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).

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.

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.

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.

Discovery and genome sequencing of a new virus related to members of the family Tymoviridae, isolated from mosquitoes of the genus Mansonia in Brazil.

A new virus, named Mutum virus, related to members of the family Tymoviridae, was isolated from mosquitoes (Mansonia spp.) in clone C6/36 cells, and its complete genome was sequenced. Its genome is 6494 nt in size with an organization resembling that of tymovirids. The isolated virus is phylogenetically related to two viruses isolated from Culex spp. mosquitoes: Ek Balam virus, reported in Mexico, and Culex-originated Tymoviridae-like virus, isolated in China. The results of this study suggest that this virus is a new member of the family Tymoviridae.

Description of the mitogenome and phylogeny of Aedes spp. (Diptera: Culicidae) from the Amazon region.

The genus Aedes (Diptera: Culicidae) includes species of great epidemiological relevance, particularly involved in transmission cycles of leading arboviruses in the Brazilian Amazon region, such as the Zika virus (ZIKV), Dengue virus (DENV), Yellow fever virus (YFV), and Chikungunya virus (CHIKV). We report here the first putatively complete sequencing of the mitochondrial genomes of Brazilian populations of the species Aedes albopictus, Aedes scapularis and Aedes serratus. The sequences obtained showed an average length of 14,947 bp, comprising 37 functional subunits, typical in animal mitochondria (13 PCGs, 22 tRNA, and 2 rRNA). The phylogeny reconstructed by Maximum likelihood method, based on the concatenated sequences of all 13 PCGs produced at least two non-directly related groupings, composed of representatives of the subgenus Ochlerotatus and Stegomyia of the genus Aedes. The data and information produced here may be useful for future taxonomic and evolutionary studies of the genus Aedes, as well as the Culicidae family.

Description of mitochon genome and phylogenetic considerations of Sabethes bipartipes, Sabethes cyaneus, Sabethes quasicyaneus, and Sabethes tarsopus (Diptera: Culicidae).

The genus Sabethes (Diptera: Culicidae) comprises species of great epidemiological relevance, particularly involved in transmission cycles of the Yellow fever virus in South America. Given the unavailability of information related to aspects of evolutionary biology and molecular taxonomy of species of this genus of mosquitoes, we report here the first sequencing of the mitochondrial genomes of Sabethes bipartipes, Sabethes cyaneus, Sabethes tarsopus, and Sabethes quasicyaneus. The sequences obtained showed an average length of 14,920 bp, comprising 37 functional genes (13 PCGs, 22 tRNA, and 02 rRNA). The phylogenies reconstructed by Maximum likelihood and Bayesian inference methods, based on the concatenated sequences of all 13 PCGs, produced similar topologies and strongly supported the monophyletic relationship between the Sabethes subgenera, corroborating the known taxonomic classification based on aspects of the external morphology of the taxa assessed. The data and information produced from the Sabethes species evaluated here may be useful for future taxonomic and evolutionary studies of the genus, as well as the Culicidae family.

Was It Chikungunya? Laboratorial and Clinical Investigations of Cases Occurred during a Triple Arboviruses' Outbreak in Rio de Janeiro, Brazil.

The co-circulation of chikungunya virus (CHIKV), dengue virus (DENV) and Zika virus (ZIKV) in Rio de Janeiro (RJ), Brazil, caused a challenging triple epidemic, as they share similar clinical signs and symptoms and geographical distribution. Here, we aimed to investigate the clinical and laboratorial aspects of chikungunya suspected cases assisted in RJ during the 2018 outbreak, focusing on the differential diagnosis with dengue and zika. All suspected cases were submitted to molecular and/or serological differential diagnostic approaches to arboviruses. A total of 242 cases suspected of arbovirus infection were investigated and 73.6% (178/242) were molecular and/or serologically confirmed as chikungunya. In RT-qPCR confirmed cases, cycle threshold (Ct) values ranged from 15.46 to 35.13, with acute cases presenting lower values. Chikungunya cases were mainly in females (64%) and the most frequently affected age group was adults between 46 to 59 years old (27%). Polyarthralgia affected 89% of patients, especially in hands and feet. No dengue virus (DENV) and Zika virus (ZIKV) infections were confirmed by molecular diagnosis, but 9.5% (23/242) had serological evidence of DENV exposure by the detection of specific anti-DENV IgM or NS1, and 42.7% (76/178) of chikungunya positive cases also presented recent DENV exposure reflected by a positive anti-DENV IgM or NS1 result. A significantly higher frequency of arthritis (p = 0.023) and limb edema (p < 0.001) was found on patients with CHIKV monoinfection compared to dengue patients and patients exposed to both viruses. Lastly, phylogenetic analysis showed that the chikungunya cases were caused by the ECSA genotype. Despite the triple arboviruses' epidemic in the state of RJ, most patients with fever and arthralgia investigated here were diagnosed as chikungunya cases, and the incidence of CHIKV/DENV co-detection was higher than that reported in other studies.

