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).

Ilheus Virus (ILHV) Resistance in Culex quinquefasciatus from the Northern Region of Brazil.

BACKGROUND: Orthoflavivirus ilheusense (ILHV) is a member of the Flaviviridae family. It was first isolated in 1944 from pools of Aedes serratus and Psorophora ferox mosquitoes; however, it has also been detected in species of the genus Culex, such as Cx. portesi and Cx. coronator. The objective of this study was to examine the vector competence of Cx. quinquefasciatus mosquitoes to ILHV infection and the subsequent transmission of the virus through their saliva during feeding on blood. METHODS: F1 generation females of Cx. quinquefasciatus (Ananindeua/PA) were orally infected with goose blood infected with strain BeH7445, and body, head and saliva samples were analyzed at 7, 14, and 21 dpi using the techniques of virus isolation in cells and indirect immunofluorescence. RESULTS: The presence of ILHV was not detected in the body and head samples of Cx. quinquefasciatus females at any of the three dpi's analyzed, indicating that the lineage of mosquitoes analyzed was resistant to ILHV. CONCLUSIONS: According to the results obtained in this study, the species Cx. quinquefasciatus proved resistant to ILHV, regardless of the virus titers to which it was exposed, which suggests the possibility that this species does not act as a vector in the ILHV transmission cycle.

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.

Genus Culex Linnaeus, 1758 (Diptera: Culicidae) as an Important Potential Arbovirus Vector in Brazil: An Integrative Review.

The genus Culex has 817 species subdivided into 28 subgenera. It has a cosmopolitan distribution, being most abundant in countries with a tropical climate. Understanding the ecology and diversity of viruses circulating in the species of this genus is important for understanding their role as arbovirus vectors in Brazil. To conduct an integrative review to identify the importance of the Culex genus as arbovirus vectors in Brazil. A search was carried out for scientific papers in the PubMed, BVSalud, Patuá-IEC and International Catalogue of Arboviruses: including certain other viruses of vertebrates databases. 36 publications describing arbovirus detections in Culex mosquitoes collected in the field in Brazil were evaluated. A total of 42 arbovirus species were detected, as well as studies analyzing the vector competence of C. quinquefasciatus for the transmission of four different arboviruses. The study of the Culex genus and its role as a vector of arboviruses in Brazil is essential for understanding transmission cycles, with the main aim of reducing cases of human infection. Thus, entomovirological surveillance guides the implementation of actions to detect circulating arboviruses among vectors to anticipate measures aimed at preventing or reducing the risk of arbovirus outbreaks in the country.

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.

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 Culex quinquefasciatus from Brazil for West Nile Virus.

West Nile virus is characterized as a neurotropic pathogen, which can cause West Nile fever and is transmitted by mosquitoes of the genus Culex. In 2018, the Instituto Evandro Chagas performed the first isolation of a WNV strain in Brazil from a horse brain sample. The present study aimed to evaluate the susceptibility of orally infected Cx. quinquefasciatus from the Amazon region of Brazil to become infected and transmit the WNV strain isolated in 2018. Oral infection was performed with blood meal artificially infected with WNV, followed by analysis of infection, dissemination, and transmission rates, as well as viral titers of body, head, and saliva samples. At the 21st dpi, the infection rate was 100%, the dissemination rate was 80%, and the transmission rate was 77%. These results indicate that Cx. quinquefasciatus is susceptible to oral infection by the Brazilian strain of WNV and may act as a possible vector of the virus since it was detected in saliva from the 21st dpi.

First Isolation and Genome Sequence Analysis of West Nile Virus in Mosquitoes in Brazil.

West Nile virus is a flavivirus transmitted by mosquitoes, mainly of the genus Culex. In Brazil, serological studies have already indicated the circulation of the virus since 2003, with the first human case detected in 2014. The objective of the present paper is to report the first isolation of WNV in a Culex (Melanoconion) mosquito. Arthropods were collected by protected human attraction and CDC light bait, and taxonomically identified and analyzed by viral isolation, complement fixation and genomic sequencing tests. WNV was isolated from samples of Culex (Melanoconion) mosquitoes, and the sequencing analysis demonstrated that the isolated strain belonged to lineage 1a. The finding of the present study presents the first evidence of the isolation and genome sequencing of WNV in arthropods 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.

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.

Serum Soluble Mediator Profiles and Networks During Acute Infection With Distinct DENV Serotypes.

