Ae. albopictus from Minas Gerais were clustered within the 2016-2018 outbreak clade, indicating YFV spread from Midwest and its infection in a main and likely novel bridging vector species. Entomo-virological surveillance is critical for YFV monitoring in Brazil, which could highlight the need to strengthen YFV surveillance, vaccination coverage, and vector control measures.

Reemergence of Dengue Virus Serotype 3, Brazil, 2023.

We characterized 3 autochthonous dengue virus serotype 3 cases and 1 imported case from 2 states in the North and South Regions of Brazil, 15 years after Brazil's last outbreak involving this serotype. We also identified a new Asian lineage recently introduced into the Americas, raising concerns about future outbreaks.

Comparative Analysis of Human Hepatic Lesions in Dengue, Yellow Fever, and Chikungunya: Revisiting Histopathological Changes in the Light of Modern Knowledge of Cell Pathology.

Arboviruses, such as yellow fever virus (YFV), dengue virus (DENV), and chikungunya virus (CHIKV), present wide global dissemination and a pathogenic profile developed in infected individuals, from non-specific clinical conditions to severe forms, characterised by the promotion of significant lesions in different organs of the harbourer, culminating in multiple organ dysfunction. An analytical cross-sectional study was carried out via the histopathological analysis of 70 samples of liver patients, collected between 2000 and 2017, with confirmed laboratory diagnoses, who died due to infection and complications due to yellow fever (YF), dengue fever (DF), and chikungunya fever (CF), to characterise, quantify, and compare the patterns of histopathological alterations in the liver between the samples. Of the histopathological findings in the human liver samples, there was a significant difference between the control and infection groups, with a predominance of alterations in the midzonal area of the three cases analysed. Hepatic involvement in cases of YF showed a greater intensity of histopathological changes. Among the alterations evaluated, cell swelling, microvesicular steatosis, and apoptosis were classified according to the degree of tissue damage from severe to very severe. Pathological abnormalities associated with YFV, DENV, and CHIKV infections showed a predominance of changes in the midzonal area. We also noted that, among the arboviruses studied, liver involvement in cases of YFV infection was more intense.

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.

Vector Competence of Aedes albopictus for Yellow Fever Virus: Risk of Reemergence of Urban Yellow Fever in Brazil.

The risk of the emergence and reemergence of zoonoses is high in regions that are under the strong influence of anthropogenic actions, as they contribute to the risk of vector disease transmission. Yellow fever (YF) is among the main pathogenic arboviral diseases in the world, and the Culicidae Aedes albopictus has been proposed as having the potential to transmit the yellow fever virus (YFV). This mosquito inhabits both urban and wild environments, and under experimental conditions, it has been shown to be susceptible to infection by YFV. In this study, the vector competence of the mosquito Ae. albopictus for the YFV was investigated. Female Ae. albopictus were exposed to non-human primates (NHP) of the genus Callithrix infected with YFV via a needle inoculation. Subsequently, on the 14th and 21st days post-infection, the legs, heads, thorax/abdomen and saliva of the arthropods were collected and analyzed by viral isolation and molecular analysis techniques to verify the infection, dissemination and transmission. The presence of YFV was detected in the saliva samples through viral isolation and in the head, thorax/abdomen and legs both by viral isolation and by molecular detection. The susceptibility of Ae. albopictus to YFV confers a potential risk of reemergence of urban YF in Brazil.

Genomic Diversity of the Rarely Observed Genotype of the Mycobacterium tuberculosis Central Asian (CAS) Lineage 3 from North Brazil.

Mycobacterium tuberculosis (Mtb) Central Asian Strain (CAS) Lineage 3 (L3) genotype is predominantly found in East-Africa, Central-Asia, Western-Asia, and South-Asia; however, a new spoligotyping CAS/SIT2545 was found in northern regions of Brazil. We aimed to characterize and describe the genetic diversity and perform a phylogenetic assessment of this novel genotype. We performed 24-MIRU-VNTR loci and Whole-genome sequencing (WGS) of six Brazilian isolates previously spoligotyped. The libraries were prepared using a Nextera-XT kit and sequenced in a NextSeq 550 Illumina instrument. We performed lineage assignment and genomic characterization. From publicly available genomes of Mtb L3 and other lineages, we created a robust dataset to run the MTBSeq pipeline and perform a phylogenetic analysis. MIRU-VNTR and WGS confirmed CAS/SIT2545 belongs to L3. Out of 1691 genomes, 1350 (79.83%) passed in quality control (genomic coverage > 95%). Strain 431 differed in 52 single nucleotide variants (SNV), confirming it does not belong to the same transmission chain. The eight genomes from a global dataset clustered closer to Brazilian strains differed in >52 SNVs. We hypothesized L3 and L1 were introduced in Brazilian Northern in the same historical event; however, there is a need for additional studies exploring the genetic diversity of Mtb Brazilian Northern.

Sequencing and description of the complete mitochondrial genome of Limatus durhamii (Diptera: Culicidae).

The genus Limatus (Diptera: Culicidae) are wild mosquitoes belonging to the Sabethini tribe that occurs in tropical countries and is related to transmission cycles of Orthobunyavirus (Bunyaviridae), particularly in the Amazon region. Given the unavailability of information related to evolutionary biology and molecular taxonomy aspects of this genus, we report here the first complete sequencing of the mitochondrial genome of Limatus durhamii Theobald, 1901. The NextSeq 500 platform was used for sample sequencing, and the mitochondrial sequence obtained was 14,875 bp long, comprising 37 functional subunits (13 PCGs, 22 tRNA and 02 rRNA). The phylogeny reconstructed by maximum likelihood based on the concatenation of all 13 PCGs corroborated the known taxonomic classification based most on aspects of the external morphology and few molecular studies. The data and information produced here may be useful in the future development of taxonomic and evolutionary studies for the genus, as well as the Culicidae family itself.