Negeviruses isolated from mosquitoes in the Brazilian Amazon.

BACKGROUND: There are several groups of viruses including Insect Specific Viruses (ISV) such as the taxon Negevirus, a group of viruses phylogenetically related to plant viruses. Negeviruses replicate in mosquito cells, but not in vertebrate cells. METHODS: Pools of hematophagous arthropods were inoculated in Vero and C6/36 cells. The cells were observed to detect possible cytopathic effect. Then, indirect immunofluorescence, RT-PCR, and nucleotide sequencing were performed. RESULTS: Seven samples which presented negative results for flaviviruses, alphaviruses and bunyaviruses, but showed cytopathic effect in C6/36 cells were sequenced. We identified the occurrence of a variety of ISVs, most of them belonging to the taxon Negevirus: The Brejeira, Negev, Cordoba and Wallerfield viruses, including a new virus for science, tentatively named Feitosa virus. CONCLUSIONS: We detected negeviruses in the Amazon region, including two viruses that were isolated for the first time in Brazil: Cordoba virus and the Negev virus and, a new virus for science: the Feitosa virus.

Whole-Genome Sequencing of Leishmania infantum chagasi Isolates from Honduras and Brazil.

This work reports on the whole-genome sequencing of Leishmania infantum chagasi from Honduras (Central America) and Brazil (South America).

First Description of the Mitogenome and Phylogeny of Culicinae Species from the Amazon Region.

The Culicidae family is distributed worldwide and comprises about 3587 species subdivided into the subfamilies Anophelinae and Culicinae. This is the first description of complete mitochondrial DNA sequences from Aedes fluviatilis, Aedeomyia squamipennis, Coquillettidia nigricans, Psorophora albipes, and Psorophora ferox. The mitogenomes showed an average length of 15,046 pb and 78.02% AT content, comprising 37 functional subunits (13 protein coding genes, 22 tRNAs, and two rRNAs). The most common start codons were ATT/ATG, and TAA was the stop codon for all PCGs. The tRNAs had the typical leaf clover structure, except tRNASer1. Phylogeny was inferred by analyzing the 13 PCGs concatenated nucleotide sequences of 48 mitogenomes. Maximum likelihood and Bayesian inference analysis placed Ps. albipes and Ps. ferox in the Janthinosoma group, like the accepted classification of Psorophora genus. Ae. fluviatilis was placed in the Aedini tribe, but was revealed to be more related to the Haemagogus genus, a result that may have been hampered by the poor sampling of Aedes sequences. Cq. nigricans clustered with Cq. chrysonotum, both related to Mansonia. Ae. squamipennis was placed as the most external lineage of the Culicinae subfamily. The yielded topology supports the concept of monophyly of all groups and ratifies the current taxonomic classification.

Whole-Genome Sequencing of Alcaligenes faecalis HZ01, with Potential to Inhibit Nontuberculous Mycobacterial Growth.

Alcaligenes faecalis is a Gram-negative rod that is ubiquitous in the environment and is an opportunistic human pathogen. Here, we report the whole-genome sequencing analysis of A. faecalis HZ01, which presents mycobacterial growth inhibitory activity and was isolated from a contaminated culture of Mycobacterium chubuense ATCC 27278.

Mitochondrial genome sequencing and phylogeny of Haemagogus albomaculatus, Haemagogus leucocelaenus, Haemagogus spegazzinii, and Haemagogus tropicalis (Diptera: Culicidae).

The genus Haemagogus (Diptera: Culicidae) comprises species of great epidemiological relevance, involved in transmission cycles of the Yellow fever virus and other arboviruses in South America. So far, only Haemagogus janthinomys has complete mitochondrial sequences available. Given the unavailability of information related to aspects of the evolutionary biology and molecular taxonomy of this genus, we report here, the first sequencing of the mitogenomes of Haemagogus albomaculatus, Haemagogus leucocelaenus, Haemagogus spegazzinii, and Haemagogus tropicalis. The mitogenomes showed an average length of 15,038 bp, average AT content of 79.3%, positive AT-skews, negative GC-skews, and comprised 37 functional subunits (13 PCGs, 22 tRNA, and 02 rRNA). The PCGs showed ATN as start codon, TAA as stop codon, and signs of purifying selection. The tRNAs had the typical leaf clover structure, except tRNASer1. Phylogenetic analyzes of Bayesian inference and Maximum Likelihood, based on concatenated sequences from all 13 PCGs, produced identical topologies and strongly supported the monophyletic relationship between the Haemagogus and Conopostegus subgenera, and corroborated with the known taxonomic classification of the evaluated taxa, based on external morphological aspects. The information produced on the mitogenomes of the Haemagogus species evaluated here may be useful in carrying out future taxonomic and evolutionary studies of the genus.