A panoramic analysis of chemokines, pro-inflammatory/regulatory cytokines, and growth factors was performed in serum samples from patients with acute DENV infection (n=317) by a high-throughput microbeads array. Most soluble mediators analyzed were increased in DENV patients regardless of the DENV serotype. The substantial increase (≥10-fold) of CXCL10, IL-6, and IFN-γ, and decreased levels of PDGF (<0.4-fold) was universally identified in all DENV serotypes. Of note, increased levels of CXCL8, CCL4, and IL-12 (≥3-9-fold) were selectively observed in DENV2 as compared to DENV1 and DENV4. Heatmap and biomarker signatures further illustrated the massive release of soluble mediators observed in DENV patients, confirming the marked increase of several soluble mediators in DENV2. Integrative correlation matrices and networks showed that DENV infection exhibited higher connectivity among soluble mediators. Of note, DENV2 displayed a more complex network, with higher connectivity involving a higher number of soluble mediators. The timeline kinetics (Day 0-1, D2, D3, D4-6) analysis additionally demonstrated differences among DENV serotypes. While DENV1 triggers a progressive increase of soluble mediators towards D3 and with a decline at D4-6, DENV2 and DENV4 develop with a progressive increase towards D4-6 with an early plateau observed in DENV4. Overall, our results provided a comprehensive overview of the immune response elicited by DENV infection, revealing that infection with distinct DENV serotypes causes distinct profiles, rhythms, and dynamic network connectivity of soluble mediators. Altogether, these findings may provide novel insights to understand the pathogenesis of acute infection with distinct DENV serotypes.

Natural Infection of Aedes aegypti by Chikungunya and Dengue type 2 Virus in a Transition Area of North-Northeast Brazil.

Dengue fever, chikungunya, and Zika are diseases caused by viruses transmitted by Aedes aegypti and Aedes albopictus. In Brazil, the number of human infections is high, but few studies are performed in mosquito vectors. This study aimed to investigate the presence of Zika, Dengue and Chikungunya viruses in Ae. aegypti and Ae. albopictus from the municipalities of Alto Alegre, Caxias, Codó, and São Mateus do Maranhão, located in the state of Maranhão, Northeast Brazil. The mosquitoes were collected with a mechanical aspirator, identified, triturated, and then submitted to RNA extraction and RT-qPCR. The positive samples were confirmed by virus isolation and genome sequencing. Three hundred and forty-eight Ae. aegypti (176 males and 172 females) and 12 Ae. albopictus (eight males and four females) were collected and tested. Ae. aegypti was the only vector positive in two municipalities-Codó, with detection of Chikungunya virus (CHIKV) belonging to the East-Central-South African genotype, and in Caxias, with detection of Dengue virus (DENV)-2 belonging to the Asian/American genotype. The detection of CHIKV and DENV-2 is evidence that those viruses are maintained in arthropod vectors, and shows the epidemiological risk in the area for chikungunya cases and a possible increase of severe dengue cases, associated with the occurrence of dengue hemorrhagic fever.

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.

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

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

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

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

Circulation of Chikungunya virus in Aedes aegypti in Maranhão, Northeast Brazil.

The simultaneous circulation of Dengue virus (DENV), Chikungunya virus (CHIKV) and Zika virus (ZIKV) arboviruses have placed Brazil among the main worldwide endemic areas. Brazilian Northeast region concentrates the highest incidence of infections caused by CHIKV and ZIKV. In Maranhão, the second biggest northeastern state, there are cases of human infections caused by these three arboviruses and presence of Aedes aegypti and Aedes albopictus vectors. In this context, this study aimed to investigate the circulation of CHIKV, DENV and ZIKV in Ae. aegypti and Ae. albopictus mosquitoes collected in urban areas of Barra do Corda, Caxias, Codó, São Luís and São Mateus do Maranhão municipalities in the state of Maranhão through Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) technique. 428 Ae. aegypti and 1 Ae. albopictus were collected, which formed 44 pools. Three of these showed positive results for CHIKV: AR832767 (five Ae. aegypti female collected in Caxias), AR832784 and AR832785 (both composed of 20 Ae. aegypti female collected in São Mateus do Maranhão). This study consolidates information about CHIKV circulation in state of Maranhão, as well as the role of Ae. aegypti in the transmission of CHIKV in urban area.

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

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

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

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