Characterization of Mycobacterium tuberculosis var. africanum isolated from a patient with pulmonary tuberculosis in Brazil.

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC), including Mycobacterium tuberculosis var. tuberculosis (MTB) and Mycobacterium tuberculosis var. africanum (MAF). While MTB is isolated worldwide, MAF is almost completely restricted to the African continent, and despite the historical proximity between Brazil and Africa during the slave trade, no case of TB being caused by MAF has been reported in Brazil to date. We hereby describe the first case of TB caused by MAF in Brazil comparing its genome against the published ones. A female patient who had never visited Africa presented with clinical symptoms typical of pulmonary TB. Based on 16S rRNA gene sequencing, the cultured isolate was identified as belonging to MTBC and partial sequence of the hsp65 gene was identical to that of MAF. This was confirmed by genotyping based on detection of Single Nucleotide Polymorphism (SNP), Region of Difference (RD) and spoligotyping. The isolate presented the Shared International Typing (SIT) 181. In the whole-genome comparison against MAF genomes available on published EMBL-EBI European Nucleotide Archive (ENA), the Brazilian genome (MAFBRA00707) was identified as belonging to Lineage 6 and clustered with isolates from The Gambia. This is the first report of the isolation of MAF from a patient from Brazil, without evidence of having any contact with an African index case.

Evolutionary Dynamics and Dissemination Pattern of the SARS-CoV-2 Lineage B.1.1.33 During the Early Pandemic Phase in Brazil.

A previous study demonstrates that most of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Brazilian strains fell in three local clades that were introduced from Europe around late February 2020. Here we investigated in more detail the origin of the major and most widely disseminated SARS-CoV-2 Brazilian lineage B.1.1.33. We recovered 190 whole viral genomes collected from 13 Brazilian states from February 29 to April 31, 2020 and combined them with other B.1.1 genomes collected globally. Our genomic survey confirms that lineage B.1.1.33 is responsible for a variable fraction of the community viral transmissions in Brazilian states, ranging from 2% of all SARS-CoV-2 genomes from Pernambuco to 80% of those from Rio de Janeiro. We detected a moderate prevalence (5-18%) of lineage B.1.1.33 in some South American countries and a very low prevalence (<1%) in North America, Europe, and Oceania. Our study reveals that lineage B.1.1.33 evolved from an ancestral clade, here designated B.1.1.33-like, that carries one of the two B.1.1.33 synapomorphic mutations. The B.1.1.33-like lineage may have been introduced from Europe or arose in Brazil in early February 2020 and a few weeks later gave origin to the lineage B.1.1.33. These SARS-CoV-2 lineages probably circulated during February 2020 and reached all Brazilian regions and multiple countries around the world by mid-March, before the implementation of air travel restrictions in Brazil. Our phylodynamic analysis also indicates that public health interventions were partially effective to control the expansion of lineage B.1.1.33 in Rio de Janeiro because its median effective reproductive number (R e ) was drastically reduced by about 66% during March 2020, but failed to bring it to below one. Continuous genomic surveillance of lineage B.1.1.33 might provide valuable information about epidemic dynamics and the effectiveness of public health interventions in some Brazilian states.

Description and phylogeny of the mitochondrial genome of Sabethes chloropterus, Sabethes glaucodaemon and Sabethes belisarioi (Diptera: Culicidae).

Mosquitoes as Sabethes chloropterus, Sabethes glaucodaemon, Sabethes belisarioi are species of medical and epidemiological importance for arboviruses transmission such as yellow fever and St. Louis encephalitis. Despite this, no information about these three species mitochondrial DNA has been found in literature. Our study presents a mitochondrial genome description, including identity, SNPs, mutation rate, and phylogeny analysis using COX1, COX2, NADH4, NADH5, CYOB genes. The Sa. chloropterus, Sa. glaucodaemon and Sa. belisaroi mitochondrial genome sizes 15.609 bp, 15.620 bp, 15.907 bp, respectively, with 37 functional genes, presenting about 4.982 single nucleotide polymorphisms and 13.291 identical sites between them, besides all genes with dN/dS < 1 ratio, and also a greater approximation between Sa. glaucodaemon and Sa. chloropterus than with Sa. belisarioi. Due to the importance of mitochondrial DNA for population structure studies, evolution, and others, we expect that this data can contribute to other studies related to these mosquitoes and their viruses.